ssc meeting in conjunction with isth 2015 toronto...
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SSC Meeting in Conjunction with ISTH 2015 Toronto Congress–
Meeting Minutes
Standing Committees
Bleeding Assessment Tool Committee ...................................................................2
Coagulation Standards Committee .........................................................................3
Subcommittees
Animal, Cellular and Molecular Models ..................................................................6
Biorheology ............................................................................................................7
Control of Anticoagulation ......................................................................................13
Disseminated Intravascular Coagulation ................................................................15
Exogenous Hemostatic Factors .............................................................................17
Factor VIII, Factor IX and Rare Coagulation Disorders ...........................................22
Factor XI and the Contact System..........................................................................27
Factor XIII and Fibrinogen ......................................................................................29
Fibrinolysis .............................................................................................................35
Genomics in Thrombosis and Hemostasis .............................................................43
Hemostasis and Malignancy ..................................................................................47
Lupus Anticoagulant/Phospholipid Dependent Antibodies ......................................48
Pediatric and Neonatal Hemostasis and Thrombosis .............................................51
Plasma Coagulation Inhibitors ................................................................................53
Platelet Immunology ..............................................................................................56
Platelet Physiology .................................................................................................58
Predictive and Diagnostic Variables in Thrombotic Disease ...................................61
Vascular Biology ....................................................................................................64
Von Willebrand Factor ............................................................................................68
Women’s Health Issues in Thrombosis and Hemostasis ........................................73
Working Groups
Working Group on Perioperative Thrombosis and Hemostasis ..............................77
ISTH-BAT Standing Committee – Business Meeting
21 June 2015 10:30 - 11:30
Francesco Rodeghiero and Barry Coller introduce the first part of the session that was focused
on the latest updates of the studies currently endorsed by the ISTH-BAT Standing Committee.
Frank Leebeek presented data about 113 children enrolled in the WiN Study: the ISTH score
was used to quantify bleeding severity in children. The score was higher in type 3 VWD than in
type 2 or 1 (17 vs. 10.5 vs. 6.5) and higher in children with severe VWD (VWF levels < 10 IU
dL).
Johanna Gebhart reported the characterization of patients with a mild to moderate bleeding
phenotype. 338 patients with a median age of 43 years were included. The median BS was 5 in
the overall patient population.
Alberto Tosetto reported the progress of the REBEL Study, that is enrolling cases with a
bleeding tendency and age and sex matched controls. 51 cases and controls are enrolled at this
moment, with a projected upper normal limit of BS ≤ 4.
Francesco Rodeghiero reviewed the achievements of the ISTH-BAT repository, particularly
focusing of the wave of interest in the last ten years but also in the light of currently ongoing and
promising developments of genomic studies. The ISTH-BAT mandate will expire on 2016.
Following this session, Willem Ouwehand presented the BRIDGE-BPD study, which is aimed at
evaluating the role of next-generation sequencing of a panel of candidate genes in the diagnosis
of bleeding disorders. The project uses a human phenotype ontology annotation to cluster
bleeding disorders with a similar presentation and may represent an attractive development in
phenotyping bleeding symptoms.
Finally, Barry Coller reviewed the modalities to access the ISTH-BAT repository and presented
data on active studies.
Approximately 50 people attended the session.
Coagulation Standards Standing Committee Minutes
21 June 2015 12:30 - 14:00
Chairman: Anthony R. Hubbard
Meeting Chairman: Craig Thelwell (on behalf of Anthony Hubbard)
Review of Lot #4 (C. Thelwell)
Lot #4 of the SSC/ISTH Secondary Coagulation Standard commenced dispatch in April 2012 at a cost of £4.00 (GBP) per vial. Between June 2014 and end May 2015 a total of 55 orders were fulfilled with 13,851 vials dispatched. Manufacturers in Europe received a total of 10,350 vials and External Quality Assurance schemes (UK NEQAS and College of American Pathologists, USA) received a total of 1,600 vials. Information on the availability of Lot #4 was distributed to 150 manufacturers in May 2014 and since then there have been 9 new customers. Five of these had received the mailshot (4 USA and 1 UK) and ordered 1,050 vials. Four were not included in the mailshot (1 Japan, 1 China, 1 France, 1 Germany) and ordered 650 vials. It was proposed to repeat the mailshot annually. No stability testing of Lot #4 was performed in the past year to retain samples for future time-points. The next time-point for the accelerated degradation study will be in January/February 2016. At the current rate of usage stocks of Lot #4 will be exhausted within 4 years. It was recommended to initiate replacement in 2016 to ensure continuity of supply by starting the tender process, inviting bids from manufacturers for the production of Lot #5.
Experience of EQA schemes with Lot #4
UK NEQAS (S. Kitchen)
Eight vials of SSC Lot #4 were used for "trouble-shooting” purposes between July 2014 and June 2015 relating to assay issues for FVIII, FIX, AT and VWF. SSC Lot #4 has also been included as an anonymous sample in one survey (UK NEQAS) which covered testing for FII, FV and FXI. For FII testing one manufacturer’s reference plasma (coded as reference B) returned a result of 105 IU/dl whereas the labelled potency for Lot#4 is 91 IU/dl, representing a difference of 15.4 % relative to the assigned potency. The result from two other reference plasmas used differed only by 5.5 % and 7.7%. Of the centers using reference B almost all used the same FII deficient plasma so the possibility that this contributed to the difference cannot be excluded, though different thromboplastin reagents were used so this is unlikely to be a contributing factor. For FV testing the median value returned for all commercial reference plasmas was 92.6 IU/dl, which is in good agreement with the assigned value of 89 IU/dl. All commercial plasmas were within 7 % of the assigned value. For FXI testing two commercial plasmas returned results with a 10.1 % and 18.0 % difference to the assigned value of 89 IU/dl (98 and 105 IU/dl for reference plasmas coded B and C). Different APTT reagents were used with reference plasma B, with good agreement between the results and so is unlikely to explain the difference. The contribution of the APTT reagent to the difference observed with plasma C is not clear. The reference plasma manufacturers have been informed by NEQAS and feedback on the outcome of any investigations has been requested.
College of American Pathologists (Dong Chen)
SSC Lot #4 was issued in the 2014 Thrombophilia Survey (CGS2B). Mean estimates for the thrombophilia associated analytes (Antithrombin activity and antigen, Protein C activity and antigen, Protein S activity and antigen) were close to the assigned values. Overall the survey results support the assigned values and stability of Lot #4.
Other business
S Kitchen raised the issue of inappropriate use of % when an IU is available. Values are often reported as a % that may or may not correspond to the IU value and it is often not made clear. This issue affects the reporting of results in peer reviewed publications as well as the labelling of reference materials in assay kits, with some manufacturers providing both IU and % values for a single reference material that conflict with each other. It was proposed that a recommendation is required from SSC on units for commercial reference plasmas and publications that the IU should be used when available.
There were 20 attendees.
Animal, Cellular and Molecular Models
21 June 2015 8:00 – 12:20
Chairman: Susan S. Smyth
Co-Chairs: Cecile V. Denis, Jose A. Diaz, Tom Knudsen, David Motto, Leslie Parise, Denise E. Sabatino
The Animal Models SSC session took place on June 21, 2015 and was a "Summit on
Preclinical Models of Venous Thrombosis”. The goal was to convene thought leaders in the
field to discuss the current state of venous thrombosis (VT) models, to understand novel
insight gained from VT models, and to identify gaps in their application that need to be
resolved to continue to move the field forward.
The first session was devoted to discussion of the most commonly employed preclinical VT
models. Jose Diaz provided an overview of considerations for selection of a particular
model. Then, Steve Grover, Brian Cooley, and Jose Diaz described the venous stenosis,
the electrolytic, and the venous stasis models and discussed strengths and weakness of
each. The second, educational session, featured presentations from Denisa Wagner,
Thomas Wakefield, Nigel Mackman, and Alisa Wolberg that highlighted the role of
inflammation, leukocyte and platelet accumulation, the formation of neutrophil extracellular
traps, tissue factor and its sources, fibrin and red blood cell accrual as essential components
to the pathophysiology of VT. A picture of the cellular and molecular events controlling the
initiation, progression / consolidation, and resolution of VT emerged. Questions and
comments from the audience and the speakers reinforced the differences in models and
their appropriate applications.
The Summit identified several current gaps, including the need to develop
Mechanism(s) to share detailed protocols from different laboratories
A repository of data from different laboratories obtained in VT models
A strategy for providing more hands-on training in VT models.
The SSC committee agreed to work the speakers and other leaders in the field to create a
consensus statement on animal models of VT.
Biorheology
20 June 2015 14:30 – 18:50
Chairman: Keith B. Neeves
Co-Chairs: Judith Cosemans, David N. Ku, Pierre Mangin, Owen J. McCarty, Mitsuhiko Sugimoto, Erik Westein
Saturday, June 20, 2015
14:30-18:50
Joint Biorheology and Platelet Physiology Education Session: Bioreactors for the study
of the biophysical mechanisms that regulate platelet function. Moderators: Keith Neeves
and Paolo Gresele.
1. Jose Lopez, United States
Dr. Lopez reviewed work from his lab on synthetic microvessels for studying VWF secretion
from endothelial cells, VWF self-association and platelet-endothelial cell-VWF interactions.
2. Shaun Jackson, Australia
Dr. Jackson reviewed work form his lab on single platelet studies using biophysical tools
including flow chamber studies with high resolution DIC and calcium dynamics imaging and
biomembrane force probe for measuring receptor-ligand kinetics.
Joint Biorheology and VWF session on acquired von Willebrand syndrome. Moderators:
Jorge Di Paola and Keith Neeves.
1. David Lillicrap, Canada, “Flow mediated interactions of VWF and ADAMTS13: shear
ecstasy,” 15:30-15:45
The interaction and cleavage of VWF by ADAMTS13 requires unfolding of the substrate (VWF).
In vitro flow studies show that VWF and ADAMTS13 interact increasingly as a thrombus grows
and the diameter of the remaining lumen gets smaller - resulting in increased flow. In addition,
tensile forces on VWF are further influenced by VWF ligands including platelet GpIb, GpIIb/IIIa
and P-selectin. Each of these ligands can contribute to the unfolding of VWF and influence
interactions and subsequent cleavage by ADAMTS13.
2. Barbara Ziegler, Germany, “Acquired von Willebrand syndrome in patients with VAD or
ECMO,” 15:45-16:00
Dr. Ziegler emphasized the expansion of Ventricular Assist Devices (VADs) over the last
decades. They are not only a transition form of therapy but are becoming more of a destination
therapy; therefore all complications may become more relevant. Dr. Ziegler discussed the 2
types of pumps in the market (centrifugal and axial). She also discussed anticoagulation and the
unusual rate of bleeding seen in patients likely due to AVWS with VADs. It is also possible that
hemolysis and free hemoglobin also influences the rate of AVWS. It is important to mention
that almost all recipients of LVAD (>90%) have higher ratios of VWF:RCo/VWF:Ag but not all of
them bleed clinically. She discussed ECMO and the high incidence of AVWS in this modality.
She also emphasized the poor correlation between VWF levels and bleeding suggesting that
many other factors may contribute to the bleeding observed.
3. David Schmidtke, USA, “Effects of high shear millisecond exposure on platelets”
The objective of this study was to replicate the VAD-induced shear-rate in a microfluidic device
and investigate the effect of the shear on platelet receptors, platelet adhesion and
aggregation. Platelet adhesion and platelet aggregate formation on collagen patterned surfaces
upstream and downstream of a transient (1-50 msec) exposure to high shear (40,000 - 100,000
s-1) were quantified. We observed that a single exposure to high shear was enough to inhibit
platelet aggregation while platelet adhesion was maintained. The defect in platelet aggregation
was dependent upon both the shear rate and exposure time. Treatment of blood with an
inhibitor to ADAMTS13 prior to the high shear exposure restored normal platelet aggregation
downstream suggesting a role of VWF in the downstream aggregation.
After these three presentations discussion ensued and several members of the SSC proposed
an AWS working group with members of the VWF and Biorheology subcommittees. The main
goal of this working group will be to study and standardize AVWS in an attempt to understand
better the presentation of disease and the mechanisms of disease as well as the factors that
contribute to bleeding. Drs. Barbara Ziegler, Augusto Federici and David Schmidtke will lead the
working group. Several members of the audience expressed interest in participating.
VWF-mediated mechanisms in complex and pathological flows. Moderators: Mitsuhiko
Sugimoto and Pierre Mangin.
1. Erik Westein, Australia, “Biomechanical regulation of VWF and thrombus formation under
complex flow conditions,” 16:50-17:05
Hydrodynamics regulate plaque development, rupture and thrombus formation. The flow in
these processes is complex in that there are areas of significant gradients in shear stresses and
recirculation flows. This work tests the hypothesis that complex blood flow induced by
intraluminal platelet aggregates is a key feature of thrombosis. The aim is characterize the
thrombotic mechanisms that are promoted by complex flows. The methods used include PDMS
microfluidic devices that model a stenotic vessel. It was found that VWF-platelet interactions are
exacerbated at the apex of the stenosis. These interactions are induced by gradients in shear
stress, which appear to be more important than the absolute magnitude of the shear stress.
Questions: What are the numbers of platelets quantified in rolling (Kinney, Dublin)? On the order
of thousands. What is the role of cellular sources of VWF (Lopez, Seattle)? Have cultured EC in
stenotic vessels, but these studies focus on plasma VWF and platelet interactions. How
activated are platelets in the poststonotic region (Ju, Sydney)? Likely intermediately activated,
working on measuring calcium dynamics. Is the poststenotic region a “black hole” due to
recirculation flows (Turitto, Chicago)? There are no recirculation flows due to low Reynolds
number in this system.
2. Keith Neeves, USA, “Update on Biorheology Subcommittee Project: Scaling in
Hemorheology” 17:05-17:20. Note this presentation replaced the scheduled talk by Matthias
Schneider (USA).
The mission and mandate of the Biorheology Subcommittee was presented. Results from our
current project on scaling in hemorheology was presented and discussed. The objective of this
project is to use the concepts from scaling analysis to allow for faithful comparisons between in
vitro models and develop new models based on in vivo blood flow/hemostasis/thrombosis.
Important dimensional and dynamic parameters were introduced and their relevance to flow
chamber studies was presented.
3. David Ku, USA, “Relative roles of VWF and platelets in formation of occlusive arterial
thrombosis,” 17:20-17:35.
The design criteria and results from a high shear microfluidic system was presented to model
occlusive thrombus formation in a stenosed vessel. Design constraints included <10 mL of
whole blood, wall shear rate of 6000 1/s in the stenosis, 5-30 minute duration and a geometry
where platelet-platelet interactions dominate (minimum height of 70 µm and aspect ratio of 5.5).
To avoid excessive shear gradients at the corners of the expansion/contraction a 15 degree
slope was used. Fabrication issues were presented using both soft lithography and
micromachining approaches. In this chamber, pulsatile flow did not affect thrombus formation.
Inter- and intra-donor variability was measured, with significant variation in occlusion times
between donors. The relative contribution of plasma VWF, platelet VWF, and platelets to
occlusive thrombus formation was measured by a series of depletion and rescue studies. In
these experiments the contribution of VWF was greater than platelets for pathologically high
shear occlusive thrombosis, and platelet VWF contributes to forming large occlusive thrombi.
Question: Previous animal studies by Bergmeir and colleagues found that the role of GP1b
exceeded its role as a receptor for VWF, do these studies contradict these studies? No, the
results are consistent with the requirement for GPIb that may be sufficient with 10% of platelets,
under the pathologically high shear conditions.
Novel flow-dependent mechanisms of platelet function, coagulation and
fibrinolysis. Moderators: Erik Westein and Judith Cosemans.
1. Elizabeth Gardiner, Australia, “Shear effects on platelet surface receptor shedding”, 17:45-
18:00.
Previous work from Gardiner and colleagues have examined the factors affecting GPVI function
and shedding including GPVI ligand binding mediated ADAM10 cleavage that yields soluble
GPVI. Pathophysiological levels of shear stress increase GPVI shedding. In these studies, the
platelet membrane levels of GPVI and GPIbα were measured during thrombus formation on
collagen in a custom flow chamber. Under steady flow (1800 1/s) the levels of GPIbα, but not
other primary receptors (GPVI, αIIb, CD9), correlates with thrombus volume. Under pulsatile
flow (mean shear 1800 1/s), platelet aggregates were smaller and GPIbα, GPVI and αIIb levels
correlated with volume indicating that levels of these receptors influenced thrombus properties.
Confocal microscopy analysis showed a loss of GPVI in the core of thrombi and a loss of
GPIbα in the thrombus shell region. A FRET substrate reporting on ADAM10 activity showed
that ADAM10 co-localized to regions of large shear gradients. These shear-dependent shedding
events have important consequences in extracorporeal devices such as LVADs and
quantification of sGPVI prior to LVAD implantation could identify patients at risk of bleeding and
aid clinical decisions relating to extent of anti-platelet therapy.
2. Paola van der Meijden, The Netherlands, “Assessment of platelet-dependent coagulation
and fibrinolysis under flow”, 18:00-18:15.
The aim of these studies was to determine how the type and density of adhesive surfaces
controls the composition and viscoelastic properties of thrombi formed under flow. The
methodology included perfusing citrated recalcified whole blood over collagen and collagen-TF
spots. Platelet and fibrin accumulation were measured by confocal microscopy. Mechanical
properties were measured by nanoindentation. Fibrinolysis was achieved by adding tPA or uPA
to the blood or rinse buffer. Lower collagen density (10 ug/mL) resulted in larger and more
dispersed thrombi than higher collagen density (50 ug/mL). Flow enhanced the accumulation of
fibrin with higher surface coverage under shear (150 1/s) compared to stasis. Thrombi formed
on collagen alone were more rigid (Young’s modulus from nanoidentation) compared to thrombi
formed on collagen/TF, where fibrin appears to decrease rigidity. Fibrin degradation under flow
showed a dose-dependence lysis for both tPA and uPA. Flow accelerates fibrinolysis compared
to static conditions, but the rate of lysis was independent to shear rate (150-1000 1/s).
Fibrinolysis is delayed proximal to the platelet surface. Questions: Why is fibrinolysis
independent of shear rate (Turitto, Chicago)? Perhaps it is reaction limited.
3. Pierre Mangin, France, “Platelet adhesion and aggregation under disturbed blood flow: A
focus on the role of pulsatility,” 18:30-18:50.
Pulsatile flow has been shown to increase, decrease, and have no effect of platelet adhesion
and aggregation in previously reported studies. This study examines steady versus pulsatile
flow in a custom flow chamber that includes a straight channel, which serves as an internal
control, and a severely stenosed (90%) channel. Microparticle velocimetry (mPIV) was used to
show in the absence of blood that the stenosis does not cause recirculation flows. A flow meter
upstream of the flow chamber was used to create flow rate waveforms that mimic those in the
common carotid artery, left coronary artery, and the portal vein. Hirudinated blood perfused over
collagen gave modest reductions in thrombus volume for both carotid and left coronary, but not
portal vein, waveforms. Pulsatility does not affect initial platelet recruitment, but does increase
platelet detachment from an evolving thrombus. Detachment occurs during the acceleration
phase of the waveform, similar observations were found in vivo. Occlusive thrombi formed in the
expansion zone of the stenosis. Under pulsatile flow there was increased thrombus formation in
the post-stenotic region relative to steady flow. ASA and cangrelor reduced thrombus formation
in the post-stenotic region. Questions: What happens in the presence of coagulation
(Cosemans, Maastricht)? Not clear, subject of future studies.
4. Discussion Session, “Challenges and needs for standardization of flow assays,” 18:30-
18:50.
An open discussion focused on the needs for standardization and best practices for flow assays
between co-chairs and audience members. There was a general consensus that a standardized
flow chamber was not a priority and could possibly stifle the creativity and mechanistic insight
that has been a result of the diverse flow chambers developed over the last 10 years. Instead,
there is a greater need to raise awareness about how rheological issues affect platelet function
and coagulation. Some of these issues are covered by the current “Scaling in hemorheology”
project. Other issues that could be addressed in a multi-center study include identifying the
sources of variability and development of internal controls that could be integrated into custom
chambers. A pamphlet or online guideline on rheological issues may be useful (Turitto,
Chicago). It is important that as we develop flow chambers and best practices to keep in mind
that ultimately we need these chambers to model what is happening in vivo, in humans (Brass,
Philadelphia). More scrupulous reporting of chamber dimensions and assays would be an
improvement in Methods sections. Co-chairs will take these comments into consideration for
future Biorheology Committee projects.
Joint Platelet Physiology and Biorheology Education Session: Bioreactors for
megakaryocyte studies and platelet production. Moderators: Paolo Gresele and Owen
McCarty.
1. Jonathon Thon, United States
Dr. Thon reviewed the evolution and state-of-the-art in the field of platelet production
bioreactors. The focus was on outlining the challenges and needs for translating recent
discoveries and devices (including his work on microfluidic reactors) into large-scale platelet
production.
2. Alessandra Balduini, Italy
Dr. Balduini reviewed the evolution and state-of-the-art in the field of megakaryocyte studies in
bioreactors. The focus was on recent advanced in bioengineering and biomaterials (including
her work on silk derived materials), phenotyping platelet produced in these bioreactors and
applications including studying diseases (myeloproliferative disorders), drug efficacy and drug
testing.
Control of Anticoagulation
21 June 2015 8:00 – 12:20
Chairman: Walter Ageno
Co-Chairs: Rebecca Beyth, Benilde Cosmi, Mark Crowther, Ismail Elalamy, Elaine M. Hylek, Pieter W. Kamphuisen, Peter Verhamme, Henry G. Watson
The meeting was introduced by the SSC chair, Walter Ageno, who presented the program and reminded the scopes of the Control of Anticoagulation subcommittee.
The first part of the morning was dedicated to the presentation of ongoing SSC projects.
The first session was chaired by Dr. Walter Ageno and was focused on registries. Dr. Alex Spyropoulos presented the proposal for a project on the perioperative management of patients treated with the direct oral anticoagulants (DOACs). Dr. Spyropoulos stressed the fact that, at present, there is little to no evidence for the periprocedural management of patients on DOACs undergoing elective procedures or surgeries. The objective of this large prospective registry is therefore to capture patient populations, management strategies, and outcomes in patients on DOACs undergoing elective or urgent/emergent surgeries or procedures. The registry is expected to accrue at least 50 centers with at least 2 patients/center per month over a 2 year time frame. Statistical analysis will be descriptive based upon type of DOAC used, patient indications, and type of procedure (major versus minor). Dr. Walter Ageno presented an update on the ongoing START-EVENTS SSC register. This observational prospective cohort study is collecting information on the management of major bleeding and thromboembolic events in patients treated with the DOACs. The registry is based on an electronic database which collects data on the index event, on the presence of risk factors, on the use of pro-hemostatic agents for the bleeding patients, on therapeutic strategies for patients with thrombosis, on the use of laboratory tests, and on the 6-month outcome of patients. An initial sample of 100 patients is planned, enrolment is currently half-way. Finally, Dr. Saskia Middeldorp presented the proposal for a registry on pregnancy outcomes in patients who become pregnant while on DOACs. This international, multicenter, prospective cohort study aims to gather experience on the outcome of pregnancy, birth defects in neonates, and late effects in children of mothers who unintendedly have been pregnant while being exposed to DOACs. Information will be collected on an electronic case report form. The study will start enrolling very soon.
The second session was chaired by Dr. Henry Watson and Pieter Kamphuisen and was focused on current SSC standardization projects. Dr. Scott Kaatz reported on the proposal for the standardization of the definition of clinically relevant non-major bleeding in studies of anticoagulants in atrial fibrillation and venous thromboembolism in non-surgical patients. The authors initially carried out a literature search for clinical trials in patients with atrial fibrillation and venous thromboembolism and extracted the definitions of clinically relevant non-major bleeding from the trial reports. Based on these definitions, they identified a number of criteria to be used, with a resulting recommended definition which was presented and discussed during the meeting, and that will be published soon on the Journal of Thrombosis and Haemostasis as an SSC communication. Dr. Nakisa Khorsand reported on the proposal for the definition of clinical outcomes to assess the effectiveness of major bleeding management. The authors carried out a systematic review of the literature to identify different criteria used in the trials and developed a definition that was agreed within the working group and with the SSC co-chairs. This definition was presented and discussed at the meeting, and will be published as an SSC
communication in the Journal of Thrombosis and Haemostasis. Dr. Geoff Barnes presented the results of a collaborative work on the nomenclature of the novel oral anticoagulants. The authors issued a survey to the leaders of 16 thrombosis and hemostasis organizations in Europe and North America. Based on the survey results, they have recommended that term Direct Oral Anticoagulant (DOAC) be used when describing oral anticoagulants with a direct target. They also recommended that a medications specific target be used when distinguishing between the various DOACs, such as for determining monitoring tools or reversal strategies. The recommendation was discussed at the meeting and is published as an SSC communication in the Journal of Thrombosis and Haemostasis. Finally, Dr. van den Besselaar presented an update on the progress of the project on the replacement of international standards for thromboplastin. There is still need to find additional contributing centers in order to complete the project in due time, and the presenter made a call for action.
The second part of the morning started with the education session, which was dedicated to the memory of Prof. Michel Meyer Samama and was chaired by Dr. Marc Samama and Dr. Jacqueline Conard. Professor Samama greatly contributed to the activities of the SSC Control of Anticoagulation and, most of all, to our knowledge on thrombosis and hemostasis. The first lecture was given by Dr. Jacqueline Conard who reviewed the contribution of Prof. Samama to the development of clinical assessment models to identify patients at increased risk of thrombosis who should benefit from thromboprophylaxis and discussed the state of the art on thromboprophylaxis in medical patients and its application in real world clinical practice. Finally, Dr. Conard reviewed the existing literature on risk assessment models for medically ill patients, their recommended use in international clinical guidelines, and their applicability in clinical practice. The second lecture was given by Dr. Ismail Elalamy on Prof. Samama studies on the laboratory measurement of antithrombotic drugs. Dr. Elalamy discussed the mechanisms of action of the DOACs, their pharmacodynamics and pharmokinetic properties, and the results of in vitro and in vivo assessment of their activity with a particular focus on the role of thrombin generation assays. This session gave us an important sense of the many achievements in basic and clinical research over the last decades in the field of anticoagulant drugs, many of which have been deeply discussed and actively promoted during the meetings of this SSC with the active contribution of Prof. Samama.
The last session was entitled "A look into future initiatives of the SSC Control of Anticoagulation” and was chaired by Dr. Elaine Hylek and Dr. Mark Crowther. The first speaker, Dr. Jan Beyer, presented the results of registries of patients treated with the DOACs in real world clinical practice, with particular focus on adherence, effectiveness, and safety issues, and discussed the strengths and weaknesses of observational studies, as well as their role in improving our knowledge on these drugs. Finally, Dr. Ismail Elalamy reviewed the role of laboratory testing for the management of patients treated with the DOACs and proposed a position paper prepared by this SSC to drive the correct use of all available tests.
Disseminated Intravascular Coagulation
21 June 2015 8:00 – 12:20
Chairman: Jecko Thachil,
Co-Chair: Marcello Di Nisio, Satoshi Gando, Takashi Ito, Bernd Jilma, Shinichiro Kurosawa, Sacha Zeerleder
This year’s SSC meeting started with an overview of DIC in obstetrics by Dr. Offer Erez from
Israel. He highlighted the difficulties in using the ISTH score in pregnancy due to the differences
in the laboratory values. A pregnancy-specific DIC score has been suggested and validated. A
call to work together in this area was suggested and was followed by a gathering of the SSC
committees for DIC and women’s health.
The next topic for discussion was the role of DAMPs or Damage Associated Molecular patterns
in DIC. Dr Patricia Liaw from Canada covered the role of various different DAMPs in sepsis and
its role in DIC. This was followed by the mechanism of generation of DAMPs by Dr. Sacha
Zeerleder from the Netherlands. It is expected that different groups will work more on this novel
marker for DIC and hopefully will identify mechanisms to use them as diagnostic markers and
therapeutic targets.
Dr. Bernd Jilma from Austria then discussed the unusual case of drowning causing
Hyperfibrinolytic afibrinogenemia in overt DIC. This was a novel concept and put forward the
need for more work on DIC from rarer causes. Prof. Toshiaki Iba from Japan related to us his
extensive experience in the use of anticoagulants, thrombomodulin and antithrombin. He
explained the timing and the dose of these agents and whether a different scoring system needs
to be used for their use.
The Educational session of DIC SSC had two fantastic and varied topics – details on basic
science from Dr. Jordan Shavit (USA) who focused on ‘zebra fish modelling in DIC’ and from the
clinical perspective from Prof. Jerrold Levy (USA) who gave his view on the management of DIC
in a critical care unit. Dr Shavit gave the presentation on zebrafish as a tool for high-throughput
genetic analysis. He showed that disruption of the zebrafish antithrombin resulted in
spontaneous venous thrombosis in larvae. Characterization of null fish revealed DIC in larvae,
which could be rescued by both human and zebrafish antithrombin complementary DNAs. Prof
Levy gave a practical overview of the clinical situations which is often encountered in critical
care from DIC point of view. He gave the diagnostic pathways and treatment algorithms which
needed to be followed in these cases to get the best outcome also elaborating on his extensive
experience with different anticoagulants and work with coagulation problems in the critically ill,
complex patients.
Prof. Satoshi Gando, a renowned DIC expert from Japan, reminded us about the
pathophysiological mechanisms of trauma-associated |DIC and stressed on the early
hyperfibrinolytic type of DIC in trauma and the differences in the coagulation activation between
inside and outside the blood vessel. Dr. Carl Erik Dempfle then covered the issues relating to
the use of D-dimer in clinical settings and the possibility of considering fibrin-related marker in
these areas.
The session ended with a call for an international registry from Dr Marcello DiNIsio who
explained the major differences in the DIC diagnosis and management in a survey under the
auspices of ISTH.
Exogenous Hemostatic Factors
20 June 2015 14:30 – 18:50
Chairman: Ivo Francischetti
Co-Chair: Kenneth J. Clemetson, Tur-Fu Huang, Manjunatha R. Kini, Francis S. Markland Jr, Robson D. Monteiro
Opening Comments and Welcome, Ivo Francischetti, USA
The mission and objectives of the SSC on Exogenous Hemostatic Factor were presented. The
educational component of the Session included talks on molecules from snake venoms and
hematophagous salivary glands affecting hemostasis, and theirs use in experimental
thrombosis. Speakers also presented recent discoveries in the areas of structural biology of
exogenous hemostatic components, and their use in drug development or as tools in
Biochemistry and Cell Biology.
Session I – Educational Session.
Manjunatha Kini, National University of Singapore, Singapore, “Factor XIa inhibitors from
snake venom” (14:30-15:00 pm).
Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and
common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being
investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk.
Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway
and (ii) characterize and further improve the properties of the identified anticoagulants. We have
isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from
the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor
that prolonged activated partial thromboplastin time without significant effects on prothrombin
time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a
slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial
thromboplastin time at ~ 3 μmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A
series of mutants were subsequently generated to improve the potency and selectivity of
recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50
~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type
inhibitor of FXIa (Ki = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times
stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice
carotid artery in FeCl3 -induced thrombosis models. We have isolated an exogenous FXIa
specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in
vitro and in vivo efficacy. These proof-of-principle data supported the further development of
Fasxiator as a novel anticoagulant candidate.
Ivo Francischetti, National Institutes of Health, USA, “Hematophagy, neutrophils, and
contact pathway” (15:00-15:30 pm)
Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sand flies, fleas, ticks,
leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis
in redundant and synergistic manners. Recent progress has been made in the identification of
salivary inhibitors affecting different aspects of hemostasis, through distinct mechanisms of
action. The contact pathways has been identified as an important player in thrombus formation,
having a peripheral role in hemostasis. Accordingly, salivary components are known to block
FIXa and FXIIa, thus affecting the intrinsic pathway and prolonging the aPTT, including a) a
FXIa anticoagulant from the Vampire Bat which inhibits arterial thrombosis without producing a
bleeding phenotype, b) a poly-phosphate binding protein from sandfly which inhibits FXI
activation by thrombin, in the presence of cofactor Poly-P, and which also prevent inflammation
in vivo, and c) a elastase inhibitor from a mosquito vector of malaria, which prevents elastase-
induced TFPI cleavage, platelet aggregation, neutrophil chemotaxis, and NETS formation, and
attenuates thrombosis. These proteins are notable examples of molecules from hematophagous
salivary secretions with antihemostatic properties. Exogenous hemostatic factors have been
employed as tools in biochemistry and cell biology and also display potential therapeutic
applications.
Session II – Subcommittee session.
Jim Huntington, University of Cambridge, Cambridge, United Kingdom, “Structure of
Pseutarin-C, a snake venom prothombin activator” (15:30-15:50 pm)
The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa,
efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the
complex assembles and its mechanism of prothrombin processing are of central importance to
human health and disease, because insufficient thrombin generation is the root cause of
hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the
crystal structure of the prothrombinase complex have been thwarted by the dependence of
complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable
prothrombinase complex preassembled in the venomgland of the Australian Eastern Brown
Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of
activated fXa from P textilis venom and the derived model of active pseutarin C. Structural
analysis supports a single substrate binding channel on fVa, to which prothrombin and the
intermediate meizothrombin bind in 2 different orientations, providing insight into the
architecture and mechanism of the prothrombinase complex-the molecular engine of blood
coagulation
Ana Marisa Chudzinski-Tavassi, Butantan Institute, Brazil, “Rational strategy applied to a
tick salivary FXa inhibitor in order to develop a potential antitumor drug candidate”
(15:50 – 16:10 pm)
Amblyomin-X is a recombinant protein, classified as a Kunitz type inhibitor, sharing 35%
homology with the TFPI Kunitz domain, identified through a transcriptome analysis of the
salivary gland from the Amblyomma cajennense tick. This recombinant protein is able to inhibit
FXa (Ki 2.2 uM) through a non-competitive pathway. In vitro assays demonstrated an increasing
time of coagulation of human, rabbit and mouse’s plasma on aPTT test using different
concentrations of Amblyomin X. The same way, aPTT increase was shown in the plasma of
rabbits and mice injected intravenously with Amblyomin X. The effect can be perceived after the
first30 minutes, has its major expression after completed 2 hours of test and ceases when
completed 4h of treatment. Besides the effect on the coagulation process, it has been shown
that Amblyomin X induces cell death by apoptosis pathway, being this action selective to tumor
cells. This way, dyne in and proteasome inhibition are targets for the Amblyomin X and play
central role in the cell death mechanism of action. In vivo assays demonstrated that the
treatment with Amblyomin X promoted regression of tumor growth and reduction of metastasis.
Acute safety pre-clinical assays in mouse and rabbits demonstrated low toxicity under 200 folds
of the effective dose. Similar results were obtained for preclinical repeated doses assays.
Robson Q. Montero, Federal University of Rio de Janeiro, Brazil, “Exogenous inhibitors
as tools for the study of new players in hemostasis and thrombosis” (16:10 - 16:30 pm)
Salivary glands from hematophagous animals constitute a major source of molecules capable of
modulating hemostasis. Neutrophil extracellular traps (NETs) have been described as web-like
structures of DNA and proteins formed through a process called NETosis. NETs have been
recently linked to thrombus formation by a variety of mechanisms which includes inactivation of
natural anticoagulants as well as platelet and contact phase activation. In this context, we have
demonstrated that salivary gland components may interfere with NETs pro-haemostatic function
by 1. Preventing thromboxane A2-dependent platelet-assisted neutrophil activation; 2. Inhibition
of elastase-mediated NETs formation or 3. Enzymatic degradation of formed NETs. Our results
provide new insight in elucidating the mechanisms of saliva to avoid host’s hemostatic
responses and innate immune system.
Kenneth Clemetson, Theodor Kocher Institute, Switzerland, “Exogenous Hemostatic
Factors as tools in platelet research” (16:50 – 17:10 pm)
Snake venoms are complex mixtures of biologically active proteins and peptides. Many affect
hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting
endothelium. Snake venom components are classified into various families, such as serine
proteases, metalloproteinases, C-type lectin-like proteins, disintegrins and phospholipases.
Snake venom C-type lectin-like proteins have a typical fold resembling that in classic C-type
lectins such as the selectins and mannose-binding proteins. Many snake venom C-type lectin-
like proteins have now been characterized, as heterodimeric structures with alpha and beta
subunits that often form large molecules by multimerization. They activate platelets by binding to
VWF or specific receptors such as GPIb, alpha2beta1 and GPVI. Simple heterodimeric GPIb-
binding molecules mainly inhibit platelet functions, whereas multimeric ones activate platelets. A
series of tetrameric snake venom C-type lectin-like proteins activates platelets by binding to
GPVI while another series affects platelet function via integrin alpha2beta1. Some act by
inducing VWF to bind to GPIb. Many structures of these proteins, often complexed with their
ligands, have been determined. Structure-activity studies show that these proteins are quite
complex despite similar backbone folding. Snake C-type lectin-like proteins often interact with
more than one platelet receptor and have complex mechanisms of action.
Heyu Ni, University of Toronto, Canada, “Anfibatide and other exogenous platelet GPIb
antagonists” (17:10 – 17:30 pm)
Ischaemic stroke is a serious disease with limited therapy options. Glycoprotein (GP)Ib binding to von Willebrand factor (vWF) exposed at vascular injury initiates platelet adhesion and contributes to platelet aggregation. GPIb has been suggested as an effective target for antithrombotic therapy in stroke. Anfibatide is a GPIb antagonist derived from snake venom and we investigated its protective effect on experimental brain ischaemia in mice. Focal cerebral ischaemia was induced by 90 min of transient middle cerebral artery occlusion (MCAO). These mice were then treated with anfibatide (4, 2, 1 μg·kg-1 ), injected i.v., after 90 min of MCAO, followed by 1 h of reperfusion. Tirofiban, a GPIIb/IIIα antagonist, was used as a positive control. Twenty-four hours after MCAO, anfibatide-treated mice showed significantly improved ischaemic lesions in a dose-dependent manner. The mice had smaller infarct volumes, less severe neurological deficits and histopathology of cerebrum tissues compared with the untreated MCAO mice. Moreover, anfibatide decreased the amount of GPIbα, vWF and accumulation of fibrin(ogen) in the vasculature of the ischaemic hemisphere. Tirofiban had similar effects on infarct size and fibrin(ogen) deposition compared with the MCAO group. Importantly, the anfibatide-treated mice showed a lower incidence of intracerebral hemorrhage and shorter tail bleeding time compared with the tirofiban-treated mice. Our data indicate anfibatide is a safe GPIb antagonist that exerts a protective effect on cerebral ischaemia and reperfusion injury. Anfibatide is a promising candidate that could be beneficial for the treatment of ischaemic stroke.
Tur-Fu Huang, National Taiwan University, Taiwan, “The second generation of platelet
GPIIb/IIIa antagonists derived from snake venom disintegrins (17:30 – 17:50 pm)
Snake venoms contain unique components that affect cell-matrix
interactions. Disintegrins represent a class of low molecular weight, Arg-Gly-Asp (RGD)-
containing, cysteine-rich peptides purified from the venom of various snakes among the
Viperidae and Crotalidae. They bind with various degrees of specificity to integrins alpha IIb
beta 3, alpha 5 beta 1 and alpha V beta 3 expressed on cells. Snake venom metalloproteases
(high molecular mass haemorrhagins) also contain disintegrin-like domains, in addition to zinc-
chelating sequences. Membrane-anchored ADAMs (A Disintegrin and Metalloprotease domain),
multidomain molecules consisting of metalloprotease, disintegrin-like, cysteine-rich, and
epidermal growth factor domains, a transmembrane domain and a cytoplasmic tail, are a new
family of proteins. In the light of the large number and wide distribution of ADAMs, they may
participate in cell-cell fusion events, including sperm-egg binding and fusion, myoblast fusion
and other cell-cell and cell-matrix interactions. The structure-function relationship of these
molecules is discussed.
Factor VIII, Factor IX & Rare Coagulation Disorders
20 June 2015 9:00 – 13:30
& 21 June 2015 8:00 – 12:20
Chairman: Guy Young
Co-Chairs: Manuel Carcao, Michael Makris, Danijela Mikovic, Flora Peyvandi, Steven W. Pipe, Elena Santagostino
Project Reports
1. Standardization of post-registration surveillance: F. Peyvandi (IT)
Dr. Peyvandi updated the SSC on her project on post-registration surveillance for efficacy and
safety of novel factor products. She described all of the registries in the various world regions
that are collecting data. The goal of the group is to recommend standards for the data collection
within these registries such that data can be compared across those databases.
2. Prophylaxis in patients without inhibitors: V. Blanchette (CA) and K. Fischer (NL)
Dr. Fischer discussed the results of her project on prophylaxis in patients without inhibitors
describing when and how to start prophylaxis in FVIII and FIX deficiency in children. The
manuscript is nearly complete and will put up on the SSC website for comment in the coming
weeks.
3. Prophylaxis in patients with inhibitors: C. Escuriola (DE)
Dr. Nadia Ewing (in place of Dr. Escuriola) presented the working group’s progress on
recommendations for prophylaxis in inhibitor patients. The group has nearly completed its work
and will develop a manuscript over the summer.
4. Bleeding Score in Hemophilia: E. Mancuso (IT)
Dr. Mancuso presented her working groups progress on this project. Of note, 37 centers have
agreed to participate in a data collection project to validate the bleeding score that was
developed. So, far 99 patients with hemophilia and B have been enrolled.
5. ITI definitions: E. Santagostino (IT)
Dr. Santagostino presented an update of her group’s project. She reviewed the recommended
definitions for successful ITI based on both pharmacokinetic as well as clinical endpoints. The
group is working on developing a manuscript which is planned for completion by next year.
6. Mild hemophilia definitions: M. Makris (GB)
Dr. Makris discussed the final results of his group’s work on defining mild hemophilia.
Specifically, he discussed modifications of the existing SSC definitions of mild hemophilia
including defining some patients with >40% levels of factor VIII as having hemophilia if they
have a DNA change consistent with hemophilia and a family member with a level of <40%. He
also discussed issues with measuring factor levels with the one-stage versus choromogenic
assay that could lead to missing the diagnosis in some patients.
7. Inhibitor reporting standardization in previously treated patients: A. Iorio (CA)
Dr. Iorio provided an interim report of his group’s work on reporting of results from trials and
other studies on the inhibitor rate in PTPs. The group has developed recommendations
regarding how to report inhibitors in PTPs in the literature.
8. Standardization of genetic assays for diagnosis of hemophilia: V. Jenkins (IE)
Dr. Jenkins reported on his group’s work on standardization and quality assurance as it applies
to genetic testing in hemophilia. First, a survey was sent out to various laboratories to determine
the types of tests that are done, the logistics of test performance, the methods of determining
the genotype. The group will use this data to develop recommendations for standardization and
quality assurance for genetic testing in hemophilia.
Education Program
1. From Alexie Romanov to Stephen Christmas to 2015: What is different about factor
IX deficiency: E. Santagostino (IT)
Dr. Santagostino provided a review of the differences between FIX and FVIII deficiency focusing
on whether or not FIX deficiency is a less severe bleeding disorder. The presentation evaluated
the literature on factor VIII and IX deficiency with respect to the frequency of prophylaxis,
hemophilic arthropathy, and bleeding rates, and in general as opposed to FVIII deficiency,
patients with FIX deficiency were less likely to be on prophylaxis, had less hemophilic
arthropathy, and had the first joint bleed at a later age, however it was not clear if this is a real
difference in terms of disease severity or epidemiologic findings that may be due to the notion
that FIX deficiency is less severe.
2. What is the “true” incidence of inhibitors in hemophilia: M. Soucie (US)
Dr. Soucie provided an epidemiological overview of inhibitor incidence including a detailed
discussion on the definitions of incidence and prevalence and elaborating on the limitations of
the statistical methods for detection of inhibitors in a low prevalence disease.
Rare Bleeding Disorders
1. Update on afibrinogenemia and dysfibrinogenemia: P. de Mooerloose and A. Casini
(CH)
Dr. Casini provided an overview of fibrinogen disorders including a detailed discussion on
genotype-phenotype correlations. Dr. Casini discussed congenital afibrinogenemia, congenital
hypofibrinogenemia, and congenital dysfibrinogenemia. All 3 conditions are associated with a
bleeding disorder with congenital afibrinogenemia clearly being the most severe of the three.
2. PRO-RBDD Project: Prospective evaluation of the intensity of bleeding episodes in
patients with fibrinogen and FXIII deficiency: F. Peyvandi (IT)
Dr. Peyvandi updated the SSC on her project of these two rare bleeding disorders including
data from a multisite registry study on bleeding events in these two rare disorders as well as
treatments given by the institutions that participated. Specifically for FXIII deficiency, she
discussed that patients with a mild deficiency exhibit bleeding symptoms as well including
mucus membrane bleeding and menorrhagia. Treatments varied throughout the world, however
most developed nations now have factor concentrates available for treatment of both and many
patients with FXIII deficiency are on prophylaxis.
Laboratory Issues 1
1. Update on Nijmegen group’s high sensitivity inhibitor assay: B. Verbruggen (NL)
Dr. Verbruggen described a novel increased sensitivity inhibitor assay based on the Nijmegen-
modified Bethesda assay. He reviewed a small validation study that his team performed which
did demonstrate that the assay correlates with the half-life, however the number of patients was
small and further validation is warranted.
2. Inhibitor measurement using SMIA: a new approach in inhibitor measurement: S.
Raut (GB)
Dr. Raut described a novel assay (South Mimms Inhibitor Assay) which is a modification of the
Classical Bethesda Assay which is aimed mostly at reducing the inter-laboratory variation of the
Bethesda assay and the Nijmegen-modified Bethesda assay. It may also improve the sensitivity.
His next step is to do a multi-laboratory study to determine the inter-laboratory coefficient of
variation.
3. Establishment of the 5th International Standard for FIX: E. Gray (UK)
Dr. Gray described the establishment of the 5th international plasma standard FIX and the
1stinternational standard for recombinant FIX. There is general agreement to have this new
standard move towards endorsement by WHO. There is less agreement about the need for an
international standard for recombinant FIX and plans for that have been deferred for now.
4. Update on EAHAD Coagulation Factor Variant Genotype Database: K. Gomez (UK)
Dr. Gomez presented the newly developed EAHAD supported database of mutations in FVIII,
FIX, and VWF. He demonstrated the utility and ease of use of the database.
5. IU vs %: Clearing up the confusion and a proposal for a position paper: S. Kitchen
(GB)
Dr. Kitchen described the issues and concerns with the use of the terms units versus IU versus
%-age. He discussed that %-age is not always equal to IU and he felt that a position paper or
recommendation from the FVIII/FIX/RBD subcommittee. Dr. Kitchen will send me a proposal for
a position paper on this topic in the next 2 months for further discussion.
6. WAPPS Project (Web-accessible population pharmacokinetics service): A. Iorio
(CA)
Dr. Iorio presented the development of his web-accessible pharmacokinetics service and
reviewed how clinicians and researchers can access it and utilize it for clinical decisions or for
developing research projects. Once the application is ready to go live, anyone will be able to
access it as a free web-based application. Once this is available, he will let me know and we
can put an announcement out on the SSC webpage.
Clinical Issues
1. Moderate hemophilia update: K. Fischer (NL)
Dr. Fischer provided an update on the clinical aspects of moderate hemophilia based on some
recent observations and large studies mostly from The Netherlands. She demonstrated data
from a large database in the Netherlands that a subset of patients with moderate hemophilia will
develop joint disease if not treated aggressively as they act more like patients with severe
hemophilia, and even those who don’t bleed like severe hemophilia are prone to developing
severe bleeds on occasion.
2. Clinical use of extended half-life factor products: G. Young (US)
Dr. Young presented a series of questions regarding the use of extended half-life factor
products to the audience and solicited audience opinions which led to an open forum discussion
on this topic. Several audience members spoke of their opinions regarding the use of these
drugs in PUPs indicating that they would not treat PUPs with these drugs until there was more
data though one person indicated that she thought PUPs with their poor venous access would
be the ideal patient group to initiate these drugs in. There was a general feeling that for patients
with poor venous access or those who have not been on prophylaxis due to the frequency of the
IV infusions are the best candidates for extended half-life drugs.
Laboratory Issues 2
1. Extended half-life factors and laboratory assays--overview of the problem: S. Pipe
(US)
Dr. Pipe provided an overview of the issues surrounding laboratory analysis of the extended
half-life factors demonstrating that this is a relevant clinical issue when treating patients with
these agents as it relates to measuring levels of the infused factor.
2. Overview of chromogenic assay basics: K. Friedman (US)
Dr. Friedman provided an overview of the mechanism for the factor VIII and factor IX
chromogenic assay. He described in detail the differences between the assays stating that the
chromogenic assay has less variability and is more reliable as it truly focuses on the action of
FVIII or FIX rather than evaluating the entire coagulation cascaded.
3. Implementation of the chromogenic assay into the clinical lab: R. Ko (US)
Dr. Ko described the implementation of the chromogenic assays for factor VIII and factor IX in
his clinical coagulation laboratory. He described the steps that he went through to make this
assay available in his institution. Briefly, the steps included making a request to the clinical
laboratory, identifying vendors for the assays, choosing a vendor, performing laboratory
calibration followed by validation, and then once ready, developing a mechanism for ordering
the laboratory tests.
Factor XI and the Contact System
21 June 2015 8:00 – 12:20
Chairman: Jonas Emsley
Co-Chairs: Jose W. Govers-Riemslag, Christine Mannhalter, Joost Meijers, James Morrissey, Ophira Salomon, Evi X. Stavrou
The factor XI and contact system was attended with 200 attendees.
Bernd Engelmann (Germany): Propagation of blood coagulation by extracellular
nucleosomes/neutrophil extracellular traps was presented. This talk emphasized the importance
of neutophils/platelets/contact system in thrombus formation; particularly in venous thrombosis
where FXIIa seems to be more important than neutophil elastase. This talk also showed the
association of DNA from NETs with factor XII and DNA as an activator of the contact system.
Jonas Emsley (UK): Data on the structure of PK and the interaction with HK was presented
together with
Ammar Majeed (Sweden): The Factor XII Registry Database. This talk reported a new
website FXII registry website URL is (www.factor12.net) where patient information will be stored
on FXII deficient. There are several reasons why this important. 1. There are few reports
characterizing factor XII deficient patients and currently no systematic collection of data. This is
important as FXII is considered an important novel target for the treatment of thrombosis and yet
there is no epidemiological data on the effects of deficiency in humans. This contrasts with the
case of Factor XI where and established website is available and >200 cases of FXI deficiency
have been characterized together with the genes sequenced. It was discussed that it would be
beneficial to have sequencing carried out for the FXII genes and that patients with a defect but
normal FXII plasma levels (CRM+) be entered into the database. The audience was encouraged
to submit information on FXII deficient patients.
Nicola Mutch (UK): Driving plasminogen activation by factor XIIa. This talk described new data on
FXII association fibrinolysis - polyp modulated the fibrinolytic side of the FXIIa activity and polyp
enhances fibrinolysis. PolyP binding characterized to FXII and plasminogen. The contribution of
platelets to fibrinolysis was characterized as platelets secrete PolyP. PolyP accumulates in fibrin
knots. A pathway was described for to contribute to fibrinolysis. FXII and fibrinogen were imaged
localized on the surface of platelets at the cap with polyp.
Keith McCrae (USA): This talk presented an update on kininogen in thrombosis and stroke models
and as an anti-angiogenic protein.
Toshitaka Sugi (Japan): Autoantibodies to FXII and kininogens in patients with recurrent
pregnancy loss were described. This talk presented data on a series of FXII deficient patients
characterized with recurrent pregnancy loss and also antibody interactions with FXII and
kininogen in the present of phospholipid. A mechanism of FXII-antibody complexes with platelets
is proposed for promoting thrombosis.
Theme: Inhibitors of FXI and the contact system: Safer anticoagulation
Maria Luiza Vilela Oliva (Brazil): Effect of plant inhibitors of the contact system on a mouse
thrombosis model. CTI is a well know plant protein that is used as an inhibitor of the contact
system. A further plan kunitz type inhibitor was presented which inhibits kallikirein and its
properties were characterized as inhibiting thrombus formation in a mouse model. It was also
proposed the inhibitor is bi-functional affecting the collagen receptor.
David Gailani (USA): New data were described for FXI anion binding sites and the effects of
mutations here on PolyP and nucleic acid activation.
Sanjay Bhanot could not attend so David Gailani presented a second talk (USA): Antisense
Reduction of FXI for Thromboprophylaxis: A Novel Therapeutic Approach. This data showed the
first clinical trial of and antisense RNA targeting FXI (N Engl J Med. 2015 Jan 15;372(3):232-40.)
This showed effective reduction in circulating FXI levels over a period of weeks such that
thrombosis was mitigated with no bleeding side effect. Data on factor XI contribution to venous
thromboembolism was also presented.
Open discussion José Govers-Riemslag (the Netherlands), and Joost Meijers, (the
Netherlands), presented in an open discussion several topics to the audience. Since a factor XII
registry was proposed, the first question was if each contact factor needs its own registry, which
was nearly unanimously accepted. A combination of registries for the 4 contact proteins was found
to be logistically unrealistic.
Furthermore, an inventory was made of the interest of the audience for the mechanisms that can
be activated by the contact system. In order of interest, the audience voted for the coagulation
system, the complement system, the kallikrein-kinin system and the fibrinolytic system. Finally,
the development of assays for the contact system was discussed. A general test that could
determine outcome of all the affected mechanisms was not found to be practical, but there was
great interest in the design and execution of specific tests that could measure contact system
mediated activation of the coagulation, kallikrein-kinin system, fibrinolytic system and complement
system. The audience felt that there was a role for the SSC subcommittee in developing such
assays.
Factor XIII and Fibrinogen
20 June 2015 14:30 – 18:50
Chairman: Helen Philippou
Co-Chairs: Zsuzsa Bagoly, Matthew J. Flick, Vytautas Ivaskevicius, Marlien Pieters, Verena Schroeder, Alisa Wolberg
Saturday 20 June 2015 | 14:30 - 18:50
At the start of the session it was estimated that there approximately 300-400 attendees. Dr
Philippou opened the session welcoming the audience.
Educational session
14.30-15:00 “Roles of the alpha chains of fibrinogen" Leonid Medved (Baltimore, USA)
Apologies were received from Prof Medved, who due to unforeseen was unable to attend the
conference.
15:00-15:30 “A Historic Recollection of Factor XIII Research" Akitada Ichinose
(Yamagata, Japan)
Prof Ichinose gave a historical overview of the discovery of FXIII and some key highlights in the
early discoveries related to FXIII and its characterization, as follows:
1) Evolution of transglutaminases, 2) Nomenclature of a plasma transglutaminase (FXIII) and
the annual numbers of its publication, 3) Discovery of FXIII through clot insolubility, 4) Real
function of FXIII; Hereditary deficiency and autoimmune hemorrhagic disease, 5) Purification of
plasma FXIII, 6) Legendary players in biochemical studies of FXIII, 7) Legendary players in
clinical studies for FXIII, 8) Cloning and molecular biology of FXIII, 9) Molecular pathological
analyses of hereditary FXIII deficiencies, 10) 3D structure of FXIII, 11) Polymorphisms of FXIII
and its relationship with thrombosis, 12) Rec. FXIII-A subunit; a very long way to the market, 13)
FXIII KO mice, 14) Modern times; active distinguished researchers and their cutting-edge
projects, 15) Remaining enigmas/issues regarding FXIII.
SSC session
15:30-15:50 “Standardization of turbidity and fibrinolysis measurements” Marlien Pieters,
South Africa
Marlien Pieters gave an update on the progress of the standardization of the fibrin clot turbidity
and lysis assay. The purpose of standardizing the assay is to improve comparison of results
between labs and to attempt to move in the direction of developing normal ranges for these
variables. In essence, the progress thus far is as follows: the protocol is being finalized by the
working group. They will also submit a funding application to the ISTH to obtain funding to
supply the necessary reagents to all participating labs. Once the protocol has been finalized and
funding obtained, labs will be invited to participate. It is envisioned that the experimental work
will take place in the second half of 2015 and that the project will be completed before the 2016
SSC meeting.
15:50-16:10 “FXIII-B standardization update” Éva Katona (Dubrecen, Hungary), Verena
Schroeder (Bern, Switzerland)
Verena Schroeder nicely introduced the session by describing the procedure necessary for
working with the NISBC and WHO for the introduction of a new standard.
Éva Katona subsequently introduced the progress made on the generation of selective
antibodies and ELISAs specific to the B-subunit measurement. The work has progressed well
and will require publication of the new ELISAs prior to being able to continue with the
standardization procedure.
16:10-16:30 “New Insights into Automated Fibrinogen Clauss Assays” Sanj Raut
(Potters Bar, UK)
The CLOTr (clot removal) method is currently the E P recommended method for assessing
fibrinogen activity in therapeutic concentrates/fibrin sealant products (used by Manufacturers
and Regulatory laboratories for Lot release purposes). This method was also used to value
assign the current 2nd International Standard for Fibrinogen Concentrate (09/242). However,
this method is slow, cumbersome, time consuming & labor intensive; not at all ideal as a high
throughput assay. There is therefore a real need for a more simple/automated alternative
method.
Clauss assay is one alternative functional assay used routinely for measuring fibrinogen in
plasma and is based on time for fibrin to clot. High concentrations of thrombin are used
(typically 100 IU/ml) to ensure clotting times are independent of thrombin concentration over a
wide range of fibrinogen levels. Fibrinogen samples are incubated with thrombin and calcium at
37ºC, and time taken to clot is compared vs reference standard. Most laboratories have an
automated Clauss method for measuring plasma (method based on photo optical end point
determination). Although, Clauss assay was shown to be suitable for measuring fibrinogen in
plasma, previous studies have shown Clauss assay to be unsuitable and highly variable for
measuring fibrinogen in concentrates.
This study assessed whether Clauss assay could be optimized to be sufficiently suitable for
measuring fibrinogen in therapeutic concentrates. Data from Claus assays (measuring
Fibrinogen concentrates) using mechanical end-point instrumentation (e.g. KC4) was found to
be comparable to data from CLOTr method and Absolute Methods (Kjeldahl & Clot
Weight). However, significantly higher (~30%) potencies were obtained from Clauss assays
using photo-optical instrumentation or when attempting to automate the assay (e.g. using auto
analyzers) & using specific commercial kits. This was still the case when pre-diluting fibrinogen
concentrates in fibrinogen deficient plasma (in order to mimic assays of plasma) to help
standardize turbidity issues, although some improvements were noticed.
As all these assays produced perfectly valid clotting curves for all dilutions, the various pre-
defined mathematical algorithms provided by the analyzers were investigated. As most auto-
analyzers are designed for assessing fibrinogen in plasma, these analyzers use predefined
“Threshold” algorithm by default, which is based on time to clot, measured in seconds. As the
analyzers were making measurement in Absorbance, a more appropriate algorithm for
measuring using photo-optical instrumentation would be one that measures in Absorbance
related units. Such algorithms exist in these analyzers, such as the “Delta” algorithm, which
uses the 2nd derivative of the clotting curve & measures in ∆ mAbs and if this algorithm is
selected then we get a very good agreement in fibrinogen potency between the CLOTr method
and the automated Clauss assay (<2% difference) and with CV <3%.
The study concluded that automated Clauss assays can be suitable for measurement of
fibrinogen concentrates using photo-optical instrumentation, as long as pre-defined algorithm
selected is based on optical (∆ mAbs) readout.
Further work is on-going to assess different fibrinogen therapeutic concentrates,
assess different Instrumentation (different algorithms), assess different Kits/reagents (specific
for analyzers) with the ultimate aim to carry out Collaborative Study to assess the suitability and
variability (between laboratories) of this assay for measurement of fibrinogen in therapeutic
concentrates.
16:50-17:15 “The Role of Fibrin(ogen) in Inflammation" Matthew Flick, Cincinnati
Children’s Hospital and Medical Center, USA
Fibrinogen has been previously implicated as a key factor in mediating host defense against
peritonitis infection by Staphylococcus aureus, a common and re-emerging gram-positive
bacterium that is a leading cause of both community and hospital-acquired infections. Dr Flick’s
laboratory has investigated the hypothesis that thrombin-mediated fibrin polymer formation is a
critical mechanistic component of the host antimicrobial response during of S. aureus
peritonitis. To establish a system to distinguish fibrinogen- and fibrin-dependent processes in
vivo, FibAEK mice were generated that have normal levels of circulating fibrinogen but lack the
chain thrombin
cleavage site. Thrombin failed to release fibrinopeptide-A from fibrinogenAEK and failed to induce
polymer formation with FibAEK plasma or purified fibrinogenAEK in 37°C mixtures regardless of
incubation time. Platelet-rich plasma from FibAEK mice supported normal platelet aggregation in
vitro, highlighting that fibrinogenAEK retains the functional capacity to support interactions with
platelets. FibAEK mice displayed both an absence of fibrin polymer formation following liver
injury, as assessed by electron microscopy, and a failure to generate stable occlusive thrombi
following FeCl3 injury of carotid arteries. Notably, FibAEK mice exhibited a profound impediment
in S. aureus clearance following intraperitoneal infection similar to fibrinogen-deficient mice, yet
FibAEK mice displayed a significant infection dose-dependent survival advantage over fibrinogen-
deficient mice following peritonitis challenge. Collectively, these findings establish for the first
time that fibrin polymer is the molecular form critical for antimicrobial mechanisms while
simultaneously highlighting biologically meaningful contributions and functions of the soluble
molecule.
17:15-17:40 “Fibrinogen Density Decreases Slightly and Fiber Stiffness Decreases
Strongly with Increasing Fibrin Fiber Diameter” Martin Guthold, Wake Forest University,
USA
The major structural component of a blood clot is a meshwork of fibrin fibers with an average
diameter of about 130 nm. The longitudinal assembly of fibrin monomers into protofibrils is well
understood; however, the radial (lateral) assembly of protofibrils into mature fibers is poorly
understood. It has long been thought that the internal structure of fibrin fibers is regular and
homogenous; that is, the protein density and the bond density between protofibrils are
uniform. Prof Guthold and colleagues performed two types of experiments to investigate the
internal structure of fibrin fibers. They formed fibrin fibers with fluorescently labelled fibrinogen
and determined the light intensity of a fiber, I, which is proportional to the number of monomers,
as a function of fiber diameter, D. They found that the intensity, I, scales as I~D1.43±0.03. This
implies that the cross-sectional monomer density also scales as D1.4, and not as D2, as would
be expected for fibers with a solid, homogeneous cross-section. They also determined the
Young’s modulus, E, as a function of fibrin fiber diameter, and found that E strongly decreases
with increasing D, as E~D-1.4. The modulus data suggest that the number of bonds per cross-
section scales as D0.6. The data are consistent with a fiber model that has a dense core and a
very loosely connected periphery. In contrast, electrospun fibrinogen fibers, used as a control,
do seem to have a homogenous cross-section, where the density scales as D2.
17:40-18:05 "Novel functions of fibrin(ogen) in liver repair and fibrosis" James P.
Luyendyk (Michigan, USA)
Experimental acute and chronic liver toxicity is associated with activation of the coagulation
cascade in rodents. This is marked by increased thrombin generation and hepatic fibrin(ogen)
deposition. Although the association between hepatotoxic responses and coagulation has been
appreciated for decades, the role of fibrin(ogen) in acute and chronic liver toxicity has not been
investigated in detail. Utilizing a combination of tools including pharmacologic modulation of
fibrinolysis and mice completely lacking fibrin(ogen) or expressing mutant fibrin(ogen) with
defective integrin-binding capacity, Dr Luyendyk and colleagues have identified previously
undescribed mechanisms whereby fibrin(ogen) can promote liver repair and inhibit liver fibrosis.
The take-home message from the presentation was that the role of fibrin(ogen) in experimental
settings of liver disease and toxicity is context and time-dependent.
18:05-18:30 “The Role of Crosslinking in Fibrin Structure and Function” Robert Ariens
University of Leeds, UK
Dr Ariëns discussed the role of cross-linking by FXIIIa in fibrin clot structure and function. He
began with explaining how FXIII activation is closely regulated by the conversion of fibrinogen to
fibrin by thrombin, and how activated FXIIIa cross-links the fibrin when it is being formed. He
described all the known FXIII cross-linking sites in fibrin and set out how gamma chain cross-
linking stabilizes the protofibril structure while alpha chain cross-linking stabilises the lateral
aggregates of protofibrils and contributes to fibrin stability through the incorporation of inhibitors
of fibrinolysis. Then he went on to discuss the binding sites for FXIII on fibrinogen, which include
sites on the fibrinogen gamma prime splice variant chain, gamma 390-396, alpha 233-425 and
alpha 389-402. These binding sites localize and direct FXIII to the reactive cross-linking sites.
Finally, Dr Ariëns discussed the effects of cross-linking by FXIIIa on fibrin clot structure and
function. Cross-linking by FXIIIa has subtle but significant effects on clot structure, by increasing
fibre number, reducing pore size and straightening fibres. Functionally, cross-linking by FXIIIa
increases stiffness of the fibrin clot, increases resistance to fibrinolysis (largely through
incorporation of inhibitors of fibrinolysis) and helps the retention of red blood cells in the clot.
18:30-18:40 “Proposal of ISTH/SSC Diagnostic Criteria 2015 for Autoimmune
Hemorrhaphilia Due to Anti-FXIII/13 Antibodies” Akitada Ichinose (Yamagata, Japan),
Hans Kohler (Bern, Switzerland), Laszlo Muszbek (Dubrecen, Hungary) and Helen
Philippou (Leeds, UK).
Professor Ichinose presented an overview of a proposal of ISTH/SSC diagnostic criteria 2015
for autoimmune hemorrhaphilia due to Anti-FXIII/13 Antibodies, as follows:
<Possible AH13>
1. Recent onset of bleeding symptoms mainly in the older adult.
2. No family history of congenital/inherited deficiency of FXIII/13.
3. Lack of previous bleeding symptoms especially in association with previous
hemostatic challenges (e.g., surgery, invasive tests, trauma, etc.).
4. Not explained by medication such as anticoagulants and antiplatelet drugs.
5. Abnormality of FXIII/13 parameter(s) on laboratory testing (FXIII/13 activity and/or antigen
<50%).
<Probable AH13>
6. Items 1-5 plus the presence of FXIII/13 inhibitors** (positive by cross-mixing tests between
patient’s and healthy control’s plasma using standard functional tests, such as an ammonia
release assay and an amine incorporation assay, after 2 hours incubation at 37°C).
<Definite AH13>
7. Items 1-5 plus the presence of anti-FXIII/13 autoantibodies (positive by immunological
methods, such as dot blot, ELISA, and immunochromatography, etc.).
*; Autoimmune hemorrhaphilia due to anti-FXIII/13 antibodies
**; are not always autoantibodies because non-immunoglobulin (antibody) inhibitors were
reported before.
The audience were asked whether there was an objection to this proposal, of which there none.
All members were in support of the proposal.
Fibrinolysis
20 June 2015 9:00 – 13:20
Chairman: Nicola Mutch
Co-Chairs: Jonathan H. Foley, Paul Y. Kim, Krasimir Kolev, Craig Thelwell, Shirley Uitte De Willige, Waander Van Heerde
Standardization of fibrinolytic proteins
Chairs: Dr Jonathan Foley & Dr Waander van Heerde
09:05-09:15 Dr Colin Longstaff, NIBSC, UK
‘Standardization of D-dimer’
CL updated the subcommittee on on-going activities to prepare a standard for D-dimer.
Previous work had identified problems of stability in D-dimer preparations, both in patient
plasma pools and when using cross-linked FDP made by lysing fibrin, after freeze drying. This
would make the development of a WHO standard impossible. However recent studies on long
term stability had identified formulations that included trehalose as more stable. It is hoped that
future work will include a collaborative study in a small number of labs to test of potential
standards, and these studies will include some patient samples as a way of addressing
questions of commutability. The results from these studies will inform future work on the
development of a definitive WHO International Standard for harmonization of D-dimer
measurements.
09:15-09:25 Dr Craig Thelwell, NIBSC, UK
‘Development of standards for plasmin, streptokinase, ancrod and Batroxobin’
Plasmin: The completed study on the calibration of the proposed WHO 4th International
Standard for Plasmin (13/206) was presented to the Fibrinolysis subcommittee in Milwaukee,
2014. The proposal was endorsed by the SSC/ISTH and the 4th IS was established by the
ECBS of WHO October 2014, with a potency of 8.0 International Units. The 4th IS is now
available to order from the NIBSC catalogue.
Streptokinase: Stocks of the WHO 3rd IS for Streptokinase (00/464) are low and a replacement
is needed. A proposal to calibrate the 4th IS as a direct replacement was presented. Potency
estimates of a candidate material, which will be a native therapeutic streptokinase preparation,
will be made in a multi-center study relative to the 3rd IS using chromogenic and fibrin-based
assay methods. Laboratories with appropriate assay methods were encouraged to contact
NIBSC to register an interest in participating in the study.
Ancrod and Batroxobin: A joint collaborative study was proposed to calibrate replacements for
the WHO 1st International Reference Reagent (IRR) for Ancrod (74/582) and the 2nd British
Standard for Batroxobin (93/526). Ancrod and Batroxobin are thrombin-like serine proteases
derived from snake venoms. Unlike thrombin ancrod and batroxobin cleave fibrinogen removing
only A-fibrinopeptides (not B-fibrinopeptides). This exerts an anticoagulant effect through the
removal of circulating fibrinogen and both are indicated for treatment of a range of thrombotic
diseases. Ancrod has been investigated in several clinical trials for treatment of acute ischaemic
stroke, with mixed results and is currently being investigated in a clinical trial for sudden
sensorineural hearing loss (SSHL). Batroxobin is used clinically to determine if prolonged
thrombin clotting times are a result of heparin contamination or abnormal fibrinogen, referred to
as "Reptilase time”. WHO/ECBS has endorsed the replacement of the 1st IRR for ancrod as the
2nd IS, and the 2nd BS for Batroxobin as the 1st IRR. Both will be calibrated relative to the
previous standard in a collaborative study using clotting and defibrinogenase assay methods.
The study is being run through the Exogenous Factors Subcommittee, but may be of interest to
members of the Fibrinolysis Subcommittee and interested laboratories are encouraged to
contact NIBSC.
09:25-09:40Dr Craig Thelwell, NIBSC, UK
‘Standardization of TAFI (CPU)’
Thrombin-activatable fibrinolysis inhibitor (TAFI, also known as proCPU or pCPB2) is a 56 kDa zymogen. TAFI is activated to TAFIa physiologically by the thrombin/thrombomudulin (TM) complex and plasmin. TAFIa is a basic carboxypeptidase that cleaves C-terminal lysines from partially degraded fibrin inhibiting fibrinolysis. TAFI/a levels have been shown to correlate with thrombotic diseases and inflammation. There are a large number of commercial and in-house methods for measuring TAFI/a in plasma. ELISA based assays are commonly used and are relatively simple to perform, however these methods rely on antibodies to distinguish between TAFI, TAFIa and TAFIai and do not allow measurement of active TAFIa. Furthermore detection for some antibodies differs between Thr/Ile325 isoforms. Functional activity based assays are also available. Direct activity based assays are complicated in plasma by carboxypeptidase N (CPN); indirect measurements require quantitative activation of TAFI and standardized methods are required due to instability of TAFIa and increased stability of Thr/Ile325, although kinetic activity is independent of 325 isoform. The activity of CPN must also be accounted for in these assays. Estimates of plasma TAFI concentration range from 73-275 nM (4-15 µg/ml) and there are a range of possible contributing factors to this apparent variation. Genotype differences in the regulatory region affect gene expression and mRNA stability and non-genetic factors can also affect gene expression. Different reactivities of antibodies in commercial kits e.g. detection of Thr/Ile325 and different plasma pools and purified TAFI used as calibrators in different methods also contribute to the apparent difference. A common reference of a normal plasma pool could help to identify the source of, and reduce some of this variability. A traditional WHO approach to calibrate a standard in International Units (IU) could follow the convention for coagulation plasma standards with 1 IU being equivalent to the amount of TAFI in normal plasma. A candidate reference plasma would be calibrated relative to local normal plasma pools arbitrarily assigned a potency of 1 IU. The study would also include local materials and a common freeze-dried plasma to determine if the variation is reduced by the inclusion of a common reference. This approach is compatible with kits currently using a normal plasma reference with a % labeled potency, where 100 % would be equivalent to 1 IU, however calibrators assigned ‘ng’ values would require a change of unit, which could be confusing. Assigning a ‘consensus ng’ value relative to local standards would not be acceptable to WHO since this is an SI unit which requires an absolute measurement with metrological traceability.
Quantitative analysis of TAFI in plasma is possible using isotope dilution mass spectrometry (IDMS) but is complicated by the high abundance plasma proteins. A tryptic peptide analysis of purified TAFI was demonstrated and two approaches to quantitate plasma TAFI were proposed. The first is to enrich TAFI by immunoprecipitation, and data was presented using a TAFI antibody magnetic bead approach. The second approach is to use multi-affinity columns to deplete the high abundance plasma proteins to improved TAFI detection. A collaborative study was proposed to include a candidate plasma reference material and a common plasma sample, both quantified for TAFI content by IDMS, to be measured by TAFI antigen and activity assays relative to local plasma pools and local reference standards. Interested laboratories were invited to contact NIBSC to participate, and collaborators were invited to contribute to the IDMS quantitation of TAFI in plasma.
Diverse roles of plasmin
Chairs: Dr Paul Kim & Dr Nicola Mutch
09:40-10:00 Dr Claudia Tersteeg, University Medical Center Utrecht, the Netherlands
‘Plasmin and Von Willebrand Factor in Thrombotic thrombocytopenic purpura’ Patients with thrombotic thrombocytopenic purpura (TTP) suffer from the presence of von Willebrand factor (VWF)/platelet-rich thrombi in the microvasculature, resulting in thrombocytopenia, hemolytic anemia, organ failure and death when left untreated. TTP is associated with a deficiency in VWF-cleaving protease ADAMTS13. However, ADAMTS13 activity levels not always predict acute TTP episodes and therefore suggest a role for other VWF-cleaving enzymes in the regulation of TTP episodes. We have investigated the role of plasmin in the cleavage of VWF in TTP. We have demonstrated that plasmin activated via uPA/uPAR is able to degrade VWF/platelet-rich microthrombi in vitro and that endogenous plasmin levels are able to control acute TTP episodes in vivo in a mouse model for TTP. Additionally, patients with acute TTP demonstrate increased levels of plasmin-alpha2-antiplasmin and PAI-1, and decreased uPA activity, suggesting regulation of plasmin activation during acute TTP. Furthermore, thrombolytic agents were demonstrated to be a possible future treatment strategy for TTP. Together, this demonstrates that plasmin cleavage of VWF is important in TTP pathology, and future research will open new doors towards new treatment strategies for this severe and life-threatening disease.
10:00-10:20 Ms. Marijke Peetermans, Katholieke Universiteit Leuven, Belgium
‘Plasmin generation by Staphylokinase during skin infections with Staphylococcus aureus’
Staphylococcus aureus (S. aureus) is a major etiologic agent of a very broad range of infections, such as skin and soft tissue infections and intravascular catheter infections. Interestingly, S. aureus produces not only coagulases that trigger fibrin formation, but also staphylokinase (SAK), a fibrin-specific plasminogen activator. The SAK-plasmin complex is protected against rapid inhibition by alpha-2-antiplasmin (α2AP). We aimed to elucidate the role of SAK-induced human plasminogen activation in infections with coagulase-positive S. aureus and understand the dynamics of the apparent opposing activation of both the coagulation and the fibrinolytic systems by S. aureus.
Skin infection: Skin infection was induced by subcutaneous injection of S. aureus Xen36 in mice
containing an adenoviral vector for human plasminogen. We observed higher bacterial loads
and increased spreading of infection in human plasminogen-expressing mice compared to wild-
type mice, indicating SAK-mediated human plasminogen activation in local invasion. In α2AP
knock-out mice (α2AP -/-) local spreading in later stages of infection was attenuated. However,
close to the initial abscess site the SAK-plasminogen complex is protected from α2AP action by
either the bacterial surface or by fibrin produced by coagulase-positive S. aureus. Fibrin-specific
SAK action and downstream gelatinase activation allows the local spreading of S.
aureus through tissue barriers and decreases bacterial clearance.
Catheter-related infection: We have previously shown the importance of S. aureus coagulase
activity in adhesion to foreign bodies and formation of biofilm matrix. However, in later stages
of in vitro biofilms, scanning electron microscopy revealed dispersal of biofilms. We analyzed
the role of SAK in biofilm structure, using S. aureus strains with different levels of SAK
production grown in diverse media with or without added prothrombin, fibrinogen or
plasminogen. High production of SAK by S. aureus LS-1 spasak interfered with bacterial-
induced fibrin formation and adhesion to foreign body surfaces. If plasminogen was omitted
from the culture medium, a thick fibrin-rich biofilm with almost no evident dispersal was seen. A
jugular vein catheter infection model was performed in human plasminogen expressing mice
using the same congenic S. aureus strains with different levels of SAK production. Indeed, the
high-SAK producing S. aureus spasak strain showed lower virulence in biofilm infection. In S.
aureus foreign body infections, SAK production leads to dispersal of biofilms and release of
bacteria from the fibrin-containing biofilm structure. The expression of SAK in S. aureus biofilms
is regulated by quorum sensing, shifting the balance from coagulase activity to fibrinolysis in
later-stage biofilms, when bacterial density increases.
Impact of cells on fibrinolysis
Chairs: Dr Krasimir Kolev & Dr Jonathan Foley
10:20-10:40 Dr Paraskevi Untiveros, University of Aberdeen, UK
‘Effect of hematocrit on clot structure and resistance to lysis’
Red blood cells (RBCs) have been considered a relatively inert bystander in coagulation and
thrombus formation, yet their sheer abundance in blood means they dominate the resulting clot.
Here we examine the effects of haematocrit (HCT) on coagulation parameters, clot firmness and
resistance to fibrinolysis. Whole blood was separated into plasma and RBC constituents by
centrifugation. Samples were reconstituted with 35% platelet rich plasma and RBCs to produce
20%, 40% and 60% final HCT. Thrombus formation and dissolution were assessed using
Thromboelastography (TEG) and the Chandler Loop Model. TEG revealed significantly shorter
clot time, clot formation time and increase in the α-angle at 20% HCT compared to 60% HCT;
indicative of faster clot formation at lower HCT. An increase in maximum clot firmness was
detected at 20% HCT, consistent with enhanced mechanical stability. Chandler model thrombi
formed with 20% HCT were significantly longer than those formed at 60% HCT (p<0.05) and
demonstrated increased resistance to lysis by both tPA (p<0.005) and uPA (p<0.005). Inclusion
of a transglutaminase (TG) inhibitor, to inhibit factor XIIIa, significantly increased lysis of 20%
HCT thrombi (p<0.001). Similarly, the TG inhibitor increased lysis of 40% HCT thrombi (p<0.01),
albeit to a lesser degree, but no change in lysis was observed at 60% HCT (p=0.113).
Interestingly, when the TG inhibitor was present all thrombi lyse at an equivalent rate,
suggesting that inhibition of factor XIIIa overcomes the stabilizing effect of HCT on lysis. IN
conclusion HCT has a dramatic impact on coagulation parameters, with lower HCT enhancing
clot formation, resulting in thrombi with increased firmness and resistance to fibrinolysis. Factor
XIIIa helps to maintain thrombus integrity and stabilizes against fibrinolytic degradation at lower
HCT, an effect that is lost at higher HCT. These observations may help to explain the increased
risk of thrombosis in anemic patients.
10:40-11:00 Dr Imre Varjú, Semmelweis University, Hungary
‘Neutrophils as key modulators of fibrin structure and lysis in thrombi’
The therapeutic modality of thrombolysis relies on administration of substances capable of
converting plasminogen to plasmin, which in turn digests the primary fibrin scaffold holding the
thrombus together. Since this approach is often ineffective, and is associated with bleeding
complications, a need for the exploration of factors influencing the effectiveness of thrombolysis
is emerging. Thrombi may contain considerable amounts of neutrophil granulocytes, which are
capable of neutrophil extracellular trap (NET) formation by catapulting their DNA with associated
histones and granular enzymes to the extracellular space as a response to thrombosis-
associated inflammatory signals. Since, according to recent studies, NETs are mandatory
components of arterial and venous thrombi, assessment of their thrombolytic consequences is
crucial. Our previous studies have shown that DNA and histones, the major components of
NETs alter the structure of clots rendering them more resistant to fibrinolysis, and recently we
have confirmed these effects using neutrophil-derived NETs in plasma clots. Mounting data
from our and others’ studies suggest that the currently used fibrin-targeted thrombolytic
protocols might be augmented by the addition of enzymes capable of NET degradation (such as
DNAses), which could open a new area of designing thrombolytic agents of the near future.
Educational Session
Chair: Dr Nicola Mutch & Dr Paul Kim
11:20-11:25 Dr Tetsumei Urano, Hamamatsu University, Japan
TU introduced the 1st joint meeting of the International Society of Fibrinolysis and Proteolysis
and the plasminogen activation workshop to take place on Oct. 17-21, 2016 in Shizuoka, Japan
11:25-11:50 Prof Guy Reed, University of Tennessee, Tennessee
‘Influence of a2AP on the pathogenesis of stroke’
Thrombotic vascular occlusion is the primary cause of ischemic stroke. Higher blood levels of
a2-antiplasmin are associated with increased stroke risk in epidemiologic studies, yet a2-
antiplasmin depletion during plasminogen activator therapy has been linked to coagulation
protein depletion and increased risk of bleeding. Therefore, we examined the contribution of a2-
antiplasmin to ischemic stroke outcomes in a model of middle cerebral artery thromboembolism
that closely simulates human stroke. We evaluated the dose-related effects of a2-antiplasmin on
stroke outcomes in mice with increased, normal or no circulating a2-antiplasmin, as well as in
mice given an a2-antiplasmin-inactivating antibody. Higher a2-antiplasmin levels were
correlated with greater ischemic brain injury, brain swelling and reduced middle cerebral artery
thrombus dissolution. In contrast, a2-antiplasmin deficiency enhanced thrombus dissolution,
increased cerebral blood flow, reduced brain infarction and decreased brain swelling. In a2-
antiplasmin-deficient mice, middle cerebral artery thromboemboli that were deficient in a2-
antiplasmin dissolved more extensively than those containing a2-antiplasmin; however, the
presence of a2-antiplasmin did not further reduce brain infarction, swelling or hemorrhage. By
comparison to tissue plasminogen activator, a2-antiplasmin inactivation hours after
thromboembolism still reduced brain infarction and hemorrhage. Microvascular thrombosis, a
process that enhances brain ischemia, was markedly reduced in a2-antiplasmin-deficient or a2-
antiplasmin-inactivated mice compared with tissue plasminogen activator-treated mice or mice
with increased a2-antiplasmin levels (all p<0.001) Matrix metalloproteinase-9 expression, which
contributes to acute brain injury, was profoundly decreased in a2-antiplasmin-deficient or a2-
antiplasmin-inactivated mice vs. tissue plasminogen activator-treated mice or mice with
increased a2-antiplasmin levels. Alpha-2-antiplasmin inactivation markedly reduced stroke
mortality vs. tissue plasminogen activator. Taken together, these data indicate that alpha-2-
antiplasmin has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage,
and survival following cerebral thromboembolism. By comparison to tissue plasminogen
activator or controls, a2-antiplasmin inactivation treatment after the onset of ischemic stroke
significantly increased survival and it reduced ischemic brain injury and hemorrhage. In part, the
protective effects of a2-antiplasmin inactivation or deficiency appear to be mediated through
reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.
11:50-12:20 Dr Manuel Yepes, Emory University, Georgia
‘Plasminogen activators in the brain’
Tissue-type plasminogen activator (tPA) is a serine proteinase found not only in the
intravascular space but also in a well-defined sub-set of neurons in the brain. We show that
overexpression of tPA in neurons protects the brain from the deleterious effects of ischemic
stroke. Early after the onset of cerebral ischemia neuronal tPA is rapidly released into the
synaptic cleft where it activates specific cell signaling pathways that promote the detection and
adaptation to metabolic stress. More specifically, the non-proteolytic interaction of tPA with N-
methyl-D-aspartate receptors (NMDARs) and a member of the LDL-receptor family on the
surface of dendritic spines activates the adenosine monophosphate-activated protein kinase
(AMPK; the sensor of cellular energy status) which leads to the recruitment to the neuronal
membrane of the glucose transporter GLUT3, and GLUT3-mediated uptake of glucose.
Together this promotes survival in neurons exposed to cerebral ischemia. These data indicate
that tPA is an endogenous neuroprotectant in the central nervous system that promotes the
detection and adaptation to metabolic stress.
Inhibitors of fibrinolysis in disease
Chair: Dr Craig Thelwell & Dr Shirley Uitte de Willige
12:20-12:40 Prof Paul Declerck, Katholieke Universiteit Leuven, Belgium
‘Thrombolysis induced by a diabody against PAI-1 and TAFI’
Endogenous fibrinolysis is hampered either through inhibition of plasmin (e.g. alpha2-
antiplasmin), through inhibition of PAs (e.g. plasminogen activator inhibitor-1, PAI-1) or through
modification of the fibrin surface (e.g. activated thrombin activatable fibrinolysis inhibitor, TAFIa).
PAI-1 and TAFI are widely studied antifibrinolytic proteins and have been linked to various
thrombotic disorders. PAI-1 is a serine protease inhibitor (SERPIN) whereas TAFIa is a
carboxypeptidase. The concerted action of PAI-1 and TAFIa by which protection of the fibrin clot
is provided, has led to the idea of dual targeting strategies. Development of antibody-
engineered inhibitors against TAFI and PAI-1 have been shown to stimulate fibrinolysis
efficiently. A heterodimer diabody, Db-TCK26D6x33H1F7, cross-reactive with human, mouse
and rat PAI-1 and TAFI was developed and was demonstrated to enhance fibrinolysis in in
vitro clot lysis and thromboelastometric experiments. Its profibrinolytic properties were further
confirmed in vivo in mouse models of thromboplastin-induced thromboembolism and in various
mouse models of stroke. Taken together all data demonstrate that dual targeting of PAI-1 and
TAFI results in a pronounced profibrinolytic effect with superior properties to those of t-PA and
minimal bleeding risks.
12:40-13:00 Mr. Travis Gould, McMaster University, Ontario
‘Cell-free DNA modulates clot structure and impairs fibrinolysis in sepsis’
Though several studies on the effects of cell-free DNA (cfDNA) and coagulation in sepsis have
been performed, little is known about the effects of cfDNA on fibrinolysis. Our studies are the
first to suggest that cfDNA impairs fibrinolysis in sepsis by preventing plasmin-mediated
cleavage of fibrin via the formation of a non-productive ternary complex of cfDNA-plasmin-fibrin.
Our studies identify cfDNA as a potential pro-fibrinolytic therapeutic target in sepsis treatment.
13:00-13:20 Ms. Dorien Leenaerts, University of Antwerp, Belgium
‘Measurement of active CPU (TAFIa) in samples from patients with acute coronary syndrome’
The presentation focused on the measurement of carboxypeptidase U (CPU, CPB2 or TAFIa) in
clinical samples and more specifically in those of patients with acute myocardial infarction (AMI).
The first part of this presentation covered the role of the proCPU (proCPB2 or TAFI) system in
arterial thrombosis. Despite the large body of literature available on proCPU, a lack of clarity
remains with regard to the exact role played by active CPU during thein vivo process of
thrombosis. This is mainly due to the difficulty of measuring ultra-low CPU concentrations in the
circulation. However, recently these challenges were overcome by the development of assays
that quantify CPU activation, instead of measuring the zymogen proCPU. During this
presentation, existing assays were discussed and particular emphasis was devoted to the
pitfalls associated with the measurement of CPU.
Secondly, data on CPU measurement in clinical samples were shown. Using a sensitive and
specific enzymatic assay, we investigated whether it is possible to detect the active enzyme CPU
in the circulation of patients with AMI. We found that CPU activity levels were significantly higher
in patients with AMI than in controls (P=0.0035). However, elevated CPU levels were not found
in all AMI patients, but in a subpopulation.
With respect to the clinical significance of these elevated CPU levels, it must be noted that CPU
activities are low, especially when comparing to the total amount of available proCPU in plasma.
However, this is probably the result of an important dilution in the circulation and rapid decay.
Most likely, detectable levels of active CPU reflect a process of ongoing generation and as a
consequence, future research (e.g. serial blood sampling on different time points) will clarify the
relevance of these findings.
13:20 Close of Meeting.
With thanks to all the speakers for excellent presentations and keeping to time. We were happy
to have such a large audience and to have such good participation which made for an
outstanding subcommittee session.
Genomics in Thrombosis and Hemostasis
21 June 2015 8:00 – 12:20
Chairman: Willem Ouwehand
Co-Chairs: Paul F. Bray, Kathleen Freson, Anne Goodeve, Michele P. Lambert, Pieter Reitsma
Part 1: NGS IN DIAGNOSTICS
Moderators: Professor Willem H. Ouwehand (United Kingdom) and Dr Michele P Lambert (United
States)
Welcome by Professor Willem H. Ouwehand
Professor Willem H. Ouwehand presented an overview of the SSC Genomics in Thrombosis and
Hemostasis.
Key people involved in the project and key activities have been presented.
Activities include:
1. Development and validation of a NGS test for rare inherited T & H disorders 2. Identification and annotation of pertinent genes and variation in these genes to clinical
standards 3. Development and maintenance of a database to provide a stable and sustainable frame
of reference for sequence information 4. Development and application of Human Phenotype Ontology terms (HPO) to patients
Speaker: Dr Char Witmer – Philadelphia, USA
Title: Inherited bleeding /coagulation disorder
Dr. Witmer has presented the limitations to coagulation testing to diagnose patients with bleeding
and coagulation disorders. She talked about some of the current applications of genetic testing
for VWF and Hemophilia A and B underlying the limitations of the available tests including the
high costs and the importance of having a new comprehensive test such as a NGS platform with
higher sensitivity and specificity.
Speaker: Professor Anne Goodeve – Sheffield, UK
Title: Hemostasis NGS panel in routine diagnostic use
The NGS gene panel currently available at Sheffield Diagnostic Genetics Service to screen
patients with bleeding and platelet disorders has been presented. Sample workflow and GATK
bioinformatics pipeline currently in use have been described including the way variants are filtered
according to frequency and pathogenicity to identify the potential causative variant.
The platform has been validated with control samples and performance assessed, and has been
in diagnostic service use for 6 months for sample from the UK and worldwide. Availability of
several genes on the panel facilitates discrimination between disorders where necessary; e.g.
hemophilia A and B, hemophilia A and type 2N VWD and also provides more rapid analysis for
disorders resulting from more than one gene e.g. FIX deficiency, Glanzmann thrombasthenia and
fibrinogen disorders.
Speaker: Dr Ilenia Simeoni, Cambridge, UK
Title: ThromboGenomics platform
An update about the ThromboGenomics (TG) project has been presented. The TG platform is
now fully validated and will become a NGS platform for routine clinical use from July 2015. A
single, comprehensive DNA based test will be offered to all the UK Hemophilia Centers to screen
patients with bleeding, thrombotic and platelet disorders. The gene panel includes so far 86 genes
and in the validation phase about 400 samples have been enrolled, sequenced and analyzed.
Results are discussed in MDT meetings and reports generated for the referring clinicians using
Sapientia, a new software developed by Congenica Ltd.
Part 2: NGS IN DIAGNOSTICS
Moderators: Professor Anne Goodeve (United Kingdom) and Dr Walter Kahr (Canada)
Speaker: Dr Dan Hampshire – Sheffield, UK
Title: Coagulation Factor Variant Databases - an update
An update about the EAHAD-DB (European Association for Haemophilia and Allied Disorders
Coagulation Factor Variant Databases) initiative has been presented. The intention of this
initiative is to gather together single gene variant databases involved in clinical bleeding disorders,
principally hemophilias A and B and von Willebrand disease, as well as other rarer coagulation
factor variants. So far F7, F8, F9 and VWF databases have been included. In future, databases
for fibrinogen and factors FII, FV, FX, FXI and FXIII will also be included. The database is free,
easily accessible and open to any new variant submission, including submission of previous
reported variants.
Speaker: Dr Andrew Paterson, Toronto, Canada
Title: Challenges in the interpretation of variants from next generation sequencing
Many false positive variants are present in current databases. NGS and the availability of whole
exome and whole genome sequencing (WES and WGS, respectively) on a large number of
controls will help to remove several of the misinterpreted variants in addition to the identification
of new variants likely to cause an inherited disease. Advantages and limitations of the Exome
Aggregation Consortium (ExAC) variant database have also been mentioned.
EDUCATIONAL SESSION
Moderators: Professor Pieter H. Reitsma (the Netherlands) and Professor Paul Bray (United
States)
Speaker: Dr Ernest Turro Cambridge, UK
Title: Methodological challenges of gene discovery by genome sequencing
An overview about sequencing strategies, variant calling and filtering of sequencing data has
been presented. One of the challenges of WGS is variant prioritization. The process includes
multiple steps and takes advantage of the use of the Human Phenotype Ontology (HPO) terms,
pathogenicity score and a novel statistical methodology call “phenotype similarity regression”.
WGS enables detection of variations also in the non-coding regions of the genome adding a
further level of complexity to the bioinformatics analysis.
Speaker: Dr Walter Kahr, Toronto, Canada
Title: Inherited platelet disorders
An overview about inherited bleeding disorders and their classification have been presented.
Glanzmann Thrombasthenia, Bernard Soulier, Hermansky Pudlak and Arthrogryposis - Renal
dysfunction – Cholestasis (ARC) syndromes have been presented in details. The recent
discovery of germline mutations in ETV6 as a cause of autosomal dominant thrombocytopenia,
red cell macrocytosis and ALL was also presented.
REPORTING OF CLINICAL VARIANTS
Moderators: Professor Kathleen Freson (Belgium) and Dr Daniel Bellissimo (United States)
Speakers: Dr Daniel Bellissimo, Pittsburgh, USA & Professor Kathleen Freson, Leuven,
Belgium
Title: International Guidelines to annotate pathogenic variants
This was a joint presentation between Dr. Dan Bellissimo, who was the first speaker, and
Professor Kathleen Freson.
The first presentation described the recently published ACMGG/AMP Guidelines for the
interpretation of sequence variant in US (Richards et al (2015 Genet in Med 17(5): 405-423).
This guideline for interpretation of sequence variants is an evidence-based scoring system that
considers the strength of the following pieces of evidence when classifying variants as
pathogenic, likely pathogenic, uncertain significance, likely benign and benign:
Population Data Computational and Predictive Data Functional Data Segregation Data De novo Data Allelic Data Other Database
Other Data
The second talk was about the Guidelines for the Interpretation of sequence variants in Europe.
NGS not only influences diagnostic outcome but also the complete organization of genome care
that can no longer be the sole responsibility of clinical geneticists but of Multi-Disciplinary Team
(MDT) that includes research specialists, clinicians, bioinformaticians and clinical geneticists.
Conclusions include the importance of variant databases, wide data sharing within and between
countries, the importance of a careful re-classification of variants of unknown significance (VUS)
and MDT for variant interpretation and reporting.
Speakers: Dr Karyn Megy, Cambridge, UK & Dan Hampshire, Sheffield, UK
Title: New large control data: opportunities & pitfalls
The outline of this joint talk included an update about the current variation databases (dbSNP,
dbVar, ClinVar, Human Gene Mutation Database (HGMD) and Locus specific databases
(LSBDs)) and the large volume of datasets coming from WES and WGS. Opportunities and pitfalls
of the available large datasets were presented. Opportunities of having large datasets to help in
classifying a variant include availability of controls, phenotypes and ethnicity data but there are
pitfalls to consider: false positive variants present in public databases, the type of sequencing
which might not be sufficient to identify the causal variant and a lack of standardization for variant
description, the reference sequence and phenotype coding.
To avoid these pitfalls, the use of multiple datasets, a standardization of the variant description in
addition to the use of a stable database such as Locus Reference Genomic (LRG) which includes
a unique stable sequence record of transcripts using IDs and no versioning.
Final Remarks
Progress
The ThromboGenomics (TG) project, which was informally initiated by Kunicki and Ouwehand at
the ISTH in Kyoto (2011) is now after extensive validation moving to the clinical stage. The global
TG network continues to expand to include new collaborators, clinicians and researchers from all
around the world.
Hemostasis and Malignancy
20 June 2015 14:30 – 18:50
Chairman: Alok A. Khorana
Co-Chairs: Marc Carrier, Howard A. Liebman, Nigel Mackman, Simon Noble, Ingrid Pabinger, Joseph S. Palumbo
Education Session
The education session was chaired by Drs Khorana and Carrier and included Drs Carrier and
Noble as speakers. Dr. Carrier spoke about occult malignancy screening after unprovoked
VTE, particularly in light of two trials to be presented at ISTH 2015. Although results of these
studies were not yet available, Dr Carrier identified study design and expectations for outcomes.
He proposed a guidance statement from SSC of ISTH after results of these studies are known
to better inform hematologists on practice issues. Dr. Noble provided an overview of qualitative
research in hematology, how it differs from generally accepted standards and sample sizes in
quantitative research and how it can inform patient care.
SSC Working Session
The session was first chaired by Drs Mackman and Pabinger. The second half was chaired by
Drs Ay and Zwicker. Drs Palumbo, Mackman and Pabinger described recent results linking
platelet activation with hemostatic factors and tumor biology. Dr. Mackman posed the question
of how therapeutic targeting of platelets could affect malignancy outcomes and potentially even
prevent thrombosis in this setting. Dr. Khorana reviewed recent data regarding clinical
predictors and biomarkers of recurrent VTE in malignancy, in context of the recent completed
CATCH study. Dr. Falanga described early findings and study design of an Italian study of
biomarker screening for cancer-associated thrombosis, the HYPERCAN study. Dr. O’Connell
described emerging data regarding prevalence and outcomes related to incidental thrombosis in
malignancy. She was followed by Dr. Di Nisio who presented the new ISTH guidance statement
on incidental thromboembolism in malignancy, recently published in JTH. Dr. Ay described how
network analysis could evaluate anticoagulants in different clinical trials indirectly. Dr. Othman
presented data suggesting a strong hypercoagulable state in prostate cancer. Dr. Zwicker
presented data on intracranial bleeding in patients with brain metastases and a guidance
proposal to better categorize such bleeds in future studies. Dr. Palumbo described draft
guidance statement proposal encompassing venous thromboembolism in pediatric cancer
patients. Finally, Dr Kamphuisen provided an update on the LONGHEVA trial which has now
transformed into an online registry and asked for SSC members to participate. The session was
adjourned at 6.40 PM.
Lupus Anticoagulant/Phospholipid Dependent Antibodies
20 June 2015 9:00 13:20
Chairman: Bas De Laat Co-Chairs: Tatsuya Atsumi, Maria Laura Bertolaccini, Katrien Devreese, Rolf T. Urbanus, Denis G. Wahl
Dr. Bas de Laat opened the session and proposed to change the title of the SSC subcommittee
from ‘Lupus coagulant / phospholipid-dependent antibodies’ to ‘Lupus
anticoagulant/antiphospholipid antibodies’.
Dr. Kumano postulated that, although the SSC recommends to use silica-based APTT assays
for LA screening over ellagic acid-based assays due to a higher sensitivity of the former, these
assays were never adequately compared with the same phospholipid concentration. In his study
he compared home-made silica-based and ellagic acid-based reagents with the same
composition and low concentration of phospholipids, with four commercial APTT reagents. Their
results on 63 LA positive and negative samples showed that the home-made ellagic acid-based
reagent was more sensitive to LA than the silica-based reagent in a low phospholipid condition
and had adequate sensitivity to detect LA. They concluded that the sensitivity of APTT reagents
for LA is dependent on the phospholipid concentration rather than the activator. In response to
the question ‘will the SSC change their guidelines based on these results?’ Dr. de Laat
emphasizes that larger multicenter studies are required and that requests for such studies can
be proposed to the SSC.
Dr. Devreese in a first presentation elaborated on the importance of the determination of cut-off
values in solid phase and automated assays used in the diagnosis of APS as these determine
the clinical performance of the tests. In this study she focused on the refinement of the cutoff
value using the HemosIL Acustar in a multicenter study in which 5 laboratories participated with
a total of 626 samples. Compared to original work in which 250 samples were used to
determine the cut-off, the multicenter approach resulted in a lower cut-off value with a stricter
confidence interval, a higher sensitivity, slightly reduced specificity and comparable predictive
value for thrombosis. Prof. Devreese concluded that the multicenter approach is a valid
alternative for the determination of cut-off values.
In a second presentation, Dr. Devreese addressed the need for a multicenter study to unravel
the importance of IgM antibodies in the diagnosis of APS. Indeed, together with Dr. Hilde
Kelchtermans she performed a meta-analysis of the available literature on the relevance of
measuring IgM antibodies. The analysis is still ongoing, but so far results are disappointing
because many of the included articles do not show individual data, nor correlations with
thrombosis or show combined data. Therefore, in a new clinical study, a minimum of 1000
patients, including APS patients, diseased controls, auto-immune diseases and healthy controls,
will be tested using solid-phase and automated assays from different suppliers. She concluded
with inviting centers to collaborate in this multicenter study. In response to the questions, she
mentions that it may be interesting to add infection disease controls, investigate the role of IgA
antibodies and to compare with LA positivity although for these determinations we will be
dependent on the values of the local labs.
Although currently the diagnosis of APS is mainly done by endpoint and quantitative assays,
Leonie Pelkmans emphasizes the need for the development of more global functional assays
to avoid false positive results due to non-pathogenic antibodies. Based on the promising results
obtained by the β2GPI-dependent LAC and earlier thrombin generation data from Katrien
Devreese, she showed an ellagic-acid triggered thrombin generation test in the absence or
presence of cardiolipin. She applied this test on normal pooled plasma supplemented with
monoclonal anti-β2GPI antibody as well as on healthy controls and APS patient plasma samples
positive or negative for reactivity against the pathogenic domain I epitope of β2GPI. Although the
results look promising, they need to be confirmed in a larger study. Therefore she asked the
SSC for help in collecting APS plasma samples.
Dr. Bertolaccini elaborated on the relevance of measuring anti-β2GPI IgA and anti-PS/PT
antibodies for the diagnosis of APS. Given the low frequency of isolated IgA positivity, she
concluded that for APS, the utility of IgA antibodies should be restricted to patients with a strong
suspicion for APS and that are negative for other antiphospholipid antibodies. As to the anti-
prothrombin antibodies, the ones that are directed against the complex PS/PT have been shown
to correlate better with thrombosis. Combined positivity for LA, anti-β2GPI and anti-PS/PT was
shown to result in a better accuracy for the diagnosis of APS and the prediction of thrombosis
and pregnancy morbidity as the current Sydney criteria. Positivity for anti-PS/PT has been
added to the GAPSS score. Furthermore, she provided evidence that the anti-PS/PT antibodies
are pathogenic in a mouse model.
Dr. Wahl aimed to determine the value of APC resistance measured by thrombin generation to
predict thrombosis in platelet-rich plasma of APS patients. In a prospective multicenter study
consisting of 137 persistent anti-phospholipid positive patients, the IC-50 APC concentration
was determined. This concentration proved to be a significant predictor for thrombosis, both in
asymptomatic patients as in patients that already showed clinical symptoms. He concluded that
there is a need for standardized protocols and cut-off determinations.
Dr. Urbanus aimed to solve the LA paradox. By using a Taipan snake venom that already
contains an FXa-FVa-like complex, he provided evidence that anti-β2GPI antibodies no longer
affect the coagulation time when the prothrombinase complex is already formed. Further
research demonstrated that anti-β2GPI antibodies inhibit the activation of FV by FXa at limiting
concentrations of phospholipids. Interestingly in patients positive for anti-β2GPI antibodies and
LA he discovered that these anti-β2GPI antibodies are not responsible for the observed LA
activity.
Dr. Rand presented data to support that resistance to annexin A5 anticoagulant activity can be
used as a biomarker for adverse clinical outcomes in APS. The value of this mechanistical
assay in the prediction of clinical events was already demonstrated in multiple clinical studies. In
a real world population of 966 patients undergoing routine thrombophilia testing, he additionally
demonstrated that annexin A5 resistance inversely correlated with anti-phospholipid antibody
assays. An increased resistance was also observed with increasing number of events. Dr. Rand
concluded that the development of such mechanistical assays may be interesting to better
predict the occurrence of thrombosis.
Dr. Bas de Laat presented the work of Dr. Pengo on Domain I and risk stratification. After an
overview on the evidence of the pathogenicity of anti-domain I antibodies, he explained the
competitive inhibition ELISA developed by Prof. Pengo to demonstrate the presence of anti-
domain I antibodies. Using this assay, a strong correlation was found between anti-domain I
antibodies and triple positivity, as well as thrombosis. Interestingly, anti-domain I antibodies
proved to be stable over time. Bas de Laat ended the presentation with the question if these
anti-domain I antibodies should be included in the APS criteria. Prof. Devreese mentioned that
this may be interesting for risk stratification, but that concerning the reduced sensitivity of the
assay, the anti-domain I assay can’t replace the anti-β2GPI assay.
Dr. Wahl presented results of clinical studies to determine if scores should be used for risk
assessments in APS and if these scores should be based on laboratory tests only or should
also include clinical parameters. In the RATIO study and a prospective multicenter study, the
GAPSS score, including both the antiphospholipid tests and clinical risk factors, proved to be
useful in the prediction of clinical events. However, the anti-domain I reactivity that doesn’t take
the clinical risk factors into account was as predictive as the GAPSS score.
Dr. Atsumi further elaborated on APS scoring and compared the anti-PL-S and GAPSS score
for their predictive value for thrombosis. In 295 patients with autoimmune diseases, 46
thrombotic events occurred during the follow up of 5 to 10 years. Both scores were predictive for
future thrombosis but the anti-PL-S score proved to be more suitable than the GAPSS score in
this cohort of patients. The question remains how to standardize each anti-phospholipid test and
to minimize the number of tests.
Dr. Arachchillage investigated if rivaroxaban influences LA assays and if anti-phospholipid
antibodies affect the activity of rivaroxaban. In vitro and ex vivo studies showed false positives
with two commercial DRVVT assays at the peak concentration of rivaroxaban. However, the
Taipan venom time/Ecarin clotting (TVT/EC) time proved to be unaffected, allowing a reliable
detection of LA. Both by thrombin generation and rivaroxaban anti-Xa levels, she demonstrated
that anti-phospholipid antibodies do not influence the activity of rivaroxaban. In reply to a
question she discourages the use of DRVVT tests in patients taking rivaroxaban but rather
advises to incorporate the alternative TVT/EC test in each laboratory.
Dr. Cohen investigated if the superior results of rivaroxaban and other direct oral anticoagulants
for the treatment of venous thrombosis can be extended to APS. The primary aim of the
prospective RAPS study was to demonstrate, in patients with APS and previous venous
thromboembolism that the intensity of anticoagulation achieved with rivaroxaban is not inferior
to that of warfarin. Secondary aims were to compare rates of recurrent thrombosis, bleeding and
the quality of life in patients on rivaroxaban with those on warfarin. Tissue factor-triggered
thrombin generation was used to compare the inhibitory effects of the anticoagulants. Results
will be presented in the late breaking abstract session on Monday.
Pediatric and Neonatal Hemostasis and Thrombosis
20 June 2015 14:30-18:50
Chairman: Anthony K. Chan
Co-Chair: Mariana Bonduel, Leonardo R. Brandao, Elizabeth A. Chalmers, Neil Goldenberg, Shoshana Revel-VilK, Heleen Van Ommen
The focus of the Subcommittee is to address issues in the area of thrombosis and hemostasis in children and neonates by developing clinical standards for evaluation, foster international collaboration in research and clinical trials, establish/maintain registries and to generate, publish and distribute reports and recommendations relating to patient care for the pediatric population. Overall, work within the Subcommittee is done through the Working Groups lead by one of the Co-chairs.
1. Position Papers:
Three position papers have been published:
Recommendations for the development of a dedicated pediatric anticoagulation service: communication from the SSC of the ISTH. Newall F, Jones S, Bauman M, Bruce A, Massicotte MP, Monagle P; Subcommittee on Perinatal and Paediatric Haemostasis. J Thromb Haemost. 2015 Jan;13(1):155-9.
Recommendations for the assessment of non-extremity venous thromboembolism outcomes: communication from the SSC of the ISTH. Rajpurkar M, Sharathkumar A, Williams S, Lau K, Ling SC, Chan AK; Subcommittee on Pediatric/Neonatal Hemostasis and Thrombosis. J Thromb Haemost. 2015 Mar;13(3):477-80.
Recommendations for the development of new anticoagulant drugs for pediatric use: communication from the SSC of the ISTH. Male C, Monagle P, Chan AK, Young G; Subcommittee on Pediatric/Neonatal Hemostasis and Thrombosis. J Thromb Haemost. 2015 Mar;13(3):481-4.
2. Administration:
Two co-chairs (Dr, Elizabeth Chalmers and Dr Mariana Bondel) will step down. Dr Fiona Newall and Dr Christoph Male are nominated to the co-chair positions. The nomination is based on their past contribution to work of the subcommittee and to avoid country or continent over-representation.
3. Ongoing Projects:
Below is the list of ongoing projects with the intention of developing a position statement or a guidance paper (to be determined by the Working Group lead by the Co-chair):
1. Diagnostic criteria for thrombosis in children: (Responsible Co-chair: Leonardo Brandao): Progress Presented at SSC meeting
2. Management of coagulopathy in liver disease. (Responsible person: Paul Monagle working with Maria Magnusson) Progress Presented at SSC meeting
3. VTE prophylaxis. (Responsible co-chair: Neil Goldenberg) Progress Presented at SSC meeting
4. Congenital Severe Purpura Fulminan Registry (Responsible person: Vicky Price , Adrian Minford ) Progress Presented at SSC meeting
5. Management of pulmonary embolism (Responsible co-chair: Neil Goldenberg) 6. Management of arterial thrombosis (Responsible co-chair: Neil Goldenberg working with
Manuela Albisetti) Progress Presented at SSC meeting 7. Antithrombin Registry with a focus on genotype and phenotype correlation ( Responsible
person : Riten Kumar) 8. Guidance paper on Catheter-related thrombosis prevention and treatment in the
pediatric population (Responsible person : Ketan Kulkarni)
Collaborations with other Subcommittees (DIC, Women’s Health Issues in T&H, Hemostasis and Malignancy) have been discussed and with some projects initiated.
1. DIC Survey (initiated) 2. Position statement on Standard and Development of Bleeding Disorder Clinic for
Women (ongoing discussion) 3. Guidance paper on Perinatal Management of hemophilia carrier and baby (ongoing
discussion) 4. Guidance paper Prevention of venous thromboembolism in pediatric cancer patients
(paper in draft form)
Anthony K C Chan Chairman, Pediatric/Neonatal Thrombosis and Hemostasis Subcommittee
Plasma Coagulation Inhibitors
20 June 2015 14:30 – 18:50
Chairman: Richard A. Marlar
Co-Chair: Ian Jennings, Jun Teruya, Hiroko Tsuda
Educational Session: Plasma Coagulation Inhibitors: Relationship of Phenotype, Plasma
Levels and Clinical Phenotype (Thrombosis).
Pieter Reitsma (The Netherlands): Molecular Defects in Protein S
Protein S is a very complex protein. It is a vitamin K-dependent protein significantly different
from the other vitamin K proteins in that it has a unique region that allows the interaction with
C4B bp Binding Protein. This binding interaction creates very complex molecular and
physiological interrelationships, hence difficulty in assessing the plasma levels and determining
the laboratory phenotype of the deficiency. Possible protein S deficient individuals were studied
from the Dutch population using a multitude of genetic methodologies to assess genetic
deficiencies. The majority of deficient patients did have identifiable genetic defects. Genetic
defects span the complete gene and have all genetic types of defects.
Anna Pavlova (Germany): Comparison of Phenotype and Genotype for Antithrombin,
Protein C and Protein S.
Clinical phenotype and laboratory values were compared to the genetic defects in AT, PC and
PS deficient patients. Evidence presented demonstrated that genetic defects were more likely to
be found in patients with lower plasma levels of the factor. In patients with only mildly abnormal
levels the potential for finding a genetic defect was significantly decreased. Patients with 40-
60% plasma levels of AT, PS or PS decreased the potential for detecting defects in only 20-30%
of the patients.
Working Session #1: On-going Projects
Project: Update and maintain genetic mutation databases for AT, PC and PS.
The committee was mandated 6 years ago to update the mutation databases for AT, PC and
PS. Over the last 6 years this has been attempted. However using only volunteer participants
with no funding, there was no significant progress toward the completion of this project. With the
creation of the Subcommittee on Genomic in Thrombosis and Hemostasis, the updating and
maintenance of a database for AT, PC and PS will be transferred to the new Subcommittee on
Genomics.
Project: Investigation into discrepancies in Protein S activity assays results.
The protein S activity assay was has been shown in External Quality Control studies to have
very significant variation among laboratories and these issues extend to patient results as well.
This study will attempt to elucidate the causes of the variation observed in the clinical PS activity
assays. Up to 40 laboratories will participate in this study. The design of the study has been
completed. The difficulty is obtaining the appropriate samples for distribution to the participating
laboratories. Both lyophilized and frozen plasma will be distributed. The results will be analyzed.
It is anticipated to have the data by the next meeting.
Project: Investigation into racial differences in genetic risk factor (AT, PC, PS) for venous
thrombosis.
The design for the study is developed and request and receipt of specimens is on-going. At this
time, the East Asian samples and some European have been received and processed. It is
anticipated that the collection of specimens and the analysis of data will be completed by the
next meeting. Then the complete set of data will be presented.
Project: Manuscript on Guidance for Clinical Testing for AT, PC, PS and APC-Resistance.
Clinical laboratory assessment of plasma levels of AT, PC, PS and APC-R is difficult and is
associated with many problems and pitfalls. The committee is producing four manuscripts on
the state-of-the-art in assessing plasma levels of the common thrombophilic risk factors. The
project leaders for these four manuscripts are:
Antithrombin: Piet Meijer
Protein C: Peter Cooper
Protein S: Richard Marlar
APC-R: Gary Moore and Dorothy Adcock
The manuscripts will be completed by December, 2015 with submission planned for May, 2016.
Working Session 2:
Jun Teruya: Other Plasma Coagulation Inhibitors
The first part of the presentation dealt with all of the plasma inhibitors that might play a role in
the coagulation process.
Heparin Cofactor II data was presented to show the possibility of the clinical relevance of this
inhibitor. Levels of this inhibitor were discussed in both adult and children. This inhibitor appears
to not play a role as a risk factor for the development of thrombosis. It may play roles in other
physiologic systems not related to coagulation.
Thrombomodulin is the cofactor for thrombin activation of protein C. The soluble form of
Thrombomodulin is being investigated as a potential drug for the treatment of sepsis. It appears
that levels of Thrombomodulin may be of benefit for increasing the survival of septic patients.
Since it is not a plasma protein, it is difficult to assess the naturally occurring protein. However
plasma levels will probably need to be assessed if the soluble form is used as treatment of
sepsis.
Tissue Factor Pathway Inhibitor (TFPI) is a regulator of factor VII and factor X. The physiology
and biochemistry of TFPI is complex which may lead to potential bleeding issues. No genetic
deficiencies of this protein have been identified. Acquired decreases and increases have been
seen in a number of clinical conditions. With this assessment, TFPI may play a role in causing
bleeding or thrombotic pathology.
Thrombophilia Testing Schemes:
Thrombophilia testing schemes vary by country and/or region. This session reviewed three
different countries standards for thrombophilia testing schemes (United Kingdom, Japan and the
United States). Testing variations occur for a variety of reasons, including mandated testing or
restricted testing (from insurance companies or national health services), racial differences (no
factor VLeidenor prothrombin 20210 testing in Japan), and clinician driven or testing availability.
The information presented showed that there are multiple approaches to thrombophilia testing
with no unified approach to testing schemes. The criteria for who to test and when the patient
should be tested are not consistent. Further studies into the variability are necessary. Other
countries and regions will also be presented at the next meeting to allow.
Platelet Immunology
20 June 2015 9:00 – 13:20
Chairman: Yves Gruel (France) Co-Chairmen: Donald Arnold (Canada), Tamam Bakchoul (Germany), Sentot Santoso (Germany), Yoshiaki Tomiyama (Japan), Chris Ward (Australia) Wednesday, 25 June (8:00-12:00) Part 1. Top Rated abstract The effects of different B-cell activating factor receptors on lymphocyte function and secretion of cytokines in immune thrombocytopenia. Presenting author: Yanan Min (China) Unfortunately, the author apparently did not attend the meeting and this presentation was therefore cancelled. Part 2. Drug-induced and autoimmune thrombocytopenia 1- During this session, the usefulness of the measurement of immature platelet fraction (IPF) by a Sysmex autoanalyzer for the diagnosis of primary ITP was discussed by Y. Tomyiama (Japan). The preliminary results revealed that XN-1000 showed better CV to measure IPF. 2- T. Bakchoul (coauthor D. Arnold) also discussed the methodological aspects related to the detection of drug-dependent antibodies. Several questions remain to be further defined such as the number of platelets, and the concentrations of drug to be used for testing. In any case, a confirmation by another laboratory is requested to ensure the responsibility of a specific drug in DITP. 3- On the other hand, T. Bakchoul (coauthor J. Fuhrmann) also discussed the optimal conditions to be used when evaluating the pathogenic effect of a platelet antibody using a NOD.SCID mice model. The methods for isolate and inject the platelets as well as the conditions of injection of the antibody have been briefly discussed. 4- Finally, A. Pecci from Italy gave a lecture on the diagnosis of inherited thrombocytopenia and recalled that many affected patients are often misdiagnosed as having ITP. In this regard, he therefore outlined how essential is the questionnaire looking at a familial history of bleeding and low platelet count and the careful analysis of platelet size and morphology. Part 3. : Alloimmune thrombocytopenia The key messages delivered during this session were: 1- The diagnosis of NAIT is confirmed when a genetic paternal/maternal incompatibility for a Human Platelet Antigen is identified and a corresponding antibody is detected in the maternal serum. 2- HPA typing should be hold using at least two methods: phenotyping and genotyping or genotyping using at least two different primers to avoid mistyping due to new variants in HPA.
3- Reference laboratories are able to diagnose only about 30% of all referred suspected cases of NAIT. The following reasons for this diagnostic gap should be taken into consideration: 1) maternal immunization against low frequency HPA antigens; 2) Low avidity antibodies; 3) some "conventional” antibodies (such as HPA-3, -15, -2) are difficult to detect, and 4) HLA antibodies may cause some cases of NAIT. 4- Modified Epitope-specific monoclonal antibodies represent a promising therapeutic approach in the management of FNAIT. While safety has already proven in human, protective efficiency still need to be verified in clinical trial.
Part 4. : Heparin-induced thrombocytopenia The key messages delivered during this session were: 1- The team of Y. Gruel (France) has developed 5B9, a new monoclonal antibody (MoAb) to PF4/heparin complexes with a human Fc fragment that fully mimics human HIT antibodies. A comparative evaluation of this antibody with 5A1, another MoAb developed in Japan and that is also a potential standard for HIT diagnosis assays showed that 5B9 was the only one to activate platelet in a heparin-dependent manner. 2- The performances of flow cytometric analysis of platelet activation for the diagnosis of HIT has also been evaluated in France in a large cohort of patients (B Tardy). Combined with and IgG-specific assay, this approach appeared as sensitive and specific than SRA. 3- The use of Platelet Microparticle Generation Assay for the diagnosis of HIT has also been discussed by V. Minet (Belgium) 4- Finally, C. Ward presented an update on the practical use of whole blood impedance assay for HIT diagnosis, but this procedure has to be prospectively evaluated.
Final discussion and perspectives Based on the presentations and the discussion of this SSC meeting, the following projects are planned for the next future. The first is to prepare an official communication of the SSC related to the laboratory testing in patients with suspected drug-induced thrombocytopenia (DITP). The second project is about to be finalized by Tamam Bakchoul (Greifswald, Germany) and will propose recommendations on the use of NOD/SCID mouse as a model for studying the pathogenesis of immune thrombocytopenia. The third ongoing project under the coordination of Chris Ward (Sydney, Australia) aims to propose a standardized procedure about the use of the whole blood impedance aggregometry (using the Multiplate analyzer) for the diagnosis of HIT. Finally, Y Gruel will propose in the next week a study coordinated by the SSC aiming to evaluate the use of 5B9 as a standard in HIT immunoassays.
Platelet Physiology
21 June 2015 8:00 – 12:20
Chairman: Paolo Gresele
Co-Chairs: Hans Deckmyn, Andrew L. Frelinger III, Paul Harrison, Diego Mezzano, Andrew D. Mumford, Patrizia Noris
The Platelet Physiology SSC Session included two 1 hour Educational Sessions, one of which was a joint session with the Biorheology SSC, and a 3 hour business SSC session.
The first educational, Saturday June 20th 2015 at 14:30-15:30, Room 716, was on “Bioreactors
for the study of the biophysical mechanisms that regulate platelet function?”
Attendance was good, scientific level of the talks excellent, and discussion from the audience
active and participated. Further details are reported in the minutes from Keith Neeves, chairman
of the Biorheology SSC.
The second educational session, Sunday, June 21st 2015, 10:00-11:00, Room 801, was on
“Bioreactors for megakaryocyte studies and platelet formation: Where do we stand?”
Jonathan Thon (United States) reviewed the evolution and state-of-the-art in the field of platelet
production bioreactors. The focus was on outlining the challenges and needs for translating recent
discoveries and devices (including his work on microfluidic reactors) into large-scale platelet
production.
Alessandra Balduini (Italy) reviewed the evolution and state-of-the-art in the field of
megakaryocyte studies in bioreactors. The focus was on recent advances in bioengineering and
biomaterials (including her work on silk derived materials), phenotyping platelet produced in these
bioreactors and applications including studying diseases (myeloproliferative disorders), drug
efficacy and drug testing.
Sunday, June 21st 2015, 8:00-12:20, Room 801
The subcommittee session started at 8:00 and was introduced by Paolo Gresele (Italy) who gave
an overview of the mission statement of the ISTH-SSC and of the specific mandate of the Platelet
Physiology SSC. The subcommittee web page was shown and audience encouraged to register
as members. A list of the concluded projects and relative publications was given (Diagnosis of
suspected inherited platelet function disorders: results of a worldwide survey - J Thromb Haemost
2014;12:1562-9; Diagnosis of inherited platelet function disorders: guidance from the SSC of the
ISTH - J Thromb Haemost 2015;13:314-22; A review of platelet secretion assays for the diagnosis
of inherited platelet secretion disorders - Thromb Haemost 2015 Epub ahead of print).
Ongoing projects and projects about to be started were listed:
Evaluation of the Bleeding Assessment Tool (BAT) for the assessment of inherited platelet function disorders (P. Gresele, P. Harrison – enrolling)
Consensus/guidance on the methods for the study of platelet secretion (D. Mezzano – work in progress)
Laboratory monitoring of P2Y12 inhibitors: a position statement (A. Frelinger – work in progress)
Generation of guidance on the measurement of platelet dimensions: methods and clinical use (P. Noris – work in progress)
Prospective evaluation of the bleeding phenotype in PT-VWD to support evidence-based diagnosis and management (M. Othman – work in progress).
Generation of guidance on the use of platelets in regenerative medicine (P. Harrison – to be started)
Validation of the diagnostic algorithm for the diagnosis of inherited platelet function disorders (P. Gresele, A. Mumford – to be started).
Paolo Gresele (Italy) then presented the preliminary, provisional results of the ongoing “Evaluation
of the Bleeding Assessment Tool (BAT) for the assessment of inherited platelet disorders” study.
The data obtained in 243 patients so far enrolled show that inherited platelet function disorders
seem to have a higher bleeding score than von Willebrand disease type 1. The data raised interest
and a call to participate in the study was made.
Diego Mezzano (Chile) then presented the ongoing project to produce a position statement on
the methods for the study of platelet secretion for the diagnosis of platelet function disorders. A
questionnaire on the evaluation of the currently available methods, to be circulated to a number
of experts outside the SSC in order to produce a consensus statement based on the RAND
methodology, was presented.
Andrew L. Frelinger III (United States) then presented a draft position statement on the laboratory
monitoring of P2Y12 inhibitors. This was preliminary discussed among the SSC members.
Literature data show that in ACS patients on dual antiplatelet therapy high on-treatment platelet
reactivity (HPR) is associated with a greater risk for ischemic outcomes while low on-treatment
platelet reactivity (LPR) with a greater risk for bleeding events, raising the possibility that altering
antiplatelet therapy based on P2Y12 monitoring would reduce ischemic and bleeding events.
Small randomized and non-randomized cohort studies demonstrated a reduction in ischemic
events when P2Y12 inhibitor therapy was altered on the basis of platelet function testing (guided
therapy). Larger randomized controlled trials, using different platelet function tests and different
therapeutic strategies, demonstrated no difference in ischemic events with vs. without guided
therapy. However, whether patient selection, choice of test, timing of test, test cut-off, and
treatment strategy in these trials was optimal has been questioned. Thus, the utility of
P2Y12 monitoring to guide antiplatelet therapy is unclear. In contrast, the limited available data
suggests that P2Y12monitoring, because of its high negative predictive value, may be useful to
identify patients who can undergo operation without elevated bleeding risk less than 5 days after
discontinuation of antiplatelet medications. Interesting discussion from the audience on the
relative weight to be given to small vs large-size guided therapy studies and about the arguments
in favor and against the questions raised on the design of the negative, large-size, guided therapy
studies took place during the session.
Maha Othman (Canada) then shortly presented the progress of the project “Prospective
evaluation of the bleeding phenotype in PT-VWD to support evidence-based diagnosis and
management”. She highlighted the importance of continuing to study PT-VWD among rare
platelet function defects and provided a brief review of current knowledge and update on the
registry. So far only 20 responses, most incomplete, were submitted. These provisional data
suggest an under-reporting of the disease worldwide; prospective information about PT-VWD
is still missing; physicians/specialists worldwide are encouraged to report information to ISTH via
the survey; survey will remain open for participation.
Patrizia Noris (Italy) gave an overview on “The measurement of platelet dimensions: Methods and
clinical use”; this will serve as the basis for the generation of a position statement on the methods
and design of clinical studies for the measurement of platelet dimensions in disease.
Finally, a talk on RNA signatures in platelets from cancer patients was given by Thomas
Wurdinger (The Netherlands) showing that platelets take up RNAs, from cancer cells that can be
measured as a reliable biomarker of tumor presence and activity in man. This subject may
represent a topic for a possible future project.
In the second part of the session, starting at 11:20 after the joint Educational session with the
Biorheology SSC, one topic, i.e. the use of platelets in regenerative medicine, was dealt with by
Harald Langer (Germany), who spoke about the pathophysiological bases of the participation of
platelets in tissue regeneration, and by Paul Harrison (United Kingdom), who discussed the state
of the art of the clinical use of platelets in regenerative medicine, underlying the lack of
standardization in the platelet preparations used and in the design of clinical trials. He also
presented the new “Platelet Rich Plasma in Achilles tendon healing” clinical study (PATH-2 trial)
he is coordinating together with Keith Willett (United Kingdom). These overviews will serve as a
basis to generate a position statement from the Platelet Physiology SSC on the use of platelets
in regenerative medicine: methods of platelet preparation and design of clinical studies.
Attendance to the session was reasonably good, ranging from approximately 150 to 350
attendees in the different phases of the SSC meeting. Discussion from the audience was active
and lively. The room session facilities were insufficient (position of the chairmen rendering
impossible to see the projection screen and lack of a monitor on the table to follow the
presentations, lack of a timer to check for adherence to the allotted times).
Predictive and Diagnostic Variables in Thrombotic Disease
20 June 2015
9:00 – 13:20
Chairman: Paul A. Kyrle
Co-Chairs: Suzanne Cannegieter, Shinya Goto, Karel Moons, Marc C. Samama, Alex C. Spyropoulos
Welcome, information on the new scope of the subcommittee, introduction of co-chairpersons and update on ongoing projects (P Kyrle)
Diagnostic variables in thrombotic disease
Chairmen: G Le Gal, P Kyrle
Dr. Geersing addressed the importance of a personalized approach to VTE diagnosis and
treatment. According to interviews in nursing homes, the majority of patients presenting with
symptoms highly suspicious for VTE do not undergo objective testing. Many patients then
receive anticoagulant treatment without VTE verification. The reason could very well be the
reluctance of doctors and elderly patients to be admitted to a hospital or to undergo
cumbersome diagnostic strategies. Dr. Geersing stressed the importance of age-adjusted D-
Dimer cut-offs and the availability of mobile ultrasound equipment that makes investigating the
patient on an out-patient basis easier.
Dr. Klok addressed issues regarding diagnosis of recurrent PE and DVT. In these instances D-
Dimer is less sensitive as compared when diagnosing a first event. There are several studies
successfully ruling out recurrent PE by algorithms including D-Dimer measuring. Therefore, a
first and a recurrent PE can be managed equally. As regards diagnosis of recurrent DVT, CUS
is less reliably and a definite diagnosis is possible in only approx. 70% of patients. MRI could be
an alternative, but it is not readily available in many hospitals and outcome studies are lacking.
The next 2 presentations were devoted to the diagnosis of “small clots”: subsegmental PE and
distal DVT. Dr. Carrier pointed out that the diagnosis of PE became more frequent over the last
years whereas PE- associated mortality remained the same. This might indicate that with the
introduction of multi-detector CTPA more PEs of unclear clinical significance are diagnosed.
There is no agreement among experts whether to treat SSPE or not and there is now an
ongoing study in France and Canada in which patients with SSPE and a negative CUS are left
untreated. A similar situation exists for distal DVT, which was covered by Dr. Righini. Studies
showing a similar frequency of recurrent VTE when the whole leg is investigated by
ultrasonography as compared to studies investigating only the proximal veins indicate, that
distal DVT might be of little clinical importance and possibly does not require anticoagulant
treatment
At the end of this session Drs. Kearon, Klok, Stevens, Di Nisio and Righini presented planned or
ongoing clinical studies: D-Dimer and diagnosis of VTE (4D, Peged), MRI to rule out recurrent
DVT (THEIA), CUS in pregnancy (CLOT3), an age-adjusted D-Dimer cutoff to rule out DVT
(ADJUST-DVT), and D-Dimer together with a modified Wells score to diagnose VTE in cancer
patients. All presentations were followed by a lively discussion.
Coffee break (20 minutes)
Education session
Chairmen: MC Samama, AC Spyropoulos
Dr. Cannegieter eluded first on the important distinction between prediction and cause of a
disease, explained confounding and bias in clinical studies and then addressed differences
between RCTs and observational studies with regard to strengths and limitations.
Dr. Moons addressed the issue of clinical prediction models, the importance to use existing data
rather than generating new ones, and how to proceed with a prediction model until it is ready for
use in clinical practice.
Predictive variables in thrombotic disease
Chairpersons: S Cannegieter, S Ghoto
Dr. Cabrera-Fuentes discussed the role of extracellular nucleic acids as potential biomarkers for
venous thrombosis. From his presentation it became evident that studies regarding its clinical
applicability are still missing although there is considerable progress in understanding the
interaction of extracellular DNA with the coagulation system.
Dr. Spyorpoulos presented a new statistical method for planning and analyzing clinical trials
which consists of combining bleeding and thrombosis as a common endpoint. This would allow
a better data interpretation and would also facilitate the conduct of clinical trials because of a
lower number of patients required.
Dr. Samama addressed the observation that over the last years due to improved surgical and
anaestheological techniques the duration of many operations has become shorter and some of
them can now be performed on a day care basis. This means that the risk of post-interventional
VTE becomes lower and in some circumstances thromboprophylaxis may no longer be required
or should be shortened. Studies addressing shorter durations of thromboprophylaxis or even
questioning the need of thrombosis prevention are needed.
Closing remarks by P Kyrle once again stressing the new scope of the subcommittee covering
now both predictive as well as diagnostic variables in arterial and thrombotic disease.
End of meeting: 13:30
Vascular Biology
20 June 2015 9:00 – 13:20
Chairman: Rienk Nieuwland
Co-Chairs: Alexander Brill, Elizabeth E. Gardiner, Chris Gardiner, Anna Randi, Florence Sabatier, Pia R. Siljander
Circulating endothelial progenitor cells; chairs Anna Randi (UK) and Florence Sabatier (France) From VSELs to endothelial progenitor cells: What is the “perfect” vasculogenic cell? David Smadja (France) In the setting of cardiovascular disease, the vascular compartment undergoes complex molecular and cellular changes that determine the range of perfusion recovery within the ischemic tissue. Thus, stimulation of vessel growth and/or remodeling has emerged as a new therapeutic option in patients with ischemic diseases. In particular, strategies based on the administration of endothelial progenitor cells (EPCs), or cell population thought to contain these vascular progenitor cells, have been shown to augment neovascularization in experimental models of ischemia and in patients with cardiovascular ischemic diseases. Several methods to obtain a better way to isolate and expand them have been developed. However, cardiovascular risk factors constitute an inhibitory environment and induce an impairment of their vasculogenic cell functions. Moreover, precise origin and identity of EPCs are controversial. Very small embryonic-like stem cells (VSELs) are multi-potent stem cells localized in adult bone marrow (BM) that may be mobilized into peripheral blood (PB) in response to tissue injury. We aimed to quantify VSELs in BM and PB of patients with critical limb ischemia (CLI) and to test their angiogenic potential in vitro as well as their therapeutic capacity in mouse model of CLI. We quantified VSELs in BM and PB from CLI patients compared to healthy controls. Our results suggest that ischemia may trigger VSELs mobilization in this patient population. Sorted BM-VSELs cultured in angiogenic media acquired a mesenchymal phenotype (CD90+, Thy-1 gene positive expression). VSEL-derived cells had a pattern of secretion similar to that of endothelial progenitor cells, as they released low levels of VEGF-A and inflammatory cytokines. Noteworthy, VSELs triggered post-ischemic revascularization in immune-deficient mice (p< 0.05 vs PBS treatment), and acquired an endothelial phenotype either in vitro when cultured in the presence of VEGF-B (Cdh-5 gene positive expression), or in vivo in Matrigel implants (human CD31+ staining in neo-vessels from plug sections). In conclusion, VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial lineage in humans. Given the potential usefulness of a cell therapy product, real ontogeny of vascular cells, their thorough characterization as well as a deep understanding of the molecular and cellular mechanisms involved in their pro-angiogenic capacities is of major importance. Blood outgrowth endothelial cells offer novel insights into von Willebrand disease. Richard Starke (UK) Blood out growth endothelial cells (BOEC) are endothelial cells that can be derived from circulating progenitors in blood. BOEC provide access to the normally inaccessible endothelium of patients. We have extensive experience in the isolation, expansion and culture of BOEC from
a variety of patients with vascular disorders, including Von Willebrand disease (VWD). VWD describes a deficiency or dysfunction of VWF in blood. Although there is some heterogeneity, BOEC from type 1 VWD patients confirmed the long-held belief that type 1 VWD is due to quantitative defects in the ability of the endothelium to produce VWF. VWF synthesis and release from type 2 VWD patients was mostly normal supporting the belief that this subtype of VWD is mainly due to dysfunctional VWF. Type 3 VWD patients are defined by the absence of plasma VWF and we demonstrated a similar absence of VWF in a type 3 patient’s BOEC. Although a good consistency is seen between repeat isolations from the same donor, some patients with the same genotype show variations in endothelial VWF production consistent with their different plasma VWF levels, suggesting that VWF production by BOEC from these patients is influenced by factors in addition to the mutation. In conclusion, BOEC can provide novel insight into the cellular biology of VWD and could be useful in the testing of novel treatment approaches on the patients’ endothelium prior to their use in the patient. Derivation of endothelial colony forming cells from pluripotent stem cells. Nattan Prasain (US) Since vascular endothelial growth factor ligand (VEGF)/receptor 2 (KDR) signaling pathway is critical in the emergence of endothelial cells during development, we have developed a novel pluripotent stem (PS) cell differentiation protocol and found neuropilin-1 (NRP-1), a VEGF co-receptor, as an early marker for identifying the emergence of ECFCs. Compared to other sub-sets of sorted cells, only NRP-1+CD31+ cells exhibited umbilical cord blood (CB) ECFC-like properties and produced a clinically relevant number of cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, formation of human vessels that inosculated with host vasculature in vivo, and made significant contributions to vascular repair of the ischaemic limb, but lacked teratoma formation potential in vivo. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders. Receptor shedding from platelets in the inflamed vasculature; chairs Elizabeth Gardiner (Australia) and Alexander Brill (UK) Platelets and the inflamed vasculature. Paul Kubes (Canada) Paul gave an elegant presentation showcasing State-of-Art imaging approaches to study the role of platelets in inflammation responses. Whilst platelets are traditionally recognized for their central role in hemostasis, numerous studies now highlight how platelets can be potent immune modulators and effectors. Platelets directly recognize, sequester and kill pathogens to activate and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behavior, enhancing their ability to phagocytose and kill pathogens. Paul highlighted a ‘touch and go’ surveillance property of platelets in liver and then demonstrated first responder roles for platelet chemokines and chemotactic gradients in mice exposed to Staphylococcus aureus where the majority of bacteria were sequestered immediately by hepatic Kupffer cells, resulting in a robust neutrophil infiltration into the liver. A marginal zone proximal to these Kupffer cells and devoid of neutrophils was described as a “necrotactic” zone. This zone displayed severe blood vessel constriction and platelet aggregation. Future work will more fully describe the molecular properties of this zone. Mechanistic insights into platelet receptor shedding. Yotis Senis (UK) Yotis provided an update on signaling properties of platelets from mice deficient in G6b-B. Mice lacking this ITIM-containing receptor exhibit macrothrombocytopenia and aberrant platelet function. The loss of response to collagen was explained by an enhanced ADAMs mediated shedding of the platelet collagen receptor GPVI. Yotis was able to demonstrate enhanced
phosphorylation of protein-tyrosine kinase Syk (a key component of platelet activation signaling pathways) in these mice and could rescue significant platelet function by crossing G6b-B deficient mice with mice bearing an arginine to alanine point mutation within Syk which modulates Syk phosphorylation events, possibly reducing the pathway that activates ADAMs-mediated removal of GPVI. Yotis also outlined some opportunities to unify and standardize protocols that measure platelet biomarkers of activation, in particular focusing on the unique platelet/megakaryocyte receptor GPVI. Glycoprotein Iba shedding and platelet clearance. Renhao Li (US) Renhao described some of the unique metalloproteinase blocking properties of an anti-GPIb antibody that recognizes the ADAM17 cleavage sequence within GPIbα. Clone 5G6, and its monomeric Fab fragment specifically bound purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα, but did not affect platelet activation and aggregation. 5G6 blocked GPIbα shedding induced by calmodulin inhibition or mitochondrial ageing reagent CCCP with similar potency to broad shedding inhibitor GM6001 in human platelets. 5G6 does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. 5G6 was shown to have utility in increasing the longevity of platelets stored for transfusion in bags, by reducing GPIbα metalloproteolysis observed as part of the platelet storage lesion. This new focus of research is significant as it could potentially extend the life of platelet transfusion bags beyond 4-5 days, and enhance platelet function in people in receipt of platelet transfusion. Microparticles and standardization; chairs Pia Siljander (Finland) and Chris Gardiner (UK) Vesicle size approximation enables inter-laboratory comparison of vesicle measurements by flow cytometry. Rienk Nieuwland (Netherlands) Measuring single extracellular vesicles by flow cytometry is a challenge. Vesicles scatter light, which is detected by flow cytometry and expressed in arbitrary units. These light scattering signals / arbitrary units are incomparable between instruments, and interpretation is hampered by differences in sensitivity, the unknown refractive index of vesicles, etc. To standardize these measurements, we (1) selected traceable reference materials (www.metves.eu), (2) developed a method to determine the refractive index of single particles < 500 nm diameter in suspension (Nano Letters 2014, E. van der Pol et al.), (3) developed reference materials and software to correct for differences in optical configurations of commercially available flow cytometers (www.exometry.com), (4) distributed reference materials, software and biological samples to 33 laboratories worldwide, (5) determined the vesicle size gate by measuring reference materials, and (6) measured and compared vesicle concentrations. Overall, 34% of included flow cytometers was too insensitive to measure any vesicles in the largest size gate (1,200-3,000 nm). In addition, marked differences in sensitivity are observed between similar instruments (brand, type) in different laboratories. Finally, a comparison of vesicle measurements in the AMC on 4 different instruments results in a CV of 20% for the largest size gate. Taken together, beads and software have been developed to set vesicle size gates in absolute units (nm) on most types of flow cytometers, the CV is improved compared to previous strategies, and 33% of included flow cytometers are too insensitive to detect vesicles. Importantly, further standardization of vesicle measurements will be done also in collaboration with ISEV (www.isev.org) and ISAC (www.isac-net.org). See also www.evflowcytometry.org for additional information. The goal of this collaboration is to develop standards and reference materials, protocols, rules for minimal requirements, education, etc. At present, a follow-up of METVES (www.metves.eu), entitled METVESII, is being written and Letters of Support are needed. Additional information is available at http://msu.euramet.org/health_2015/SRTs/SRT-h03.pdf and please contact Rienk Nieuwland ([email protected]) for questions.
Studies of the microparticle cargo and its functional components. Eric Boilard (Canada) Platelets are anucleated blood element highly potent at generating extracellular vesicles (EV) called microparticles (MPs). Whereas EVs appear as an important means of intercellular communication, the complexity of the platelet MP cargo is starting to be delineated. Studies show that platelet MPs contain functional enzymes and a broad repertoire of nucleic acid (e.g. microRNA). A subset of platelet MPs also bears organelles, such as respiratory-competent mitochondria. Including specific mitochondrial markers for MP assessment thus permits to reveal the diversity of platelet MPs. Furthermore, mitochondria are thought to originate from the endosymbiosis of the Alphaproteobacterium Rickettsia prowazekii during the development of eukaryotic cells. As mitochondria are released concomitantly with MPs from activated platelets, they can trigger highly potent pro-inflammatory signals. The platelet MP cargo is thus complex, and its contribution to physio(patho)logical conditions must be considered. Progress of endothelial microparticle detection and characterization by flow cytometry. Romaric Lacroix (France) Endothelial-derived microparticles (EMPs) are a useful marker of endothelium injury in vascular disorders. However, measuring EMP by flow cytometry remains a challenge, which explains the diversity of (CD) markers used in the literature for identification. Therefore the objective of the presented work was to evaluate the capacity of antibodies used in the literature to detect EMPs. Several clones of antibody were selected for each specificity, and their detection capacity was evaluated on EMPs from primary endothelial cells of different territories both in unstimulated and inflammatory conditions. This work showed that among published markers CD31 (PECAM), CD54 (ICAM1) and CD146 have the best capture efficiency by immune-magnetic separation and detection capacity by flow cytometry to detect EMPs. Interestingly, despite the expression on leukocytes, these markers were not detected on leukocyte-derived MPs in the conditions of the study. It was also demonstrated that combining markers is an interesting approach to improve the signal to noise ratio, the repeatability and lowered the detection limit of the EMP detection. Finally, a strategy was proposed to evaluate and select antibodies for the different subpopulation of MPs whose performances cannot be extrapolated from cellular data.
von Willebrand Factor
20 June 2015 14:30 – 18:50
Chairman: Jorge A. DiPaola
Co-Chair: Sandra Haberichter, Daniel J. Hampshire, Koichi Kokame, Johanna Kremer Hovinga, Frank W. Leebeek, Alberto Tosetto
1) EDUCATION SESSION
Peter Lenting, PhD (France)
Factor VIII and VWF, rekindling a longstanding relationship
In his presentation Dr. Lenting discussed the cellular origin of FVIII and VWF. Interestingly
DDAVP response may be independent from the liver pool. He also presented the controversy of
the requirement of FVIII for ADAMTS 13 cleavage. He also presented the overexpression of
FVIII in a wild type mouse that shows that the FVIII/VWF molar ratio >6 is associated with
bleeding. Discussed clearance. About 95% of FVIII is bound to VWF. Knowing VWF, VWF pp
and blood group you can calculate the half-life of infused FVIII in hemophiliacs. LRP 1 mediates
clearance of both FVIII and VWF. Other receptors also do. And most of them bind both VWF
and FVIII. This information in the future may help to improve the half-life of FVIII. Interestingly,
pegylated VWF had a 4-5 X longer half-life.
Johanna Kremer Hovinga, MD (Switzerland)
ADAMTS13 deficiency, TTP and beyond
Dr. Kremer Hovinga discussed pathophysiology of TTP and its diagnostic criteria. She pointed
out that the dramatic increased in survival with plasmapheresis. Clinical cases were presented
and different clinical situations associated with ADAMTS13 deficiency. Also carriers of
mutations in ADAMTS13 (heterozygote state) might present with AHA associated with clinical
situations of stress (pregnancy, sepsis, etc.). The relapse rate appears to be less in Rituximab
treated patients. Long-term survivors appear to have increased hypertension, depression and
autoimmune diseases. This long-term chronicity might be associated with the presence of
immune complexes of ADAMTS13/ADAMTS13Ab. These immune complexes may persist up to
6 years. Spleen from long term chronic patients show that specific memory cells can generate
anti ADAMTS13 Ab.
2) JOINT BIORHEOLOGY & VWF SESSION ON ACQUIRED VON WILLEBRAND
SYNDROME
David Lillicrap (Canada)
Flow mediated interactions of VWF and ADAMTS13: shear ecstasy
The interaction and cleavage of VWF by ADAMTS13 requires unfolding of the substrate
(VWF). In vitro flow studies show that VWF and ADAMTS13 interact increasingly as a thrombus
grows and the diameter of the remaining lumen gets smaller - resulting in increased flow. In
addition, tensile forces on VWF are further influenced by VWF ligands including platelet
GpIb, GpIIb/IIIa and P-selectin. Each of these ligands can contribute to the unfolding of VWF
and influence interactions and subsequent cleavage by ADAMTS13.
Barbara Zieger (Germany)
Acquired von Willebrand syndrome in patients with VAD or ECMO
Dr. Zieger emphasized the expansion of Ventricular Assist Devices (VADs) over the last
decades. They are not only a transition form of therapy but are becoming more of a destination
therapy; therefore all complications may become more relevant. Dr. Zieger discussed the 2
types of pumps in the market (centrifugal and axial) in continuous flow VAD. She also discussed
anticoagulation and the unusual rate of bleeding seen in patients likely due to AVWS with VADs.
It is also possible that hemolysis and free hemoglobin also influences the rate of AVWS. It is
important to mention that almost all recipients of VAD (>90%) have lower ratios of
VWF:CB/VWF:Ag and ratio of VWF:RCo/VWF:Ag, respectively, but not all of them bleed
clinically. The ratio of VWF:CB/VWF:Ag (in-house test with collagen type 1) was more sensitive
to the loss of HMW multimers than the ratio of VWF:RCo/VWF:Ag. She discussed ECMO and
the high incidence of AVWS in this modality. She also emphasized the poor correlation between
VWF levels and bleeding suggesting that many other factors may contribute to the bleeding
observed. In addition, she showed that patients with VAD or ECMO develop thrombocytopenia
and thrombocytopathy.
David Schmidtke (USA)
Effects of high shear millisecond exposure on platelets
The objective of this study was to replicate the VAD-induced shear-rate in a microfluidic device
and investigate the effect of the shear on platelet receptors, platelet adhesion and
aggregation. Platelet adhesion and platelet aggregate formation on collagen-patterned surfaces
upstream and downstream of a transient (1-50 msec) exposure to high shear (40,000 - 100,000
s-1) were quantified. We observed that a single exposure to high shear was enough to inhibit
platelet aggregation while platelet adhesion was maintained. The defect in platelet aggregation
was dependent upon both the shear rate and exposure time. Treatment of blood with an
inhibitor to ADAMTS13 prior to the high shear exposure restored normal platelet aggregation
downstream suggesting a role of VWF in the downstream aggregation.
After these three presentations discussion ensued and several members of the SSC proposed
an AWS working group with members of the VWF and Biorheology subcommittees. The main
goal of this working group will be to study and standardize AVWS in an attempt to understand
better the presentation of disease and the mechanisms of disease as well as the factors that
contribute to bleeding. Drs. Barbara Zieger, Augusto Federici and David Schmidtke will lead the
working group. Several members of the audience expressed interest in participating.
3) Update of VWF functional assays
Sandy Haberichter (USA)
Update on VWF functional assays
Dr. Haberichter presented the current nomenclature for activity assays recently published in the
Journal of Thrombosis and Haemostasis as an SSC communication (Bodo et al.). She
discussed advantages and disadvantages of this nomenclature. One of the advantages is that
some of the assays prevent the issues raised by genetic variants and their influence on
ristocetin based assays. New automated techniques show higher precision than the ones
previously used. Still she emphasized the fact that standardization is needed. In the clinical
laboratory not all the tests that report VWF activity are the same. She mentioned that influence
of collagen binding discusses specifically 1-3 and 4-6 including the fact that some genetic
variants appear to exhibit lower collagen binding.
Koichi Kokame (Japan)
Update on ADAMTS13 functional assays
Summarized 3 studies on ADAMTS13 assays including the Italian and Swiss studies. He
emphasized that this is an ideal time to perform a new multicenter study on ADAMTS13 assays
since many assays are being used. The WHO 1st ADAMTS13 standard is ready to be used;
however other studies are needed since for example it is unclear the role of immune complexes
on these assays. A new standardization sample study was proposed. Dr. Federici suggested
the use of lyophilized plasma to decrease cost. Also Dr. Leebeek proposed the use of antigen
and activity.
4) Development and validation of assessment tools for clinical trials in VWD
Paula James (Canada)
Dr. James presented the evolution of the BATs over the last decade. She discussed a newer
and shorter version of what is called a “self-BAT” which is administered to people (in lay
language) and compared to the ISTH BAT in a crossover study. Actually individuals received
the questionnaire and self-administer it. There was excellent agreement between scores.
Normal ranges were determined and those were in agreement with the ISTH-BAT. It was also
tested for VWD screening. NPV was 100% for women but lower for men. Carriers of hemophilia
were tested and the NPV was 0.75. Dr. Leebeek emphasized the issue of quality of life and
correlation with the bleeding score.
Alberto Tosetto (Italy)
There was a proposal by Dr. Tosetto for the formation of a new working group on the
standardization of diagnostic criteria of VWD. This will attempt to (re) define VWD since the last
communication on VWD classification from the SSC dates back to 2005. He argues that several
advances in the field including more comprehensive genetics, better assays and the use of
bleeding scores have changed the way we categorize the disease and therefore we might be
ready for a new classification. The working group will spend 2 years attempting this task with the
intention of reporting to the SSC in Berlin in 2017. The use of bleeding assessment tools
emerged 10 years ago, but the considerable amount of diagnostic and prognostic data available
have never translated into clinical guidelines. As a first step he proposed the preparation and
distribution of a first survey as an SSC activity in the next months.
5) Studying VWF under flow
Maria Brehm (Germany)
Studying VWF mutations under flow
Dr. Brehm discussed VWF mutants she studied under flow. Most of these mutations have been
traditionally associated with VWD 2A. Dr. Brehm showed the intracellular processing of these
mutants by immunofluorescence. Then she studied these mutants under flow. The shear rate
for most of the experiments was between 500s -1 and 10000s-1 on a VWF surface. The onset of
string formation occurs at 2500s -1. Dr. Brehm showed three different phenotypes of platelet
adhesion/aggregation for the specific mutations. She finally emphasized on the importance of
studying VWF under shear flow conditions.
Chris Ng (USA)
Studying VWD under flow
Dr. Ng described the use of Microfluidic (MF) devices to study platelet adhesion under flow in
individuals with VWD. He presented data from approximately 40 individuals either diagnosed
with VWD or with mucocutaneous bleeding. A microfluidic device was used in a cohort of
individuals with von Willebrand disease and mucocutaneous bleeding to determine if it could
better characterize VWD/mucocutaneous bleeding in the context of currently available clinical
labs and potentially predict clinical bleeding. This quantitative outputs from this device correlate
with common laboratory assays and demonstrates a more linear relationship with VWF:Ag and
VWF:RCo than the PFA100 in all patients and those with Type 1 VWD. Output of this device did
not correlate with clinical bleeding as measured by the ISTH Bleeding assessment score.
6) Visualizing VWF
Walter Kahr (Canada)
VWF in megakaryocytes
Dr. Kahr focused his presentation on VWF in megakaryocytes. First he discussed the formation
of Weibel Palade bodies containing VWF in endothelial cells. He then mentioned the unique
characteristics of megakaryocyte (MK) and platelet alpha granules when compared to Weibel
Palade bodies. He explained that 20% of plasma VWF originates from platelets, that there is no
uptake of plasma VWF into MK or platelets, that platelet VWF is characterized by enrichment of
HMWM, and that platelet VWF has altered VWF glycosylation. He went ahead and discussed
several molecules involved in alpha granule formation including VPS33B, VPS16B and
NBEAL2. He presented a Nbeal2-/- mouse model where VWF instead of being packaged into
developing alpha granules in MKs, is being externalized to the outside. Finally he showed the
results of a collaboration with Dr. P. James of a patient with type 3 VWD containing a
homozygous frameshift mutation at position c.8418_8419 resulting from a TCCC insertion
therewith adding 17 amino acids to the C-terminal of VWF. This patient had no VWF in their
plasma but did have VWF in their platelets, where using high resolution confocal IF microscopy
the VWF was at the periphery of the platelets instead of being localized in platelet alpha
granules.
Simon Webster (United Kingdom)
Optimization of immunocytochemical staining and high-resolution imaging of VWF in
HEK293 cells
Simon Webster discussed the issues he had previously trying to get HEK293 cells to make WPB
when we transfected them with VWF. This was optimized by reducing the amount of DNA used
for transfections, increasing the post transfection culture time and using different batches of
HEK293 cells. Fluorescent microscopy imaging was improved initially by using a widefield
deconvolution microscope. He then presented recent imaging using a super resolution Structured
Illumination Microscope (SIM), which was able to resolve WPB membranes and show distinct
Rab27a localization. He aimed to highlight the importance of generating high quality images in
order to make any useful comparisons between wild-type and mutant VWF biosynthesis and
storage.
Jeroen Eikenboom (the Netherlands)
Visualizing VWF exocytosis
Dr. Eikenboom described new studies using correlative light and volume scanning electron
microscopy to determine the budding of Weibel Palade bodies from the Golgi network. Multiple
connections of the WP bodies with the Golgi were identified facilitating content delivery indicating
that the Golgi apparatus may provide a framework that determine size and content of WP bodies.
Women's Health Issues in Thrombosis and Hemostasis
20 June 2015 9:00 – 13:20
Chairman: Rezan A. Abdul-Kadir
Co-Chair: Claudia Chi, Hannah Cohen, Ida Martinelli, Saskia Middeldorp, Maha Othman, Rochelle Winikoff
Welcome and introduction (Rezan Abdul-Kadir)
Rezan Abdul-Kadir opened the session and welcomed the attendees, new co-chairs and members. Rezan Abdul-Kadir presented history of women subcommittee and reported that the subcommittee membership increased over the last year from 26 members to 138. Rezan Abdul-Kadir also summarized the Mandate of the subcommittee; produce guidance, promote collaboration, identify gaps in knowledge. Rezan Abdul-Kadir encouraged further participation.
Benjamin Brenner gave a report on WHITH Berlin 2015 summarizing number of delegates and range of topics spoken on. Tributes to Victor Marden and Meyer Michel Samama. Invited all to attend next WHITH meeting in February 2017.
Updates on registries and projects (Moderated by Saskia Middeldorp and Maha Othman)
DOAC and pregnancy (Saskia Middeldorp) - Registry was first presented to ISTH in Milwaukee 2014. Protocol has been written over past year and steering committee appointed. Guidance document currently being drafted. Question of can we reassure women who conceive whilst taking DOAC? Outline of web-based international registry. Project under auspices of Women's and Anticoagulation ISTH SSCs. Protocol has been submitted to Amsterdam REC. Primary objective to record fetal and maternal outcomes from DOAC exposure in pregnancy. Secondary objective is to record late effects in children. Data collection has started, web based registry to launch soon. Invitation extended for volunteers for national coordinators.
Thrombophilia screening after repeated failures in assisted reproductive techniques (Elvira Grandone) - FIRsT registry introduced. A prospective registry aiming to collect data on whether there is benefit in giving LMWH to improve ART outcomes. Plan to collect and evaluate data on the 1st cycle after 2 or more ART failures. In addition aim to collect and evaluate thrombophilia screening data if available. OTTILIA registry update presented. OTTILIA is an observational study on antithrombotic prevention in thrombophilia and pregnancy loss. To date 114 centers involved with 122 women recruited (143 pregnancies). The attendees were invited for participation in both.
Thrombophilia and outcomes in assisted reproduction technologies: Summary of systematic review and meta-analysis (Marcello Di Nisio) - Systematic review of 33 studies including around 6000 patients. Quality assessment procedure explained. Criticism of poor overall methodological quality in studies discussed. No robust evidence of benefit of using LMWH in any condition. Further criticisms of research were highlighted to include underpowered, heterogeneity, lack of correction for other risk factors. One study even suggested a protective effect for Factor V Leiden and lupus anticoagulant.
YEARS diagnostic study in PE (Menno Huisman) - Current algorithms for diagnosis of PE not validated for pregnancy. Ongoing debate of CTPA vs VQ and respective risks of radiation exposure to both mother and fetus discussed. YEARS involves D-dimer and clinical algorithm for use in pregnant population. Evidence of 11% reduction in use of CT. Currently 78 patients registered, invitation for participation extended to all.
The use of thrombo-elastography in pregnancy: Registry (Maha Othman) - Fact that currently guidance only exists for use of TEG/ROTEM in hemophilia (ISTH), insufficient evidence to produce other guidelines. Aims of registry included: recording access to technology problems, protocols used, problems encountered. Questionnaires have been distributed, so far 71 responses but only 18 complete. From these: ROTEM used more than TEG; 43% had no access; 50% clinical use only; 50% access in local lab; 47% no training; most using manufacturers reference ranges (not pregnancy specific). Mostly used in DIC/PPH/transfusion. Main barrier to use is expense.
Obstetric issues in mothers of children with severe protein C deficiency (Adrian Minford) - Severe protein C deficiency explained with significant long-term complications for children (visual and neurological). Potential benefit of elective early delivery to prevent complications if fetus known to be affected. Based on carrier rates should be 135 cases in UK but actually only 10 known - hypothesis that affected couples suffer excess rates of fetal loss and neonatal death. Suggestion that screening couples with recurrent miscarriage, unexplained stillbirth or neonatal death may be of benefit. Aim to collect data on obstetric history of couples with affected children.
Thrombosis issues (Moderated by HC and I. Martinelli)
Management of Direct Oral Anticoagulants (DOAC) in women of childbearing age (Deepa Arachchillage) - Potential reproductive toxicity is unknown. Animal model studies have suggested increased miscarriage, skeletal and cardiac vessel abnormalities. No effect on fertility. Radiolabeling studies show lower level in fetal tissues compared to maternal but high levels are expressed in milk. Individual drug properties (e.g. molecular weight, drug binding and pKa) are likely to affect extent of transfer across placenta. Gestational age at which exposure occurs also affects type risk likely. Comment that human data is limited and incomplete (reliance on data from drug companies). Current recommendations outlined including that in pregnancy DOAC should immediately be switched to LMWH, anomaly scan and fetal echo by fetal medicine specialist, regular scan and specialist follow-up. Inadvertent exposure not grounds for TOP. In women of childbearing age importance of counselling and contraception highlighted. Recommendation is to avoid DOAC in breastfeeding. ISTH guidance document is in progress.
Management of obstetric anti-phospholipid syndrome: New developments (Beverley Hunt) - Lack of data on the prevalence of specific complications highlighted. Risk factors identifying women with APS likely to suffer obstetric morbidity were discussed (evidence from PROMISSE study, Rufatti et al). Treatment regime used at St Thomas's explained including use of aspirin, LMWH and steroids. Steroids used in women with recurrent first trimester loss. New potential treatment option of hyroxychloroquine discussed. Dr Hunt planning prospective study on use of hydroxychloroquine in pregnant women with APS (HYPATIA); will be presented at ISTH 22/6/15. Invitation is open for all to participate.
Management of thrombotic thrombocytopenic purpura in pregnancy (Marie Scully) - Most recent UK registry shows 10% cases TTP associated with pregnancy and of these 56% are late onset congenital TTP. Overlap between patients with diagnoses of TTP, PET, HELLP and HUS. UK registry shows higher number of presentation in 3rd trimester/postpartum. Good evidence of
benefit of treatment in reducing obstetric morbidity in congenital TTP, acquired TTP presenting in pregnancy less consistent benefit of treatment seen. Treatment regime used by Dr Scully for each type of TTP in pregnancy explained. Case study of use of Rituximab in TTP in pregnancy was also presented.
Education Session (Moderated by Rezan Abdul-Kadir and Andra James)
Preeclampsia - the role of hemostasis in its pathophysiology (Chris Gardiner) - Origins of PET in placenta. Differentiation of characteristics of early- vs late-onset PET was presented as well as role of increased tissue factor in placenta and platelet activation in PET. Overview of current and ongoing research projects looking at novel treatments. Upcoming study is to report on use of Pravastatin in PET. Good evidence for low dose aspirin in reducing risk of early-onset PET, study looking at 150mg dose ongoing. PRESERVE-1 study (antithrombin to treat established PET) ongoing. Some evidence of benefit of activated protein C for early-onset PET. Case study of successful use of Eculizumab to prolong pregnancy in HELLP, price of drug is barrier.
Women and bleeding disorders - the role of bleeding assessment tools (Paula James) - Overview of BATs currently in use was presented. Case study used to illustrate importance of appropriate selection of BAT to reach correct diagnosis. Validation of self-BAT BS (assessment tool developed by her group) against ISTH BAT-BS in carriers of hemophilia was discussed. Project highlighted group of women with normal factor levels but increased bleeding score, and of these 11% reported experiencing haemarthrosis. Need for further research in this area.
Hemostasis issues (Moderated by Rochelle Winikoff and Peter Kouides)
Women and platelet function defects (Sarah O'Brien) - Detailed her unit's experience of using electron microscopy in diagnosis of bleeding disorders in teenagers referred with HMB. Correlation with platelet aggregation tests and analysis of distribution of EM results in her unit showing over-diagnosis and prompt for her unit's lab to establish own reference ranges. Conclusions: importance of site-specific reference ranges for EM, larger studies needed to determine value of repeat testing for diagnosis, difficulty of diagnosis in patients with discordant results for EM and platelet aggregation.
Can carriers of hemophilia with normal factor levels bleed? (Robert Sidonio) - Plug I et al 2006 showed women carriers with levels at 40-60% showed increased bleeding. Severity of genetic mutation correlates with severity of phenotype in carriers (Miesbach study). Paroskie and Sidonio BJH 2015 FVIII level not good predictor for bleeding in carriers. Olsson et al 2014 BAT score does not correlate with factor levels. Hypothesis is that perhaps these carriers are not able to mobilize factor appropriately.
Postpartum hemorrhage in carriers of hemophilia and women with VWD (Jeroen Eikenboom) - International guidelines suggest factor replacement in 3rd trimester if levels <0.50, but dose is not specified. Dr Jeroen Eikenboom presented his study showing that VWD and hemophilia B carriers had excess rates of PPH despite factor replacement when indicated. Proposed changes in guidelines for management of these patients: use of antifibrinolytics, double uterotonics, and studies to look at what is the appropriate dose of factor replacement
Abnormal uterine bleeding in adolescents: Patterns, diagnoses and clinical outcomes (Ayesha Zia) - Concern that underdiagnosing bleeding disorders in adolescents with HMB as very often just labelled as due to anovulatory cycles. Study focused on these patients, management algorithm explained. Interim results shared - 36% seen in the emergency department, 20%
needed transfusion, 50% iron deficient, 1/3 anemic. 44% found to have VWD. Evidence that for girls with HMB and inherited bleeding disorder management algorithm discussed leads to significant improvement in quality of life. Invitation for collaborators to enter data to the SSC registry.
Foundation for women and girls with blood disorders (FWGBD) (Ann-Marie Nazzaro) - Need to educate healthcare providers about consequences and effects of blood disorders in women. FWGBD devised at ISTH 2009. Strategy includes presenting at medical society meetings. Website (fwgbd.org). Assistance with establishing 'Clinics of Excellence' in USA, census of specialist services provision in USA. International collaboration is needed to reach out all women with bleeding disorders and improve their lives wherever they live.
Meeting closed - Rezan Abdul-Kadir thanked all the attendees and then speaker and emphasized the importance of collaboration to achieve the goals of the subcommittee in producing good quality data for aspects of care with lack of evidence. Rezan Abdul-Kadir also invited the attendee to provide feedback on the today session on the website and send proposal for what should be discussed in future meetings. In addition, Rezan Abdul-Kadir asked for the members and all attendees to participate by leading / send suggestions and proposals for projects and guidance papers.
Working Group on Perioperative Thrombosis and Hemostasis
21 June 2015 8:00 – 10:00
Chairman: James Douketis
1) Review of the Session:
This 2-hour session began with an Introduction by Jim Douketis, outlining the goals of the Perioperative Working Group, its multidisciplinary make-up and ways for ISTH members to become involved.
The first presentation by Pierre Albadalejo outlined the challenges in the perioperative management of patients who are receiving dual antiplatelet therapy. This was followed by a presentation by Jerry Levy, outlining the biological basis for managing anticoagulant-associated bleeding.
After a discussion period, the third presentation by Marc Samama identified gaps in knowledge relating to laboratory monitoring and administration of different pro-hemostatic agents in the perioperative period, outlining a call-to-action for the Working Group to lead. The final presentation, by Alex Spyropoulos, reviewed and critiqued recently completed and ongoing randomized trials addressed perioperative anticoagulation.
Overall, the session was very well-received, with standing-room only attendance of ~150 people.
2) Future leadership and activities of (applied for) Subcommittee on Perioperative
Thrombosis and Hemostasis:
Originally, group was formed with a non-hierarchical structure with Jim Douketis as the de facto chair due to linkage with the SSC.
With proposed application to a bona fide Subcommittee of the SSC, there is a need for a more structured organization, with a Chair and Co-chairs.
There was unanimous agreement that Marc Samama would be the proposed Subcommittee Chair and other current Working Group members would be Co-chairs.
3) Guidance and guideline development:
There was consensus that the field of perioperative thrombosis and hemostasis was a fertile area for guidance document development, including guidance on the topics presented during the educational session.
The new Chair, with the assistance of the Co-Chairs would identify and prioritize potential guidance projects, also identifying more junior ISTH members as co-authors.
There was general agreement that development of formal practice guidelines was beyond the scope of this group and, indeed, not a direction (at this this time) that was being pursued by the ISTH.
4) Research activities:
The Working Group identified 2 broad areas of research involvement:
1) Facilitating research led by Working Group members or other colleagues by
providing input and expertise. For example, members could be called up to serve as
Steering Committee members or act in another advisory capacity. In addition, members
would serve a conduit to collaborative research networks in their home country.
2) Developing de novo multi-center clinical trials would be an important goal of this
group, building on research networks developed by individual member; there would be the
potential for concurrent applications to funding agencies across countries.
5) Potential future studies:
Yaron Shargall, a thoracic surgeon at McMaster University, Hamilton, Canada discussed a proposed randomized trial assessing VTE prevention after thoracic surgery, with feedback provided. The group encouraged Dr. Shargall to continue this research, where evidence is lacking about best practices, and would provide ongoing feedback and, if needed, participation in the study organization.