venom induced consumption snake bite… coagulopathy
TRANSCRIPT
Venom Induced Consumption Venom Induced Consumption CoagulopathyCoagulopathy
Pathophysiology and implications for antivenom
therapyGeoff IsbisterNewcastle, Australia
Snake bite…
• 22 year old male bitten by a brown snake
• Holding snake and attempting to kill himself
– Initial collapse at the scene with spontaneous recovery
• Brought in by police and ambulance
• Blood alcohol = 0.24
Snake bite…
• Emergency Department
• Clotting studies done
• 2 vials given empirically on arrival2 hr 6 hr 9 hr
How much antivenom?
When would you give more antivenom?
What is Snake Bite Coagulopathy?
• Venoms contain a procoagulant toxin:
– Prothrombin activator
– Causes a clot in vitro
• Venom‐induced consumption coagulopathy (VICC)
– Clotting factor consumption in vivo
• Specific factor deficiencies:– Fibrinogen
– Factors V and VIII
• Most Australasian elapids:
– Brown snake, tiger snake group and taipan
VICC ... factor deficienciesAustralasian elapids
Normal Mild VICC Severe VICC
INR 0.9 – 1.3 1.7 (1.2 – 10) >12‡ (1.3 – >12)
aPTT (seconds) 26 – 38 44 (30 – 140) >180‡ (30 – >180)
Fibrinogen (g/L) 1.5 – 4.0 0.6 (0.3 – 1.2) <0.2* (<0.2 – 2.5)
Factor II (%) 50 - 150 76 (28 – 95) 58 (0.1 – 97)
Factor V (%) 50 - 150 53 (14 – 91) 7.1 (0 – 119)
Factor VIII (%) 50 - 150 194 (3 – 82) 10.1 (0.4 – 118)
d-Dimer (mg/L) < 0.25 140 (12 – 746) 826 (1.7 – 906)
Isbister et al J Thromb Haem 2010
severe haemophilia +
afibrinogenaemia+
severe factor V deficiency
Isbister et al J Thromb Haem 2010
Recovery of Factors
32 hours~14 hours
Recovery to INR<2 = median 14.4h (IQR 11.5‐ 17.5 h)
XIIIa
XaIXX
IX
Plasmin
VaIXV
ThrombinIXProthrombin (II)
Fibrinogen Fibrin XDPsCross Linked
Fibrin
VIIIaVIII
ATIII
Prothrombin
activators
Thrombin-like enzymes
FDPs
Brown Snakevenom
[Xa, Va]
Xa:Va complex
Protein CProtein C Protein S
C:S complexThrombomodulin
No fibrinogen
No factor V
No factor VIII
Very high D-dimer
Model of snakebite coagulopathy• Homeostasis ‐ simulate• Activated system with a ‘virtual’ elapid bite• Superimposed elapid bite data from PNG (n=78)• Ask questions of the model
– What’s the maximum likely half‐life of procoagulant taipan toxin?– How quickly must antivenom be given to effect VICC recovery?
TF VII VIIa
VIIa:TF VII:TF
II
IIa
V
Va
TAT
Xa
Xa:Va
IIa:Tmod
Tmod
PCAPC
PS
APC:PS
X
Pg
PFg
F
XF
DP
XIII XIIIa
XI XIa
XIIa
VIII
VIIIa
IX IXa
IXa:VIIIa
TFPI
Xa:TFPI
VIIa:TF:Xa:TFPI
r31
r32
r36
VKH2
VKO VK
Warfarin
IXa:AT-III:Heparin
Xa:AT-III:Heparin
AT-III:Heparin
r1
r2 r3
r4
r5
r6
r7 r8
r9
r10
r11
r12
r13
r14
r15
r16
r17
r18
r19 r20
r21
r22
r23
r24
r25
r26
r27
r28
r29 r30
r33
r35
r37
r34
r44
r45 r48
r47
pII
pVII
pIX
pX
pPC
pPS
K
Pk
CA
XII
IIa:AT-III:Heparin
r38 r39 r40
r41
r42
r43
r46
Activation process, reaction or complex formation
Stimulation of reaction or production
Stimulation of degradation
Inhibition of reaction
VK_p
Tanos et al Toxicon 2008, Wajima et al Clin Pharm Ther 2009
Honours/Post Doc
Procoagulant toxins
Prothrombin ActivatorsAustralasian Elapids
Echis spp.
Factor X activator
Factor V activator
Russell’s Viper
Thrombin‐like Enzymes
Malaysian Pit ViperAmerican vipers
Crotalus spp., Bothrops spp.
Sri Lankan Russell’s Viper ... factor deficiencies
Venom contains factor X and factor V activators
Normal Russells Viper Australian VICC
INR 0.9 – 1.3 6.3 (1.6 - > 12) >12‡ (1.3 – >12)
aPTT (seconds) 26 – 38 145 (40 - >180) >180‡ (30 – >180)
Fibrinogen (g/L) 1.5 – 4.0 0.3 (0.1 – 2.4) <0.2* (<0.2 – 2.5)
Factor II (%) 50 - 150 63 (34 – 93) 58 (0.1 – 97)
Factor V (%) 50 - 150 <2.5 (<2.5 – 57) 7.1 (0 – 119)
Factor VIII (%) 50 - 150 32 (3 – 135) 10.1 (0.4 – 118)
Factor X (%) 50 - 150 31 (7 – 96) 54 (37 – 117)
d-Dimer (mg/L) < 0.25 46 (2 – 896) 826 (1.7 – 906)
Scorgie et al HAA 2010
Other Snakes … factor deficiencies
Table 5. Pretreatment levels of clotting factors and FDP in a group of 18 patients bitten by E. carinatus Warrell et al QJM 1977
Carpet Viper Echis sppSouth American Rattlesnake Crotalus
Sano‐Martins et al QJM 2001
Antivenom and coagulopathy:…what do we really know?
1. Does it WORK?
a) Efficacy: i. In vitro: Does it bind toxins? Does it neutralise activity?
ii. In vivo: Does it bind free venom? Does it increase elimination of toxins?
b) Clinical effectiveness: Does it improve patient outcomes?
2. Dosing?
a) Initial dose: How do we determine this?
b) Re‐dosing: Do we need to ?
Efficacy vs. Effectiveness
Antivenom efficacy:
Ability to bind toxins/venoms
...to neutralise venom effects IN VITRO
...to reverse venom effects IN VITRO
...to neutralise venom effects IN VIVO
...to reverse venom effects IN VIVO
...to change patient outcomes from envenoming: Either preventing further or persistent effects OR reversing established effects
Antivenom effectiveness
Central CompartmentEg. coagulant toxin
Peripheral CompartmentEg. neurotoxin, myotoxin
• Increasing doses of antivenom being used– Commence with a larger dose of antivenom
• Antivenom needs to be titrated:– Repeat coags 1 to 3 hours post‐AV, fibrinogen best
• IF abnormal repeat dose antivenom
– Regular coagulation testing
– Continue antivenom until coagulation normal
• Does NORMAL = measurable fibrinogen, normal INR and aPTT, or even normal D‐dimer?
Australia … “Previous / Current” practice Australia … Increasing Antivenom Dose
• CSL initially recommended 500 U
– Now equivalent to ½ vial
• CSL antivenom handbook:
– 3 to 4 vials
• Toxicologists in 2006:
– 5 vials (Eastern States)
– 10 vials (WA) = 20 x initial CSL dose
Increasing Dose of Antivenom
• Animal work suggesting insufficient:– Tibballs 1990: up to 25 x dose needed
• N=1 studies
– Sprivulis 1996: unable to neutralise 20 x• Massive amounts of venom (100 x clinical)
• Insufficient antivenom being tested
– Madaras 2005
• Clinical studies:– Yeung et al 2004:
• Variable fibrinogen assays (derived, LOQ)
• Did not consider time for clotting recovery
In Australia…
• A re‐assessment:– Antivenom dose
– Concept of titration
– Role of antivenom:• Prevention
• Reversal of coagulopathy
• Improvement in recovery of coagulopathy
• Key change: measurement of venom concentration in envenomed patients
Why had previous in vitro studies suggested larger doses?
‐ Assumed much higher venom concentrations i.e. 50,000 ng/mL
VICC... antivenom dose and efficacy
• Dose with binding efficacy:
– 1 vial binds all venom in vitro
– 1 vial binds all venom in vivo
• In vitro efficacy:– 1 vial neutralises procoagulant activity in vitro
• Clinical Effectiveness:
– Antivenom does not prevent coagulopathy occurring:
• need to give <30 minutes
– Antivenom does not speed recovery
Y Y Y Y
Venom assay: Enzyme Immunoassay
Y AntibodyAnti-snake venom IgG
Venom
Biotinylated AntibodyAnti-snake venom IgG
Streptavidin Horseradish Peroxidase conjugate
Serum
Microplate reader – 405nm
Sandwich ELISA
Toxicon 2010
Venom binding
0%
25%
50%
75%
100%
0 100 200 300 400 500
Antivenom Concentration (microg/mL)
Pe
rce
nt
Fre
e V
en
om
12.5ng
50ng
100ng
0 5 10 15 200
1
2
3
4
5Tiger snake
Rough-scaled snake
Stephen's banded snake
Antivenom Concentration (mU/mL)
Fre
e v
en
om
co
nc
en
tra
tio
n (
ng
/mL
)
[Venom] = 4ng/mL
Antivenom concentration in patients after 1 vial of antivenom
Clinically relevant venom concentrations
In vivo binding...after antivenom
• Brown snake (27 patients)
– No venom detected in any sample; 9 given only 1 vial
– Rough‐scale snake (20 patients)
– No venom detected except 1 patient given small amount of polyvalent; 5 given only 1 vial
– Tiger snake (46 patients)
– No venom detected after antivenom; 7 given only 1 vial
– Taipan (17 patients)
– No venom detected after antivenom; 7 given only 1 vial
Isbister et al Toxicon 2007
Gan et al Med J Aust 2009
Kulawickrama et al Toxicon 2010
Our patient...
15:25 16:20 20:20 Day 3
Venom (ng/mL) 121 0 0 0
Antivenom (U/L) 0 1044 685 137
2 vials of antivenom
1hr 2hr 6hr 60hr
Procoagulant Activity
Antivenom efficacy...neutralisation
0.0
0.2
0.4
0.6
0.8
1.0
0 200 400 600 800
CT (venom alone)/CT
Antivenom Concentration (mU/mL)
Brown snake venom
Taipan venom
Tiger snake venom
Rough‐scale snake
venom
Stephens banded snake
venom
Antivenom concentration in patients after 1 vial or less
• In vitro clotting studies
Venom added to plasma
Time to cloudiness (clotting) recorded
Venom induces clot formation in vitro
Venom + antivenom mixtures added to plasma
Increasing time to cloudiness
Antivenom prevents clot formation
venom
Brown Snake 1 vial
Tiger Snake/Rough Scale 1 vialBlack Snakes:Mulga Snake 1 vial
Red-bellied Black Snake 1 vial tiger/black
Taipan 1 vialDeath Adder 1 vial
Antivenom Dose
• Can we trust the science OR do we just give more because it makes us feel better– False attribution of FAILURE to not enough antivenom and SUCCESS to giving the antivenom
Brown Snake 4 to 10 vials
Tiger Snake/Rough Scale 4 vials
Black Snakes:Mulga Snake 1 vial
Red-bellied Black Snake 1 vial tiger/black
Taipan 3 vials
Death Adder 1 vial
Australia: Do we need to re‐dose?
• Once all venom (toxins) bound no further doses are required:
– i.e. no venom detectable in serum
• Assumes:
– No ongoing absorption of venom
– Can only be determined with venom concentrations:
• Not possible in real time
• Establish dose in large prospective studies (ASP)
Our patient...
15:25 16:20 20:20 Day 3
Venom (ng/mL) 121 0 0 0
Antivenom (U/L) 0 1044 685 137
2 vials of antivenom
1hr 2hr 6hr 60hr
16:20 20:20
>180 >180
>180 >180
PKPD delay between venom removal and recovery of coagulopathy
Recovery of coagulopathy
• Delay between venom neutralisation and coagulopathy resolving:– Dependent on resynthesis of clotting factors
– Will antivenom speed this process? NO
• INCORRECT ASSUMPTION:– Ongoing coagulopathy = insufficient antivenom
– BASED ON:• Antivenom will treat the coagulopathy
• SO, no reason to re‐dose
Other snakes: Dose and re‐dose?Russells viper: Venom Concentrations
Issues:•Venom concentrations:
oVenom not all boundoDecreased venom without re‐dose
• 3rd dose not required
Questions:
Can we assume that all venom is absorbed???
1. Is antivenom binding and immediately
neutralising venom
OR
2. Is antivenom assisting in elimination of venom
Does venom have to be undetectable in serum for
antivenom to be EFFECTIVE ?
Sri Lanka: Russell’s viperAustralian Elapid Coagulopathy
Post‐bite
Sri Lankan Russell’s viper
ELIMINATION
NEUTRALISATION
2nd AV Dose Median (IQR)
How do we determine dose?• Australia:
– Snakes with small fangs delivering small doses• Small range of doses 1 to 200ng/mL
– Most likely rapidly and completely absorbed– Highly potent venoms– Free venom levels go to ZERO– DOSE can be determined in a cohort study
• Sri Lanka:– Snakes with very large fangs (vipers) deliver large doses
• Large range of doses 2 to 5000ng/mL
– Absorbed over 24+ hours– Less potent venoms– Free venom remains DETECTABLE– More difficult to establish dose
Antivenom: proposing a mechanism of action?
• Binding of all venom
– No detectable venom on ELISA
– Probable immediate neutralisation (coagulant toxins)
– Requires excess antivenom ie. AV:venom > ? 5
• Increasing venom elimination
– Free venom still detectable by ELISA
– Delay in neutralisation (coagulant toxins)
– Requires at least 1:1 ratio of AV: venom
X
PTAProthrombin
Elimination
PTA
Prothrombin
X
Neutralisation
NOT detectable with ELISA for free venom
DETECTABLE with ELISA for free venom
Toxin in central compartmente.g. coagulant toxin
Appears to be the case in Australia
Might be the case in Sri Lanka
Are ‘free’ venom assays enough?
• Free venom assay:
– “0” means there is no free venom
= neutralisation
– “non‐zero” does NOT mean that antivenom is not bound to venom
•Venom can probably still be detected when there is a 1:1 binding of venom‐antivenom
• Can we measure the venom‐antivenom complex?
Y Y Y Y
[venom – AV] assay: Enzyme Immunoassay
Y AntibodyAnti-snake venom IgG
Venom – AntivenomComplex
Anti-Horse IgG Antibody Conjugated
Serum
Microplate reader – 405nm
VAV Assay
Y
YYY Y
Measuring the Venom‐Antivenom complex
Venom‐Antivenom Assay
Y Y Y Y
Y
Y Y Y YY
Y Y Y
Y Y Y Y
Y
Y YY Y
Y Y Y Y
Y YYYY
Y
Y Y Y Y
Y
Y
Y
Y
Y
Y
Y
Y Y
Y
YY
Y
Free venom assay
Venom‐Antivenom Assay
n – 1 AV Ab per toxin
Slope = 0.075 mU AV / ng venom This implies that 0.075 mU of AV is required for each nanogram of venom (at VAVmax)
ie 0.075 U for each microgram of venom
ie 0.75 U for 10 micrograms of venom
This is very close to CSL definition of 1 U required for 10 micrograms of venom
Antivenom:venom ratio ?
Antivenom:venom ratioCan we use this for other antivenoms ?
11E1Feb23 Plate2 : Measurement of VAV complex (solid lines) and of free RVV (dotted lines) in mixtures of RVV and RVVAV
0.0
0.5
1.0
1.5
2.0
2.5
3.0
100502512.56.253.131.560
conc RVVAV ug/ml in well
abs
at 4
50n
m
RVV = 200 ng/ml
RVV = 100 ng/ml
RVV = 50 ng/ml
RVV = 200 ng/ml
RVV = 100 ng/ml
RVV = 50 ng/ml
EFFECTIVENESS ?
• Antivenom – toxin interactions
– Effectiveness per se?
– Effective dose
• Toxin target:
– Peripheral
– Central
• Toxin reversibility
• Question does antivenom shorten the course of the coagulopathy?– Not possible to do a placebo controlled trial of AV
• Objective: to investigate predictors of VICC recovery– Antivenom:
• timing of antivenom• dose of antivenom
– Factor replacement– Other covariates: snake, age, sex
• Main outcome measure: – Time until INR < 2 = ‘VICC recovery’
Q J Med 2009Recovery of VICC
Antivenom dose – no effectAntivenom time – no effectFFP use: large highly probable positive effect
Interpretation
• Recovery of VICC
– Administration of FFP within 4 hours only important determinant
– EARLIER administration of antivenom or INCREASED dose of antivenom had NO effect
• Suggests antivenom has no effect
– ONLY for Australian elapids
• Recovery of VICC
– Administration of FFP within 4 hours only important determinant
– EARLIER administration of antivenom or INCREASED dose of antivenom had NO effect
• Suggests antivenom has no effect
– ONLY for Australian elapids
VICC … Echis spp.
• Prothrombin activator toxin
– Snake venom metalloproteinase (not like human factor Xa)
– Also a haemorhaggin vascular injury
• Bleeding/Coagulopathy:
– Spontaneous bleeding
– Prolonged coagulopathy: reported to continue for up to 10 days untreated
Warrell QJM 1977
Am J Emerg Med 2011
• 12 year period
• 71 envenomed viper bites
– 62 got antivenom
• 39 got early antivenom < 24 hours post‐bite
• 23 got delayed antivenom > 24 hours post‐bite
– 9 did not get antivenom
Echis … recovery of VICC
Faster recovery of VICC with antivenomHOWEVER …. They all did recover
Echis … recovery of VICC
• No relationship between time of AV and recovery of VICC ?– Authors: delayed antivenom is still beneficial
• Difficult for this to be biologically plausible –
• i.e. if antivenom is given late then VICC must recover even faster in these patients ?
• Assume antivenom works – supported by 9 patients not getting antivenom
– Cynic: Does antivenom speed the recovery at all?• Not disimilar to Australian elapids
• Are the 9 untreated patients convincing (small no. etc)
HOW DO WE EXPLAIN THIS?http\\: star.ferntree.com
ASP investigators
Geoff Isbister Simon BrownNick Buckley Bart CurrieJulian White Colin PageDavid Spain Alan TankelOvidu Pascui Helen MeadRichard Whitaker Peter GarrettRandall Greenberg Anna HoldgateMark Miller Yusuf NagreeKate Porges Kent McGregorLambros Halkidis Gary WilkesMark Coghlan Andrew ParkinShane Curran Rod EllisMichael Taylor Katie MillsPeter Miller Rob BonninGreg Treston Chris GavaghanChris Barnes Nick RyanBen Close Chris TrethewyJustin Yeung
Newcastle Toxicology UnitMargaret O’Leary, Leonie Calver, Renai KearneyAustralian Clinical ToxicologistsAndis Graudins, Lindsay Murray, Ian Whyte, Rob Dowsett, Frank Daly, Naren Gunja, Mark Little, Sam Alfred, Shaun Greene, David McCoubrie, Andrew Dawson, Michael DownesAustralian Laboratories Sarah Just, Tony Ghent, Vaughan Williams, Nick Michalopoulos, Mario KyriacouPKPD Toxicology GroupStephen Duffull, Toshihiro Wajima, Panayiota Tanas, AbhishekHaematology Research:Michael Seldon, Lisa Lincz, Fiona Scorgie, Lesley Survela, Tina NoutsosMonash LabWayne Hodgson, Nicki KonstanakopoulousNSW, Qld and Victorian Poison Centres
Acknowledgements