24175488artif cell

Lyophilized Platelets: Fifty Years in the Making

Arthur P. BodeDepartment of Pathology and Laboratory Medicine, East Carolina University,

The Brody School of Medicine, Greenville, NC, USA

Thomas H. FischerDepartment of Pathology, The University of North Carolina at Chapel Hill,

Medical School, Chapel Hill, NC, USA

Abstract: Starting with the work of Klein et al. in the early 1950s, there has beena concerted effort to apply the process of freeze-drying for the preservation ofplatelets in order to provide hemorrhagic patients with a stable infusible hemo-static agent to stop bleeding. The original attempts did not preserve platelet struc-tural integrity and proved to be of little clinical benefit. However, it was knownthat fixation by various cross-linking agents rendered platelets able to withstandstructurally intact the stresses of lyophilization but with (assumed) complete lossof functionality. Read and coworkers showed that fixed and freeze-dried plateletscould respond to ristocetin-induced agglutination, and thus devised a widelyaccepted assay for von Willebrands factor that demonstrated that reconsti-tuted platelets participated well in this in vitro model of an important interactionin primary hemostasis. This review chronicles the efforts of the authors to refinethe fixation process so that the freeze-dried and reconstituted platelets retainfundamental hemostatic properties necessary to stop bleeding. The resultant pro-duct has demonstrated correction or reduction of the bleeding times in animalmodels with platelet deficits including the thrombocytopenic rabbit model ofBlajchman and coworkers, a canine cardiopulmonary bypass model of open-heartsurgery at East Carolina University (ECU), and a porcine trauma model at

The authors would like to recognize the collective contribution of the manyscientific collaborators and advisors that have been involved in this project,and specifically thank Dr. Marjorie Read (UNC-CH) for leading this effort inits earlier stages. This work has been most generously supported by The US Officeof Naval Research since 1989. The authors also acknowledge the support andcontinued development efforts of Entegrion Inc. and its investors.

Address correspondence to Arthur P. Bode, Department of Pathology andLaboratory Medicine, The Brody School of Medicine, East Carolina University,Greenville, NC 27858, USA. E-mail: [email protected]

Artificial Cells, Blood Substitutes, and Biotechnology, 35: 125–133, 2007

Copyright Q Informa Healthcare

ISSN: 1073-1199 print/1532-4184 online

DOI: 10.1080/10731190600974962

125

The University of North Carolina at Chapel Hill (UNC-CH) involving exsangui-nation and complete blood exchange with a hemoglobin-based oxygen carrier(HBOC). In addition, it has been shown that the fixation process kills virusesand bacteria spiked into the platelet suspension, indicating that the final materialmay indeed be the first truly sterile cellular transfusion product. The initial goalfor clinical benefit is to prevent exsanguination and hypovolemic shock in combatcasualties of armed services personnel, for whom platelet transfusions are mostoften unavailable. Commercial interests are being brought to bear by EntegrionInc. (formerly known as Hemocellular Therapeutics Corporation) to transfer thistechnology to a scaleable manufacturing platform for the production of StasixTM,a pharmaceutical preparation of fixed and freeze-dried platelets for intravenousor topical use in the arrest of active hemorrhage in a wide variety of patients witha platelet-related bleeding diathesis. It has taken fiftyþ years from the firstattempt at making a clinically useful freeze-dried platelet preparation to get tothe rapidly-approaching clinical trials of StasixTM; stabilization of the plateletshas been the key to realizing this advance.

Keywords: Freeze-drying; Hemorrhage; Hemostatic agent; Lyophilization; Plate-lets; Platelet adhesion; Transfusion medicine

INTRODUCTION

In 1935, freeze-drying (vacuum lyophilization of a previously frozenmaterial to remove water by sublimation) was applied by Flosdorf [1]and others to preservation of biological materials. This same technologybecame a real lifesaver when applied to the preservation of blood plasmafor use as a resuscitation fluid on the battlefields of World War II. Thefreeze-drying of protein solutions without wholesale denaturation is farfrom a simple process. However, successful dehydration and rehydrationof cells is much more complicated, so it was not too surprising that thefirst attempts to freeze-dry the cellular elements of blood resulted in lossof their structural integrity and function. In 1955, Klein and coworkers[2] reported on their first experiments in testing the procoagulant proper-ties of a freeze-dried preparation made from an extracted platelet suspen-sion. The material was later infused into pediatric leukemia patients withthrombocytopenia in an effort to restore hemostasis [3,4]. The resultswere controversial. Klein claimed a therapeutic benefit was obtained,stating that the clinical results were ‘‘suggestive’’ of a positive hemostaticeffect. Others then tried to reproduce this work with infusion of a similarfreeze-dried platelet preparation in a model of irradiated thrombocytope-nic rats [5] or dogs [6], but no reduction in bleeding was observed.Further development of this material was discontinued.

Platelets are responsible for adhering to sites of vascular injury tostop hemorrhage in the primary step of hemostasis. The platelet surface

126 A. P. Bode and T. H. Fischer

membrane must be intact to accomplish this task efficiently. The lackof convincing efficacy of the Klein preparation was most likely due tostructural disintegration of the platelets in their freeze-dried state. How-ever, platelet structure can be well-preserved in a dry vacuum, like in anelectron microscope, by appropriate use of cross-linking fixation agents[7]. In 1975, Allain and coworkers at UNC-CH demonstrated that plate-lets cross-linked with 3.6% paraformaldehyde for 48 hours at 4�Cretained the ability to agglutinate in the presence of vonWillebrand Fac-tor (vWF) and ristocetin [8]. These platelets appeared to be structurallyintact under the microscope, but most likely were bereft of metabolicactivity and incapable of the release reaction. Far from just being a lab-oratory curiosity, the fixed platelets became an important reagent in thediagnostic assay for quantitation of vWF activity in clinical testing. Pres-ervation of ristocetin response and binding of vWF indicated an intactGPIb receptor, and suggested that some primary elements of hemostasismight be well supported by fixed platelets.

These preparations of fixed platelets were stable in terms of ristoce-tin response for several weeks refrigerated. Read and Brinkhous wentfurther and adapted a benchtop freeze-drying protocol to extend thestorage of the fixed platelets [9]. The dried platelets remained intactand ristocetin response was now preserved for a year or more in thereconstituted product, making this an even more useful diagnosticreagent for the clinical lab. However, the question of their utility asan in vivo hemostatic agent remained untested until 1989 when Readand Bode began to evaluate these preparations further. It was apparentthat the platelets as currently processed where incapable of otherexpressions of platelet responsiveness [unpublished data], and thusrefinements of the fixation procedure where investigated to improvenative function. The following sections of this review describe thechanges in process and their effects as this project progressed to devel-opments with commercial potential.

FIXATION PROTOCOL

After much trial and correction experimentation to target the ‘‘lightest’’fixation conditions that still maintain cell integrity through the stressesof dehydration=rehydration, we settled on exposure of washed plateletsat 1� 109=mL to 1.8 g=dL paraformaldehyde at pH ¼ 6.8–7.0 for 45–60 minutes at room temperature, followed by further washing and finalbulking in 5g=dL bovine serum albumin. Yields depended somewhaton age of the input platelets and the method of washing platelets outof their native plasma milieu (centrifugation or gel filtration); typically

Lyophilized Platelets: Fifty Years in the Making 127

we recovered 50–70% through the wet process steps. Recovery of intactplatelets after freeze-drying and reconstitution was 90–100%. Oneindication of under-fixation was poor recoveries of platelet count and sizeafter rehydration. The main indications of over-fixation were irreversibleclumping of platelets during washing after the paraformaldehydeexposure and complete abrogation of metabolic activity in the reconsti-tuted product.

The correctly processed preparation was capable of spreading on con-tact with glass surfaces albeit slower than fresh platelets [10], expressedprocoagulant activity that increased upon stimulation with thrombinand=or collagen [11], adhered to physiologic thrombogenic surfaces underhigh shear through GPIb [12], generated thromboxanes through inhibita-ble mechanisms [12], and demonstrated intracellular phosphorylation reac-tions [13] or activation neoantigen expression [12] upon stimulation. Thesepreparations have not been shown to aggregate macroscopically withagonists other than ristocetin, although the number of singlet plateletsdecreases with addition of ADP or collagen [data not published], and thereis no clot retraction response as measured in classical assays. The real issue,though, was whether infusion of the reconstituted platelets could correctthe bleeding time in animal models of platelet dysfunction.

ANIMAL MODELS

The thrombocytopenic rabbit model of Morris Blajchman (McMasterUniversity) has gained worldwide acceptance for testing hemostatic effi-cacy of platelet preparations, and has the unique feature of supportingxenographic infusion of human platelet materials. Since the mid–1970s,data have been produced in this model for platelets stored under a varietyof conditions, or for platelet derivatives or substitutes [14]. Our initialstudies in 1994 with fixed, freeze-dried platelets in this model producedvery encouraging data that showed correction of the bleeding time nearlyto the extent as seen with fresh human platelets [15]. In this model we alsosaw that overfixed platelets did not stay in circulation very long and oftencaused extraordinary stress on the recipient subject [unpublished data].For platelets fixed 45–60 minutes, the best results were obtained in thismodel with pools of preparations to reduce variability, suggesting thatpooling of materials would be appropriate when dealing with pharmaceu-tical preparations at larger scale. Concurrently, Marjorie Read demon-strated in her laboratory that these preparations corrected the toenailbleeding times in rats made thrombocytopenic by anti-platelet antiserum[10]. The positive results of these two models strongly advanced thenotion that the fixed freeze-dried platelets should be developed further


as a novel hemostatic agent for arrest of active bleeding in hemorrhagicthrombocytopenic patients, such as those treated in the historical trialsconducted by Klein et al. [3].

Soon after these initial successes in thrombocytopenic models, thefixed freeze-dried platelets were tested in a surgical setting. The modelwas one of open-heart surgery in dogs; this was primarily a medicalpractice regimen at ECU for training of cardiothoracic surgery residentsin cardiopulmonary bypass (CPB) procedures. CPB is known to producea platelet function deficit in human surgical patients [16] and did so inthese animal subjects as well. Our introduction of fixed freeze-dried can-ine platelets into this surgery model to correct the abnormal bleeding timecaused by CPB was without known precedent but appeared to be areasonable test of hemostatic efficacy in a clinical setting that is likelyto benefit from the availability of a novel hemostatic agent. We infusedthe lyophilized platelets under a variety of surgical and supportconditions in normal canine subjects and found that a large single bolusgiven just prior to weaning from the CPB pump gave an immediate cor-rection of the bleeding time test that was sustained in continued testingfor at least a 3–hour post-op period [17]. This persistence of effect wasremarkable in that it was not strictly dependant on circulating plateletcount. Moreover, the subjects were splenectomized prior to CPB so itis unlikely that endogenous platelets played a role in the sustained hemos-tasis recovery we observed. In a very limited number of CPB procedures,lyophilized human platelets were introduced in place of lyophilized can-ine platelets and also produced a correction of the bleeding time, but theeffect did not persist for more than 1–2 hours in the post-op phase. Com-parison of effectiveness of the lyophilized platelets to fresh canine orhuman platelets or other preparations has not yet been undertaken.

Other in vivo tests of fixed freeze-dried platelets continued, especiallyto assess the effects of major changes in platelet washing procedure, orvariation of input platelet sources, or for other information on circu-lation dynamics. Further testing in the Blajchman thrombocytopenicrabbit model demonstrated an effect on the bleeding time even afterthe infused lyophilized platelets had been in circulation for three hoursand the count was as low as 40,000=mL [18]. Read and colleagues usedinfusions of fixed freeze-dried porcine platelets to staunch the exsangui-nating bleeding of an injured pig in the UNC von Willebrands animalcolony [unpublished]. Tracer studies were performed in normal caninesubjects with lyophilized canine platelets labeled with a fluorescent mem-brane dye to reveal aggregation at sites of intravascular injury but not inthe systemic circulation [10]. Multiple infusions of lyophilized canineplatelets were initiated longterm in two normal canine subjects to see ifnew antibodies arose; none were observed [19].


FURTHER DEVELOPMENT

For a period of time, commercial development of the fixed freeze-driedplatelets was pursued by Centeon Corporation (formerly Armour Phar-maceuticals) under a license agreement with ECU and UNC. Theirexpertise in handling blood components in a GMP environment turnedattention to issues of product safety and scale-up. Experiments conductedby Centeon demonstrated that the paraformaldehyde fixation procedurewas completely microbicidal for at least eight test viruses spiked at highconcentrations into washed platelet suspensions [17]. Read and Becker atUNC also found a strong bacteriostatic effect of the fixation with strainsrelevant to transfusion concerns [18]. These findings together were parti-cularly striking because they demonstrated the possibility of creatingfor the first time a cellular transfusion product that was truly sterile.Centeon’s timeline was set for initiating human clinical trials in the year2000 with their pharmaceutical preparations, but commercial develop-ment was halted when their manufacturing facility came under a consentdecree due to new safety concerns by the FDA for plasma-derivedproducts in general. The technology license returned to ECU and UNCand further academic development was undertaken.

Fischer then began work with a new animal model in pigs under-going controlled exsanguination and blood exchange using a hemoglo-bin-based oxygen carrier (HBOC) in collaboration with EmergencyMedicine at UNC. This model offered a unique clinical scenario inwhich native hemostasis was compromised by hemodilution and hypo-volemic shock. When fixed, freeze-dried porcine platelets were intro-duced in these subjects as the hematocrit neared zero, the bleedingtime was corrected and continuing hemorrhage from prior bleeding timetests was arrested [20]. The positive results in this model suggested thatpharmaceutical preparations of lyophilized platelets would be of use incombination with new HBOC solutions in the resuscitation of traumapatients.

Thus the fixed, freeze-dried platelets showed hemostatic efficacy inanimal models representing a broad spectrum of clinical hemorrhage sce-narios: (a) excessive bleeding due to thrombocytopenia; (b) abnormalhemostasis caused by CPB in open-heart surgery; (c) excessive bleedingin vonWillebrands disease; and (d) restoration of normal hemostasis inextreme hemodilution resuscitation. It seemed appropriate to pursuefurther commercial development to bring this promising product tofruition. For this reason, a start-up company named HemocellularTherapeutics Corporation was incorporated in 2002 as a joint ventureby ECU and UNC so that advancement of the fixed freeze-dried plateletsto clinical trials could continue.


REMAINING ISSUES

In order to bring any new hemostatic agent into FDA-approved clinicaluse, safety issues must be addressed in addition to the demonstration ofefficacy. Some safety-related information can be inferred already fromthe foregoing academic research on the fixed, freeze-dried platelets, butHemocellular developed a plan to specifically assess clinical effects. Prelimi-nary and unpublished findings from their efforts include evidence that HLAantigens are ablated to a large extent by the fixation process. Also, there isno evidence as yet in serum samples from diverse human subgroups for anypre-existing antibodies to novel antigens induced by fixation on the freeze-dried platelets, and there is no detectable formaldehyde remaining in thefinal product after GMP manufacture. Registration toxicity and thrombo-genicity studies have yet to be completed, but no toxic or thrombogenicevent has been observed in the >200 subjects of the several animal modelsreceiving infusions of the correct formulation. If the impending INDsubmission is approved by the FDA, the first human exposure should bein late 2007.

The advantages presented by a freeze-dried cellular hemostatic agentover the current blood bank therapeutic of stored platelet concentrates

Figure 1. Summary of interests from a survey of physicians for important desiredattributes in a new cellular hemostatic agent; comparison of those attributes instandard of care blood bank stored platelet concentrates versus preclinicalimpressions of the lyophilized platelets. The comments used to describe bloodbank concentrates should be regarded as opinions rooted in experiences and=orpublished statements of respondents in the survey.


will be numerous and impressive. Platelet concentrates are not alwaysavailable for bleeding patients, especially in scenarios involving armedservice personnel in field or mass casualty events in civilian trauma,and stored blood bank platelets may not be fully effective in stoppingactive hemorrhage [21]. Blood bank components at present are notguaranteed to be sterile, and are known to have unfavorableimmunomodulatory effects [22]. Alloimmunization of multiply trans-fused patients can lead to refractoriness against any beneficial effects ofplatelet concentrates in preventing spontaneous bleeding. A material thatis sterile, immediately effective, non-immunogenic, safe and easy to use inemergent clinical situations to treat the hemorrhagic patient will have avery significant impact on transfusion medicine (Fig. 1). These are theattributes we hope to firmly establish in the fixed, freeze-dried plateletsas clinical trials proceed, 50 years since the first recorded clinical use ofa lyophilized platelet preparation.

REFERENCES

1. Flosdorf, E.W., Mudd, S. (1935). Procedure and apparatus for preservationin ‘‘Lyophile’’ form of serum and other biological substances. J. Immunol. 29:389–425.

2. Klein, E., Farber, S., Freman, G. (1955). Effects of dried fraction from plate-lets on Ca clotting time of normal plasmas and plasma in thrombocytopeniaand hemophilia. Fed Proc. 14: 410–1324.

3. Klein, E., Farber, S., Djerassi, I., Toch, R., Freeman, G., Arnold, P. (1956).The preparation and clinical administration of lyophilized platelet materialto children with acute leukemia and aplastic anemia. J. Pediatrics 49: 517–522.

4. Klein, E., Farber, S., Djorassi, I., Arnold, P. (1956). Hemostasis followingthe administration of lyophilized platelet material in secondary thrombocyto-penia. Sixth Intl. Congr. Hematology 47.

5. Fliedner, T.M., Sorensen, D.K., Bond, V.P., Cronkite, E.P., Jackson, D.P.,Adamik, E. (1958). Comparing effectiveness of fresh and lyophilized plateletsin controlling irradiation hemorrhage in the rat. Proc. Soc. Exp. Biol. 99:731–733.

6. Jackson, D.P., Sorensen, D.K., Cronkite, E.P., Bond, V.P., Fliedner, T.M.(1959). Effectiveness of transfusions of fresh and lyophilized platelets in con-trolling bleeding due to thrombocytopenia. J. Clin. Invest. 38: 1689–1697.

7. Murphy, M.J., Jr. (1972). The shape of blood platelets: An application oflyophilisation and scanning electron microscopy. Thrombos. Diath. Hae-morrh. 28: 237–243.

8. Allain, J.P., Cooper, H.A., Wagner, R.H., Brinkhous, K.M. (1975). Plateletsfixed with paraformaldehyde: A new reagent for assay of von WillebrandFactor and platelet aggregating factor. J. Lab. Clin. Med. 85: 328–335.


9. Brinkhous, K.M., Read, M.S. (1978). Preservation of platelet receptors forplatelet aggregating factor=von Willebrand Factor by air drying, freezingor lyophilization: New stable platelet preparations for von Willebrand Factorassays. Thromb. Res. 13: 591–597.

10. Read, M.S., Reddick, R.L., Bode, A.P., Bellinger, D.A., Nichols, T.C.,Taylor, K., Smith, S.V., McMahon, D.K., Griggs, T.R., Brinkhous, K.M.(1995). Preservation of hemostatic and structural properties of rehydratedlyophilized platelets: Potential for long-term storage of dried platelets fortransfusion. Proc. Natl. Acad. Sci. 92: 397–401.

11. Read, M.S., Sanders, W., Fischer, T.H., Merricks, E.P., Nichols, T.C., Bode, A.P.,Russel, K.E., Bellinger, D.A., Reddick, R.L., Khandelwal, G. (2002). Thrombusformation with rehydrated, lyophilized platelets. Hematology 7: 359–369.

12. Bode, A.P., Read, M.S., Reddick, R.L. (1999). Activation and adherence oflyophilized human platelets on canine vessel strips in the Baumgartner per-fusion chamber. J. Lab. Clin. Med. 133: 200–211.

13. Fischer, T.H., Merricks, E.P., Russell, K.E., Raymer, R.A., White, G.C.,Bode, A.P., Nichols, T.C., Read, M.S. (2000). Intracellular function in rehy-drated, lyophilized platelets. British Journal of Haematology 111: 167–174.

14. Blajchman, M.A., Lee, D.H. (1997). The thrombocytopenic rabbit bleedingtime model to evaluate the in vivo hemostatic efficacy of platelets and plateletsubstitutes. Transf. Med. Rev. 11: 95–105.

15. Bode, A.P., Blajchman, M.A., Bardossy, L., Read, M.S. (1994). Hemostaticproperties of human lyophilized platelets in a thrombocytopenic rabbit modeland a simulated bleeding time device. Blood 84(10): Suppl 1, #1840, 464a.

16. Woodman, R.C., Harker, L.A. (1990). Bleeding complications associatedwith cardiopulmonary bypass. Blood 76(9): 1680–1697.

17. Bode, A.P., Read, M.S., Summaria, L., Lust, R.M. (1997). Lyophilized plate-lets: Nearing clinical trials. Platelets 8: 436–437.

18. Bode, A.P., Read, M.S. (2000). Lyophilized platelets for transfusion medi-cine, in Platelet Therapy: Current Status and Future Challenges, ElsevierScience: Amsterdam, pp. 131–167.

19. Fischer, T.H., Merricks, E.P., Raymer, R.A., Nichols, T.C., Bode, A.P.,Read, M.S. (2000). Multiple infusions of rehydrated, lyophilized plateletsdoes not induce antiplatelet antibodies or thrombocytopenia in a caninemodel. Blood 96(Suppl): 106b.

20. Fischer, T.H., Merricks, E.P., Nichols, T.C., Raymer, R.R., Hayes, P.M.,Pearce, L.B., Bode, A.P., Xu, S-Z. (2001). The co-infusion of rehydrated, lyo-philized platelets with HBOC-201 for hemostasis in dilutional thrombocyto-penia. Blood 98(Suppl): #2250.

21. Khuri, S., Healey, N., MacGregor, H., Barnard, M., Szymanski, I., Birjiniuk,V., Michelson, A., Gagono, D., Valeri, C. (1999). Comparison of the effectsof transfusions of cryopreserved and liquid-preserved platelets on hemostasisand blood loss after cardiopulmonary bypass. J. Thorac Cardiovasc. Surg.117: 172–184.

22. Spiess, B.D. (2001). Blood transfusion: The silent epidemic. Annals of Tho-racic Surgery 72(5): S1832–7.


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