Prethrombin-1 as a Drug Substance Promoting Hemostasis with Reduced Risk of Thrombosis

Abstract

Introduction Prethrombin-1 is a Gla-domain lacking enzymatically inactive split product that results from the cleavage of fragment 1 from prothrombin by thrombin in a feedback reaction.

Methods A prethrombin-1 preparation derived from human plasma was tested for its hemostatic and thrombogenic properties. Animal models of nail clipping (for rabbits) and tail clipping (for mice) were developed to measure blood loss in FVIII-inhibitor or rivaroxaban anticoagulated rabbits and mice, respectively. A modified Wessler test was used in rabbits to assess the thrombogenic potential by Wessler score and clot weight. Studies were performed in groups of three to six for prethrombin-1 dose escalation and comparison with prothrombin, Beriplex®, FEIBA®, and saline as a control. Data were analyzed using t-statistics or the Mann Whitney U test as applicable.

Results Prethrombin-1 has excellent hemostatic properties in anticoagulated mouse and rabbit bleeding models. Wessler tests suggest that in contrast to activated and nonactivated prothrombin complexes, prethrombin-1 has negligible thrombogenic potential.

Conclusion The thrombin zymogen prethrombin-1 promotes hemostasis with reduced risk of thrombosis. Prethrombin-1 may have potential to become a life-saving treatment for patients who bleed or are at risk of bleeding.

Keywords

coagulation - vitamin K-dependent proenzyme - clotting factors - prothrombin - prethrombin-1

Authors' Contribution

J.G.G. wrote the first draft, conceived the project, and supervised experiments. J.G.G., M.P., H.B., C.M., A.T., and R.G. designed and performed experiments, analyzed, and interpreted data. J.G.G., M.P., H.B., C.M., A.T., R.G., M.K., and W.S. contributed to the concept, literature search, and conclusions. All authors edited the final version.

Publikationsverlauf

Eingereicht: 27. Dezember 2023

Angenommen: 23. Mai 2024

Artikel online veröffentlicht:
12. Juni 2024

• References

• 1 Downing MR, Butkowski RJ, Clark MM, Mann KG. Human prothrombin activation. J Biol Chem 1975; 250 (23) 8897-8906
• 2 Nesheim ME, Mann KG. The kinetics and cofactor dependence of the two cleavages involved in prothrombin activation. J Biol Chem 1983; 258 (09) 5386-5391
• 3 Mackman N, Tilley RE, Key NS. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arterioscler Thromb Vasc Biol 2007; 27 (08) 1687-1693
• 4 Kalafatis M, Egan JO, van 't Veer C, Cawthern KM, Mann KG. The regulation of clotting factors. Crit Rev Eukaryot Gene Expr 1997; 7 (03) 241-280
• 5 Wood JP, Silveira JR, Maille NM, Haynes LM, Tracy PB. Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity. Blood 2011; 117 (05) 1710-1718
• 6 Haynes LM, Bouchard BA, Tracy PB, Mann KG. Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism. J Biol Chem 2012; 287 (46) 38647-38655
• 7 Heldebrant CM, Mann KG. The activation of prothrombin. I. Isolation and preliminary characterization of intermediates. J Biol Chem 1973; 248 (10) 3642-3652
• 8 Heldebrant CM, Butkowski RJ, Bajaj SP, Mann KG. The activation of prothrombin. II. Partial reactions, physical and chemical characterization of the intermediates of activation. J Biol Chem 1973; 248 (20) 7149-7163
• 9 Stojanovski BM, Di Cera E. Role of sequence and position of the cleavage sites in prothrombin activation. J Biol Chem 2021; 297 (02) 100955
• 10 Mann KG, Elion J, Butkowski RJ, Downing M, Nesheim ME. Prothrombin. Methods Enzymol 1981; 80 (Pt C): 286-302
• 11 Lanchantin GF, Friedmann JA, Hart DW. On the occurrence of polymorphic human prothrombin. Electrophoretic and chromatographic alterations of the molecule due to the action of thrombin. J Biol Chem 1968; 243 (03) 476-486
• 12 Chen Z, Pelc LA, Di Cera E. Crystal structure of prethrombin-1. Proc Natl Acad Sci U S A 2010; 107 (45) 19278-19283
• 13 Bukys MA, Orban T, Kim PY, Nesheim ME, Kalafatis M. The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase. Thromb Haemost 2008; 99 (03) 511-522
• 14 Butenas S, van't Veer C, Mann KG. “Normal” thrombin generation. Blood 1999; 94 (07) 2169-2178
• 15 Dusel CH, Grundmann C, Eich S, Seitz R, König H. Identification of prothrombin as a major thrombogenic agent in prothrombin complex concentrates. Blood Coagul Fibrinolysis 2004; 15 (05) 405-411
• 16 Hansson KM, Lindblom A, Elg M, Lövgren A. Recombinant human prothrombin (MEDI8111) prevents bleeding in haemophilia A and B mice. Haemophilia 2016; 22 (03) 453-461
• 17 ClinicalTrials.gov. To assesssafety, tolerability and pharmacodynamics of intravenous MEDI8111 after single ascending doses. Study Results. Accessed 28 May 2024 at: https://classic.clinicaltrials.gov/ct2/show/NCT01958645
• 18 Chandra S, Brummelhuis HG. Prothrombin complex concentrates for clinical use. Vox Sang 1981; 41 (5-6): 257-273
• 19 Varadi K, Tangada S, Loeschberger M. et al. Pro- and anticoagulant factors facilitate thrombin generation and balance the haemostatic response to FEIBA(®) in prophylactic therapy. Haemophilia 2016; 22 (04) 615-624
• 20 Salter B, Crowther M. A historical perspective on the reversal of anticoagulants. Semin Thromb Hemost 2022; 48 (08) 955-970
• 21 Backus B, Beyer-Westendorf J, Body R. et al. Management of major bleeding for anticoagulated patients in the emergency department: an European experts consensus statement. Eur J Emerg Med 2023; 30 (05) 315-323
• 22 Levy JH, Spyropoulos AC, Samama CM, Douketis J. Direct oral anticoagulants: new drugs and new concepts. JACC Cardiovasc Interv 2014; 7 (12) 1333-1351
• 23 Wessler S, Reimer SM, Sheps MC. Biologic assay of a thrombosis-inducing activity in human serum. J Appl Physiol 1959; 14: 943-946
• 24 Giles AR, Johnston M, Hoogendoorn H, Blajchman M, Hirsh J. The thrombogenicity of prothrombin complex concentrates: I. The relationship between in vitro characteristics and in vivo thrombogenicity in rabbits. Thromb Res 1980; 17 (3–4): 353-366
• 25 Seegers WH, Hassouna HI, Novoa E. Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas. Thromb Res 1977; 11 (05) 633-642
• 26 Butenas S, Mann KG. Blood coagulation. Biochemistry (Mosc) 2002; 67 (01) 3-12
• 27 Friedmann AP, Koutychenko A, Wu C. et al. Identification and characterization of a factor Va-binding site on human prothrombin fragment 2. Sci Rep 2019; 9 (01) 2436