A Fully Recombinant Partial Prothrombin Complex Effectively Bypasses fVIII In Vitro and In Vivo

Michèle Himmelspach; Günter Richter; Evelyn Muhr; Katalin Varadi; Peter L. Turecek; Friedrich Dorner; Hans Peter Schwarz; Uwe Schlokat

doi:10.1055/s-0037-1613347

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2002; 88(06): 1003-1011
DOI: 10.1055/s-0037-1613347

Involvement of Thrombin Receptors in the Subject-dependent Variability in Ca2+ Signal Generation

Schattauer GmbH

A Fully Recombinant Partial Prothrombin Complex Effectively Bypasses fVIII In Vitro and In Vivo

Michèle Himmelspach

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Günter Richter

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Evelyn Muhr

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Katalin Varadi

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Peter L. Turecek

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Friedrich Dorner

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Hans Peter Schwarz

¹Baxter BioScience, Vienna and Orth/Donau, Austria

,

Uwe Schlokat

¹Baxter BioScience, Vienna and Orth/Donau, Austria

› Author Affiliations

Abstract

Summary

The development of inhibitory antibodies is a serious complication in hemophilic patients, severely compromising therapeutic success. Bleeding episodes in affected patients are controlled by treatment with a plasma-derived prothrombin complex concentrate (PCC), activated PCC (APCC) or recombinant activated factor VII. We hypothesized that a recombinant two-component agent consisting of recombinant prothrombin (rfII) and activated factor X (rfXa) would have substantial fVIII bypassing activity and could be a safe alternative therapeutic option. To test this hypothesis we assembled an agent in vitro solely consisting of rfII and rfXa at a molar ratio of 37,500:1. These factors are believed to be responsible for the activity of APCC preparations. Recombinant fX, used as the source for fXa generation, and rfII were purified from serum-free and protein-free conditioned media of stably transfected CHO and BHK tissue culture cells, respectively. Activation of rfX to rfXa was accomplished by the plant protease ficin, obviating the need for a protease derived from a human or animal source. We found that in vitro the complex reduced the abnormally prolonged activated partial thromboplastin time (APTT) of a high-titer fVIII inhibitor plasma similar to an APCC preparation. Furthermore, addition of increasing amounts of rfII/rfXa to inhibitor plasma resulted in a linear dose-dependent increase in the rate of thrombin generation. In a rabbit fVIII inhibitor model, treatment with rfII/rfXa statistically significantly reduced the intensity of the abnormal cuticle bleeding. In the Wessler test, rfII/rfXa showed no thrombogenicity. These data show that a well-defined, particularly safe and efficacious agent with fVIII bypassing activity can be generated from recombinant fII and fXa.

Keywords

Recombinant fII - recombinant fXa - fII/fXa complex - fVIII bypassing activity - hemophilic patient with inhibitors - ficin

Full Text

References

References
1 Hay CRM. Factor VIII inhibitors in mild and moderate-severity haemophilia A. Haemophilia 1998; 04: 558-63.
2 Bray GL, Gomperts ED, Courter S, Gruppo R, Gordon EM, Manco-Johnson M, Shapiro A, Scheidel E, White 3rd G, Lee M. and the recombinant study group. A multicenter study of recombinant factor VIII (recombinate): safety, efficacy and inhibitor risk in previously untreated patients with hemophilia A. Blood 1994; 83: 2428-35.
3 Lusher JM, Arkin S, Abildgaard CF, Schwartz RS. Kogenate previously untreated patients study group. Recombinant factor VIII for the treatment of previously untreated patients with hemophilia A. N Engl J Med 1993; 238: 453-9.
4 Ehrenforth S, Kreutz W, Scharrer I, Linde R, Funk M, Gungor T. Incidence of development of factor VIII and fIX inhibitors in hemophiliacs. Lancet 1992; 339: 594-8.
5 Addiego J, Kasper CK, Adildgaard C, Hilgartner M, Lusher J, Glader B, Aledort J. Frequency of inhibitor development in haemophiliacs treated with low-purity factor VIII. Lancet 1993; 342: 462-3.
6 Sultan Y. and the French Hemophilia Study Group. Prevalence of inhibitors in a population of 3435 hemophilia patients in France. Thromb Haemost 1992; 67: 600-2.
7 Schwaab R, Brackmann HH, Meyer C, Seehafer J, Kirchgesser M, Hack A. et al. Haemophilia A: mutation type determines risk of inhibitor formation. Thromb Haemost 1995; 74: 1402-6.
8 Hoyer LW. The incidence of factor VIII inhibitors in patients with severe hemophilia A. In: Inhibitors to coagulation factors. Aledort LM, Hoyer LW, Lusher JM, Reisner HM, White GCI. eds. New York: Plenum Press; 1995: 35-45.
9 Lechner K. Aktivierte Prothrombinkomplexkonzentrate. Hämostaseologie. 1982 03. 42/110-48/115.
10 Macik BG. Treatment of fVIII inhibitors: Products and strategies. Semin Thromb Hemost 1993; 19: 13-24.
11 Lusher JM. Use of prothrombin complex concentrates in management of bleeding in hemophiliacs with inhibitors: benefits and limitations. Semin Hematol 1994; 31 (2 suppl 4): 49-52.
12 Roberts HR. The use of agents that by-pass factor VIII inhibitors in patients with Haemophilia. Vox Sang 1999; 77 (Suppl. 01) 38-41.
13 Hay CR, Negrier C, Ludlam CA. The treatment of bleeding in acquired heamophilia with recombinant factor VIIa: a multicenter study. Thromb Haemost 1997; 78: 1463-7.
14 Scharrer I. Recombinant factor VIIa for patients with inhibitors to factor VIII, or IX or factor VII deficiency. Haemophilia 1999; 05: 253-9.
15 Monroe DM, Hoffman M, Oliver JA, Roberts HR. Platelet activity of highdose factor VIIa is independent of tissue factor. Br J Haematol 1997; 99: 542-7.
16 Lundblad RL, Bergtrom J, De Vreker R, Bray G, Gomperts E, Baker D, Kingdon HS, Mann KG, Hartshorn J, Jenny RJ. Measurement of active coagulation factors in Autoplex T with colorimetric active site-specific assay technology. Thromb Haemost 1998; 80: 811-5.
17 Elsinger F. Laboratory tests of activated prothrombin complex preparations. In: Activated prothrombin complex concentrates, Managing Hemophilia with Factor VIII Inhibitor. Mariani G, Russo MA, Mandelli F. eds. Praeger Publisher; 1982: 77-87.
18 Vinazzer H. Comparison between two concentrates with factor VIII inhibitor bypassing activity. Thromb Res 1982; 26: 21-9.
19 Turecek PL, Varadi K, Gritsch H, Schwarz HP. Activated prothrombin complex concentrates induce hemostasis by a balanced effect at multiple sites of the coagulation pathway. Haemophilia 1998; 04 (Suppl. 01) 197.
20 Hedner U, Kisiel W. Use of human factor VIIa in the treatment of two hemophilia A patients with high-titer inhibitors. J Clin Invest 1983; 71: 1836-41.
21 Giles AR, Kenneth MG, Nesheim ME. A combination of fXa and phosphatidylcholine-phosphatidylserine vesicles bypasses factor VIII in vivo. Br J Haematol 1988; 69: 491-7.
22 Turecek PL, Varadi K, Gritsch H, Auer W, Pichler L, Eder G, Schwarz HP. Factor Xa and Prothrombin: Mechanism of action of FEIBA. Vox Sang 1999; 77 (Suppl. 01) 72-9.
23 Richter G, Schwarz HP, Turecek PL. Activation and inactivation of human factor X by proteases derived from Ficus carica . Br J Haematol. In Press.
24 Fischer BE, Schlokat U, Mitterer A, Savidis-Dacho H, Grillberger L, Reiter M, Mundt W, Dorner F, Eibl J. Rational Design, recombinant preparation, and in vitro and in vivo characterization of human prothrombin-derived hirudin antagonists. J Biotechnol Biol Chem 1996; 271: 23737-42.
25 Schlokat U, Fischer B, Herlitschka S, Antoine G, Preininger A, Mohr G, Himmelspach M, Kistner O, Falkner F, Dorner F. Production of highly homogeneous and structurally intact von Willebrand Factor multimers by furin-mediated propeptide removal in vitro. Biotechnol Appl Biochem 1996; 24: 257-67.
26 Palmiter RD, Behringer RR, Maxwell F, Maxwell IH, Brinster RL. Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell 1987; 50: 435-43.
27 Himmelspach M, Pfleiderer M, Fischer BE, Plaimauer B, Antoine G, Falkner FG, Dorner F, Schlokat U. Recombinant human factor X: High yield expression and the role of furin in proteolytic maturation in vivo and in vitro. Thromb Res 2000; 97: 51-67.
28 Preininger A, Schlokat U, Mohr G, Himmelspach M, Stichler V, Kyd-Rebenburg A, Plaimauer B, Turecek PL, Schwarz HP, Wernhart W, Fischer BE, Dorner F. Strategies for recombinant furin employment in a biotechnological process: complete target protein precursor cleavage. Cytotechnology 1999; 30: 1-16.
29 Herlitschka SE, Falkner FG, Schlokat U, Dorner F. Overexpression of human prothrombin in permanent cell lines using a dominant selection/ amplification fusion marker. Prot Exp Purif 1996; 08: 358-64.
30 Van der Eb AJ, Graham FL. Assay of transforming activity of tumor virus DNA. Methods Enzymol 1980; 65: 826-39.
31 Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 1989; 77: 4216-20.
32 Fischer B, Mitterer A, Dorner F. Purification of recombinant human coagulation factors II and IX and protein S expressed in recombinant Vaccinia virus-infected Vero cells. J Biotechnol 1995; 38: 129-36.
33 Brummelhuis HGF. Preparation of the prothrombin complex. In: Methods of plasma protein fractionation. Curling JM. ed. New York: Academic Press; 1980: 117-28.
34 Englund PT, King TP, Craig LC, Walti A. Studies on ficin. Its isolation and characterization. Biochemistry 1968; 07: 163-75.
35 Varadi K, Siekman J, Turecek PL, Schwarz HP, Marder VJ. Phospholipidbound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation. Thromb Haemost 1999; 82: 1673-9.
36 Turecek PL, Gritsch H, Richter G, Auer W, Pichler L, Schwarz HP. Assessment of bleeding for the evaluation of therapeutic preparations in small animal models of antibody-induced hemophilia and von Willebrand disease. Thromb Haemost 1997; 77: 591-9.
37 SAS Institute Inc. 2000 SAS Online Doc, Version 8. February 2000 Cary, NC, USA..
38 Wessler S, Reimer SM, Sheps MC. Biologic assay of a thrombosis-inducing activity in human serum. J Appl Physiol 1959; 14: 943-6.
39 Philippou H, Adami A, Lane DA, MacGregor IR, Tuddenham EG, Lowe GD, Rumley A, Ludlam CA. High purity factor IX and prothrombin complex concentrate (PCC): pharmacokinetics and evidence that factor IXa is the thrombogenic trigger in PCC. Thromb Haemost 1996; 76: 23-8.
40 Tans G, Janssen-Claessen T, Hemker HC, Zwaal RFA, Rosing J. Meizothrombin formation during factor Xa-catalyzed prothrombin activation. J Biol Chem 1991; 266: 21864-73.
41 Giles AR, Nesheim ME, Hoogendoorn H, Tracy PB, Mann KG. The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex concentrates in rabbits. Blood 1982; 59: 401-7.
42 Giles AR, Johnston M, Hoogendoorn H, Blajchman MA, Hirsh J. The Thrombogenicity of Prothrombin Complex Concentrates (PCC): I. The Relationship between In Vitro Characteristics and In Vivo Thrombogenicity in Rabbits. Thromb Res 1980; 17: 353-66.
43 Houdebine LM. Transgenic animal bioreactors. Transgenic Res 2000; 09: 305-20.
44 Lubon H. Transgenic animal bioreactors in biotechnology and production of blood proteins. Biotechnol Annu Rev 1998; 04: 1-54.
45 Van Cott KE, Butler SP, Russell CG, Subramanian A, Lubon H, Gwazdauskas FC, Knight J, Drohan WN, Velander WH. Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland. Genet Anal 1999; 15: 155-60.
46 Drew R, Paleyanda RK, Lee TK, Chang RR, Rehemtulla A, Kaufman RJ, Drohan WN, Lubon H. Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin. Proc Natl Acad Sci USA 1995; 92: 10462-6.