Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide

Summary

We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv_59D8). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clotbound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications. TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for antifXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni²⁺ and β_15-22-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv_59D8-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv_59D8-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.

Keywords

Coagulation inhibitors - drug design - fibrinogen / fibrin

^* These authors contributed equally to this study

• References

• 1 Mukherjee D, Topol EJ. The role of lowmolecular-weight heparin in cardiovascular diseases. Prog Cardiovasc Dis 2002; 45: 139-56.
• 2 Coussement PK, Bassand JP, Convens C. et al. A synthetic factor-Xa inhibitor (ORG31540/ SR9017A) as an adjunct to fibrinolysis in acute myocardial infarction. The PENTALYSE study. Eur Heart J 2001; 22: 1716-24.
• 3 Bauer KA, Hawkins DW, Peters PC. et al. Fondaparinux, a synthetic pentasaccharide: the first in a new class of antithrombotic agents – the selective factor Xa inhibitors. Cardiovasc Drug Rev 2002; 20: 37-52.
• 4 Abendschein DR, Baum PK, Martin DJ. et al. Effects of ZK-807834, a novel inhibitor of factor Xa, on arterial and venous thrombosis in rabbits. J Cardiovasc Pharmacol 2000; 35: 796-805.
• 5 Abendschein DR, Baum PK, Verhallen P. et al. A novel synthetic inhibitor of factor Xa decreases early reocclusion and improves 24-h patency after coronary fibrinolysis in dogs. J Pharmacol Exp Ther 2001; 296: 567-72.
• 6 Dyke CK, Becker RC, Kleiman NS. et al. First experience with direct factor Xa inhibition in patients with stable coronary disease: a pharmacokinetic and pharmacodynamic evaluation. Circulation 2002; 105: 2385-91.
• 7 Waxman L, Smith DE, Arcuri KE. et al. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 1990; 248: 593-6.
• 8 Schaffer LW, Davidson JT, Vlasuk GP. et al. Antithrombotic efficacy of recombinant tick anticoagulant peptide. A potent inhibitor of coagulation factor Xa in a primate model of arterial thrombosis. Circulation 1991; 84: 1741-8.
• 9 Sitko GR, Ramjit DR, Stabilito II. et al. Conjunctive enhancement of enzymatic thrombolysis and prevention of thrombotic reocclusion with the selective factor Xa inhibitor, tick anticoagulant peptide. Comparison to hirudin and heparin in a canine model of acute coronary artery thrombosis. Circulation 1992; 85: 805-15.
• 10 Vlasuk GP, Ramjit D, Fujita T. et al. Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis. Thromb Haemost 1991; 65: 257-62.
• 11 Mao SS, Huang J, Welebob C. et al. Identification and characterization of variants of tick anticoagulant peptide with increased inhibitory potency toward human factor Xa. Biochemistry 1995; 34: 5098-103.
• 12 Weitz JI, Hudoba M, Massel D. et al. Clotbound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-91.
• 13 Herault JP, Bernat A, Pflieger AM. et al. Comparative effects of two direct and indirect factor Xa inhibitors on free and clot-bound prothrombinase. J Pharmacol Exp Ther 1997; 283: 16-22.
• 14 Eisenberg PR, Siegel JE, Abendschein DR. et al. Importance of factor Xa in determining the procoagulant activity of whole-blood clots. J Clin Invest 1993; 91: 1877-83.
• 15 Lynch Jr. JJ, Sitko GR, Mellott MJ. et al. Maintenance of canine coronary artery patency following thrombolysis with front loaded plus low dose maintenance conjunctive therapy. A comparison of factor Xa versus thrombin inhibition. Cardiovasc Res 1994; 28: 78-85.
• 16 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science 1983; 222: 1129-32.
• 17 Bode C, Hanson SR, Schmedtje JF. et al. Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting. Circulation 1997; 95: 800-4.
• 18 Peter K, Graeber J, Kipriyanov S. et al. Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa. Circulation 2000; 101: 1158-64.
• 19 Jordan SP, Mao SS, Lewis SD. et al. Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide. Biochemistry 1992; 31: 5374-80.
• 20 Wei A, Alexander RS, Duke J. et al. Unexpected binding mode of tick anticoagulant peptide complexed to bovine factor Xa. J Mol Biol 1998; 283: 147-54.
• 21 Rand MD, Lock JB, van’t Veer C. et al. Blood clotting in minimally altered whole blood. Blood 1996; 88: 3432-45.
• 22 McKenzie CR, Abendschein DR, Eisenberg PR. Sustained inhibition of whole-blood clot procoagulant activity by inhibition of thrombus-associated factor Xa. Arterioscler Thromb Vasc Biol 1996; 16: 1285-91.
• 23 Zoldhelyi P, Bichler J, Owen WG. et al. Persistent thrombin generation in humans during specific thrombin inhibition with hirudin. Circulation 1994; 90: 2671-8.
• 24 Nicolini FA, Lee P, Malycky JL. et al. Selective inhibition of factor Xa during thrombolytic therapy markedly improves coronary artery patency in a canine model of coronary thrombosis. Blood Coagul Fibrinolysis 1996; 07: 39-48.
• 25 Prager NA, Abendschein DR, McKenzie CR. et al. Role of thrombin compared with factor Xa in the procoagulant activity of whole blood clots. Circulation 1995; 92: 962-7.
• 26 Prasa D, Svendsen L, Sturzebecher J. Inhibition of thrombin generation in plasma by inhibitors of factor Xa. Thromb Haemost 1997; 78: 1215-20.
• 27 Iino M, Takeya H, Takemitsu T. et al. Characterization of the binding of factor Xa to fibrinogen/fibrin derivatives and localization of the factor Xa binding site on fibrinogen. Eur J Biochem 1995; 232: 90-7.
• 28 Brandjes DP, Heijboer H, Buller HR. et al. Acenocoumarol and heparin compared with acenocoumarol alone in the initial treatment of proximal-vein thrombosis. N Engl J Med 1992; 327: 1485-9.
• 29 Agnelli G, Renga C, Weitz JI. et al. Sustained antithrombotic activity of hirudin after its plasma clearance: comparison with heparin. Blood 1992; 80: 960-5.
• 30 Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003; 23: 17-25.
• 31 Rezaie AR. DX-9065a inhibition of factor Xa and the prothrombinase complex: mechanism of inhibition and comparison with therapeutic heparins. Thromb Haemost 2003; 89: 112-121.
• 32 Krishnaswamy S, Vlasuk GP, Bergum PW. Assembly of the prothrombinase complex enhances the inhibition of bovine factor Xa by tick anticoagulant peptide. Biochemistry 1994; 33: 7897-907.
• 33 Dunwiddie CT, Neeper MP, Nutt EM. et al. Site-directed analysis of the functional domains in the factor Xa inhibitor tick anticoagulant peptide: identification of two distinct regions that constitute the enzyme recognition sites. Biochemistry 1992; 31: 12126-31.
• 34 St Charles R, Padmanabhan K, Arni RV. et al. Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor. Protein Sci 2000; 09: 265-72.
• 35 Wei A, Smallwood A, Alexander RS. et al. Crystallization and preliminary X-ray diffraction data of the complex of recombinant tick anticoagulant peptide (rTAP) and bovine factor Xa. Acta Crystallogr D Biol Crystallogr 1999; 55: 862-4.
• 36 Antuch W, Guntert P, Billeter M. et al. NMR solution structure of the recombinant tick anticoagulant protein (rTAP), a factor Xa inhibitor from the tick Ornithodoros moubata. FEBS Lett 1994; 352: 251-7.