Human Factor VIIai(K62E) Is a More Effective Inhibitor of Coagulation Than Wild-Type FVIIai.

Abstract

Abstract Background: In previous work we provided evidence for a linked conformational change of the recombinant factor VII(rFVII) first epidermal growth factor-like(EGF1) domain associated with chloromethylketone(ck) covalent modification of the wild-type(WT) rFVIIa active site (J Biol Chem275:34894,2000). All naturally-occurring mutations of the human FVII gene hitherto described encode plasma FVII with both reduced activity and affinity for their cell-surface receptor tissue factor (TF). We have recently characterized a mutant rFVII protein, rFVIIa(K62E), which has both increased enzymatic activity and a 5-fold greater affinity for TF than wild-type rFVIIa (J Thromb Haemost3:1250, 2005). In the present report we provide data about the TF-binding characteristics and conformational state of rFVIIa(K62E) before and after inactivation with a variety of chloromethylketones. Methods: Purification of both the rFVIIa(K62E) mutant protein and rFVIIa(WT) and their ck-inactivated derivatives was achieved by anion-exchange chromatography. FVII antigen concentration was determined by ELISA and biological activity by modified prothrombin time (PT) assay. Affinity of inactivated rFVIIa(rFVIIai) for both TF and a FVII EGF1 domain conformation-specific monoclonal antibody(Moab 231–7) was determined by competitive ELISA (IC50). Results: The affinity of rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR for TF were increased 24.4-fold and 7.7-fold respectively versus rFVIIa(WT)[shown below]. Notably, the enhanced affinity of both rFVIIai(K62E) and unmodified rFVIIa(K62E) for TF was associated with substantially increased affinity for Moab 231–7 as compared to rFVIIa(WT). The clotting time of diluted human plasma was significantly more prolonged by rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR than rFVIIai(WT). Comparison of rFVIIai(K62E) and rFVIIai(WT) rFVIIa Preparation Affinity for TF(IC50) Relative Increase in TF Affinity Relative Increase in Affinity for Moab 231–7 Prolongation of Plasma Clotting time(sec) Reduction of Coagulant Activity(%) Affinity of rFVII for TF (IC50 expressed as molar excess of rFVII) was determined by competitive ELISA. Plasma clotting times were determined at 2.5-fold molar excess of each rFVIIai inhibitor. Control plasma clotting time was 81 sec. rFVIIai(K62E)DEGR 0.13 24.4 3.1 68 90 rFVIIai(K62E)FFR 0.41 7.7 4.9 57 86 rFVIIa(K62E) 0.46 6.9 4.1 ND ND rFVIIai(WT)DEGR 0.98 3.2 3.5 1 4 rFVIIai(WT)FFR 0.62 5.1 1.2 27 65 rFVIIa(WT) 3.17 1 1 ND ND Conclusions: We provide evidence that both active-site modification and the substitution K→E in human rFVIIa(K62E) causes conformational change(s) within the FVII EGF1 domain associated with a substantially increased affinity of rFVIIa(K62E) and rFVIIai(K62E) for TF. Both rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR show considerable promise as more effective inhibitors of coagulation than rFVIIai(WT).