Differences between human and rabbit coagulation factor X—implications for in vivo models of thrombosis

Abstract

The activation of factor X (fX) to factor Xa (fXa) marks the penultimate step in the coagulation cascade and modulating fXa activity may be effective for antithrombotic therapy. Even though fXa inhibitors are screened using in vitro inhibition of human fXa (HfXa) while subsequent evaluation uses in vivo rabbit models, there is limited knowledge of species differences between the coagulation proteins. When comparing amino acid sequences for the human (HfX) and rabbit (RafX) protein, differences are found in the activation peptide and active site regions. In order to study the relative functional characteristics of HfX and RafX, we asked (1) whether fX from the two species is immunologically related, (2) whether the two proteins are activated to fXa in a similar manner, (3) whether HfXa and rabbit factor Xa (RafXa) have similar catalytic activities toward tripeptide substrates. To answer (1), we expressed RafX–glutathione S-transferase (RafX–GST) fusion protein in bacteria and purified the protein for use as an antigen. The resulting monoclonal antibodies were suitable for affinity purification of plasma RafX and for effective anticoagulation in rabbit plasma clotting assays. We found two antibodies (mAb 214 and mAb 290) that anticoagulated rabbit plasma in a dose responsive manner but did not cross-react with human plasma. At a concentration of 500 nM, mAb 214 attained a two-fold extension of rabbit plasma activated partial thromboplastin time (aPTT). To answer (2), we purified plasma RafX and compared the activation of HfX and RafX with Russell's viper venom (RVV-X). Under equivalent reaction conditions, conversion was 30% slower for the rabbit protein. To answer (3), amidolytic activity of HfXa and RafXa were assayed by cleavage of three para-nitroanilide (pNA) substrates (S2222 [Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA·HCl], S2765 [ Z- d-Arg-Gly-Arg-pNA·HCl] and Spectrozyme Xa [MeO-CO- d-CHG-Gly-Arg-pNA·AcOH]). Michaelis constants ( K m) for the rabbit protein were 187, 72 and 69 μM, respectively, and for the human analog, 255, 63 and 135 μM, respectively. Comparing the extent of substrate turnover ( V max) for HfXa and RafXa, the latter was shown to cleave all three substrates at a reduced rate. Based on these observations, it can be speculated that the relative antithrombotic potency of active site directed fXa inhibitors might be different between the two species. Predicted human therapeutic doses derived from in vivo results in rabbit models should therefore take species variation into consideration.