Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.

Prothrombinase is the surface-bound complex in which factor Xa (Xa) converts prothrombin to thrombin in vivo. Studies have shown that Xa within prothrombinase is resistant to inhibition by antithrombin + heparin (AT+H). Previously we found that, unlike AT+H, a covalent conjugate of AT and H (ATH) was able to neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa in prothrombinase. Mixtures of CaCl₂, PCPS vesicles, factor Va (Va) and prothrombin in TSP buffer were combined with either AT+H or ATH. Following addition of Xa, time samples were neutralized with Na₂EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k₂) were calculated from plots of activity versus time. Results were compared to those in corresponding experiments with Xa alone. Inhibition of Xa in prothrombinase by AT+H had a k₂ (x 10⁸ M⁻¹min⁻¹) of 0.688 +/− 0.030. Conversely, free Xa neutralization by AT+H was significantly more rapid (k₂ = 1.53 +/− 0.35, p = 0.041). ATH reaction with prothrombinase Xa proceeded at a rate that was insignificantly different from that with Xa alone (2.36 +/− 0.31 and 2.83 +/− 0.83, respectively) and similar to AT+H reaction with free Xa. Varying concentrations of prothrombinase components showed similar effects. We conclude that covalently linked complexes of AT + H undergo unhindered reaction with Xa in prothrombinase, while non-covalently linked AT+H encounters resistance. It is possible that ATH may effectively prevent plasma thrombin generation by neutralization of Xa in prothrombinase and thus may prohibit feedback activation of coagulation.