Inhibitory Mechanism of the Protein C Pathway on Tissue Factor-induced Thrombin Generation

Abstract

The effects of the components of the protein C pathway on thrombin generation were studied in a reconstituted model in which thrombin is generated by factor VIIa and relipidated tissue factor (TF) via the activation of the purified coagulation factors X, IX, VIII, V, and prothrombin. The influence of protein C and soluble thrombomodulin on thrombin generation was correlated with factor Xa generation, factor V(a) and factor VIII(a) formation/inactivation, and protein C activation. Thrombin generation initiated by low concentrations of factor VIIa·TF (1.25 pM) occurs in an explosive fashion during a propagation phase which occurs after an initiation phase of ∼1 min in which only traces of thrombin are formed. In the absence of other inhibitors, protein C (65 nM) in combination with high concentrations of soluble thrombomodulin (10 nM) resulted in a reduced rate of thrombin generation during the propagation phase without affecting the initiation phase; the activated protein C generated failed to neutralize prothrombinase activity and did not prevent prothrombin consumption. In the presence of plasma levels of the tissue factor pathway inhibitor (2.5 nM recombinant TFPI), the protein C pathway reduced the rate of thrombin generation, initiated by 1.25 pM factor VIIa·TF, and completely eliminated prothrombinase activity at soluble thrombomodulin concentrations of ≥1 nM. The neutralization of prothrombinase activity coincided with cleavages at Arg-506 and subsequent cleavage at Arg-306 of the factor Va heavy chain by activated protein C. Thus, the protein C pathway combined with TFPI creates a minimal inhibitory potential required to shut down TF-initiated thrombin generation. The protein C pathway constituents did not influence factor Xa generation or factor VIIIa degradation over the interval in which prothrombinase activity was neutralized. Our data thus suggest that the protein C pathway regulates thrombin generation solely by the inactivation of factor Va. At low initiating factor VIIa·TF (1.25 pM) and high thrombomodulin concentrations (10 nM), the factor Va heavy chain is cleaved before significant amounts of light chain are generated. The ability of the protein C pathway to inhibit thrombin generation was greatly reduced when the reaction was initiated in the presence of factor Va, supporting the hypothesis that effective down-regulation of thrombin generation by the protein C pathway, in reactions initiated with the procofactor, occurs by prevention of the coexistence of the factor Va heavy and light chains.