INHIBITORY MECHANISM OF THE PROTEIN-C PATHWAY ON TISSUE FACTOR-INDUCED THROMBIN GENERATION - SYNERGISTIC EFFECT IN COMBINATION WITH TISSUE FACTOR PATHWAY INHIBITOR

The effects of the components of the protein C path way on thrombin ge neration were studied in a reconstituted model in which thrombin is ge nerated by factor VIIa and relipidated tissue factor (TF) via the acti vation of the purified coagulation factors X, IX, VIII, V, and prothro mbin. The influence of protein C and soluble thrombomodulin on thrombi n generation was correlated with factor Xa generation, factor V(a) and factor VIII(a) formation/inactivation, and protein C activation, Thro mbin generation initiated by low concentrations of factor VIIa . TF (1 .25 pM) occurs in an explosive fashion during a propagation phase whic h occurs after an initiation phase of similar to 1 min in which only t races of thrombin are formed, In the absence of other inhibitors, prot ein C (65 nM) in combination with high concentrations of soluble throm bomodulin (10 nM) resulted in a reduced rate of thrombin generation du ring the propagation phase without affecting the initiation phase; the activated pro 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 recom binant TFPI), the protein C pathway reduced the rate of thrombin gener ation, initiated by 1.25 pM factor VIIa . TF, and completely eliminate d prothrombinase activity at soluble thrombomodulin concentrations of greater than or equal to 1 nM. The neutralization of prothrombinase ac tivity 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 pot ential required to shut down TF-initiated thrombin generation, The pro tein C pathway constituents did not influence factor Xa generation or factor VIIIa degradation over the interval in which prothrombinase act ivity was neutralized, Our data thus suggest that the protein C pathwa y regulates thrombin generation solely by the inactivation of factor V a. At low initiating factor VIIa . TF (1.25 pM) and high thrombomoduli n concentrations (10 nM), the factor Va heavy chain is cleaved before significant amounts of light chain are generated, The ability of the p rotein C pathway to inhibit thrombin generation was greatly reduced wh en 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 li ght chains.