REGULATION OF TISSUE FACTOR INITIATED THROMBIN GENERATION BY THE STOICHIOMETRIC INHIBITORS TISSUE FACTOR PATHWAY INHIBITOR, ANTITHROMBIN-III, AND HEPARIN COFACTOR-II

The effects of the stoichiometric inhibitors tissue factor pathway inh ibitor (TFPI), antithrombin-III (AT-III) and heparin cofactor-II (HC-I I) on thrombin generation were evaluated in a reaction system composed of coagulation factors VIIa, X, IX, VIII, and V and prothrombin initi ated by tissue factor (TF) and phospholipids. Initiation of the reacti on in the absence of inhibitors resulted in explosive thrombin generat ion for factor VIIa-TF concentrations varying from 100 to 0.25 pM with the lag time or initiation phase of thrombin generation increasing fr om 0 to 180 s with decreasing factor VIIa-TF concentrations, During th e propagation phase, prothrombin is quantitatively activated to 1.4 mu M alpha-thrombin. At normal plasma concentration (2.5 nM) full-length recombinant TFPI prolonged the initiation phase of thrombin generatio n 2-fold, and the rate of thrombin generation in the propagation phase of the reaction was 25-50% that of the uninhibited reaction when the reaction was initiated with 1.25-20 pM factor VIIa-TF. Inhibition of t he reaction by TFPI is associated with a delay in factor V activation, In the presence of TFPI no explosive thrombin generation was observed when factor VIII was omitted from reactions initiated by factor VIIa- TF concentrations less than or equal to 20 pM. This indicates that in the presence of TFPI the factor Ma factor VIIIa pathway becomes essent ial at low factor VIIa-TF concentrations. In the reconstituted system, AT-III (3.4 mu M) did not prolong the initiation phase of thrombin ge neration when the reaction was initiated with 1.25 pM factor VIIa-TF, nor did AT-III delay factor V activation. The rate of thrombin formati on in the presence of AT-III was reduced to 30% that of the uninhibite d reaction, and the cu-thrombin formed was rapidly inhibited subsequen t to its generation, The addition of HC-II alone at its physiological concentration (1.38 mu M) to the procoagulant mixture did not alter th e rate or extent of thrombin generation. Subsequently, the thrombin fo rmed was slowly inhibited by HC-II. The slow inactivation of thrombin by HC-II does not contribute to thrombin inhibition in the presence of AT-III. In contrast, the combination of physiological levels of AT-II I and TFPI inhibited explosive thrombin generation initiated by 1.25 p M factor Wa TF completely, The absence of prothrombin consumption indi cated that the combination of TFPI and AT-III is able to prevent the f ormation of prothrombinase activity at low factor VIIa-TF concentratio ns, The data indicate that TFPI potentiates the action of AT-III by de creasing the rate of formation and thus the amount of catalyst formed in the reaction, enabling AT-III to effectively scavenge the limited t races of factor IXa and factor Xa formed in the presence of TFPI, The initiation of thrombin generation by increasing factor VIIa-TF concent rations in the presence of physiological concentrations of TFPI and AT -III showed dramatic changes in the maximal rates of thrombin generati on over small changes in initiator concentration, These data demonstra te that significant thrombin generation becomes a ''threshold-limited' ' event with regard to the initiating factor VIIa-TF concentration in the presence of TFPI and AT-III.