A model for the tissue factor pathway to thrombin. I. An empirical study.

The activation of prothrombin, factor V, factor VIII, factor IX, and factor X by the tissue factor-factor VIIa complex, in vitro, in a system in which each precursor protein was present at plasma concentration, was evaluated using a combination of activity assays, immunoblots, active-site blots, and autoradiography. The thrombin generation curves observed were distinctly nonlinear and typically displayed a time lag in which little or no thrombin was observed. This was followed by an almost linear propagation phase of thrombin formation. The lag was a function of tissue factor/factor VIIa concentration and represented primarily the interval of factor V and factor VIII activation. The postlag propagation phase of thrombin generation was nearly independent of the initial activator (factor VIIa or tissue factor) concentration over a 10(3)-fold range in factor VIIa-tissue factor concentration. Maximum thrombin generation rates were observed when less than 1% of the factor IX and X present was activated but when nearly 100% activation of the cofactors, factor V and factor VIII, was achieved. Analyses of the activation pattern of factor V indicated that the cofactor is activated by both factor Xa and thrombin which are formed at low levels during the lag phase of the reaction. When the initial reaction mixture contained factor Va instead of factor V, the lag was substantially reduced. When factor V was deleted from the reaction mixture, no thrombin formation was observed. When either factor VII or factor IX was deleted from the reaction system, the propagation phase of thrombin formation (at 5 pM tissue factor-factor VIIa complex) was only one-third that observed for reactions which contained factor VIII and factor IX. The addition of factor XI to the experimental system increased the rate of thrombin formation by 15% during the propagation phase but had no effect upon the lag phase of the reaction. Our data suggest that normal hemostasis may be initiated by the factor VIIa-tissue factor complex and support the concept of multiple feedback reactions which amplify and propagate the hemostatic response.