A MODEL FOR THE TISSUE FACTOR PATHWAY TO THROMBIN .1. AN EMPIRICAL-STUDY

The activation of prothrombin, factor V, factor VIII, factor IX, and f actor X by the tissue factor factor Wa complex, in vitro, in a system in which each precursor protein was present at plasma concentration, w as evaluated using a combination of activity assays, immunoblots, acti ve site blots, and autoradiography. The thrombin generation curves obs erved were distinctly nonlinear and typically displayed a time lag in which little or no thrombin was observed. This was followed by an almo st linear propagation phase of thrombin formation. The lag was a funct ion of tissue factor/factor VIIa con centration and represented primar ily the interval of fac tor 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 facto r IX and X present was activated but when nearly 100% activation of th e cofactors, factor V and factor VIII, was achieved. Analyses of the a ctivation 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 conta ined factor Va instead of factor V, the lag was substantially reduced. When factor V was deleted from the reaction mixture, no thrombin form ation was observed. When either factor VIII or factor IX was deleted f rom the reaction system, the propagation phase of thrombin formation ( at 5 pM tissue factor-factor VIIa complex) was only one third that obs erved for reactions which contained factor VIII and factor IX. The add ition of factor XI to the experimental system increased the rate of th rombin formation by 15% during the propagation phase but had no effect upon the lag phase of the reaction. Our data suggest that normal hemo stasis may be initiated by the factor VIIa-tissue factor complex and s upport the concept of multiple feedback reactions which amplify and pr opagate the hemostatic response.