Evaluation of the initiation phase of blood coagulation using ultrasensitive assays for serine proteases

The initiation phase of enzyme generation in a reconstituted model of the tissue factor (TF) pathway to thrombin was evaluated. At 1.25 pM added TF, no thrombin generation was observed in the absence of factor V. The substitution of factor Va for factor V increased the rate of thrombin generation. Factor X activation during the initiation phase was not influenced by the absence of factor VIII or thrombin, leading to the conclusion that initially factor Xa is generated exclusively by the factor VIIa-TF complex. When thrombin was eliminated from the system, no contribution of the factor IXa-factor VIIIa complex to factor X activation was observed during the propagation phase. Similarly, factor V activation was also not observed in the absence of thrombin, indicating that thrombin is the only enzyme responsible for factor V and factor VIII activation. Only subnanomolar amounts of factor VII were activated when prothrombin activation was almost complete. In the absence of coagulation inhibitors, factor XI did not influence thrombin generation initiated by 1.25 pM factor VIIa-TF complex. The termination of factor XIa generation by added hirudin in the factor XI experiment indicates that factor XI activation occurs exclusively by thrombin.