Requirement of Factor VIII for the Generation of Xa in a Purified System and in Plasma.

The requirements of purified Factors IXa, VIII and X for Factor Xa generation were studied by quantitating the Factor Xa produced using the amidolytic action of Xa on the synthetic chromogenic substrate S-2222. The rate of Factor Xa generation was found to be dependent on the initial concentrations of Factors VIII, IXa and X. The exposure of Factor VIII to thrombin (0.001 U/ml) for up to 10 minutes increased the rate of Factor Xa generation tenfold. Longer exposures (>15 minutes) led to inactivation of Factor VIII. Factor Xa production was demonstrably slower in plasma, even under conditions where the Factor IXa inhibitory activity of plasma had been neutralized by excess Factor IXa. Using purified clotting factors, calcium and phospholipid, no Factor Xa was formed in the absence of Factor VIII. In contrast, the addition of Factor IXa to plasma obtained cither from severe hemophiliacs or normal plasma which had been immunodepleted of coagulant and antigenic Factor VIII and had no demonstrable Factor VIII coagulant activity resulted in the generation of Factor Xa. These findings suggest that Factor VIII is required for the activation of Factor X by Factor IXa in the purified system but that mechanisms appear to be present in plasma which enable Factor IXa to participate in the activation of Factor X even when Factor VIII is absein.