FACTOR-V ENHANCES THE COFACTOR FUNCTION OF PROTEIN-S IN THE APC-MEDIATED INACTIVATION OF FACTOR-VIII - INFLUENCE OF THE FACTOR V-R506Q MUTATION

Factor V and protein S are cofactors of activated protein C (APC) whic h accelerate APC-mediated factor VIII inactivation. The effects of fac tor V and protein S were quantitated in a reaction system in which pla sma factor Vm was inactivated by APC and the loss of factor Vm activit y was monitored in a factor X-activating system in which a chromogenic substrate was used to probe factor Xa formation. Factor V increased t he rate of APC-mediated factor Vm inactivation in a dose-dependent man ner in representative plasma samples with protein S or factor V defici ency, abnormal factor V (heterozygous or homozygous for factor V-R506Q ), or a combination of heterozygous protein S deficiency and heterozyg ous factor V-R506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response i n these plasmas was corrected by addition of protein S, indicating tha t both factor V and protein S are required for optimal inactivation of factor WI by APC. The effects of factor V and protein S were also stu died in a reaction system with purified proteins. APC-catalysed factor Vm inactivation was enhanced 3.7-fold in the presence of 1.1 nM facto r V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was ii- fold. Factor V is a more potent cofactor than protein S, as can be con cluded from the fact that 0.04 nM factor V gave the same stimulation a s 2.4 nM protein S. Protein S lost its cofactor function after complex ation with C4b binding protein, which indicates that it is free protei n S that acts as a cofactor. To investigate the effect of the R506Q mu tation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor Vm inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lo st when factor V was activated with thrombin or with the factor V acti vator from Russell's viper venom. These data indicate that optimal ina ctivation of factor Vm by APC requires the presence of an intact facto r V molecule and free protein S.