POSTTRANSLATIONAL SULFATION OF FACTOR-V IS REQUIRED FOR EFFICIENT THROMBIN CLEAVAGE AND ACTIVATION AND FOR FULL PROCOAGULANT ACTIVITY

Factor VIII and factor V function as cofactors in the blood coagulatio n cascade to accelerate the rate of activation of factor X and prothro mbin, respectively. Both cofactors require proteolytic activation by e ither activated factor X or thrombin for functional activity. Human fa ctor VIII and factor V expressed in mammalian cells are both modified by posttranslational sulfation of tyrosine residues. In the present st udy, the posttranslational addition of sulfate in factor V expressed i n transfected Chinese hamster ovary (CHO) cells was demonstrated by [S -35] sulfate incorporation into the thrombin-cleaved 94-kDa heavy chai n and the 150-kDa activation peptide. The presence of tyrosine sulfate in recombinant factor V was confirmed by barium hydroxide hydrolysis and two-dimensional thin-layer electrophoresis. The importance of sulf ation for factor V secretion and activity was evaluated by characteriz ing factor V expressed in Chinese hamster ovary cells grown in the pre sence of sodium chlorate, a potent inhibitor of posttranslational sulf ation in intact cells. Increasing concentrations of sodium chlorate in hibited the incorporation of [S-35]sulfate into factor V but did not i nhibit the synthesis or secretion of factor V. However, the specific a ctivity of factor V secreted in the presence of sodium chlorate was re duced 5-fold, The reduced activity was attributed to (1) slower cleava ge and activation by thrombin and (2) a reduced intrinsic activity of factor Va. In contrast, sulfation of factor V did not affect the rate of activation mediated by factor Xa. These results show that sulfation of factor V is required for efficient thrombin activation but not for activation by factor Xa. In addition, factor V isolated from a patien t with homozygous combined factor V and factor VIII deficiency is not defective in posttranslational sulfation.