Revised Manuscript Received February 24, 1994* abstract: Factor VIII and factor V function as cofactors in the blood coagulation cascade to accelerate the rate of activation of factor X and prothrombin, respectively. Both cofactors require proteolytic activation by either activatedfactor X or thrombin for functional activity. Human factor VIII and factor V expressed in mammalian cells are both modified by posttranslational sulfation of tyrosine residues. In the present study, the posttranslational addition of sulfate in factor V expressed in transfected Chinese hamster ovary (CHO) cells was demonstrated by [35S] sulfate incorporation into the thrombin-cleaved 94-kDa heavy chain 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 sulfation for factor V secretion and activity was evaluated by characterizing factor V expressed in Chinese hamster ovary cells grown in the presence of sodium chlorate, a potent inhibitor of posttranslational sulfation in intact cells. Increasing concentrations of sodium chlorate inhibited the incorporation of [35S] sulfate into factor V but did not inhibit the synthesis or secretion of factorV. However, the specific activityof factor V secreted in the presence of sodium chlorate was reduced 5-fold. The reduced activity was attributed to (1) slower cleavage 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 patient with homozygous combined factor V and factor VIII deficiency is not defective in posttranslational sulfation.

Coagulation factor V is a 330-kDa single-chain plasma glycoprotein that plays a critical role in hemostasis as a cofactor for the factor Xa dependent proteolytic cleavage of prothrombin to thrombin (Esmon, 1979; Nesheim et al., 1979; Dahlback, 1980; Kane & Majerus, 1981). The amino acid sequence deduced from the cDNA showed that factor V is synthesized as a 2224 amino acid precursor containing a 28 amino acid signal peptide (Kane & Davie, 1986; Jenny et al., 1987). Intact single-chain factor V has little intrinsic cofactor activity (Katzman et al., 1981; Monkovic & Tracy, 1990). Factor V is cleaved by thrombin after residues 709, 1018, and 1545 to generate activated factor Va that is composed of a 94-kDa amino-terminal-derived fragment in a metal-ion association with a carboxy-terminal-derived 74-kDa fragment