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 coagulation cascade to accelerate the rate of activation of factor X and prothrombin, respectively. Both cofactors require proteolytic activation by either activated factor 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 factor V. However, the specific activity of 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Pittman
D D
DD
Department of Molecular and Cellular Genetics, Genetics Institute, Cambridge, Massachusetts 02140.
Tomkinson
K N
KN
Michnick
D
D
Selighsohn
U
U
Kaufman
R J
RJ
D000818:Animals; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D004586:Electrophoresis / Q000379:methods; D005165:Factor V / Q000378:metabolism*; D005166:Factor V Deficiency / Q000150:complications / Q000378:metabolism; D006467:Hemophilia A / Q000150:complications / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D011994:Recombinant Proteins; D013431:Sulfates; D005165:Factor V; D013917:Thrombin