IDENTIFICATION OF INDIVIDUAL TYROSINE SULFATION SITES WITHIN FACTOR-VIII REQUIRED FOR OPTIMAL ACTIVITY AND EFFICIENT THROMBIN CLEAVAGE

Factor VIII functions as an essential cofactor in the blood coagulatio n cascade for the factor IXa-mediated activation of factor X. Factor V III contains 6 tyrosine residues at positions 346, 718, 719, 723, 1664 , and 1680 that are modified by post-translational sulfation. This mod ification is required for full factor VIII procoagulant activity. We h ave employed site-directed mutagenesis to identify the individual sulf ated tyrosines within factor VIII that influence activity. The molecul es were expressed in COS-1 monkey cells by transient transfection, and the resultant proteins were characterized. Metabolic incorporation of [S-35]sulfate demonstrated that all 6 tyrosine residues are sulfated in factor VIII. Sulfation at residues 346 and 1664 was required for fu ll activity in a factor VIII clotting assay but did not affect factor VIII activity monitored by a factor Xa generation assay. The Tyr(346) --> Phe and Tyr(1664) --> Phe mutants displayed played delayed thrombi n activation that correlated with delayed cleavage at residues 372 and 1689, respectively. In contrast, these mutants were efficiently activ ated by factor Xa. A triple Tyr to Phe mutant at residues 718, 719, an d 723 displayed both reduced factor VIII clotting activity and factor Xa generation activity. Finally, a Tyr(1680) --> Phe mutant factor VII I displayed a 5-fold reduced affinity for von Willebrand factor. The r esults demonstrate that 1) sulfation at tyrosine residues 346 and 1664 increases factor VIII activity by increasing the rate of thrombin act ivation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF. In addition, the results implicate that thrombin interacts with three distinct sit es within factor VIII, two of which are required for proteolytic activ ation. The results demonstrate that the six sites of tyrosine sulfatio n modulate factor VIII activity through different mechanisms.