Proteolysis at Arg740 facilitates subsequent bond cleavages during thrombin-catalyzed activation of factor VIII

Abstract

Thrombin activates factor VIII by proteolysis at three P1 residues: Arg372, Arg740, and Arg1689. Cleavage at Arg372 and Arg1689 are essential for procofactor activation; however cleavage at Arg740 has not been rigorously studied. To evaluate the role for cleavage at Arg740, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, respectively, cleavage at this site. Specific activity values for the variants were ∼50 and 20%, respectively, that of wild-type factor VIII. Activation of factor VIII R740H by thrombin showed an ∼40-fold reduction in the rate of A2 subunit generation, which reflected an ∼20-fold reduction in cleavage rate at Arg372. Similarly, a ∼40-fold rate reduction in cleavage at Arg1689 and consequent generation of the A3-C1-C2 subunit were observed. Rate values for A2 and A3-C1-C2 subunit generation were reduced by >700-fold and ∼140-fold, respectively, in the R740Q variant. These results suggest that initial cleavage at Arg740 affects cleavage at both Arg372 and Arg 1689 sites. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed more modest rate reductions (<10-fold) in generating A2 and A3-C1-C2 subunits from either variant, suggesting that factor Xa-catalyzed activation of factor VIII was significantly less dependent upon prior cleavage at residue 740 than thrombin. Overall, these results support a model whereby cleavage of factor VIII by thrombin is an ordered pathway with cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689, which result in procofactor activation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.