Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Abstract 847

Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are ³⁷⁰QIR*^↓SVA³⁷⁵, ¹⁶⁸⁷SPR*^↓SFQ¹⁶⁹², and ⁷³⁸EPR*^↓SFS⁷⁴³, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor.