Residues Surrounding Arg336 and Arg562 Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C.

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg336 and Arg562 within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate ∼25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3′ sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3′)562, 336(P4-P2)562, and 336(P1′-P3′)562 mutants, where indicated residues surrounding the Arg336 site were replaced with those surrounding Arg562, were similar to wild type (WT) factor VIII; whereas 562(P4-P3′)336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced ∼6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg562. Cleavage rates at Arg336 were reduced ∼100-fold for 336(P4-P3′)562, and ∼9-16-fold for 336(P4-P2)562 and 336(P1′-P3′)562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3′)562 variant. Alternatively, the 562(P4-P3′)336 variant showed a modest increase in cleavage rate (∼4-fold) at Arg562 compared with WT, whereas these rates were increased by ∼27- and 6-fold for 562(P4-P3′)336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3′ residues surrounding Arg336 and Arg562 make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg336 by APC is attributed to favorable P4-P3′ residues at this site, whereas cleavage at Arg562 can be accelerated following replacement with more optimal P4-P3′ residues. [ABSTRACT FROM AUTHOR]