Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates the coagulation cofactors, factors (F) Va and VIIIa. FVIIIa is a non-covalent heterotrimer consisting of A1, A2 and A3-C1-C2 subunits. APC-catalyzed inactivation of FVIIIa results from proteolysis at the P1 residues Arg336 and Arg562 within the A1 and A2 subunits, respectively, with cleavage at Arg336 representing the dominant reaction. We recently showed that replacement of the P4-P3′ residues surrounding the Arg336 site with the corresponding residues flanking Arg562 resulted in an ∼100-fold reduction in cleavage rate at Arg336 (

Varfaj et al,
J. Biol. Chem.
). Comparison of the P4-P3′ residues at the slow-reacting site in FVIIIa (Arg562) with the corresponding residues at the fast-reacting site in FVa (Arg506) revealed that these sequences differ primarily at the P2 and P2′ positions. This observation suggested an important contribution by these residues to cleavage efficiency. The role of the P2 and P2′ residues in the proteolysis of FVIIIa by APC was investigated by preparing recombinant FVIII proteins possessing mutations at Leu335 (P2) and Lys338 (P2′) flanking the P1 Arg336. B-domainless FVIII proteins were stably expressed in BHK cells and purified. Leu335Arg and Leu335Gln mutants were constructed based upon the P2 residues preceding Arg506 in FVa and Arg562 in FVIIIa, respectively. The Lys338Ile mutant was based upon the P2′ residue following Arg506 in FVa. Specific activity values for all FVIII variants were similar to wild type (WT) FVIII. APC-catalyzed inactivation rates for FVIIIa were determined using a FXa generation assay, and rates for proteolysis at the scissile bonds within the A1 and A2 subunits were determined by SDS-PAGE and Western blotting. Rates for APC-catalyzed cleavage of the A1 subunit for the FVIIIa variants Leu335Arg and Leu335Gln were reduced ∼2- and ∼4-fold, respectively, as compared to WT. However, the rate of cleavage of the A1 subunit in the Lys338Ile FVIIIa variant was enhanced ∼3-fold compared to WT. Cleavage rates at Arg562 in all the variants were unaffected by mutation at either residue 335 or 338. These relative values for rates of proteolysis paralleled the observed rates for inactivation of the FVIIIa forms. Overall, these results suggest that both P2 and P2′ residues are important in the efficient proteolysis at Arg336 in FVIIIa. Furthermore, Leu appears more optimal than either Arg or Gln at the P2 position, whereas Ile is preferred over Lys at the P2′ position in this macromolecular substrate for APC.

Author notes

Disclosure: No relevant conflicts of interest to declare.