The Mutation Leu335Pro in Factor VIII Results in Its Rapid Inactivation by Thrombin-Catalyzed Proteolysis at Arg336.

Activation of the heterodimeric factor VIII procofactor is achieved by proteolysis of the heavy chain at residues Arg740 and Arg372 and of the light chain at Arg1689 in reactions catalyzed by thrombin or factor Xa. The active cofactor, factor VIIIa, is a heterotrimer consisting of the A1, A2, and A3-C1-C2 subunits. Factor VIIIa inactivation occurs by two mechanisms, spontaneous dissociation of the A2 subunit from factor VIIIa and proteolytic inactivation catalyzed by activated protein C (APC) as well as factor Xa. APC-catalyzed inactivation of factor VIIIa 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. Factor Xa-catalyzed inactivation of factor VIIIa occurs following cleavage only at the former site. While investigating the role for residues in the P2 position (residue 335) in the proteolytic mechanism for factor VIIIa inactivation, we stably expressed and purified several recombinant B-domainless factor VIII mutants. One variant, Leu335Pro, was based upon the optimal residue (Pro) at the P2 position for thrombin. Specific activity of the Leu335Pro factor VIII was ∼75% that of WT factor VIII. A time course of thrombin-catalyzed activation of the Leu335Pro factor VIII, as monitored by a one-stage clotting assay, yielded a similar activity increase to that of WT, consistent with similar rates of cleavages converting the procofactor to cofactor. However, this activity was highly unstable in the variant and quickly decayed such that at 10 min following thrombin addition, the residual activity of the variant was reduced ∼40-fold compared to WT. To investigate the reason for this rapid rate of inactivation of the newly generated cofactor, we monitored cleavages in the factor VIII heavy chain following reaction with thrombin, as well as APC and factor Xa by Western blot analysis. As predicted from the activity data, thrombin efficiently cleaved both the WT and Leu335Pro at Arg740 and Arg372, generating the A1 and A2 subunits of factor VIIIa. In WT factor VIIIa, we also observed a relatively slow rate of cleavage in the A1 subunit generating a product consistent with proteolysis at the APC-sensitive Arg336 site. This material represented <20% of the total A1 subunit at an extended time point (40 min) in the reaction suggesting that thrombin-cleavage at this site is a minor pathway. However, thrombin cleavage of the A1 subunit derived from Leu335Pro generated the truncated A1 product at a >50-fold increased rate compared with WT. Since Pro represents an optimal residue at the P2 position for thrombin action, these results suggest that the presence of this residue facilitated cleavage at Arg336. While the presence of Pro335 did not appreciably affect cleavage of Arg336 by APC compared to the cleavage rate observed in the WT factor VIII, this rate was ∼5-fold less than thrombin-catalyzed cleavage at Arg336 in the Leu335Pro variant. Furthermore, the rate of factor Xa-catalyzed cleavage at this site in the variant was accelerated ∼10-fold relative to WT. Overall, these data suggest that replacement of Leu335 with Pro markedly increases proteolytic inactivation of factor VIIIa by both thrombin, and to a lesser extent factor Xa by creating a more optimal region for active site engagement at Arg336.