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

Abstract

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg 336 and Arg 562 within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate ϳ25-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. variant showed a modest increase in cleavage rate (ϳ4-fold) at Arg 562 compared with WT, whereas these rates were increased by ϳ27-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 Arg 336 and Arg 562 make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg 336 by APC is attributed to favorable P4-P3 residues at this site, whereas cleavage at Arg 562 can be accelerated following replacement with more optimal P4-P3 residues.Factor VIII plays a critical role in the coagulation cascade evident from hemophilia A occurring in individuals with deficient or defective factor VIII. Factor VIII is synthesized as a single chain precursor (1, 2) consisting of three A domains homologous to the A domains in factor V, a unique B domain, and two homologous C domains (1, 3). A region rich in acidic amino acid residues designated a1 (residues 337-372), a2 (residues 711-740), and a3 (residues 1649 -1689) borders each A domain. Thus, the factor VIII domain organization is represented as NH 2 -A1-a1-A2-a2-B-a3-A3-C1-C2-COOH. Intracellular proteolytic processing generates the factor VIII procofactor composed of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains) that are associated via a metal ion-dependent linkage. Thrombin activates the procofactor by limited proteolysis generating the active cofactor, factor VIIIa, a heterotrimer composed of A1, A2, and A3-C1-C2 subunits (4, 5). Factor VIIIa associates with the serine protease factor IXa in a phospholipid membrane-dependent interaction, forming the intrinsic factor Xase complex that efficiently activates factor X during the propagation phase of coagulation (see Ref. 6 for a review).Down-regulation of the intrinsic factor Xase complex is largely due to inactivation of factor VIIIa, which is thought to occur by two independent mechanisms. The first reflects dissociation of the A2 subunit from the A1/A3-C1-C2 dimer, a result of a weak affinity electrostatic interaction (5, 7). The second mechanism results from proteolytic inactivation of the cofactor catalyzed by APC.3 APC is a potent anticoagulant that proteolytically inactivates factor Va and factor VIIIa, and deficiencies in this proteinase are linked to thrombosis (see Ref. 8 for a review). APC cleaves both procofactor factor VIII and factor VIIIa, although the latter represents the more relevant, physiologic substrate, at Arg 336 (A1 subunit) and at Arg 562 (A2 subunit) (9, 10). Cleavage at the former site alters the interactions ...

Sequences Flanking Arg336 in Factor VIIIa Modulate Factor Xa-catalyzed Cleavage Rates at this Site and Cofactor Function

“…In an earlier study examining APC-catalyzed cleavage and inactivation of factor VIIIa, which like factor Xa, attacks Arg 336 initially and Arg 562 second- (15). This rate reduction was attributed to replacement of critical flanking residues with residues less optimal for cleavage.…”

“…Rates of factor VIIIa inactivation, and A1 cleavage were calculated by dividing the absolute value of B by factor Xa concentration and results are expressed in nanomolar FVIIIa/min/nM factor Xa or nanomolar A1/min/nM factor Xa (15).…”

“…The factor VIII variants 336(P4-P3Ј)562 (15) and R336Q (19) were made by replacing P4-P3Ј residues flanking Arg 336 with those flanking Arg 562 and by mutating Arg 336 to Gln, respectively. Mutants were introduced into Bluescript II K/SϪ factor VIII cloning vector using the Stratagene QuikChange Site-directed Mutagenesis Kit and the mutated factor VIII coding region was ligated into the B-domainless factor VIII expression vector at XhoI/NotI restriction sites as described previously (20).…”

“…age at these sites (15,16). Given the homologies of APC and factor Xa as well as the common bonds cleaved during cofactor inactivation, we initiated a study to assess the roles of scissile bond flanking sequences in factor Xa-catalyzed inactivation of factor VIIIa.…”

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

“…Cleavage at the former site, which correlates with loss of function, appears to alter the orientation of A2 subunit with the A1/ A3C1C2 dimer (15), while cleavage at the latter site destroys a FXa-interactive site (16). These cleavages that inactivate FVIIIa occur at a slower rate than the activating cleavages catalyzed by FXa (12,17,18) and this property favors FXa as initially serving to activate FVIII. We have previously shown that P4-P3' residues flanking the Arg336 and Arg562 sites contribute towards determining rates of activated protein C (APC) or FXa cleavage at these sites (17)(18)(19).…”