Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala‐activated protein C mediated by factor Va

Abstract

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

“…The present study supports the conclusion that APC(S360A) efficiently and completely down-regulates FXa and thrombin formation in the absence of proteolysis of its cofactor substrates, FVa and FVIIIa, both in model systems and in human plasma (37). Binding of APC to FVa or FVIIIa, the first step in their proteolytic inactivation, is sufficient to inhibit thrombin and FXa formation.…”

“…Interactions between APC and its substrates (FVa and FVIIIa) are largely dependent on three exposed surface loops (the 37 233(78) ) that together form an electropositive exosite on APC (5-9). Protein C numbering is used throughout the text, followed by chymotrypsinogen numbering in parentheses.…”

Recombinant human activated protein C inhibits integrin-mediated neutrophil migration

“…Unlike rhAPC, rhPC failed to block neutrophil adhesion and migration on FN ( Figure 2C,G). The recombinant APC variant S360A-APC, however, which lacks proteolytic activity, 27 successfully inhibited neutrophil migration ( Figure 2G). Thus, these data suggest that the RGD sequence in rhAPC is an essential feature of its direct interaction with the neutrophil integrins and for inhibition of neutrophil migration on matrix proteins but that APC's enzymatic proteolytic activity is not necessary for the inhibition.…”

Activated Protein C Mutant with Minimal Anticoagulant Activity, Normal Cytoprotective Activity, and Preservation of Thrombin Activable Fibrinolysis Inhibitor-dependent Cytoprotective Functions

“…The concentration of S360A-APC was determined by enzyme-linked immunosorbent assay (American Bioproducts) (26). Amidolytic assays (S-2366, Chromogenix, Spectrozyme aPC, American Diagnostica and Pefachrome PCa, Pentapharm) were performed as described before (21,27).…”