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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 \u201cactivated\u201d 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\u2010derived and wild\u2010type 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 \u03b1<jats:sub>1<\/jats:sub> antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half\u2010life 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\u2010FVa compared with normal Arg 506\u2010FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.<\/jats:p>","DOI":"10.1002\/pro.5560060115","type":"journal-article","created":{"date-parts":[[2010,7,12]],"date-time":"2010-07-12T04:07:32Z","timestamp":1278907652000},"page":"132-140","source":"Crossref","is-referenced-by-count":31,"title":["Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala\u2010activated protein C mediated by factor Va"],"prefix":"10.1002","volume":"6","author":[{"given":"Andrew J.","family":"Gale","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary 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