CLEAVAGE OF HUMAN VONWILLEBRAND-FACTOR BY PLATELET CALCIUM-ACTIVATED PROTEASE

Abstract

In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous Ca-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross-reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of Ca, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent MW of the reduced monomer from 230,000-205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical MW 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin or .epsilon.-aminocaproic acid (none of which inhibits platelet CAP), proteolysis of vWF observed in this study apparently is a direct effect of CAP and is not mediated by way of secondary proteases.