Cleavage of human von Willebrand factor by platelet calcium-activated
protease
TJ Kunicki, RR Montgomery and J Schullek
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 calcium-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 calcium, radiolabeled
purified plasma vWF was also degraded by the protease in question, with a
decrease in the apparent molecular weight of the reduced monomer from
230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded
plasma vWF by preparative CIE was shown to be composed of an identical mol
wt 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), we
conclude that proteolysis of vWF observed in this study is a direct effect
of CAP and is not mediated by way of secondary proteases.
Volume 65,
Issue 2,
pp. 352-356,
02/01/1985
Copyright © 1985 by The American Society of Hematology
