Limited proteolysis of human von Willebrand factor by Staphylococcus aureus V-8 protease: isolation and partial characterization of a platelet-binding domain.
Purified human von Willebrand factor (vWF) was digested with Staphylococcus aureus V-8 protease, and specific domains interacting with platelets were isolated and characterized. Amino acid sequence analysis and sodium dodecyl sulfate gel electrophoresis demonstrated that the digestion proceeded primarily by a single cleavage of the native 270K subunit between an internal Glu-Glu peptide bond. This produced an integral stepwise degradation of the multimers of vWF with a concomitant accumulation of bands with mobility similar to that of the smaller molecular weight vWF multimers. The immediate precursor of the final products contained equimolar amounts of 270K subunit and of two polypeptides (170K and 110K). The cleavage of the remaining 270K subunit converted vWF into two main fragments (fragments II and III). These fragments were isolated by ion exchange chromatography, characterized, and assayed for platelet binding in the presence of ristocetin. Fragment III is a dimer of 315K composed primarily of two chains of 170K. Amino acid sequence analysis indicated that it originated from the amino-terminal portion of the 270K subunit and contained 11% of the original ristocetin cofactor activity. Also, it binds to platelets at the same specific sites as native vWF and shows a platelet binding pattern similar to that of partially reduced vWF (500K). Fragment II is a dimer of 235K composed of two identical chains of 110K. Amino acid sequence analysis indicated that it originated from the carboxyl-terminal portion of the 270K subunit and lacked ristocetin cofactor activity. Also, it does not bind to platelets or inhibit the binding of 125I-vWF in the presence of ristocetin.(ABSTRACT TRUNCATED AT 250 WORDS)
D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012697:Serine Endopeptidases; D014841:von Willebrand Factor / Q000378:metabolism*
D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase