抗von Willebrand因子(vWF)モノクローナル抗体NMC-4による血小板膜糖蛋白(GP)Ⅰb結合ドメインの解析

Abstract

An anti-von Willebrand factor (vWF) mouse monoclonal antibody designated as NMC-4 was shown to induce the inhibition of both the ristocetin- and botrocetin-induced vWF bindings to platelet glycoprotein (GP)Ⅰb, as well as the block of desialylated vWF (AS-vWF) binding to GPⅠb. Using NMC-4 coupled Sepharose 4B column, a 97 kDa fragment was immunopurified from a tryptic digest of native vWF in nonreduced condition. The 97 kDa fragment showed a doublet polypeptide with a M. W. 52/48 kDa after reduction with dithiothreitol on SDS-polyacrylamide gel. The NH＿2-terminal sequence and amino acid analysis of the 97 kDa fragment indicated that it was a homodimer composed of vWF peptide (amino acid residue 449-728). These results demonstrated the possible presence of one or three interchain disulfide-bonds involving the cysteine residues 459, 462, and/or 464. This fragment competitively inhibited both the ristocetin- and botrocetin- induced vWF bindings to GPⅠb as well as AS-vWF binding to GPⅠb. On Western blotting, NMC-4 reacted with the reduced 97 kDa fragment with less intensity than the nonredued one. Two synthetic peptides, Cys 474-Pro 488 and Leu 694-Pro 708, inhibited ristocetin-induced binding of 97 kDa fragment to GPⅠb. But neither of them inhibited botrocetin-induced binding of 97 kDa fragment to GPⅠb or its direct binding to GPⅠb. These results clearly indicate that the GPⅠb binding domain expressed by either ristocetin or botrocetin resides on a different portion within the 97 kDa fragment.