von Willebrand因子(vWF)フラグメント(97 kDa，52/48 kDa，及びFⅢ-T2) と抗vWFモノクローナル抗体NMC-4の免疫反応性

Abstract

We have shown that an anti-von Willebrand factor (vWF) monoclonal antibody designated as NMC-4 blocks both the ristocetin- and botrocetin-induced (¹²⁵Ⅰ) vWF bindings to platelet glycoprotein (GP)Ⅰb. Using NMC-4 coupled Sepharose 4B column or electroelution apparatus, a 97 kDa fragment and a 130 kDa fragment were distinctively purified from a whole tryptic digest of vWF. The 97 kDa fragment, which was shown to be a homodimer of the amino acid residue 449-728 of vWF subunit, retained the activity to inhibit both the ristocetin- and botrocetin-induced (¹²⁵Ⅰ) vWF binding to GPⅠb as found in the parent molecule. Another vWF fragment, the FⅢ-T2 fragment, which is a twin heterodimer composed of two H-chains (residue 273-511) and two L-chains (residue 674-728) held together by disulfide-bonds, was also purified by high pressure liquid chromatography. FⅢ-T2 fragment showed no inhibitory effect on botrocetin-induced vWF binding to GPⅠb, whereas it blocked the ristocetin-induced vWF binding. On Western blotting analysis and dot blot assay, NMC-4 failed to react with both the reduced and nonreduced FⅢ-T2 fragment. These results indicate that the vWF binding domains to GPⅠb expressed by either ristocetin or botrocetin are different and the amino acid residue Leu512-Lys673 is important for both botrocetin-induced vWF binding and NMC-4 binding.