Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib.
Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.
Fujimura
Y
Y
Department of Blood Transfusion, Nara Medical College, Kashihara City, Japan.
Usami
Y
Y
Titani
K
K
Niinomi
K
K
Nishio
K
K
Takase
T
T
Yoshioka
A
A
Fukui
H
H
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015153:Blotting, Western; D003435:Crotalid Venoms / Q000494:pharmacology*; D006388:Hemagglutinins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D012310:Ristocetin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D006388:Hemagglutinins; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin