Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate.
A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.
Andrews
R K
RK
Department of Medicine, University of Sydney, Westmead Hospital, NSW, Australia.
Gorman
J J
JJ
Booth
W J
WJ
Corino
G L
GL
Castaldi
P A
PA
Berndt
M C
MC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003435:Crotalid Venoms / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006388:Hemagglutinins / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D003435:Crotalid Venoms; D006388:Hemagglutinins; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D013388:Succinimides; D014841:von Willebrand Factor; C030961:botrocetin; D003094:Collagen; C035760:bis(sulfosuccinimidyl)suberate; D010450:Endopeptidases; C023139:dispase