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.

Abstract

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.

• Authors: R K Andrews, J J Gorman, W J Booth, G L Corino, P A Castaldi, M C Berndt
• Journal: Biochemistry (Biochemistry) Vol. 28 Issue 21 Pg. 8326-36 (Oct 17 1989) ISSN: 0006-2960 [Print] United States
• PMID: 2690940 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Amino Acids
• Cross-Linking Reagents
• Crotalid Venoms
• Hemagglutinins
• Peptide Fragments
• Platelet Membrane Glycoproteins
• Succinimides
• von Willebrand Factor
• botrocetin
• Collagen
• bis(sulfosuccinimidyl)suberate
• Endopeptidases
• dispase

Topics

• Amino Acid Sequence
• Amino Acids (analysis)
• Blood Platelets (metabolism)
• Chromatography, Affinity
• Chromatography, High Pressure Liquid
• Collagen (metabolism)
• Cross-Linking Reagents (pharmacology)
• Crotalid Venoms (pharmacology)
• Electrophoresis, Polyacrylamide Gel
• Endopeptidases (metabolism)
• Hemagglutinins (pharmacology)
• Humans
• Molecular Sequence Data
• Peptide Fragments (analysis, isolation & purification, metabolism)
• Platelet Membrane Glycoproteins (metabolism)
• Succinimides (pharmacology)
• von Willebrand Factor (analysis, isolation & purification, metabolism)