Cross-Linking of a Monomelic 39/34-kDa Dispase Fragment of von Willebrand Factor (Leu-480/Val-481–Gly-718) to the N-Terminal Region of the α-Chain of Membrane Glycoprotein Ib on Intact Platelets with Bis(sulfosuccinimidyl) Suberate

Robert K. Andrews, Jeffrey J. Gorman, William J. Booth, Gary L. Corino, Peter A. Castaldi, Michael C. Berndt

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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 α-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.

Original languageEnglish
Pages (from-to)8326-8336
Number of pages11
JournalBiochemistry
Volume28
Issue number21
DOIs
Publication statusPublished - 1 Jan 1989
Externally publishedYes

Cite this

@article{7855e44a1c03451780b34c1be68ee993,
title = "Cross-Linking of a Monomelic 39/34-kDa Dispase Fragment of von Willebrand Factor (Leu-480/Val-481–Gly-718) to the N-Terminal Region of the α-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 α-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.",
author = "Andrews, {Robert K.} and Gorman, {Jeffrey J.} and Booth, {William J.} and Corino, {Gary L.} and Castaldi, {Peter A.} and Berndt, {Michael C.}",
year = "1989",
month = "1",
day = "1",
doi = "10.1021/bi00447a010",
language = "English",
volume = "28",
pages = "8326--8336",
journal = "Biochemistry",
issn = "0006-2960",
number = "21",

}

Cross-Linking of a Monomelic 39/34-kDa Dispase Fragment of von Willebrand Factor (Leu-480/Val-481–Gly-718) to the N-Terminal Region of the α-Chain of Membrane Glycoprotein Ib on Intact Platelets with Bis(sulfosuccinimidyl) Suberate. / Andrews, Robert K.; Gorman, Jeffrey J.; Booth, William J.; Corino, Gary L.; Castaldi, Peter A.; Berndt, Michael C.

In: Biochemistry, Vol. 28, No. 21, 01.01.1989, p. 8326-8336.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Cross-Linking of a Monomelic 39/34-kDa Dispase Fragment of von Willebrand Factor (Leu-480/Val-481–Gly-718) to the N-Terminal Region of the α-Chain of Membrane Glycoprotein Ib on Intact Platelets with Bis(sulfosuccinimidyl) Suberate

AU - Andrews, Robert K.

AU - Gorman, Jeffrey J.

AU - Booth, William J.

AU - Corino, Gary L.

AU - Castaldi, Peter A.

AU - Berndt, Michael C.

PY - 1989/1/1

Y1 - 1989/1/1

N2 - 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 α-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.

AB - 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 α-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.

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U2 - 10.1021/bi00447a010

DO - 10.1021/bi00447a010

M3 - Article

VL - 28

SP - 8326

EP - 8336

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 21

ER -