Destabilization of the A1 domain in von Willebrand factor dissociates the A1A2A3 tri-domain and provokes spontaneous binding to glycoprotein Ibα and platelet activation under shear stress

Matthew Auton, Katie E. Sowa, Scott M. Smith, Erik Sedlák, K. Vinod Vijayan, Miguel A. Cruz

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ibα (GPIbα). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIbα without ristocetin with an apparent KD of 85 ± 34 nM, comparable with that of WT (36 ± 12 nM) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s-1 shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s -1, platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIbα and αIIbβ3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of Adomain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.

Original languageEnglish (US)
Pages (from-to)22831-22839
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number30
DOIs
StatePublished - Jul 23 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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