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

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 Ibalpha (GPIbalpha). 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 GPIbalpha without ristocetin with an apparent K(D) 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 GPIbalpha and alphaIIbbeta3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.

SUBMITTER: Auton M

PROVIDER: S-EPMC2906274 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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

Auton Matthew M Sowa Katie E KE Smith Scott M SM Sedlák Erik E Vijayan K Vinod KV Cruz Miguel A MA

The Journal of biological chemistry 20100524 30

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 Ibalpha (GPIbalpha). 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 resu ...[more]

PMID: 20498367

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Project description:Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIb?. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIb? with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix ?1, loops ?1?2 and ?3?2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIb?-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIb?. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIb?, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

Dataset Information

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

Publications

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

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