Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ib alpha and Platelet Activation under Shear Stress

作者：Auton Matthew; Sowa Katie E; Smith Scott M; Sedlak Erik; Vijayan K Vinod; Cruz Miguel A^*

来源：Journal of Biological Chemistry, 2010, 285(30): 22829-22837.

DOI：10.1074/jbc.M110.103358

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 Ib alpha (GPIb alpha). 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 GPIb alpha 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 GPIb alpha and alpha IIb beta 3 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.

• 出版日期2010-7-23