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
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
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DOI：
10.1074/jbc.m110.103358
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发表时间：
2010-07-23
影响因子：
4.8
通讯作者：
Cruz, Miguel A.
中科院分区：
文献类型：
--
作者：
Auton, Matthew;Sowa, Katie E.;Cruz, Miguel A.
本研究使用重组A1 A2 A3三结构域蛋白来证明血管性血友病因子（VWF）中的A结构域缔合调节与血小板糖蛋白Ib α（GPIb α）的结合。我们进行了野生型（WT）A1结构域和R1450 E的变体，解离的三域复合物不稳定的A1结构域之间的比较研究。使用尿素变性和差示扫描量热法，我们证明了A1结构域结构的不稳定伴随着三个A结构域之间的相互作用减少的结果。这种解离导致R1450 E自发结合GPIb α，而没有利托那韦，表观KD为85 +/- 34 nM，与WT（36 +/- 12 nM）与利托那韦相当。突变体阻断100%瑞斯托霉素诱导的血小板凝集，而WT未能抑制。突变体增强剪切诱导的血小板聚集在500和5000 s（-1）的剪切速率，分别达到42%和66%。在WT存在下，剪切诱导的血小板聚集不超过18%。A1 A2 A3变体在灌注前加入纤维蛋白（原）包被的表面。在1500 s（-1）时，含WT的血液中的血小板粘附
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