Molecular mechanisms of shear induced platelet activation

本项目的重点是研究血小板信号对剪切应力的响应。初步数据表明，完整的血小板受到病理性剪切应力的反应，在两个独特的和功能上重要的方式。这些反应是1）肌动蛋白结合蛋白（ABP）-280的vWF非依赖性磷酸化和2）14-3- 3 zeta衔接蛋白从GpI α的胞质结构域的vWF依赖性释放。所提出的实验将检查vWF介导的血小板粘附和聚集是否由ABP-280磷酸化触发，ABP-280磷酸化作为剪切细胞表面的直接效应发展，并将建立ABP-280磷酸化作为剪切细胞表面的直接效应发展的机制，并将建立ABO-280磷酸化作为剪切细胞表面的直接效应发展的机制，并将建立ABP-280磷酸化调节由内向外和由外向内信号传导的机制。这项建议的具体目标如下。1)检验剪切应力诱导的肌动蛋白结合蛋白280磷酸化调节其与血小板GpIb-IX-V复合物的关系的假设。我们将确定ABP-280磷酸化是否影响GpIbalpha结合的亲和力和/或化学计量，并鉴定指导ABP-280磷酸化的激酶。2)检验ABP-280与GpI α胞质结构域结合调节由内而外信号传导（vWF结合）和由外向内信号传导（GpIIb-IIIa激活）的假设。我们将确定这些反应如何受到GpIbalpha胞质尾内结构域的影响，如何受到GpIbalpha与14-3- 3 zeta衔接蛋白的结合的影响，以及如何受到磷酸肌醇3-激酶，3）检验剪切应力诱导的vWF结合导致Gp-IX-V-IV的寡聚化的假设。V导致调节GpIIb-IIIa的Syk活化。我们将确定剪切诱导的vWF与GpIb-IX-V的结合是否会导致基于GpI α的信号复合物组织，从而导致Syk激活，导致PI-3激酶激活，影响GpIIb- IIIa功能。这些研究的结果应使我们能够确定剪切诱导的血小板粘附和聚集的触发机制，并开始建立导致病理性动脉血栓形成的血小板生化反应的顺序。

This project focuses on investigations of platelet signaling in response to shear stress. Preliminary data indicate that intact platelets subjected to pathological shear stress respond in two unique and functionally significant ways. These responses are 1) the vWF-independent phosphorylation of actin binding protein (ABP)- 280 and 2) the vWF- dependent release of a 14-3-3zeta adaptor protein from the cytoplasmic domain of GpIbalpha. The experiments proposed will examine if vWF- mediated platelet adhesion and aggregation are triggered by ABP-280 phosphorylation that develops as a direct effect of shearing the cell surface, and will establish the mechanism by which ABP-280 phosphorylation that develops as a direct effect of shearing the cell surface, and will establish the mechanism by which ABO-280 phosphorylation that develops as a direct effect of shearing the cell surface, and will establish the mechanism by which ABP-280 phosphorylation modulates both inside-out and outside-in signaling. The specific aims of this proposal are as follows. 1) Test the hypothesis that shear stress-induced phosphorylation of actin binding protein 280 modulates its association with the platelet GpIb-IX-V complex. We will determine if ABP-280 phosphorylation affects the affinity and/or stoichiometry of GpIbalpha binding, and identify the kinases that are directing ABP-280 phosphorylation. 2) Test the hypothesis that ABP-280 binding to the cytoplasmic domain of GpIbalpha modulates both inside- out signaling (vWF binding) and outside-in signaling (GpIIb-IIIa activation). We will determine how these responses are influenced by domains within the cytoplasmic tail of GpIbalpha, by the association of GpIbalpha with a 14-3-3zeta adaptor protein, and by dynamic molecular associations between phosphoinositide 3-kinase, 14-3-3zeta and GpIb-IX- V. 3) Test the hypothesis that shear stress-induced vWF binding causes the oligomerization of Gp-IX-V resulting in Syk activation that modulates GpIIb-IIIa. We will determine if shear-induced vWF binding to GpIb-IX-V causes a GpIbalpha-based signal complex to organize that causes Syk activation leading to PI-3-kinase activation affecting GpIIb- IIIa function. Results of these studies should allow us to identify trigger mechanisms for shear-induced platelet adhesion and aggregation, and begin to establish the sequence of platelet biochemical responses leading to pathological arterial thrombosis.