Signaling mechanisms of platelet GPIb-IX

描述（由申请人提供）：血管性血液病因子（VWF）的血小板受体，糖蛋白（GP） Ib-IX-V复合物（GPIb-IX），对于血小板粘附损伤血管壁的初始过程至关重要，特别是在剪切应力下。VWF结合GPIb-IX还启动信号传导，导致血小板整合素aIIbb3的激活，介导血小板稳定粘附、血小板聚集和血栓形成。在目前的资助期内，我们在了解调节GPIb受体功能的信号机制和vwf诱导的GPIb信号导致血小板活化方面取得了重大进展。特别是，我们已经证明了Rac1在介导VWF/ gpib - ix介导的早期信号传导中的重要作用。我们还证明了潜在的下游Rac1效应物LIM激酶1 （LIMK1）在选择性刺激GPIb- ix依赖性血栓素（TX） A2合成和信号放大中的意想不到的作用。GPIb-IX也与凝血酶相互作用，在低剂量凝血酶诱导的血小板活化中发挥重要作用。然而，目前尚不清楚GPIb- ix是促进凝血酶更有效地切割蛋白酶活化受体（PAR），还是GPIb信号传导促进凝血酶诱导的血小板活化。根据我们的初步数据，我们假设Rac1-和limk1介导的GPIb-IX信号通路促进par依赖性血小板对低剂量凝血酶的反应。在这项竞争性更新应用的目的1中，我们将研究GPIb-IX信号通路在促进凝血素诱导的血小板活化中的作用，以及介导VWF和凝血素诱导的GPIb-IX信号通路的Rac1和LIMK1信号通路。活化血小板在促进凝血中发挥重要作用，主要是通过提供外化磷脂酰丝氨酸（PS）。然而，在目前公认的检测条件下，即使是极高浓度的生理性血小板激动剂胶原蛋白和凝血酶也只能诱导一小部分血小板外化PS，这一悖论的原因尚不清楚。我们发现，在与正常血流相关的剪切力下，血小板不表达促凝活性（尽管极高的剪切应力可能诱导血小板释放促凝剂MP，而这需要GPIb-IX- VWF相互作用）。然而，激动剂需要生理水平的剪切应力才能有效地诱导PS外化和MP从血小板中释放。重要的是，我们发现Rac1在剪切依赖性血小板PS外化和不依赖gpib的血小板激动剂诱导的MP释放中起关键作用。因此，我们假设血小板促凝功能的激活需要剪切力诱导的信号，其中Rac1起着重要作用。因此，在Aim 2中，我们将研究剪切力、Rac1和GPIb-IX信号在血小板促凝功能激活中的作用。这些研究有助于我们理解低浓度的激动剂凝血酶或初始血小板粘附如何引起强烈的血小板反应，不仅导致血小板血栓形成，还导致血管损伤部位的凝血，并有助于开发治疗血栓性疾病的新抑制剂。

DESCRIPTION (provided by applicant): The platelet receptor for von Willebrand factor (VWF), the glycoprotein (GP) Ib-IX-V complex (GPIb-IX), is vital for initial platelet adhesion to injured blood vessel wall, especially under shear stress. VWF binding to GPIb-IX also initiates signaling resulting in the activation of the platelet integrin aIIbb3, which mediates stable platelet adhesio, platelet aggregation, and thrombus formation. In the current funding periods, we have made significant progress in understanding the signaling mechanisms that regulates the receptor function of GPIb and VWF-induced GPIb signaling leading to platelet activation. In particular, we have demonstrated an important role for Rac1 in mediating VWF/GPIb-IX-mediated early signaling. We also demonstrated an unexpected role of a potential downstream Rac1 effector, LIM kinase 1 (LIMK1) in selectively stimulating GPIb- IX-dependent thromboxane (TX) A2 synthesis and signal amplification. GPIb-IX also interacts with thrombin, and is important in low dose thrombin-induced platelet activation. However, it is unclear whether GPIb-IX serves facilitate more efficient thrombin cleavage of protease-activated receptors (PAR), or GPIb signaling promote thrombin-induced platelet activation. Based on our preliminary data, we hypothesize that the Rac1- and LIMK1-mediated GPIb-IX signaling pathways promotes PAR-dependent platelet response to low dose thrombin. In Aim 1 of this competitive renewal application, we will investigate the role of GPIb-IX signaling in promoting thrombin-induced platelet activation and the Rac1 and LIMK1 signaling pathways that mediates VWF- and thrombin-induced GPIb-IX signaling. Activated platelets play an important role in facilitating coagulation, mainly by providing externalized phosphotidylserine (PS). However, under currently accepted assay conditions, even extremely high concentrations of physiological platelet agonists collagen and thrombin only induce a small fraction of platelets to externalize PS, the reason for this paradox remains unclear. We show that under the shear force relevant to normal blood flow, platelets do not express procoagulant activity (although the extremely high shear stress may induce platelet procoagulant MP release that requires GPIb-IX- VWF interaction). However, physiological levels of shear stress are required for agonists to efficiently induce PS externalization and MP release from platelets. Importantly, we discovered that Rac1 plays a critical role in shear-dependent platelet PS externalization and MP release induced by GPIb-independent platelet agonists. Thus, we hypothesize that activation of platelet procoagulant function requires shear force-induced signaling in which Rac1 plays an important role. Therefore, in Aim 2, we will investigate the role of shear force, Rac1 and GPIb-IX signaling in the activation of platelet procoagulant function. These study should facilitate our understanding how low concentrations of agonists thrombin or initial platelet adhesion will elicit strong platele response leading to not only platelet thrombus formation but also clotting at the site of vascular injury, and help develop new inhibitor for treating thrombotic diseases.