The roles of protein kinase G in platelet activation

描述(申请人提供)：血小板黏附和聚集在心脏病发作和中风等血栓性疾病的发展中起关键作用。在正常循环中，血小板处于“静息”状态。在血管损伤或动脉粥样硬化斑块的部位，血小板暴露于可溶的血小板激动剂或内皮下黏附蛋白可触发血小板激活。血小板活化的一个常见后果和特征是血小板整合素AIIBβ3的激活，它介导了血小板的黏附、扩散和聚集。在过去的20年里，人们已经接受了cGMP依赖的蛋白激酶(蛋白激酶G，PKG)抑制血小板激活的观点。然而，最近有报道称，增强cGMP的药物西地那非与一些患者的心脏病发作和血栓形成有关。在我们的研究中，我们发现重组人PKG在重组的整合素激活模型中的表达促进了GPIB-IX介导的整合素αIIbbeta3的激活。此外，vWF或小剂量凝血酶诱导的整合素依赖的血小板聚集可被各种PKG抑制剂抑制，并被PKG激活剂增强。此外，我们还发现西地那非促进了vWF或凝血酶诱导的血小板聚集。因此，cGMP-PKG可能在血小板活化中起到刺激作用。有趣的是，我们发现cGMP在激动剂模拟后立即升高时是刺激性的，但在与血小板长时间预孵育后却是抑制的。因此，我们假设，当cGMP在止血过程中被血小板激动剂升高时，会诱导双相血小板反应：最初阶段的刺激反应导致血小板激活和血栓形成，第二阶段的抑制反应使血小板减敏并防止血栓过度生长。为了验证这一假说，我们建议(1)研究cGMP-PKG通路在血小板黏附和激活中的刺激作用；(2)研究cGMP对血小板第二时相抑制反应的机制；(3)确定鸟苷环化酶在血小板激活过程中的作用和机制；(4)研究PKG的结构-功能关系和地形调控机制；(5)启动PKG介导的整合素激活下游通路的初步研究。了解cGMP-PKG通路在血小板中的双相作用，将有助于对血小板活化的分子机制以及常用的cGMP增强剂如西地那非引起血栓形成的机制提供新的认识。

DESCRIPTION (provided by applicant): The platelet adhesion and aggregation play critical roles in the development of thrombotic diseases such as heart attack and stroke. In normal circulation, platelets are in a "resting" state. At sites of vascular injury or atherosclerotic plagues, exposure of platelets to soluble platelet agonists or sub endothelial adhesive proteins triggers platelet activation. A common consequence and characteristic of platelet activation is the activation of the platelet integrin aIIb beta3, which mediates platelet adhesion, spreading and aggregation. Over the last 20 years, it has been accepted that platelet activation is inhibited by the cGMP-dependent protein kinase (protein kinase G, PKG). However, there have been recently reports that a cGMP-enhancing drug, sildenafil, was associated with heart attack and thrombosis in some patients. In our study, we have found that expression of recombinant human PKG in a reconstituted integrin activation model promotes GPIb-IX-mediated integrin alphaIIbbeta3 activation. In addition, integrin dependent platelet aggregation induced by vWF or low dose thrombin was inhibited by various PKG inhibitors and enhanced by PKG activators. Furthermore, we found that sildenafil promoted vWF- or thrombin-induced platelet aggregation. Thus, cGMP-PKG may play a stimulatory role in platelet activation. Interestingly, we found that cGMP is stimulatory when elevated immediately following agonist simulation, but is inhibitory after a prolonged preincubation with platelets. Thus, we hypothesize that, when elevated by the platelet agonists during hemostasis, cGMP induces biphasic platelet responses: an initial phase stimulatory response leading to platelet activation and thrombus formation, and a secondary phase of inhibitory responses that desensitizes platelets and prevent overgrowth of thrombus. To test this hypothesis, we propose (1) to investigate the stimulatory roles of cGMP-PKG pathway in platelet adhesion and activation; (2) to investigate the mechanisms of the second phase platelet inhibitory response to cGMP; (3) to identify the roles and mechanisms of guanyl cyclase regulation during platelet activation; (4) to investigate the structure-function relationship and topographic regulatory mechanisms of PKG; and (5) to initiate preliminary studies on the downstream pathways of PKG-mediated integrin activation. Understanding the biphasic roles of cGMP-PKG pathway in platelets should provide new insight into molecular mechanisms of platelet activation and the mechanisms of thrombosis associated with popularly used cGMP enhancing drugs such as sildenafil.