The roles of protein kinase G in platelet activation

蛋白激酶 G 在血小板活化中的作用

• 批准号: 6760893
• 负责人: Xiaoping Du
• 金额: $ 31.17万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760893
• 关键词: CHO cells; G protein coupled receptor kinase; cGMP dependent protein kinase; clinical research; cyclic AMP; cyclic GMP; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate; gene targeting; genetically modified animals; guanylate cyclase; human tissue; integrins; laboratory mouse; mitogen activated protein kinase; nitric oxide; nitric oxide synthase; phosphorylation; platelet activation; platelet aggregation; protein kinase A; protein localization; protein protein interaction; thrombosis; thromboxanes

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.

描述（由申请人提供）：血小板粘附和聚集在血栓性疾病（如心脏病发作和中风）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，血小板暴露于可溶性血小板激动剂或内皮下粘附蛋白会触发血小板活化。血小板活化的一个共同结果和特征是活化血小板整合素aIIb beta3，它介导血小板粘附、扩散和聚集。在过去的20年里，人们已经接受了血小板活化被cgmp依赖性蛋白激酶（蛋白激酶G， PKG）抑制。然而，最近有报道称，一种cgmp增强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在重组整合素激活模型中的表达促进了gpib - ix介导的整合素alphaIIbbeta3的激活。此外，vWF或低剂量凝血酶诱导的整合素依赖性血小板聚集可被多种PKG抑制剂抑制，并可被PKG激活剂增强。此外，我们发现西地那非促进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具有刺激性，但在与血小板长期预孵育后具有抑制性。因此，我们假设，在止血过程中，当血小板激动剂使cGMP升高时，cGMP诱导两期血小板反应：初始阶段的刺激反应导致血小板活化和血栓形成，第二阶段的抑制反应导致血小板脱敏并防止血栓过度生长。为了验证这一假设，我们提出(1)研究cGMP-PKG通路在血小板粘附和活化中的刺激作用；(2)探讨cGMP第二期血小板抑制反应的机制；(3)明确鸟苷环化酶在血小板活化中的作用和机制；(4)研究PKG的结构-功能关系和地形调节机制；(5)对pkg介导的整合素激活的下游途径进行初步研究。了解cGMP- pkg通路在血小板中的双相作用，将为血小板活化的分子机制以及与常用的cGMP增强药物（如西地那非）相关的血栓形成机制提供新的见解。