The cGMP-dependent protein kinase pathway in platelets

描述（由申请方提供）：血小板活化在血栓性疾病（如心脏病发作和中风）的发展中起关键作用。血小板在血栓形成中的作用主要涉及两个方面：第一，血小板粘附、聚集和颗粒物质的分泌在动脉粥样硬化的发展中起重要作用，导致慢性血管损伤。其次，血管损伤部位的血小板活化在引发动脉血栓形成中至关重要。在血小板活化过程中合成细胞内第二信使分子cGMP。自cGMP发现以来，cGMP在血小板活化中的作用一直存在争议。cGMP通过激活cGMP依赖性蛋白激酶（PKG）抑制血小板活化是近30年来的流行观点。然而，这一概念受到了挑战，我们最近发现，cGMP和PKG发挥双相作用，在血小板活化，我们表明，cGMP起着刺激作用时，低浓度的cGMP产生血小板激动剂刺激，但成为抑制在高浓度的cGMP。在上一个资助期间，我们在了解cGMP途径在血小板活化中的双相作用机制方面取得了重大进展。我们已经发现，cGMP-PKG途径在刺激血小板分泌和大多数血小板激动剂诱导的分泌依赖性血小板聚集中起着普遍作用。我们已经获得的证据支持一个新的上游信号通路的cGMP在血小板活化过程中涉及磷酸肌醇3-激酶，各种形式的Akt和一氧化氮（NO）合酶。我们还获得了证据支持一种新的下游信号转导机制的cGMP涉及PKG和PKG依赖的p38和ERK丝裂原活化蛋白激酶途径的各种亚型的顺序激活。这些发现使我们能够假设一种新的刺激血小板分泌的信号通路。在该途径中，血小板激动剂通过激活PI 3 K、Akt、N 0 S3、可溶性鸟苷酸环化酶和PKG的不同亚型，并经由p38和ERK途径诱导血小板分泌，其放大并稳定血小板聚集。为了检验这一假设，我们提出了以下具体目标：1。研究不同Akt亚型在血小板活化过程中作为cGMP通路上游激活剂的作用。2.确定激动剂诱导的血小板NO产生的机制、其调节及其在血小板分泌和聚集中的作用。3.研究PKG不同亚型在血小板分泌和聚集中的作用及其信号转导机制。

DESCRIPTION (provided by applicant): Platelet activation plays critical roles in the development of thrombotic diseases such as heart attack and stroke. The roles of platelets in thrombosis involve two major aspects: Firstly, platelet adhesion, aggregation and secretion of granule contents are important in the development of atherosclerosis, which causes chronic vascular injury. Secondly, platelet activation at the site of vascular injury is critical in initiating arterial thrombosis. An intracellular secondary messenger molecule, cGMP, is synthesized during platelet activation. The role of cGMP in platelet activation has been controversial since the discovery of cGMP. It has been a prevailing concept in the past 30 years that cGMP, by activating the cGMP-dependent protein kinase (PKG), inhibits platelet activation. This concept, however, has been challenged by our recent finding that cGMP and PKG play a biphasic role in platelet activation, We show that cGMP plays a stimulatory role when low concentrations of cGMP is produced upon platelet agonist stimulation, but becomes inhibitory at high cGMP concentrations. During the last funding period, we have made significant progress in understanding the mechanisms of the biphasic roles of the cGMP pathway in platelet activation. We have found that the cGMP-PKG pathway plays a general role in stimulating platelet secretion and secretion-dependent platelet aggregation induced by most platelet agonists. We have obtained evidence supporting a new upstream signaling pathway of cGMP during platelet activation involving phosphoinositide 3-kinase, various forms of Akt and nitric oxide (NO) synthase. We have also obtained evidence supporting a novel downstream signaling mechanism of cGMP involving various isoforms of PKG and PKG-dependent sequentially activation of p38 and ERK mitogen-activated protein kinase pathways. These discoveries allow us to hypothesize a novel signaling pathway that stimulates platelet secretion. In this pathway, platelet agonists, by activating different isoforms of PI3K, Akt, NOS3, soluble guanylyl cyclase, and PKG, and via the p38 and ERK pathways, induce platelet secretion, which amplifies and stabilizes platelet aggregation. To test this hypothesis, we propose following specific aims: 1. To investigate the role of different Akt isoforms as upstream activators of the cGMP pathway during platelet activation. 2. To determine mechanisms of agonist-induced platelet NO production, its regulation, and its role in platelet secretion and aggregation. 3. To characterize the roles and the signaling mechanisms of different isoforms of PKG in platelet secretion and aggregation.