The cGMP-dependent protein kinase pathway in platelets

描述（由申请人提供）：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包括两个方面：第一，血小板粘附、聚集和颗粒内容物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是引发动脉血栓形成的关键。在血小板活化过程中，细胞内次级信使分子cGMP被合成。自cGMP被发现以来，其在血小板活化中的作用一直存在争议。在过去的30年里，cGMP通过激活cGMP依赖性蛋白激酶（PKG）来抑制血小板活化一直是一个流行的概念。然而，我们最近发现cGMP和PKG在血小板激活中起双相作用，这一概念受到了挑战。我们发现，当血小板激动剂刺激产生低浓度cGMP时，cGMP起刺激作用，但在高浓度cGMP时，cGMP起抑制作用。在上一个资助期内，我们在了解cGMP途径在血小板激活中的双相作用机制方面取得了重大进展。我们发现cGMP-PKG通路在大多数血小板激动剂诱导的血小板分泌和分泌依赖性血小板聚集中起一般作用。我们已经获得证据支持血小板活化过程中cGMP的一个新的上游信号通路，涉及磷酸肌肽3-激酶、各种形式的Akt和一氧化氮（NO）合成酶。我们还获得了支持新的cGMP下游信号传导机制的证据，该机制涉及PKG的各种亚型以及PKG依赖的p38和ERK有丝分裂原激活的蛋白激酶途径的顺序激活。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。在这一途径中，血小板激动剂通过激活PI3K、Akt、NOS3、可溶性关酰环化酶和PKG的不同亚型，并通过p38和ERK途径诱导血小板分泌，从而放大和稳定血小板聚集。为了验证这一假设，我们提出以下具体目标：探讨不同Akt亚型在血小板活化过程中作为cGMP通路上游激活因子的作用。2. 探讨激动剂诱导血小板NO生成的机制、调控及其在血小板分泌和聚集中的作用。3. 探讨PKG不同亚型在血小板分泌和聚集中的作用及信号机制。

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.