Novel strategies to prevent FcgRIIA-dependent platelet activation and thrombosis

描述(申请人提供)：血小板Fc受体Fc？RIIA的激活是几种免疫介导性血小板减少症和血栓形成(ITT)综合征发病机制的核心，包括肝素诱导的血小板减少症/血栓形成。这些血栓综合征成功治疗的障碍之一是，针对血小板激活途径的治疗靶向预防血栓形成要么无效，要么伴随着出血并发症的固有风险。这项建议的目的是a)确定优先促进Fc？RIIA介导的血小板激活的关键信号分子，以及b)开发治疗抗血小板的策略，以防止ITT的病理性血栓形成，同时最大限度地减少出血并发症。我们的初步研究发现，酪氨酸激酶Syk、丝氨酸/苏氨酸激酶Akt和钙传感器CalDAG-GEFI有望成为ITT抗血小板治疗的新靶点。在Fc？RIIA等ITAM偶联受体的下游，Syk对磷脂酶C？2的激活以及第二信使钙离子和二酰甘油的产生具有重要作用。CalDAG-GEFI将细胞内钙离子的增加与调节整合素激活、血栓素生成和颗粒释放的信号通路联系起来。对Akt的初步研究表明，Akt在Fc？RIIA诱导的钙离子通量和整合素激活中起重要作用，从而将其与Syk和CalDAG-GEFI联系起来。初步结果以及我们和其他人的工作进一步表明，Syk、CalDAG-GEFI和Akt对Fc？RIIA/ITAM介导的血小板激活特别重要。通过拟议的研究，我们将加深对CalDAG-GEFI(Aim 1)和Akt(Aim 2)促进Fc？RIIA介导的血小板激活的分子机制的理解。在目标3中，我们将验证Syk和Akt，并探索CalDAG-GEFI作为ITT的治疗靶点。Syk和Akt用于治疗和预防ITT的药物抑制的安全性和有效性将在我们建立的小鼠模型中进行测试。CalDAG-GEFI或Akt2作为治疗ITT的治疗靶点的有效性将在转人Fc？RIIA基因的小鼠和剔除CalDAG-GEFI或Akt2的小鼠之间的杂交中进行评估。这项提案的成功完成将加速免疫介导性血小板疾病新疗法的合理设计。

DESCRIPTION (provided by applicant): Activation of the platelet Fc receptor, Fc?RIIA, is central to the pathogenesis of several immune- mediated thrombocytopenia and thrombosis (ITT) syndromes, including heparin-induced thrombocytopenia/thrombosis. One of the barriers to successful treatment of these thrombotic syndromes is that therapeutic targeting of platelet activation pathways to prevent thrombosis is either not effective or comes with an inherent risk of bleeding complications. The purpose of this proposal is a) to identify critical signaling molecules, which preferentially contribute to Fc?RIIA- mediated platelet activation and b) to develop therapeutic anti-platelet strategies to prevent pathologic thrombosis in ITT while minimizing bleeding complications. Our preliminary studies identify the tyrosine kinase Syk, the Ser/Thr kinase Akt, and the Ca2+ sensor CalDAG-GEFI as promising new targets for antiplatelet therapy in the setting of ITT. Downstream of ITAM-coupled receptors like Fc?RIIA, Syk is important for the activation of phospholipase C?2 and thus the generation of the second messengers Ca2+ and diacylglycerol. CalDAG-GEFI links an increase in intracellular Ca2+ to the signaling pathways regulating integrin activation, thromboxane generation, and granule release. Preliminary studies on Akt demonstrate that it is important for Fc?RIIA-induced calcium flux and integrin activation, thus linking it to Syk and CalDAG-GEFI. Preliminary results as well as work by us and others further demonstrate that Syk, CalDAG- GEFI, and Akt are particularly important for Fc?RIIA/ITAM-mediated platelet activation. With the proposed studies, we will improve our understanding of the molecular mechanisms by which CalDAG-GEFI (aim 1) and Akt (aim 2) contribute to Fc?RIIA-mediated platelet activation. In aim 3, we will validate Syk and Akt and explore CalDAG-GEFI as therapeutic targets in ITT. The safety and efficacy of pharmacologic inhibition of Syk and Akt for the treatment and prevention of ITT will be tested in our established mouse models. The utility of CalDAG-GEFI or Akt2 as therapeutic targets for the treatment of ITT will be evaluated in crosses between mice transgenic for the human Fc?RIIA and mice knockout for CalDAG-GEFI or Akt2. The successful completion of this proposal will accelerate the rational design of novel therapeutics for immune-mediated platelet disorders.