Crosstalk between membrane traffic proteins and integrin activation

描述（由申请人提供）：血小板是止血的核心，因为它们对血管损伤作出反应并分泌各种颗粒货物分子，这些颗粒货物分子对血栓形成及其后遗症至关重要。血小板分泌是一个多步骤的过程，包括提供收缩力的细胞质细胞器的集中、由膜运输蛋白控制的膜融合和颗粒内容物的释放。血小板整合素αIIbβ3与纤维蛋白原、纤维蛋白等黏附配体相互作用，介导血小板黏附和聚集，在心脏病、中风等血栓性疾病的发生发展中起关键作用。本提案的总体目标是建立膜运输蛋白和整合素之间的联系，这涉及血小板功能的两个关键事件，血小板货物释放和整合素激活。VPS33B是Sec1/Munc18蛋白家族的成员，在胞吐过程中发挥多种作用。VPS33B在关节挛缩、肾功能不全、胆汁淤积（ARC）综合征患者中存在缺陷。这些患者的血小板缺乏α颗粒。为了确定α颗粒在血小板功能和血栓形成中的作用，我们建立了α颗粒缺乏的小鼠模型，其中VPS33B仅在巨核细胞和血小板中缺失。在Specific Aim 1中，我们将确定VPS33B在αIIbβ3依赖性内吞作用和αIIbβ3外向内信号传导中的作用。在缺乏VPS33B的血小板中，α颗粒含量vWF和纤维蛋白原显著降低。令人惊讶的是，VPS33B-/-血小板不能收缩凝块，并且在纤维蛋白原上的扩散有缺陷。通过CHO重组α ib β3激活模型，我们发现过表达VPS33B显著增强了细胞在纤维蛋白原和肌动蛋白聚合上的扩散。因此，我们假设在血小板中，膜运输蛋白和整合素激活之间存在串扰，VPS33B是α ib β3外向内信号传导的关键贡献者。这一假设将通过血小板特异性VPS33B条件敲除小鼠和其他VPS33B敲除小鼠（如全身敲除小鼠和中国仓鼠卵巢（CHO）整合素激活模型）进行验证。我们的初步数据表明，通过共聚焦显微镜检测，VPS33B与αIIbβ3共定位，通过共免疫沉淀检测，VPS33B与αIIbβ3形成复合物。在Specific Aim 2中，我们将使用多种方法来表征VPS33B和α细胞质结构域的结合决定因素。在Specific Aim 3中，我们将确定vps33b依赖性αIIbβ3外向内信号传导的分子机制。这些研究的完成不仅将鉴定出在αIIbβ3 outside-in信号通路中起重要作用的新型αIIbβ3结合伙伴，而且将首次在血小板中建立膜转运蛋白与整合素激活之间的联系。

DESCRIPTION (provided by applicant): Crosstalk between membrane traffic proteins and integrin activation DESCRIPTION (provided by applicant): Platelets are central to hemostasis because they respond to vascular damage and secrete a variety of granule cargo molecules, which are critical to thrombosis and its sequellae. Platelet secretion is a multistep process involving centralization of cytoplasmic organelles that provides a contractile force, membrane fusion controlled by membrane traffic proteins, and release of granule contents. The platelet integrin, αIIbβ3 interacts with adhesive ligands such as fibrinogen and fibrin and mediates platelet adhesion and aggregation and thus plays a critical role in the development of thrombotic diseases such as heart attack and stroke. The overall goal of this proposal is to establish a link between the membrane traffic proteins and integrins that involve the two key events to platelet function, platelet cargo release and integrin activation. VPS33B is a member of the Sec1/Munc18 protein family that has multiple roles in exocytosis. VPS33B is defective in patients with arthrogryposis, renal dysfunction, cholestasis (ARC) syndrome. Platelets from these patients lack α granules. In an attempt to define the roles of α granules in platelet functio and thrombosis, we produced a mouse model of α granule deficiency, in which VPS33B was deleted only in megakaryocytes and platelets. In Specific Aim 1, we will determine the roles of VPS33B in αIIbβ3 dependent endocytosis and αIIbβ3 outside-in signaling. α granule contents vWF and fibrinogen are significantly reduced in the platelets lacking VPS33B. Surprisingly, VPS33B-/- platelets fail to retract a clot and are defective in spreading on fibrinogen. Using the CHO recombinant αIIbβ3activation model, we showed that overexpression of VPS33B markedly potentiates cell spreading on fibrinogen and actin polymerization. Thus, we hypothesize that in platelets, there exists a crosstalk between the membrane traffic proteins and integrin activation and that VPS33B is a key contributor to αIIbβ3 outside-in signaling. This hypothesis will be tested using the platelet- specific VPS33B conditional knockout mice and other VPS33B knockout mice such as whole-body knockout mice and a Chinese Hamster Ovary (CHO) integrin activation model. Our preliminary data demonstrated that VPS33B co-localizes with αIIbβ3 as detected by confocal microscopy and forms a complex with αIIbβ3 as detected by co-immunoprecipitation. In Specific Aim 2 we will use multiple approaches to characterize the binding determinants in VPS33B and theα cytoplasmic domains for each other. In Specific Aim 3 we will identify the molecular mechanisms of VPS33B-dependent αIIbβ3 outside-in signaling. Completion of these studies will not only identify a novel αIIbβ3 binding partner that plays an important role in αIIbβ3 outside-in signaling, but also for the first time establish a lin between membrane traffic proteins and integrin activation in platelets.