Impaired integrin-dependent function of WASP-deficient platelets

描述（由申请人提供）：Wiskott-Aldrich综合征（WAS）患者因固有缺陷血小板加速丢失而发生严重血小板减少症。血小板丢失的确切机制尚不清楚。受影响的基因产物WASP是一种血细胞细胞骨架调节蛋白，被认为存在于静息细胞的细胞质中。我们最近发现，WASP池（总数的1/4）位于膜骨架中的血小板中，膜骨架是支撑和稳定脂质双层的支架结构。在用凝血酶活化血小板并搅拌时，这些WASP分子表现得像真正的膜骨架蛋白，经历磷酸化活化、改变对细胞骨架部分的分配和蛋白水解失活，这些变化在整联蛋白aIIb 3缺陷型血小板和用整联蛋白拮抗剂处理的正常血小板中被消除。这些发现确定了一个WASP池，该池与容易动员的aIIb 3整合素分子紧密并列，该整合素分子参与需要整合素由外向内信号传导的血小板细胞结构重排。拟议的R21项目将测试血小板WASP的中心功能是调节依赖于整合素由外向内信号传导的生理反应的假设。由于WAS患者的血小板作为研究模型具有固有的局限性，该项目的次要目标是证明黄蜂-/-小鼠血小板复制功能性疾病缺陷。我们将测试WAS患者和黄蜂缺陷小鼠的血小板是否受损（相对于各自的对照血小板）在固定的纤维蛋白原上的粘附和扩散，一个典型的整合素由外向内依赖的功能，通过使用荧光显微镜和光度板测定（目的1a）。我们还将在静脉剪切率和小动脉剪切率下，在平行板灌注系统中研究流动条件下血小板粘附（与纤维蛋白原和胶原蛋白）（目的1b）。为了推进血小板WASP通常调节整联蛋白依赖性功能的假设，将测试患者和黄蜂-/-小鼠的血小板的已知需要信号传导外的整联蛋白的其他功能的异常。这些是促凝血表面的生成，通过FITC-乳凝集素结合检测暴露的磷脂酰丝氨酸进行评估（目的2a），在小鼠尾部切割模型中再出血以评估凝块稳定性（目的2b）和纤维蛋白凝块收缩（目的2c）。我们预计，该试点项目的结果将（i）确定血小板WASP的中心功能为整联蛋白外向依赖性生理反应的调节，（ii）建立血小板疾病缺陷的小鼠模型，（iii）产生基准发现以支持假设-RO 1项目主要依赖于小鼠模型，以确定正常血小板中的WASP信号通路和导致患者血小板减少的分子机制。血小板减少症公共卫生相关性：患有Wiskott-Aldrich综合征（WAS）的患者遗传了一种称为WASP的缺陷基因，因此，这种蛋白质（也称为WASP）在他们的白色血细胞和血小板中缺失。血小板是血液凝固和伤口愈合所需的微小细胞。由于未知的缺陷，患者的血小板以异常快的速度从血液中去除，导致血小板计数低（血小板减少症）并导致患者遭受出血。本项目的目的是确定WASP在血小板中的正常功能以及Wiskott-Aldrich患者血小板丢失的原因。

DESCRIPTION (provided by applicant): Patients with Wiskott-Aldrich syndrome (WAS) suffer from profound thrombocytopenia due to accelerated loss of intrinsically defective platelets. The exact mechanism of platelet loss is unknown. The affected gene product, WASP, is a blood cell cytoskeletal regulatory protein thought to reside in the cytoplasm of resting cells. We recently found that a pool of WASP (1/4 of the total) is localized in platelets in the membrane skeleton, the scaffold structure that underlies and stabilizes the lipid bilayer. On activating platelets with thrombin plus stirring, these WASP molecules behaved like genuine membrane skeletal proteins, undergoing activation by phosphorylation, altered partitioning to the cytoskeletal fraction and de- activation by proteolysis, changes that were abrogated in integrin aIIb¿3 deficient platelets and in normal platelets treated with integrin antagonist. The findings identify a pool of WASP in close juxtaposition to readily mobilized aIIb¿3 integrin molecules that participates in platelet cytoarchitectural re-arrangements requiring integrin outside-in signaling. The proposed R21 project will test the hypothesis that the central function of platelet WASP is regulating physiological responses that are dependent on integrin outside-in signaling. Because platelets of WAS patients have inherent limitations as a study model, a secondary goal of the project is to demonstrate that wasp-/- mouse platelets replicate the functional disease defects. We will test whether platelets of WAS patients and wasp-deficient mice are impaired (relative to respective control platelets) in adhesion and spreading on immobilized fibrinogen, a classic integrin outside-in dependent function, by the use of fluorescence microscopy and a photometric plate assay (aim 1a). We will also study platelet adhesion (to fibrinogen and to collagen) under flow conditions in a parallel plate perfusion system under venous shear rate and arteriolar shear rate (aim 1b). To advance the hypothesis that platelet WASP regulates integrin-dependent functions generally, platelets of patients and wasp-/- mice will be tested for aberrancies of additional functions known to require integrin outside in signaling. These are generation of procoagulant surfaces, to be assessed as exposed phosphatidylserine detected by binding of FITC-lactadherin (aim 2a), rebleeding in a mouse tail cut model to assess clot stabilization (aim 2b) and fibrin clot retraction (aim 2c). We anticipate that the results of this pilot project will (i) identify the central function of platelet WASP as regulation of integrin outside-in dependent physiological responses and (ii) establish a mouse model of the platelet disease defect and (iii) generate benchmark findings to support a hypothesis-driven RO1 project relying primarily on the mouse model to define the WASP signaling pathway in normal platelets and the molecular mechanism responsible for the patients' thrombocytopenia. PUBLIC HEALTH RELEVANCE: Patients with the Wiskott-Aldrich syndrome (WAS) inherit a defective gene called WASP, and as a result, the protein (also called WASP) is missing in their white blood cells and platelets. Platelets are the tiny cells required for blood clotting and wound healing. Due to an unknown defect, platelets of the patients are removed from the blood at an abnormally rapid rate, causing low platelet count (thrombocytopenia) and causing the patients to suffer from bleeding. The goal of this project is to identify the normal function of WASP in platelets and the cause of platelet loss in Wiskott-Aldrich patients.