Impaired integrin-dependent function of WASP-deficient platelets

描述(申请人提供)：Wiskott-Aldrich综合征患者由于先天缺陷的血小板加速丢失而患上严重的血小板减少症。血小板丢失的确切机制尚不清楚。受影响的基因产物WASP是一种血细胞骨架调节蛋白，被认为驻留在静止细胞的细胞质中。我们最近发现，一个WASP池(占总数的1/4)定位于膜骨架的血小板中，膜骨架是支撑和稳定脂质双层的支架结构。在用凝血酶和搅拌激活血小板时，这些WASP分子表现为真正的膜骨架蛋白，通过磷酸化激活，改变对细胞骨架部分的分配，通过蛋白分解使其失活，这种变化在整合素AIIB？3缺陷的血小板和整合素拮抗剂治疗的正常血小板中被消除。这些发现确定了WASP池与容易动员的AIIB？3整合素分子密切并列，这些分子参与了需要整合素由外向内信号的血小板细胞结构重排。建议的R21项目将检验一种假设，即血小板WASP的中心功能是调节依赖于整合素由外向内信号的生理反应。由于AS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/小鼠的血小板复制功能性疾病缺陷。我们将通过使用荧光显微镜和光度平板分析(AIM 1a)来测试风湿病患者和黄蜂缺陷小鼠的血小板在固定的纤维蛋白原(一种经典的整合素由外向内依赖的功能)上的黏附和扩散方面是否受损(相对于各自的对照血小板)。我们还将在平行平板灌流系统中研究静脉切变率和小动脉切变率下的血小板粘附性(与纤维蛋白原和胶原的粘附性)(目标1b)。为了推进这一假设，即血小板WASP一般调节整合素依赖的功能，将测试患者和黄蜂/小鼠的血小板是否存在已知需要外部整合素参与信号传递的额外功能的异常。这些是促凝剂表面的产生，通过FITC-乳粘附素结合检测暴露的磷脂酰丝氨酸(目标2a)，小鼠尾部切割模型中的再出血以评估凝块稳定性(目标2b)和纤维蛋白凝块回缩(目标2c)。我们预计，这一试点项目的结果将：(I)确定血小板WASP的中心功能是整合素由外向内依赖的生理反应的调节；(Ii)建立血小板疾病缺陷的小鼠模型；以及(Iii)产生基准结果，以支持主要依赖于小鼠模型的假说驱动的RO1项目，以确定正常血小板中的WASP信号通路以及导致患者血小板减少的分子机制。公共卫生相关性：Wiskott-Aldrich综合征患者遗传了一种名为WASP的缺陷基因，结果是，这种蛋白质(也称为WASP)在他们的白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。由于一种未知的缺陷，患者的血小板以异常快的速度从血液中取出，导致血小板计数低(血小板减少)，并导致患者出血。该项目的目标是确定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.