Impaired integrin-dependent function of WASP-deficient platelets

描述（由申请人提供）：Wiskott-Aldrich 综合征 (WAS) 患者由于内在缺陷血小板的加速损失而患有严重的血小板减少症。血小板损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 库（占总数的 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。在用凝血酶加搅拌激活血小板时，这些 WASP 分子的行为类似于真正的膜骨架蛋白，通过磷酸化进行激活，改变细胞骨架部分的分配并通过蛋白水解去激活，这些变化在整合素 aIIb-3 缺陷血小板和用整合素拮抗剂处理的正常血小板中被消除。研究结果确定了一组 WASP 与易于动员的 aIIb-3 整合素分子紧密并列，这些分子参与需要整合素由外而内信号传导的血小板细胞结构重排。拟议的 R21 项目将检验血小板 WASP 的核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，因此该项目的第二个目标是证明黄蜂/小鼠血小板能够复制功能性疾病缺陷。我们将通过使用荧光显微镜和光度板测定（目标 1a）来测试 WAS 患者和黄蜂缺陷小鼠的血小板在固定纤维蛋白原上的粘附和扩散是否受损（相对于各自的对照血小板），这是一种经典的整合素由外向内依赖的功能。我们还将研究在静脉剪切速率和小动脉剪切速率下平行板灌注系统中流动条件下的血小板粘附（与纤维蛋白原和胶原蛋白）（目标 1b）。为了推进血小板 WASP 一般调节整合素依赖性功能的假设，将测试患者和 wasp-/- 小鼠的血小板是否存在已知需要整合素在信号传导之外的其他功能的异常。这些是促凝血表面的生成，通过 FITC-乳粘素的结合检测暴露的磷脂酰丝氨酸（目标 2a），在小鼠断尾模型中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）。我们预计该试点项目的结果将（i）确定血小板 WASP 的核心功能是调节整合素由外而内依赖的生理反应，（ii）建立血小板疾病缺陷的小鼠模型，以及（iii）产生基准研究结果以支持主要依靠小鼠模型来定义正常血小板中的 WASP 信号通路和导致患者血小板减少症的分子机制的假设驱动的 RO1 项目。公共健康相关性：Wiskott-Aldrich 综合征 (WAS) 患者继承了一种名为 WASP 的缺陷基因，因此，他们的白细胞和血小板中缺少这种蛋白质（也称为 WASP）。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）并导致患者出血。该项目的目标是确定 Wiskott-Aldrich 患者血小板中 WASP 的正常功能以及血小板丢失的原因。

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.