Impaired integrin-dependent function of WASP-deficient platelets

WASP 缺陷血小板的整合素依赖性功能受损

• 批准号: 8605265
• 负责人: EILEEN REMOLD-O'DONNELL
• 金额: $ 6.43万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8605265
• 关键词: Impaired; integrin; dependent; function; WASP

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.

描述（由适用提供）：Wiskott-Aldrich综合征患者（被）患有严重的血小板减少症，原因是由于本质上有缺陷的血小板的加速丧失。血小板损失的确切机制尚不清楚。受影响的基因产物黄蜂是一种被认为驻留在静息细胞细胞质中的血细胞骨骼调节蛋白。我们最近发现，黄蜂（总数的1/4）池位于膜骨架中的血小板中，膜骨架是脚手架结构，其底座是构成并稳定脂质双层的脚手架结构。在用凝血酶加搅拌的激活血小板时，这些黄蜂分子的表现像真正的膜骨骼蛋白，通过磷酸化而受到激活，改变了对细胞骨骼分数的分配，并通过蛋白水平的蛋白质水溶性来激活，蛋白质水溶性，蛋白质水溶性，在整合素AIIB中消除了综合素AIIIB和正常的血小板中消除的变化。这些发现确定了一个与WASP的密切并置的池，以容易动员AIIB课程3整合蛋白分子，该分子参与了血小板细胞划界的重新分配，需要整合素外部信号传导。提出的R21项目将检验以下假设：血小板黄蜂的中心功能正在调节依赖整联蛋白外部信号传导的物理反应。由于患者的血小板作为研究模型具有继承的局限性，因此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。我们将测试是否通过使用荧光显微镜和光度板测定法（AIM 1A）来测试粘合剂和固定纤维蛋白原（一种经典的外部依赖性功能）上固定纤维蛋白原（一种经典的内整联蛋白）（一种经典的整合素外部功能）（AIM 1A）的固定纤维蛋白原（AIM 1A），是否会受损（相对于相对对照组）的患者和黄蜂缺陷小鼠（相对于相对对照组）。我们还将在静脉剪切速率和艺术层剪切速率（AIM 1B）下在平行板灌注系统的流动条件下研究血小板粘合剂（至纤维蛋白原和胶原蛋白）。为了推动血小板软糖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，以测试已知在信号传导外需要整合素的其他功能的异常。这些是通过FITC-乳肽的结合（AIM 2A）检测到的proc凝表面的产生，可以评估为暴露的磷脂酰丝氨酸，在小鼠尾部切割模型中均可评估凝块稳定（AIM 2B）和纤维蛋白凝块撤回（AIM 2C）。我们预计该试验项目的结果将（i）确定血小板WASP的主要功能作为整联蛋白外部依赖性物理反应的调节，并且（ii）建立一个小鼠模型的血小板疾病缺陷的模型，（iii）生成基准测试结果，以支持假设驱动的RO1项目，主要依赖于鼠标的模型，以确定WASP信号的机制，以定义WASP信号的机制。血小板减少症。公共卫生相关性：Wiskott-Aldrich综合征（WAS）的患者继承了一个称为WASP的缺陷基因，因此，其白细胞和血小板中缺少蛋白质（也称为WASP）。血小板是血液凝结和伤口愈合所需的微小细胞。由于缺陷，以绝对快速的速率从血液中清除血小板，导致血小板计数低（血小板减少症），并导致患者出血。该项目的目的是确定Wiskott-Aldrich患者的血小板中黄蜂的正常功能和血小板损失的原因。