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.

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.