Crosstalk between membrane traffic proteins and integrin activation

University Of Kentucky, Lexington KY

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Abstract

DESCRIPTION (provided by applicant): Platelets are central to hemostasis because they respond to vascular damage and secrete a variety of granule cargo molecules, which are critical to thrombosis and its sequellae. Platelet secretion is a multistep process involving centralization of cytoplasmic organelles that provides a contractile force, membrane fusion controlled by membrane traffic proteins, and release of granule contents. The platelet integrin, ?llbß3 interacts with adhesive ligands such as fibrinogen and fibrin and mediates platelet adhesion and aggregation and thus plays a critical role in the development of thrombotic diseases such as heart attack and stroke. The overall goal of this proposal is to establish a link between the membrane traffic proteins and integrins that involve the two key events to platelet function, platelet cargo release and integrin activation. VPS33B is a member of the Sec1/Munc18 protein family that has multiple roles in exocytosis. VPS33B is defective in patients with arthrogryposis, renal dysfunction, cholestasis (ARC) syndrome. Platelets from these patients lack ?-granules. In an attempt to define the roles of ?-granules in platelet functio and thrombosis, we produced a mouse model of ?-granule deficiency, in which VPS33B was deleted only in megakaryocytes and platelets. In Specific Aim 1, we will determine the roles of VPS33B in ?llbß3-dependnet endocytosis and ?llbß3 outside-in signaling. ?-granule contents vWF and fibrinogen are significantly reduced in the platelets lacking VPS33B. Surprisingly, VPS33B-/- platelets fail to retract a clot and are defective in spreading on fibrinogen. Using the CHO recombinant ?llbß3 activation model, we showed that overexpression of VPS33B markedly potentiates cell spreading on fibrinogen and actin polymerization. Thus, we hypothesize that in platelets, there exists a crosstalk between the membrane traffic proteins and integrin activation and that VPS33B is a key contributor to ?llbß3 outside-in signaling. This hypothesis will be tested using the platelet- specific VPS33B conditional knockout mice and other VPS33B knockout mice such as whole-body knockout mice and a Chinese Hamster Ovary (CHO) integrin activation model. Our preliminary data demonstrated that VPS33B co-localizes with ?llbß3 as detected by confocal microscopy and forms a complex with ?llbß3 as detected by co-immunoprecipitation. In Specific Aim 2 we will use multiple approaches to characterize the binding determinants in VPS33B and the ß3 cytoplasmic domains for each other. In Specific Aim 3 we will identify the molecular mechanisms of VPS33B-dependent ?llbß3 outside-in signaling. Completion of these studies will not only identify a novel ?llbß3 binding partner that plays an important role in ?llbß3 outside-in signaling, but also for the first time establish a lin between membrane traffic proteins and integrin activation in platelets.