Crosstalk between membrane traffic proteins and integrin activation 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, ?IIb?3 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 ?IIb?3 dependent endocytosis and ?IIb?3 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 ?IIb?3activation 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 ?IIb?3 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 ?IIb?3 as detected by confocal microscopy and forms a complex with ?IIb?3 as detected by co-immunoprecipitation.
In Specific Aim 2 we will use multiple approaches to characterize the binding determinants in VPS33B and the? cytoplasmic domains for each other.
In Specific Aim 3 we will identify the molecular mechanisms of VPS33B-dependent ?IIb?3 outside-in signaling. Completion of these studies will not only identify a novel ?IIb?3 binding partner that plays an important role in ?IIb?3 outside-in signaling, but also for the first time establish a lin between membrane traffic proteins and integrin activation in platelets.

Public Health Relevance

Blood platelets are essential for acute thrombotic events, such as heart attacks and strokes, and also contribute to local and systemic inflammation. This research project seeks to understand how the membrane traffic proteins such as VPS33B regulate platelet integrin activation and thrombosis. Completion of the work will provide new insights into pathways that could be targeted to prevent and treat arterial thrombosis, a leading cause of death in the United States.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL123927-01A1
Application #
8837170
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
2014-09-25
Project End
2018-06-30
Budget Start
2014-09-25
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$375,000
Indirect Cost
$125,000
Name
University of Kentucky
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506