Mechanisms and functions of CRACR2A-L Rab GTPase in vascular endothelial cells

Thrombus formation (blood clotting) is an essential process to maintain vascular integrity and prevent excessive bleeding after vessel damage. However abnormal thrombi formation leads to blood vessel occlusions and development of atherosclerotic plaques with subsequent hypertension, stroke or ischaemia. Von-Willebrand Factor (vWF)-mediated platelet adhesion is a major regulator of thrombus formation, therefore vWF secretion from endothelial cells is highly regulated. Increased plasma levels of vWF have been proposed as a risk factor for cardiovascular disease, especially in high risk groups such as diabetics. vWF is contained in special storage vesicles called Weibel Palade Bodies (WPBs) which rapidly release their constituents upon stimulation. Agonist induced vWF secretion is a Ca2+-dependent process and is regulated by molecular switches called Rab GTPases. The protein that couples the rise in agonist-induced intracellular Ca2+ to Rab GTPase activation has remained elusive despite being the focus of several research groups. However, we recently identified a novel Rab GTPase expressed in endothelial cells that, unlike the majority of the Rab GTPase family, contains Ca2+ binding domains. This research proposes that the Ca2+ binding Rab GTPase, CRACR2A-L, has important functions and could potentially be offered as a target for novel therapeutics for athero-thrombotic diseases.

Objective 1: To determine the contribution of CRACR2A-L to Weibel Palade body trafficking. The general strategy for this project will be to use high resolution imaging to quantify and measure the distribution of CRACR2A-L and vWF in endothelial cells. Contribution of CRACR2A-L to Weibel Palade body trafficking in response to thrombotic stimuli will be assessed by overexpressing wtCRACR2A-L or GTP/Ca2+-binding mutants or alternatively, cellular levels of protein will be reduced by short interfering RNA. Secretion of vWF as a measure of Weibel Palade body exocytosis will be assayed using ELISA. The effect of CRACR2A-L on other endothelial functions such as apoptosis and proliferation will also be assessed.
Objective 2: To discover the fundamental mechanisms by which CRACR2A-L regulates Weibel Palade bodies. Affinity chromatography followed by LC-MS/MS will be used to identify downstream effector proteins. Potential effector proteins will be identified and ranked according to peptide abundance and pathway analysis. Confirmed effectors will be verified by co-immunoprecipitation and rescue of function experiments using high resolution imaging. To be able to understand the structural mechanisms of CRACR2A-L action, structural models will be created by X-ray crystallography and homology modelling.
Objective 3: To verify the in vivo role of CRACR2A-L using an endothelial specific knockout mouse. First, production of a CRACR2A global 'knockout first' (with conditional potential) mouse will be outsourced. Subsequent breeding with FLP deleters and endothelial specific Cre recombinase mice will generate an endothelial specific CRACR2A-L knockout mouse. The in vivo contribution of global and endothelial CRACR2A-L to Weibel-Palade body exocytosis will be analysed by measuring clotting in response to tail injury. In addition, real time intravital imaging of clot formation will be performed in the femoral artery and in the mouse ear. Angiogenesis will also be assessed.

Primarily the impact flows from the basic understanding of cellular trafficking to translational research in the vasculature where novel approaches could be devised to manipulate the secretion of vWF to improve cardiovascular health.
The research on a fundamental mechanism in Rab GTPase regulation of cellular trafficking will be: publishable in text books to enhance student learning; used to encourage further funding applications from vascular biologists, cellular biologists and researchers of many disease areas; result in the publication of several peer reviewed papers for international dissemination. In addition, spin-off companies could specialise in drug discovery programmes using findings from the structural analysis.
Clinically, there is a potential to enhance the treatment of thrombosis in cardiovascular disease, especially in susceptible populations such as diabetics by manipulating vWF release. This would foster further collaborations and investment with clinicians and translational scientists and could involve the exploitation of intellectual property and patenting.