Towards a novel platelet inhibitor: Targeting the C1 domain of CalDAG-GEFI to inhibit Rap1b

Univ Of North Carolina Chapel Hill, Chapel Hill NC

Investigators

Abstract

? DESCRIPTION (provided by applicant): Towards a novel platelet inhibitor: Targeting the C1 domain of CalDAG-GEFI to inhibit Rap1b Abstract Several anti-platelet drugs on the market such as Plavix protect patients against heart attacks, but also cause excessive bleeding in patients undergoing surgery, due to irreversible platelet activation. CalDAG-GEFI however, a regulator of the small GTPase Rap1b, reversibly activates platelets and prevents excessive bleeding. Biochemical and biophysical studies of Rap1b and CalDAG-GEFI interactions lay the foundation for future anti-platelet drug design that can takes advantage of the unique anti-thrombotic potential of CalDAG- GEFI. This project aims to build a fundamental molecular model of C1-Rap1b interactions for future drug design efforts. Hypothesis: C1 domain of CalDAG-GEFI critically regulates Rap1b activation through (1) CalDAG-GEFI membrane localization and, (2) initial Rap1b binding for subsequent GEF activation. In Aim 1, we investigate the role of Rap1b-C1 binding in CalDAG-GEF activation. Absence of C1 domain has been shown to reduce Rap1b activation by CalDAG-GEFI. We propose initial recruitment of Rap1b by C1 promotes GEF binding and activation of Rap1b. We will delineate this mechanism by determining nucleotide dependence of C1-Rap1b interactions, mapping sites of interaction using protein NMR, and generating defective C1 mutants to characterize structural changes within each protein that ultimately promote Rap1b binding to GEF domain. We have been able to generate HSQCs for both C1 domain and Rap1b, establishing an NMR-tractable system for studies proposed. In parallel, we investigate the role of lipid binding to the C1 domain for CalDAG-GEFI membrane association and GEF regulation in Aim 2. We propose IPL signaling to C1 is needed for membrane localization of CalDAG-GEFI. We have preliminary data for acidic PhosphatidylInositolPhosphates (PIP) lipids binding to the C1 domain. We will probe the specificity of PIP to C1 domain through in vitro lipid co-sedimentation assays that mimic platelet membrane composition. We will confirm in vitro data regarding Rap1b activation through C1 binding and CalDAG-GEFI localization in a physiological environment. We propose both C1 initial binding to Rap1b and CalDAG-GEFI membrane localization are physiologically relevant phenomena. We will test the functionality and localization of generated CalDAG-GEFI mutants in a platelet-like, megakaryocytic, cell line. Fibrinogen binding and ?IIb?3 binding assays will e used to test functionality of CalDAG-GEFI mutants, and confocal microscopy to monitor CalDAG-GEFI localization.