RAP1-GTPase signaling and platelet function

Abstract

Platelets are critical for hemostasis, i.e., the body's ability to prevent blood loss at sites of vascular injury. They patrol the vasculature in a quiescent, non-adhesive state for approximately 10 days, after which they are removed from circulation by phagocytic cells of the reticulo-endothelial system. At sites of vascular injury, they promptly shift to an activated, adhesive state required for the formation of a hemostatic plug. The small GTPase RAP1 is a critical regulator of platelet adhesiveness. Our recent studies demonstrate that the antagonistic balance between the RAP1 regulators, CalDAG-GEFI and RASA3, is critical for the modulation of platelet adhesiveness, both in circulation and at sites of vascular injury. The RAP1 activator CalDAG-GEFI responds to small changes in the cytoplasmic calcium concentration and thus provides sensitivity and speed to the activation response, essential for efficient platelet adhesion under conditions of hemodynamic shear stress. The RAP1 inhibitor RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI-dependent RAP1 activation. Upon cellular stimulation, it is turned off by P2Y12 signaling to enable sustained RAP1 activation, required for the formation of a stable hemostatic plug. This review will summarize important studies that elucidated the signaling pathways that control RAP1 activation in platelets.

Keywords: GTPase; Platelets; RAP1; Signaling; Thrombosis.

Fig.1. RAS/RAPs, GEFs and GAPs protein expression in platelets

Estimated protein levels based on the most comprehensive quantitative proteomic analysis of human[7] (full bars) and mouse[29] (striped bars) platelets. CalDAG-GEFI and RASA3 are the most abundant RAP regulators expressed in both human and mouse platelets.

Fig.2. Domain structure of the RAP1 regulators, CalDAG-GEFI and RASA3

CalDAG-GEFI consists of a RAP-specific catalytic guanine nucleotide exchange factor (GEF) domain, a pair of Ca²⁺ binding EF hand domains (EF) and a C1-like domain of hitherto unknown function that is necessary for optimal RAP-GEF activity. RASA3 consists of a catalytic GTPase-activating protein (GAP) domain specific for RAS and RAP, flanked by a pair of C2 domains and by a PH/Btk domain that binds phosphatidylinositol 4,5-diphosphate (PIP₂) and phosphatidylinositol 3,4,5-triphosphate (PIP₃). The Rasa3^hlb mutant mouse strain is characterized by a single point mutation (H794L) in the C-terminal region of RASA3. GTP (guanosine triphosphate), GDP (guanosine diphosphate).

Fig.3. Schematic model of RAP1-dependent platelet regulation

Left panel. In platelets circulating healthy vessels RASA3 is active to restrain uncontrolled RAP1 activation and maintain a quiescent, non-adhesive state (no integrin activation). Central panel. At sites of vascular injury, platelets stimulation results in an increase in the cytosolic Ca²⁺ levels and the release of ADP from dense granules. Ca²⁺ activates CalDAG-GEFI, which mediates rapid GTP-loading of RAP1. ADP stimulates the P2Y12 receptor, which leads to decreased RASA3 function and sustained RAP1 signaling. Fast and sustained RAP1/integrin activation results in the formation of stable three-dimensional thrombi over a broad range of hemodynamic shear conditions. Right panel. Inhibitors to P2Y12, such as Clopidogrel, prevent the inactivation of RASA3, prohibit sustained RAP1 signaling and destabilize the growing thrombus.