Novel small G protein pathway regulators of GPVI signaling in platelets

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Project summary Platelet activation by sub-endothelial collagen is crucial not only for hemostatic plug formation but also for thrombotic events leading to heart attack and stroke. Whereas several signaling cascades downstream of heterotrimeric G proteins that regulate platelet functional responses have been characterized, little attention has been paid towards the signaling cascades that involve monomeric small G proteins. We have previously published that the small GTPase RhoG selectively regulates GPVI signaling events in platelets and functional responses. However, the mechanisms of RhoG activation and regulation are not known in platelets. We have strong preliminary data indicating that multiple Ras family small G protein pathways are activated by GPVI receptors and that they play an important role in the regulation of GPVI pathways and hence platelet function. We bring together our expertise with platelet biology and small G protein biochemistry to understand the functional roles of small G protein pathways in platelet function. In this project our overall hypothesis is that some Ras family monomeric small G protein pathways specifically regulate GPVI pathways. Our specific aims are as follows: 1) Evaluate the Role of ELMO1 in the regulation of platelet functional responses. We have identified that a small G protein, RhoG selectively regulates GPVI pathways. We propose that RhoG regulates GPVI signaling through an associated protein, ELMO1, in platelets. We will evaluate the function of ELMO1 in platelets using murine platelets deficient in ELMO1. Our preliminary data show that platelets express ELMO1 and CRP-induced platelet secretion and aggregation are potentiated in ELMO1-null mice. We will test the hypothesis that ELMO1 negatively regulates RhoG activity in platelets through GAPs (GTPase activating proteins), by identifying associated GAPs and by evaluating the function of Arl4, known associating GTPase of ELMO1, using knockout mice. 2) Investigate the functional roles of TC21/R-Ras2 in platelets. TC21 has been shown to be involved in integrin activation, but the full effects and mechanisms are poorly characterized. We have found that TC21 is expressed in murine and human platelets, is activated by GPVI agonists, and that TC21 is required for integrin activation and platelet function downstream of the GPVI receptor. We hypothesize that TC21 potentiates GPVI receptor signaling to regulate integrin IIb3 activation in platelets. We will investigate the roles of TC21 in GPVI signaling, integrin activation and platelet function using ex vivo platelets and in vivo thrombosis models, in human platelets and in platelets derived from mice deleted for TC21. 3) Delineate the mechanisms of activation of TC21/R-Ras2 and RhoG in platelets. We will utilize ex vivo human platelets to investigate the mechanisms of TC21 and RhoG activation. We will test the hypothesis that TC21 regulates RhoG activation through PI3kinase/PIP3/GEF (guanine nucleotide exchange factor) pathways. Our preliminary data identified four GEFs that associate with PIP3 in platelets. The studies proposed in this application will enhance our understanding of the signaling networks and their regulation of functional responses in platelets with a special emphasis on collagen signaling.