RASA3 is a critical inhibitor of RAP1-dependent platelet activation
Lucia Stefanini, … , Luanne L. Peters, Wolfgang Bergmeier
Lucia Stefanini, … , Luanne L. Peters, Wolfgang Bergmeier
Published February 23, 2015
Citation Information: J Clin Invest. 2015;125(4):1419-1432. https://doi.org/10.1172/JCI77993.
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Research Article Hematology

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

Abstract

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Authors

Lucia Stefanini, David S. Paul, Raymond F. Robledo, E. Ricky Chan, Todd M. Getz, Robert A. Campbell, Daniel O. Kechele, Caterina Casari, Raymond Piatt, Kathleen M. Caron, Nigel Mackman, Andrew S. Weyrich, Matthew C. Parrott, Yacine Boulaftali, Mark D. Adams, Luanne L. Peters, Wolfgang Bergmeier

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Figure 7

Schematic illustrating the regulation of RAP1 signaling in platelets.

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Schematic illustrating the regulation of RAP1 signaling in platelets. RA...
RAP1 activation in platelets is tightly controlled by the antagonistic balance between the calcium-sensitive RAP-GEF, CalDAG-GEFI, and the RAP-GAP, RASA3. In quiescent platelets, RASA3 restrains unwanted CalDAG-GEFI/RAP1 signaling to ensure platelet homeostasis. At sites of vascular injury, platelet stimulation through ITAM-coupled (collagen receptor GPVI, podoplanin receptor CLEC-2, and IgG receptor FcγRIIA) and G protein–coupled (thrombin receptors PAR1/3/4, ADP receptor P2Y1, and TxA2 receptors) receptors leads to the activation of PLCγ2 and PLCβ2/3, respectively. PLCs convert PIP2 to DAG and inositol 1,4,5-trisphosphate (IP3). IP3 mediates Ca2+ store release and a rise in the cytosolic Ca2+ concentration, which triggers rapid CalDAG-GEFI-dependent RAP1 activation. DAG leads to PKC activation, which contributes to nucleotide exchange on RAP1 and the release of platelet granules. CalDAG-GEFI signaling eventually subsides and RASA3 must be inactivated in order to maintain RAP1 in an activated state. RASA3 inactivation depends on PI3 kinase signaling, which in platelets is under the control of Gαi-coupled receptors, such as P2Y12 (ADP) or α2A-R (epinephrine). Both rapid and sustained RAP1 signaling are critical for integrin inside-out activation and the formation of a hemostatic plug. P2Y12 inhibitors prevent the inactivation of RASA3 and thus destabilize the growing thrombus.