XXXIII Congreso Nacional de la Sociedad Española de Trombosis y Hemostasia

XXXIII Congreso Nacional de la Sociedad Española de Trombosis y Hemostasia 100 Platelets are critical in the body’s response to vascular injury (hemostasis). To form hemostatic plug platelets need to be able to respond to trace amounts of agonists produced at injury sites, and to rapidly transition to an adhesive state (integrin inside-out activation) required for plug formation. However, if unbalanced, this highly sensitive signaling machinery can lead to premature platelet activation and thrombocytopenia (low platelet count) and/ or thrombosis (1). The small GTPase RAP1 is a critical regulator of platelet adhesiveness (2). RAP1 cycles between a GDP-bound “off-state” and a GTP-bound “on-state”, the latter being required for integrin activation. In platelets, the guanine nucleotide exchange factor (GEF), CalDAG-GEFI (RasGRP2), is the primary GEF mediating RAP1 activation. CalDAG-GEFI activity is regulated by a pair of EF hand domains, which have high sensitivity towards calci- um (K D ~80nM) and thus sense minor changes in the cytoplasmic calcium concentration (3,4). Platelets lacking functional Cal- DAG-GEFI show strongly impaired sensitivity towards threshold concentrations of agonists, both in vitro and in vivo . In addition to sensitivity, CalDAG-GEFI is also critical for the near-immediate activation of RAP1, triggered by rapid calcium mobilization from stores in the endoplasmic reticulum. Both RAP1 activation and integrin inside-out activation are delayed in platelets from mice and humans lacking functional CalDAG-GEFI (5,6). Our recent work identified the RAP-GAP, RASA3 (GAP1 IP4bp , R-RAS-GAP) as a critical negative regulator of RAP1 signaling in platelets (7). RASA3 is anchored to the plasma membrane by a unique PH/Btk domain and thus is perfectly positioned to pre- vent premature integrin activation in resting platelets. Mice lacking functional RASA3 exhibit severe thrombocytopenia due to prema- ture platelet activation in circulation. Concomitant deletion of Cal- DAG-GEFI reversed this phenotype in Rasa3 mutant mice, strongly suggesting that RASA3 is critical to restrain the highly sensitive CalDAG-GEFI/RAP1 signaling pathway. Thus, RASA3 serves as a “hand brake” in circulating platelets, which is required to maintain these cells in a quiescent state. At sites of vascular injury, this break needs to be released to allow for hemostatic plug formation. The Critical switches of platelet signaling and function W. Bergmeier Department of Biochemistry and Biophysics. McAllister Heart Institute. University of North Carolina at Chapel Hill. Chapel Hill, North Carolina. USA signal for RASA3 inactivation is provided by PI3 kinase signaling, downstream of the platelet receptor for ADP, P2Y12. Together, our work identified an antagonistic balance between CalDAG-GEFI and RASA3 signaling in platelets, which is criti- cal for the fine-tuning of platelet adhesiveness, both in the circu- lation and at sites of vascular injury. Genetic and environmental factors that shift the balance towards RAP1 activation may lead to thrombocytopenia and thrombosis, while those that lead to impaired RAP1 activation may cause bleeding in patients. The latter is impressively shown by the marked platelet adhesion defect and the moderate to severe bleeding diathesis observed in the various (> 10) patients with mutations in the gene for CalDAG-GEFI. In my presentation I will review the literature on RAP1 signaling in platelets and I will discuss unpublished work on structure-function studies for CalDAG-GEFI and best treatment practices for patients with platelet function disorders. References 1. Jackson SP. Arterial thrombosis--insidious, unpredictable and dead- ly. Nat Med 2011;17:1423-36. 2. Stefanini L, Bergmeier W. RAP1-GTPase signaling and platelet function. J Mol Med 2015. 3. Crittenden JR, Bergmeier W, ZhangY, Piffath CL, LiangY, Wagner DD, et al. CalDAG-GEFI integrates signaling for platelet aggrega- tion and thrombus formation. Nat Med 2004;10:982-6. 4. Iwig JS, Vercoulen Y, Das R, Barros T, Limnander A, Che Y, et al. Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1. eLife 2013;2 e00813-3. 5. Stolla M, Stefanini L, Roden RC, Chavez M, Hirsch J, Greene T, et al. The kinetics of alphaIIbbeta3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy. Blood 2011;117:1005-13. 6. Kato H, NakazawaY, KurokawaY, Kashiwagi H, MorikawaY, Mori- ta D, et al. Human CalDAG-GEFI deficiency increases bleeding and delays α IIb β 3 activation. Blood 2016; 128:2729-33. 7. Stefanini L, Paul DS, Robledo RF, Chan ER, Getz TM, Campbell RA, et al. RASA3 is a critical inhibitor of RAP1-dependent platelet activation. J Clin Invest 2015;125(4):1419-32.