Lucia Stefanini, PhD1,
Moritz Stolla, MD*,1,
Sean F Maloney*,2,
Timothy Daniel Ouellette, BS*,1,
Scott L. Diamond, PhD*,3 and
Wolfgang Bergmeier, PhD1
1 Cardeza Foundation and Dept. of Medicine, Thomas Jefferson University, Philadelphia, PA, USA, 2 Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA, 3 Department of Chemical Engineering, Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
Poster Board II-968
The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrelbisulfate (Plavix), currently the most successful anti-plateletstrategy used in the clinic. In a recent study, we have shownthat the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI,and P2Y12 represent the major signaling pathways leading toRap1 and integrin activation in platelets (Cifuni et al., 2008,Blood). In the present study, we have further evaluated theimportance of CalDAG-GEFI signaling and Rap1 activation forvarious aspects of platelet activation, and we have comparedthrombus formation of CalDAG-GEFI–/– and WT/clopidogrelplatelets under static and flow conditions in vitro. Our studiesestablish a revised model for platelet activation by collagen.In platelets activated with threshold concentrations of GPVIagonists, CalDAG-GEFI serves as a highly sensitive responseelement to Ca2+ that allows for the rapid activation of Rap1.CalDAG-GEFI-mediated Rap1 activation triggers a first wave ofintegrin activation and ERK (MAPK) signaling, followed by TxA2release. TxA2 provides crucial feedback for the activation ofPKC and granule/ADP release. ADP in turn triggers the second,P2Y12-dependent wave of Rap1-mediated signaling events, leadingto the sustained activation of integrins and further releaseof TxA2. Higher concentrations of GPVI agonists lead to theconcomitant activation of CalDAG-GEFI and PKC, facilitatingplatelet aggregation independent of feedback by endogenous TxA2.Under physiological flow conditions, CalDAG-GEFI-dependent plateletactivation (clopidogrel-treated WT platelets) allowed for theformation of small but unstable thrombi, which rapidly disintegratedat high shear rates. In contrast, CalDAG-GEFI–/–platelets (P2Y12-dependent platelet activation) in anticoagulatedblood firmly adhered to the thrombogenic surface but failedto form thrombi, even at high concentrations of collagen. Additionof exogenous TxA2 to anticoagulated CalDAG-GEFI–/–blood did not restore thrombus formation under flow. However,small thrombi were observed with non-anticoagulated CalDAG-GEFI–/–blood perfused at venous but not arterial shear rates, suggestingthat a) locally generated thrombin facilitates the recruitmentof free flowing CalDAG-GEFI–/– platelets to alreadyadherent platelets, and b) the slow kinetics of P2Y12-dependentRap1 activation only supports thrombin-induced platelet-plateletcohesion at low shear conditions. In conclusion, our studiesdemonstrate that CalDAG-GEFI/Rap1 signaling plays a criticalrole for the first wave of integrin activation and TxA2 generationimportant for platelet adhesion to a thrombogenic surface. Signalingby P2Y12/Rap1 is essential for sustained platelet activation/thrombusstabilization and partially compensates for CalDAG-GEFI/Rap1-mediatedplatelet adhesion under low flow conditions.
Disclosures: No relevant conflicts of interest to declare.
* Asterisk with author names denotes non-ASH members.