The involvement of the small GTPase Rap1b in platelet integrin ␣ 2 ␤ 1 -dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin ␣ 2 ␤ 1 , monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A 2 production but was critically regulated by phospholipase C (PLC)-derived second messengers. Both Ca 2؉ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-␣ 2 ␤ 1 , which were completely blocked by inhibition of PLC with U73122. Moreover, integrin ␣ 2 ␤ 1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca 2؉ and diacylglycerol, despite normal phosphorylation and activation of PLC␥2. In addition, CalDAG-GEFI-deficient platelets showed defective integrin ␣ 2 ␤ 1 -dependent adhesion and spreading. We found that outside-in signaling through integrin ␣ 2 ␤ 1 triggered inside-out activation of integrin ␣ IIb ␤ 3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrindependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors. (Blood.

Stimulation of G q -coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca 2؉ mobilization and protein kinase C (PKC) activation. We found that ADPinduced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G q -coupled P2Y1 receptor but also upon blockade of the G i -coupled P2Y12 receptor. The role of G i on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G q stimulation by the thromboxane A 2 analogue U46619. Importantly, activation of phospholipase C and intracellular Ca 2؉ mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G i -coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.It is generally accepted that, with a very few exceptions, platelet response to soluble agonists originates from the convergence of at least two different signal transduction pathways typically initiated by the heterotrimeric G-proteins G q and G i . Several receptors for platelet agonists are coupled to G q , including the P2Y1 receptor for ADP, the TP␣ receptor for the thromboxane A 2 (TxA 2 ), 3 and the PAR1 and PAR4 receptors for thrombin (1, 2). Signaling downstream G q involves the stimulation of members of the ␤ subfamily of phospholipase C (PLC), which generate the second messengers inositol 1,4,5-trisphosphosphate (IP 3 ) and diacylglycerol (DAG), responsible for Ca 2ϩ mobilization from intracellular stores and protein kinase C (PKC) activation, respectively (3). This pathway is necessary but not sufficient to elicit a full platelet response, and the concomitant activation of a G i -coupled receptor is absolutely mandatory (4). To fulfill this requirement, some agonists, like ADP, directly stimulate multiple G-protein coupled receptors, one of which is associated to G i (5, 6), whereas others, such as TxA 2 , rely on the auxiliary action of secondary messengers released by activated platelets to stimulate a G i -coupled receptor (7). Despite their essential role, there are very few G i -coupled receptors on the platelet surface, and these include the ␣2-adrenergic receptor for epinephrine and the P2Y12 receptor for ADP (8, 9). Therefore, ADP, which is also one of the major component of the platelet releas...

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner.