Thrombin is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Thrombin activates platelets mainly through protease-activated receptor 1 (PAR1), PAR4, and glycoprotein Ib. Because adenosine diphosphate and thromboxane A 2 have been shown to cause platelet aggregation by concomitant signaling through G q and G i pathways, we investigated whether coactivation of G q and G i signaling pathways is the general mechanism by which PAR1 and PAR4 agonists also activate platelet fibrinogen receptor (␣IIb3). APAR1-activating peptide, SFLLRN, and PAR4-activating peptides GYPGKF and AYPGKF, caused inhibition of stimulated adenylyl cyclase in human platelets but not in the presence of either Ro 31-8220, a protein kinase C selective inhibitor that abolishes secretion, or AR-C66096, a P2Y12 receptor-selective antagonist; ␣-thrombin-induced inhibition of adenylyl cyclase was also blocked by Ro 31-8220 or AR-C66096. In platelets from a P2Y12 receptor-defective patient, ␣-thrombin, SFLLRN, and GYPGKF also failed to inhibit adenylyl cyclase. In platelets from mice lacking the P2Y12 receptor, neither ␣-thrombin nor AYPGKF caused inhibition of adenylyl cyclase. Furthermore, AR-C66096 caused a rightward shift of human platelet aggregation induced by the lower concentrations of ␣-thrombin and AYPGKF but had no effect at higher concentrations. Similar results were obtained with platelets from mice deficient in the P2Y12. We conclude that (1) thrombin-and thrombin receptoractivating peptide-induced inhibition of adenylyl cyclase in platelets depends exclusively on secreted adenosine diphosphate that stimulates G i signaling pathways and ( IntroductionPlatelet activation plays a major role in hemostasis and thrombosis. Several agonists, including adenosine diphosphate (ADP), thrombin, and thromboxane A 2 , can activate platelets. 1 These agonists cause platelets to change their shape, to aggregate, and to release the contents of granules. Thrombin, generated at the site of vascular damage by extrinsic and intrinsic coagulation cascades, is an important agonist for platelet activation. Thrombin mediates its cellular effects primarily through a family of G protein-coupled protease-activated receptors (PARs). These receptors are activated by a unique mechanism in which the protease creates a new extracellular amino-terminus that functions as a tethered ligand, resulting in intramolecular activation. 2,3 Three of the 4 known PARs, PAR1, PAR3, and PAR4, are activated by thrombin. PAR1 is detected in human platelets and has a major role in activation of human platelets by thrombin, but it plays no role in mouse platelets. 4 PAR2 functions as a receptor for trypsin but not for thrombin. 5 PAR3 is necessary for mouse platelets to be activated by lower concentrations of thrombin. 6 PAR3 functions as a cofactor for the activation of PAR4 by thrombin in mouse platelets. 7 There is another receptor, PAR4, which appears to function in both mouse and human platelets. 4,8 PAR4 also mediates platelet respo...
Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A 2 , another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-␥-dichloromethylene adenosine 5-triphosphate (AR-C67085), a P2Y12 receptor-selective antagonist, and adenosine-2-phosphate-5-phosphate (A2P5P), a P2Y1 receptor-selective antagonist, inhibited ADP-induced thromboxane A 2 generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A 2 production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid-induced platelet aggregation, it failed to inhibit thromboxane A 2 generation induced by arachidonic acid. Thus, ADPinduced arachidonic acid liberation, but not subsequent conversion to thromboxane A 2 , requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site-6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin ␣ IIb  3 , caused both platelet aggregation and thromboxane A 2 generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A 2 generation, indicating that these signaling molecules have no significant role in phospholipase A 2 activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A 2 , suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A 2 activation, resulting in arachidonic acid liberation and thromboxane IntroductionAdenosine diphosphate (ADP) is an important platelet agonist that plays a role in hemostasis and pathophysiological arterial thrombosis. 1 ADP causes platelets to undergo shape change, release granule contents, and aggregate. [2][3][4] Upon exposure to activating agonists, such as ADP, platelets hydrolyze arachidonic acid from phospholipid and convert it into thromboxane A 2 by sequential oxygenation via cyclo-oxygenase and thromboxane A 2 synthase. 5 The released thromboxane A 2 acts as a positive feedback mediator in the activation and recruitment of more platelets to the primary hemostatic plug. 6 ADP also causes adhesion of platelets to vitronectin or osteopontin, which might play an important role in anchoring platelets to disrupted atherosclerotic plaques and to the walls of the injured arteries. 7 All the ADP-induced intracellular signaling events, viz, rapid influx of calcium, activation of phospholipase C, mobilization of calcium from intracellular stor...
Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin αIIbβ3) is activated by agonist-mediated G q stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5h-dimethyl-bis-(o-aminophenoxy)ethane-N,N,Nh,Nh -tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADPand U46619-induced aggregation and anti-αIIbβ3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially
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