15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and cangrelor

The effects on platelet function of temperatures attained during hypothermia used in cardiac surgery are controversial. Here we have performed studies on platelet aggregation in whole blood and platelet-rich plasma after stimulation with a range of concentrations of ADP, TRAP, U46619 and PAF at both 28 degrees C and 37 degrees C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28 degrees C compared with 37 degrees C. Aggregation induced by ADP was also enhanced. Similar results were obtained in hirudinised blood. There was no spontaneous aggregation in PRP but ADP-induced aggregation was enhanced at 28 degrees C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28 degrees C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28 degrees C compared with 37 degrees C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28 degrees C, but reversal of aggregation was seen at 37 degrees C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28 degrees C compared with 37 degrees C. Expression was significantly increased at 28 degrees C after stimulation with ADP, PAF and TRAP but not after stimulation with U46619. Overall, our results demonstrate an enhancement of platelet function at 28 degrees C compared with 37 degrees C, particularly in the presence of ADP.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adenosine Monophosphate; Anticoagulants; Antigens, CD; Aspirin; Blood Platelets; Blood Specimen Collection; Citrates; Cold Temperature; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Hirudins; Humans; Hypothermia, Induced; In Vitro Techniques; P-Selectin; Platelet Activating Factor; Platelet Activation; Platelet Aggregation; Platelet Function Tests; Platelet Membrane Glycoproteins; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Receptors, Thrombin; Tetraspanin 30; Time Factors

Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state.. Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation.. Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adenosine Monophosphate; Antibodies, Monoclonal; Blood Platelets; Dose-Response Relationship, Drug; Humans; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Purinergic P2 Receptor Antagonists; rap GTP-Binding Proteins; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Signal Transduction; Thrombin

The stimulation of platelets by low doses of collagen induces extracellular signal-regulated kinase 2 (ERK2) activation. In this report, we demonstrate that collagen-induced ERK2 activation depends on thromboxane A(2) (TXA(2)) formation and ADP release. The collagen-induced ERK2 activation was inhibited by indomethacin (88%) and by AR-C69931MX (70%), a specific antagonist of P2Y12, a Gi-coupled ADP receptor. AR-C69931MX (10 microM) inhibition was overcome by epinephrine (1 microM), an agonist of the Gi-coupled alpha(2A)-adrenergic receptor, suggesting that the Gi-coupled receptor was necessary for ERK2 activation by collagen. By contrast, MRS 2179 (10 microM), a specific antagonist of P2Y1, a Gq-coupled ADP receptor, did not affect collagen-induced ERK2 activation. Little or no ERK2 activation was observed with ADP alone (10 microM). By contrast, U46619 (10 microM), a stable analog of TXA(2), induced ERK2 activation in an ADP-dependent manner, via the P2Y12 receptor. These results suggest that the Gi-dependent signaling pathway, stimulated by ADP or epinephrine, was not the only pathway required for ERK2 activation by collagen. Costimulation of the specific G(12/13)-coupled TXA(2) receptor with a low dose of U46619 (10 nM) and of Gi- and Gq-coupled ADP receptor (10 microM) induced very low levels of ERK2 activation, similar to those observed with ADP alone, suggesting that G(12/13) is not involved or not sufficient to induce the additional pathway necessary for ERK2 activation. The Gq-coupled TXA(2) receptor was required for ERK2 activation by U46619 (10 microM) and low doses of collagen, clearly showing that a coordinated pathway through both Gq from TXA(2) and Gi from ADP was necessary for ERK2 activation. Finally, we demonstrate that ERK2 activation is involved in collagen-induced aggregation and secretion.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adenosine Monophosphate; Animals; Cattle; Collagen; Enzyme Activation; Epinephrine; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Integrin alpha2beta1; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Phosphorylation; Platelet Aggregation; Platelet Membrane Glycoproteins; Protein Kinase C; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction; Thromboxane A2

Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcgammaRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca(2+) concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcgammaRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the G(i)-coupled ADP receptor P2Y12, but was not affected by blockade of the G(q)-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca(2+) mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca(2+) signaling. In platelets stimulated by cross-linking of FcgammaRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G(i)-coupled alpha(2A)-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G(q)-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G(q)-coupled thromboxane A(2) receptor by was completely inhibited by ADP scavengers under conditions in which intracellular Ca(2+) mobilization was unaffected. Inhibition of -induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G(i)-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adenosine Monophosphate; Antigens, CD; Blood Platelets; Calcium; Chromatography, Gel; Cross-Linking Reagents; Dimerization; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epinephrine; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Platelet Aggregation Inhibitors; Protein Binding; rap GTP-Binding Proteins; Receptors, IgG; Recombinant Proteins; Serotonin; Signal Transduction; Time Factors

Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Monophosphate; Animals; Apyrase; Blotting, Western; Cyclic AMP; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Heterotrimeric GTP-Binding Proteins; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; rac GTP-Binding Proteins; rho GTP-Binding Proteins

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adenosine Monophosphate; Analysis of Variance; Annexin A5; Aspirin; Blood Platelets; Calcium; Collagen; Cytosol; Fibrinolytic Agents; Flow Cytometry; Humans; Membrane Proteins; P-Selectin; Peptide Fragments; Platelet Activation; Platelet Aggregation; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Serotonin; Stimulation, Chemical; Thromboxane A2