P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

In thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi(2)-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin alpha(IIb)beta(3) outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA(2)-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA(2) receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA(2) signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled alpha(2A)-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI(2) or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.     

Effects of inhibition of P2Y(1) and P2Y(12) on whole blood clotting,coagulum elasticity and fibrinolysis resistance studied with free oscillation rheometry

Introduction: In vivo, initial platelet activation is likely caused by platelet contacts with collagen in the subendothelium or from the small amounts of thrombin formed by the tissue factor/factor VIIa complex. Our aim was to study the coagulative role of ADP released by the platelets after activation with strong stimuli such as collagen and/or thrombin, and the relative importance of the platelet ADP receptors P2Y₁ and P2Y₁₂. Materials and methods: We used 10 Hz free oscillation rheometry to measure clotting time, clot elasticity and fibrinolysis resistance of non-anticoagulated whole blood. The platelets were activated with a collagen-related peptide (CRP), with the PAR1 thrombin receptor activating peptide TRAP-6 or by thrombin, the latter generated by small amounts of thromboplastin. To inhibit the platelet ADP receptors, we used the P2Y₁ antagonist MRS2179 and the P2Y₁₂ antagonist AR-C69931MX. Results: Both antagonists significantly retarded the clotting induced by CRP. The effects were most pronounced with AR-C69931MX. For TRAP-6, the same trend was seen, but the retardation was only significant with AR-C69931MX. Clotting induced by small amounts of thromboplastin was not affected by any ADP-receptor antagonist. Addition of both antagonists did not change the results as compared to samples with AR-C69931MX alone. Nor did the antagonists, one at a time or in concert, effect fibrinolysis or the elastic properties of the clot. Conclusion: We conclude that ADP-receptor inhibition prolongs the clotting time for whole blood activated by CRP, but that it does not affect the properties of the subsequently formed coagulum.     

Autoinhibition of transmitter release from PC12 cells and sympathetic neurons through a P2Y receptor-mediated inhibition of voltage-gated Ca2+ channels

Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.     

Open multicentre study of the P2T receptor antagonist AR-C69931MX assessing safety, tolerability and activity in patients with acute coronary syndromes

Platelet aggregation is the central process in the pathophysiology of acute coronary syndromes. ADP contributes to thrombosis by activating platelets, and AR-C69931MX is a specific antagonist of this process acting at the P2T receptor. At 5 hospitals, 39 patients with unstable angina or non-Q wave myocardial infarction, who were receiving aspirin and heparin, were administered intravenous AR-C69931MX with stepped dose increments over 3 h to a plateau of either 2 microg/kg/min for 21 h (Part 1; n = 12) or up to 69 h (Part 2; n = 13) or 4 microg/kg/min for up to 69 h (Part 3: n = 14). Safety parameters, platelet aggregation (PA) induced by ADP 3 micromol/L (impedance aggregometry), bleeding time (BT) and plasma concentrations of AR-C69931XX were assessed. AR-C69931MX was well tolerated. 33 patients completed the study. There were no deaths at 30 days and no serious adverse events attributed to AR-C69931MX. Trivial bleeding (56%) was common. At 24 h, mean inhibition of PA was 96.0 +/- 8.6, 94.9 +/- 14.4 and 98.7 +/- 2.1% and BT was 9.5 +/- 8.4, 14.0 +/- 9.7 and 16.0 +/- 11.1 min for Parts 1, 2 and 3 respectively. At 1 h post-infusion, mean inhibition of PA was 36.2 +/- 39.2, 20.7 +/- 25.9 and 40.7 +/- 36.7% respectively. 90% patients had a plasma half-life for AR-C69931XX of <9 min. In conclusion, AR-C69931MX is a potent, short-acting platelet ADP receptor antagonist suitable for further studies as an antithrombotic agent.     

Inhibition of ADP-induced P-selectin expression and platelet-leukocyte conjugate formation by clopidogrel and the P2Y12 receptor antagonist AR-C69931MX but not aspirin

Platelet-leukocyte interactions are recognised to have pro-inflammatory effects, which may be important in the pathophysiology of ischaemic heart disease. Clopidogrel and the novel intravenous antithrombotic agent AR-C69931MX act at the level of the platelet P2Y12 receptor, which is known to amplify platelet activation, aggregation and other responses induced by numerous platelet agonists. We studied the effects of clopidogrel and aspirin on ADP-induced platelet-leukocyte conjugate formation and P-selectin expression in healthy volunteers. The effects of clopidogrel and AR-C69931MX administered to patients with ischaemic heart disease were also assessed. AR-C69931MX and aspirin were also studied in vitro. Clopidogrel and AR-C69931MX suppressed ADP-induced platelet aggregation, P-selectin expression and platelet-leukocyte conjugate formation whereas aspirin had no inhibitory effect. These effects of clopidogrel and AR-C69931MX may confer therapeutic benefits in the management of acute coronary syndromes.     

Glycoprotein Ibalpha inhibition and ADP receptor antagonists, but not aspirin, reduce platelet thrombus formation in flowing blood exposed to atherosclerotic plaques

Anti-platelet drugs are used to prevent intra-arterial thrombus formation after rupture of atherosclerotic plaques. Until now, the inhibitory effect of present and future anti-platelet drugs such as aspirin, ADP receptor P2Y(1)/P2Y(12) antagonists and glycoprotein (GP) Ibalpha inhibitors on the interaction of platelets with human plaques is not known. To study those effects we obtained human atherosclerotic plaques by surgical endarterectomy. Plaques induced maximal platelet aggregation in hirudinized platelet-rich plasma (PRP) and blood that was effectively inhibited by aspirin, the P2Y(1) antagonist MRS2179 and the P2Y(12) antagonist AR-C69931MX, but not by GPIbalpha blockade with the mAB 6B4. Inhibition of platelet aggregation by MRS2179 was 74 +/- 37% and 68 +/- 20%, by AR-C69931MX 94 +/- 7% and 80 +/- 6%, and by aspirin 88 +/- 19% and 64 +/- 28%, in PRP and blood, respectively (mean +/- SD; n = 6-12 with plaques from 6 patients). The combination of both ADP receptor antagonists completely inhibited plaque-induced platelet aggregation in hirudinized PRP and blood. Under arterial flow conditions (1,500s(-1)), blockade of platelet GPIbalpha resulted in a strong decrease of plaque-stimulated platelet adhesion/aggregate formation of 77 +/- 5% (mean +/- SD; n = 4). Furthermore, MRS2179, AR-C69931MX and their combination reduced plaque-dependent platelet aggregate formation by 35 +/- 14%, 32 +/- 13% and 58 +/- 12% (mean +/- SD; n = 5), respectively. Aspirin was without significant effect. In conclusion, a GPIbalpha-blocking antibody, as well as P2Y(1) and P2Y(12) receptor antagonists, alone or in combination, reduce in contrast to aspirin human plaque-induced platelet thrombus formation under arterial flow. Although these new anti-platelet agents inhibit platelet thrombus formation after plaque rupture, more efficient platelet blockers are required.     

The relative importance of the ADP receptors, P2Y12 and P2Y1, in thrombin-induced platelet activation

The objective of this study was to test the relative importance of the two adenosine diphosphate (ADP) receptors P2Y(1) and P2Y(12) in thrombin-induced platelet activation using specific receptor antagonists. Blood from healthy volunteers was incubated with MRS2179, a reversible P2Y(1) antagonist, or AR-C69931, a reversible P2Y(12) antagonist prior to activation with different concentrations of ADP or thrombin. Platelet function in whole blood was assayed by flow cytometry using the antibody PAC-1 to estimate the expression of conformational active alpha(IIb)beta(3), the fibrinogen receptor. Complete inhibition of P2Y(12) or P2Y(1) abolished the ADP response, but only inhibition of P2Y(12) reduced the thrombin-induced response. The relative inhibition of the thrombin response by complete inhibition of P2Y(12) was most pronounced at thrombin concentrations just enough for complete PAR1 cleavage, which is sufficient to release all ADP, giving 70-86% inhibition. Above this concentration, the relative importance of P2Y(12) inhibition decreased due to activation of ADP independent pathways. This study supports P2Y(12) as a drug target compared with P2Y(1).     

Evidence that the rat is not an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation

Abnormal platelet aggregation seen in experimentally induced diabetic, hypercholesterolemic and spontaneously hypertensive rats (SHR) has been linked with increased prostaglandin synthesis. The present study was conducted to examine the role of prostaglandins in rat platelet activation using normal Wistar Kyoto (WKY) and SHR rats. Up to 30 microM ADP did not induce secondary phase of platelet aggregation in rat PRP and up to 30 microM epinephrine did not produce any response in rat PRP. In other experiments ADP (1.0 microM) and epinephrine (2.0 microM) induced typical biphasic aggregation responses in human PRP. Up to 20 microM U46619, a stable analog of prostaglandin H2, did not induce platelet aggregation in rat PRP or washed rat platelets. In contrast 2.0 microM U46619 caused maximal aggregation in human PRP and washed human platelets. Arachidonic acid (1.5-2.0 mM) induced aggregation in washed rat platelets. However, this was associated with excessive (67% and 94%) loss of cytoplasmic LDH. The low concentrations of thrombin (0.04 and 0.05 U/ml), induced two to three-fold increase in aggregation response in SHR platelets as compared to WKY platelets. Higher concentrations of thrombin (0.1 and 0.3 U/ml) induced similar aggregation responses in SHR and WKY platelets. Thrombin (0.04-0.3 U/ml) induced serotonin secretion in a concentration dependent manner. The extent of secretion was the same in SHR and WKY platelets at all concentrations. Thrombin-induced synthesis of thromboxane A2 (TXA2) in WKY and SHR platelets was quantified using a radioimmunoassay for TXB2. Thrombin (0.04-0.3 U/ml) produced TXB2 in WKY and SHR platelets in a concentration dependent manner. The SHR platelets produced significantly larger amounts of TXB2 as compared to WKY platelets. In other experiments aspirin (500 microM) inhibited thrombin (0.05 U/ml) induced TXB2 synthesis by 75% in both WKY and SHR platelets but failed to inhibit aggregation or secretion in either WKY or SHR platelets. Based on these data it is suggested that: (a) rat platelets inspite of their ability to synthesize TXA2 do not require TXA2 for aggregation; and (b) the rat may not be an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation.     

Akt activation is involved in P2Y12 receptor-mediated chemotaxis of microglia

Microglia play a variety of significant roles in the central nervous system (CNS), and in one of those roles they undergo morphological change in response to neural injury and migrate to the injured region. We previously reported that ATP/ADP promotes microglial chemotaxis via the Gi/o-coupled P2Y12 receptor; however, the intracellular signaling underlying P2Y12-receptor-mediated microglial chemotaxis is not fully understood. In this study, we examined the role of phospholipase C (PLC) and calcium signaling in ADP-induced microglial chemotaxis. A PLC inhibitor, U73122, significantly suppressed the chemotaxis and completely blocked the ADP-evoked intracellular calcium response, and a calcium chelator, BAPTA-AM, inhibited the chemotaxis. These results indicate that ADP-induced microglial chemotaxis is regulated by a PLC-mediated calcium pathway. ADP stimulation induced Akt phosphorylation in microglia, and the phosphorylation was inhibited by a P2Y12 receptor antagonist, AR-C69931MX. The Akt phosphorylation was blocked by U73122 and BAPTA-AM as well as by a phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, and inhibition of the Akt activation resulted in failure of chemotaxis. These results indicate that Akt activation is dependent on the PI3K pathway and a PLC-mediated increase in intracellular calcium and suggest that Akt activation is involved in ADP-induced microglial chemotaxis.     

Pharmacodynamic profile of antiplatelet agents: marked differences between single versus costimulation with platelet activators

BACKGROUND: In pharmacodynamic studies with antiplatelet agents, platelets are usually activated in vitro with single agonists (e.g., ADP) solely. We questioned whether differences occur between single and combined stimulation of platelets [involving the major thrombin-receptors, protease-activated receptors (PAR)1 and PAR4], and whether the pharmacodynamic response to common antiplatelet drugs vary when a combined stimulus is applied instead of a single agonist. METHODS: We investigated the influence of different antiplatelet agents (aspirin [500 mg]) in vivo, the P2Y12-antagonist AR-C 69931MX (4 nM) and the GPII/IIIa-antagonist (abciximab ([5 microg/ml] in vitro) on the degranulation response (CD62) and expression of the activated GPIIb/IIIa-receptor (PAC-1) after stimulation with ADP (2 microM), collagen (4 microg/ml), a PAR1-activating peptide (3 microM TRAP) and a PAR4-activating peptide (200 microM AYPGKF) alone or in a combination of each two agonists by flow cytometry in healthy subjects. RESULTS: (1) Combined activation of TRAP with AYPGKF resulted in synergistic CD62 and PAC-1 expression. Only AYPGKF but neither TRAP nor ADP acted synergistically with collagen. (2) AR-C 69931MX inhibited platelet degranulation (CD62) in all inducer combinations with ADP or the combination TRAP with AYPGKF. The effect was considerably smaller or absent for the combination of collagen with a second inducer. (3) Aspirin intake reduced platelet degranulation and PAC-1 expression only for AYPGKF costimulation with collagen. CONCLUSION: Because a variety of different agonists influence platelet activation and its distinct functions at a time, investigations which regard the concert of these agonists might be closer to the in vivo situation and better reflect the pharmacodynamic profile of an antiplatelet agent than using one single inducing agent.     

Regulation of tissue factor-induced coagulation and platelet aggregation in flowing whole blood

Photochemically induced thrombosis (a thrombin-dependent process) was measured in rats treated with moderate doses of anticoagulants, but which appeared to be unchanged. We considered the possibility that platelet-inhibiting agents, which also indirectly inhibit coagulation, would act as more potent antithrombotic agents. Inhibitors used as such were prostaglandin E1 (PGE1), which elevates cyclic AMP levels, and the P2Y12 ADP-receptor antagonist, AR-C69931MX. Effects of these agents were investigated in an ex vivo model system, in which whole blood under coagulant conditions was perfused over fibrinogen at defined wall shear rate. Perfusion of blood (rat or human) in the presence of tissue factor resulted in deposition of activated platelets and subsequent aggregate formation, along with exposure of procoagulant phosphatidylserine (PS) on the platelet surface and formation of fibrin fibers. In the presence of PGE1 aggregation was completely inhibited, but platelet adhesion and PS exposure were only party reduced, while fibrin formation was hardly affected. Treatment with AR-C69931MX caused similar, but less complete effects. These results indicate that in tissue factor-triggered blood under conditions of flow: (i) the platelet procoagulant response is independent of aggregate formation; (ii) the platelet-inhibiting effect of PGE1 and AR-C69931MX is sufficient to suppress aggregation, but not platelet adhesion and coagulation. These platelet inhibitors thus maintain their aggregation-inhibiting effect at sites of thrombin formation.     

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75 antagonists

Two-pore potassium (K_2P) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Gα_i-coupled hP2Y₁₂ receptor, ADP and ATP significantly inhibited hK_2P2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y₁₂ antagonist AR-C69931MX, the hP2Y₁ antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Gα_q/11-coupled hP2Y₁ receptor, ADP and ATP also inhibited hK_2P2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K_2P2.1 channels, ADP-induced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Gα_q/11- and Gα_i-coupled P2Y receptors inhibit K_2P channels and the action of hP2Y₁₂ receptors appears to involve co-activation of endogenous hP2Y₁ receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity.     

Characterization of a new compound, S-145, as a specific TXA2 receptor antagonist in platelets

S-145, a newly synthesized thromboxane A2 (TXA2) receptor antagonist, was investigated for its effects on platelet TXA2 receptors. In rat washed platelets, S-145 (100 nM) completely suppressed both U46619-induced shape change and collagen-induced shape change and aggregation, whereas aggregation responses provoked by ADP and thrombin were unaffected by S-145. S-145 had no effect on the cAMP levels in rat platelets as well as no inhibitory activity for the bindings of 3H-PGE1, 3H-Iloprost, 3H-PGD2 and 3H-PGF2 alpha to rat platelet membranes. TXB2 formation was not affected by S-145 (10 microM) in rat washed platelets. S-145 antagonized not only the collagen-induced aggregation but also the binding of 3H-U46619 in rat washed platelets with stereospecificity and high potency, which exerted an IC50 of 4.7 nM or a Ki of 2.5 nM, respectively. The potent activity of S-145 in inhibition of 3H-U46619 binding to crude platelet membranes was seen in rat as well as rabbit and human. These results demonstrate that S-145 is a highly potent and selective antagonist for platelet TXA2 receptors.     

Potentiation of platelet aggregation by heparin in human whole blood is attenuated by P2Y12 and P2Y1 antagonists but not aspirin

INTRODUCTION: Unfractionated heparin (UFH) potentiates platelet aggregation induced by some agonists. P2Y12 and P2Y1 receptors play a major role in amplifying platelet aggregation. We assessed the ability of cangrelor, a selective P2Y12 antagonist, A2P5P, a selective P2Y1 antagonist, and aspirin to block the potentiating effects of heparin. MATERIALS AND METHODS: Whole blood from healthy human volunteers was anticoagulated with either hirudin or UFH 10 IU/ml. Some tubes anticoagulated with hirudin also contained UFH 1 or 10 IU/ml. The low-molecular-weight heparin dalteparin was also assessed. Platelet aggregation was performed using whole blood single-platelet counting. Dense granule release was assessed using 14C-5HT-labelled platelets. RESULTS: UFH and, to a lesser extent, dalteparin potentiated platelet aggregation induced by ADP, PAF, 5HT, U46619, epinephrine and TRAP in a concentration-dependent manner but inhibited aggregation induced by collagen. Cangrelor effectively opposed the potentiating effects of heparins on sustained aggregation induced by ADP, PAF, 5HT, U46619 and TRAP but had less effect on epinephrine-induced aggregation, whereas A2P5P was more effective at blocking both the initial phase of ADP-induced aggregation and the aggregation response to epinephrine, reflecting the differences in G protein coupling between the agonist receptors. Aspirin had no effect on potentiation by heparin. Heparins did not increase ADP- or TRAP-induced 14C-5HT release. CONCLUSIONS: Heparins potentiate platelet responses to ADP and numerous other agonists. This potentiation is attenuated by cangrelor and A2P5P, and is not mediated by increased dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins.     

Antagonism of U46619-induced aggregation of human and canine platelets by four TXA2 receptor antagonists

We compared the abilities of four TXA2/PGH2 receptor antagonists, AH 23,848, SQ 29,548, BM 13.177 and BM 13.505, to inhibit aggregation of human and canine platelet rich plasma (PRP) induced by the stable cyclic endoperoxide analog, U46619, alone (human) or in combination with epinephrine (dog). The rank orders of potency of these antagonists, which correlated well between human (h) and canine (c) preparations were [IC50]: SQ 29,548 [28 nM (h) and 92 nM (c)] greater than AH 23,848 [0.5 microM (h) and 0.6 microM (c)] congruent to BM 13.505 [0.4 microM (h) and 0.8 microM (c)] greater than BM 13.177 [3.9 microM (h) and 4.4 microM (c)]. The second wave of aggregation of human PRP induced by epinephrine and platelet activating factor (PAF) was abolished by similar concentrations of the TXA2/PGH2 antagonists, whereas aggregation of canine PRP induced by ADP, serotonin plus epinephrine, or PAF was unaffected by these concentrations of the TXA2/PGH2 antagonists. Epinephrine plus U46619-stimulated aggregation of canine PRP was abolished by RX 781094 (1 microM) but not by prazosin (10(-4) M), selective alpha 2- and alpha 1-adrenoceptor antagonists, respectively. Thus, four selective TXA2/PGH2 receptor antagonists, compared in two species, yield IC50's ranging from 28 nM to 4 microM and nearly identical rank orders of potency.     

Investigation on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.     

Potentiation of thromboxane A2-induced platelet secretion by Gi signaling through the phosphoinositide-3 kinase pathway

Platelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3'phosphate-5'-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T(AC) receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T(AC) receptor or the alpha2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2T(AC) receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.     

Ticlopidine selectively inhibits human platelet responses to adenosine diphosphate.

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.     

Effects of the NHE-1 inhibitor cariporide alone or together with the P2Y12 antagonist AR-C 69331 MX on CD62p expression and formation of platelet-leukocyte aggregates

The sodium-hydrogen exchanger isoform 1 (NHE-1) contributes to platelet activation at elevated pH. Effects of NHE-1 inhibitors on platelet degranulation and formation of proinflammatory and procoagulatory platelet-leukocyte aggregates (PLA) and possible interactions with P2Y(12) inhibitors--which also affect platelet degranulation--have not been investigated. Whole blood from healthy human subjects was incubated with the NHE-1 inhibitor cariporide and the P2Y(12) inhibitor AR-C 69331 MX at clinically reasonable concentrations, in the presence of normal pH or in a propionate model to activate the NHE-1 (approximately pH 7.0). The degranulation marker CD62p, the expression of the activated GPIIb/IIIa receptor (PAC-1), and formation of platelet-leukocyte (monocyte) aggregates (PLA) was assessed by flow cytometry. Cariporide at concentrations up to 20 microg/ml had no effects on ADP- (5 microM) or TRAP- (2 microM) induced CD62p expression or PLA formation at normal pH. At pH 7.0 and stimulation with ADP, PLA decreased from 64+/-24% (control) to 47+/-23% under cariporide at 2 microg/ml (p<0.05), and the MFI of PLA (i.e. the platelet mass attached at monocytes) decreased from 547+/-203 to 360+/-96 units (p<0.05). PAC-1 MFI decreased from 66+/-23 to 34+/-18 units (p<0.05) after ADP and from 74+/-29 to 42+/-17 units (p<0.05) after TRAP, respectively. AR-C 69331 MX (10 nM) had inhibitory effects on all parameters irrespectively of the pH, and the combination of both agents at pH 7.0 shows additive effects. In conclusion, our investigation points to-perhaps clinically relevant-effects of NHE-1 inhibition on the degranulation of platelets and formation of platelet-leukocyte aggregates.     

Characterisation of species differences in the platelet ADP and thrombin response

INTRODUCTION: A number of animal models are used to study platelet-dependent diseases. In the present investigation, we have used a simple flow cytometry assay to evaluate platelet function in man, rat, mouse, guinea pig and dog. MATERIALS AND METHODS: Platelet activation was evaluated in diluted whole blood by measuring fibrinogen binding to activated platelets using a polyclonal anti-human fibrinogen antibody that cross-reacts with fibrinogen from all species tested. The assay was used to evaluate platelet function with respect to ADP and thrombin sensitivity. The relative importance of the two platelet ADP receptors and total ADP in the thrombin response was also studied by using receptor-specific antagonists and apyrase, respectively. RESULTS: Mouse platelets were most sensitive to both agonists. Unlike in man and dog the maximal response to ADP was greater than to thrombin in mouse, rat and guinea pig. P2Y(12) blockade was in all species equally effective as ADP removal in inhibiting thrombin-induced platelet activation whereas P2Y(1) blockade was almost ineffective. CONCLUSION: The present study describes a simple platelet function test that can be used to evaluate platelet function in man, rat, mouse, guinea pig and dog. Platelets from the tested species differed in their sensitivity to ADP and thrombin. In contrast to human and canine platelets, mouse, rat and guinea pig platelets displayed a stronger maximal response to ADP than to thrombin. In terms of the relative contribution of P2Y(1) and P2Y(12) in the thrombin response, the P2Y(12) receptor was the key receptor in all species and its blockade gave equal effect as total removal of ADP.