ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets

Stimulating human platelets with thrombin induces the activation of the extracellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 microM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 microM) and MRS2179 (100 microM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 microM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 microM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via alpha(2A)-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthio-ADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2 -receptor with U46619 (10 microM), which leads to ADP secretion and P2Y12 receptor-dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.     

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.     

Superoxide dismutase cooperates with prostacyclin to inhibit platelet aggregation: a comparative study in washed platelets and platelet rich plasma.

The role of superoxide anions (O2-) in human platelet aggregation in Krebs' buffer or plasma was investigated. In indomethacin (10 microM)-treated washed platelets superoxide dismutase (SOD; 60 U/ml) or ferricytochrome c (FCC; 70 microM) inhibited platelet aggregation by thrombin but not that by collagen or ADP. In addition, in indomethacin (10 microM)-treated washed platelets, SOD significantly potentiated the anti-aggregatory activity of prostacyclin (PGI2) or iloprost when thrombin but not collagen was used as the aggregating agent. In platelet rich plasma, SOD (60 U/ml) did not inhibit platelet aggregation nor did it potentiate the anti-aggregatory activity of iloprost when ADP, collagen or thrombin were used as aggregating agents. Thus, O2- participate in the aggregatory activity of thrombin but not collagen or ADP and PGI2 or iloprost, by reducing the sensitivity of platelets to thrombin, co-operate with SOD to inhibit thrombin-induced platelet aggregation. The interpretation of the use of SOD in experiments involving endothelium-derived relaxing factor (NO) is discussed.     

Antithrombogenic effects of calcium channel blockers: synergism with prostacyclin and thromboxane synthase inhibitors

Four calcium channel blockers (nimodipine, nifedipine, verapamil and diltiazem) of three chemical classes were tested in vitro for inhibition of platelet aggregation using heparinized human platelet rich plasma. Both ADP- and thrombin-induced aggregation were inhibited as was the biosynthesis of thromboxane A2 in response to ADP or thrombin. However, the IC50's for the calcium channel blockers were greater than or equal to 110 microM. Nimodipine was also tested in combination with prostacyclin, the potent platelet antiaggregatory agent, or with a thromboxane synthase inhibitor, U63557A. At concentrations at which neither nimodipine or prostacyclin inhibited platelet aggregation greater than or equal to 10%, the two compounds is combination synergistically inhibited both ADP- and thrombin-induced platelet aggregation. U63557A inhibited biosynthesis of thromboxane A2 by platelets in response to ADP or thrombin, but did not inhibit either ADP- or thrombin-induced platelet aggregation. However, U63557A in combination with a threshold inhibitory concentration of nimodipine resulted in a synergistic inhibition of platelet aggregation induced by ADP or thrombin. These results suggest that calcium channel blockers may be of therapeutic value as a new class of antithrombogenic agents when used in combination with agents that inhibit either platelet aggregation or synthesis of platelet thromboxane A2.     

Pharmacological profile of G619, a new platelet aggregation inhibitor.

G619, a 4-OH-isophthalic acid derivative, was studied for its capacity to inhibit platelet aggregation. G619 dose-dependently inhibited U46619, collagen, ADP, PAF, thrombin and epinephrine-induced platelet aggregation in vitro. The IC50 values for inhibition of U46619-induced human and rabbit platelet aggregation were 39 and 43 microM, respectively. G619, at 100 microM, inhibited high concentration collagen (10 micrograms/ml)-induced aggregation of rabbit platelets pretreated with indomethacin and increased the level of cAMP in washed rabbit platelets by 30% (p less than 0.01 vs basal). However, G619, did not inhibit fibrinogen binding to GPIIb/IIIa receptor, phosphodiesterase, U46619-induced contractile responses on canine saphenous vein or rabbit aorta, calcium-induced vasoconstriction and thrombin or PAF-induced elevation of [Ca++]i in platelets in vitro. In vivo, the U46619-induced maximal thrombocytopenia in rats was reduced from 40% (vehicle) to 22% and 18% by 10 and 30 mg/kg of G619 i.v., respectively. G619 (30 mg/kg) had no effect on the U46619-induced vasopressor response or sudden death in rats, and had no effect on TxB2 formation. Our results indicate that G619 is a broad-spectrum platelet aggregation inhibitor and may have its effect on a common mechanism for platelet aggregation besides an effect on the thromboxane A2 receptor.     

Effect of hyperosmolarity on agonist-induced increases of intracellular calcium in human platelets

BACKGROUND: Hypertonic solutions are useful for the management of hypovolemic shock but cause impairment in platelet function. This effect would reduce the ischemia/reperfusion damage caused by activated platelets, but it could be the cause of aggravating blood loss in case of uncontrolled hemorrhage. In this paper, it was studied if osmotic shrinkage of platelets affects the changes in intracellular calcium concentration ([Ca(2+)](i)) induced by thrombin or adenosine 5' diphosphate (ADP). Furthermore, it was investigated if hypertonic solutions change the mobilization from intracellular stores or the calcium entry. Previous reports from our laboratory described that the capacitative calcium entry is increased by alkalization and also that hyperosmolarity has an alkalinizing effect on human platelets so it was hypothesized that hyperosmolarity would be able to modify agonist-induced calcium entry. MATERIAL AND METHODS: To study the response to agonists, platelets loaded with 2'7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (FURA 2) were incubated at 37 degrees C for 500 s in a N-2-hidroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution with 1 mM CaCl(2). The osmolarity of the solution was elevated by the addition of 200 mM mannitol or sucrose and after the stimulation with 0.1 IU/ml of thrombin or 100 microM ADP, [Ca(2+)](i) increases were compared. Platelets incubated in zero calcium/EGTA were stimulated with these agonists or with 0.1 microM thapsigargin to separately study the effect of hyperosmolarity on both calcium mobilization from intracellular stores and extracellular calcium entry. RESULTS: It was found that hypertonic solutions decrease the [Ca(2+)](i) peak induced by the agonist: The increase of [Ca(2+)](i) in the presence of 200 mM mannitol produced by 100 microM ADP was 62+/-6% and response to 0.1 IU/ml thrombin was 74+/-7% of the increase in isotonic control solution. In the case of ADP, both mobilization and calcium entry were reduced to 66+/-3% and 65+/-6% of isotonic control, respectively. In the case of thrombin, only the mobilization showed a significant change (79+/-2% of parallel control). In platelets depleted by thapsigargin, the capacitative calcium entry was increased in hypertonic mannitol (174+/-25% of the isotonic control). Similar results were obtained with hypertonic sucrose solutions. CONCLUSIONS: In spite of the stimulatory effect of hyperosmolarity on capacitative calcium entry observed in platelets in which the calcium stores were depleted with thapsigargin, the full response of intracellular calcium to the agonists tested (ADP and thrombin) was reduced by an increase in osmolarity. The decreased Ca(2+) mobilization observed may play a role in the reduction in hypertonic media of accompanying platelets responses such as aggregation or shape change.     

Ouabain affects platelet reactivity as measured in vitro

Ouabain, a digitalis glycoside and an inhibitor of the Mg2+-dependent Na+-K+ ATPase, was used to probe the role of intracellular Na+ levels in the regulation of platelet reactivity. Platelets preincubated with 10 to 150 microM ouabain exhibited a potentiated aggregation response to collagen (14.4 to 180 micrograms/mL), ADP (4 to 12 microM) and thrombin (0.03 to 0.10 unit/mL). Ouabain markedly decreased the time interval between addition of collagen and the onset of shape change. At submaximal concentrations of collagen, thrombin and ADP, preincubation with ouabain increased the rate and amplitude of the aggregation response. Irreversible aggregation was achieved in ouabain-treated platelets by using concentrations of ADP which induced only reversible aggregation in the absence of ouabain. In addition, chelation of extracellular calcium with EGTA or EDTA (2 mM) failed to block reactivity to collagen, ADP or thrombin in ouabain-treated platelets. These results suggest that ouabain induces a "preactivation state" in platelets, perhaps via modulation of intracellular Na+ levels.     

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.     

In vitro studies of a new synthetic thrombin inhibitor

(2R, 4R)4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8-quinoline- sulfonyl)-L-arginyl]-2-piperidine carboxylic acid monohydrate (MCI-9038) was found to be a potent synthetic inhibitor of thrombin. In concentrations as low as 1 microM, the thrombin time, prothrombin time, and partial thromboplastin time were more than doubled. The venom (Bothrops atrox) time was similarly prolonged. The drug also inhibited the thrombin-induced activation of factors VIII and XIII. While MCI-9038 in concentrations of 10(-4) M had no effect on platelet aggregation induced by collagen, ADP, epinephrine, and arachidonate, nanomolar concentrations inhibited thrombin-induced platelet aggregation and the release of platelet ADP. The drug also significantly inhibited the adhesion of thrombin-treated platelets to cultured bovine aortic endothelial cells. We conclude that MCI-9038 is an extremely potent inhibitor of the effects of thrombin on platelets and clotting factors.     

Inhibitory mechanisms of dantrolene on platelet aggregation

The antiplatelet effect of dantrolene and possible inhibitory mechanisms were studied in rabbit platelets. Preincubation of rabbit washed platelets with dantrolene (50-300 microM) inhibited the platelet aggregation and adenosine triphosphate release induced by arachidonic acid (100 microM), collagen (10 microg/mL), or thrombin (0.1 U/mL) in a dose-dependent manner. The thromboxane B2 formation caused by collagen or thrombin was inhibited by dantrolene, while formation of thromboxane B2 and prostaglandin D2 induced by arachidonic acid were not inhibited. In addition, the formation of phosphoinositide breakdown and the rise of intracellular calcium level induced by collagen or thrombin were also inhibited in a dose-dependent manner by dantrolene in the presence of indomethacin (2 microM). However, the platelets cyclic AMP level was not affected by dantrolene. In conclusion, the present study demonstrates that dantrolene inhibits platelet activation mainly due to suppression of phosphoinositide breakdown.     

Effect of dipyridamole on spontaneous platelet aggregation in whole blood decreases with the time after venepuncture: evidence for the role of ADP

Spontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40, and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37 degrees C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 microM at each time interval; (b) 0.5-100 microM at 3 and 30 minutes and (c) 15 microM in combination with 100 microM adenosine, 8 microM 2-chloroadenosine (2Clad, an ADP receptor blocker) and 50 microM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 microM Dipy and 10 microM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 microM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.     

The novel effect of a new prostacyclin analogue ZK36374 on the aggregation of human platelets in whole blood

Platelet aggregation was studied at 37 degrees C in citrated whole human blood, using the Ultra Flo 100 Whole Blood Platelet Counter. Aggregation was measured as a fall in the number of single platelets following addition of an aggregating agent. At peak aggregation, the fall in the number of platelets induced by ADP (10 microM), collagen (1 microgram/ml) or thrombin (0.2 U/ml) was about 90%. When blood was incubated with the prostacyclin-analogue ZK36374, the aggregation responses to ADP, collagen and thrombin were reduced with IC50's = 0.5, 1.5 and 3 nM respectively and the corresponding IC100's were: 1, 3 and 12 nM. When ZK36374 was added at peak aggregation, the number of single platelets increased significantly due to disaggregation of preformed platelet aggregates. It is concluded that the present technique represents a rapid, sensitive and more physiological approach for investigating the effects of pharmacological agents on platelet aggregation.     

Inhibition of platelet aggregation by ethanol in vitro shows specificity for aggregating agent used and is influenced by platelet lipid composition

Ethanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.     

Differential effects of dietary supplementation with fish oil or soy lecithin on human platelet adhesion

To investigate the possible regulating role of omega-6 and of omega-3 fatty acids on platelet adhesiveness, we randomised 60 volunteers into three groups to take 20 ml (equivalent to 0.3 g omega-6, 3.6 g omega-3; omega-6/omega-3 ratio 0.1) per day of a fish oil supplement, or to take 25 g (equivalent to 1.5 g omega-6, 0.5 g omega-3; omega-6/omega-3 ratio 3) per day of a soy lecithin supplement, or to continue on their usual diet without any supplement (control group) for a period of 15 days. Platelet adhesion on fibrinogen-coated 96-well microtitre plates was evaluated in the resting condition and after stimulation with 2 microM ADP or 0.02 U/ml thrombin. Compared to the values before the experimental period, the fish oil group showed a significant reduction in stimulated adhesion (with ADP: from 18.8% to 15.6%, p<0.01; with thrombin: from 24.4% to 20.8%, p<0.005), whereas no difference was noted in the resting condition (from 3.6% to 3.5%, NS). In the soy lecithin group, platelet adhesion was increased in all test conditions (with ADP: from 18.7% to 23.2%, p<0.001; with thrombin: from 24.0% to 29.9% p<0.001; resting: from 3.5% to 6.6%, p<0.001). No significant changes were observed in the control group. A good correlation was found between platelet adhesion data and the changes in the platelet fatty acid omega-6/omega-3 ratio caused by the different supplementations. Our results indicate an inhibitory effect of fish oil rich in omega-3 fatty acids on stimulated human platelet adhesiveness and a stimulatory effect of soy lecithin rich in omega-6 fatty acids on resting and stimulated adhesion. They suggest moreover that the omega-6/omega-3 ratio is a determinant of platelet adhesion.     

Antiplatelet effects of protopine isolated from Corydalis tubers

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.     

gammaA/gamma' fibrinogen inhibits thrombin-induced platelet aggregation

The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.     

Synergism between platelet aggregating agents: the role of the arachidonate pathway.

Aggregation of platelets by low concentrations of ADP is augmented by non-aggregating concentrations of collagen, thrombin, arachidonate or the divalent cation ionophore A23,187. Release-inducing agents act synergistically with ADP and with each other. Both collagen and thrombin cause aggregation by releasing ADP and by freeing platelet arachidonate to form prostaglandin endoperoxides which give rise to thromboxane A₂. In these experiments the role of the arachidonate pathway in the synergism between pairs of aggregating and release-inducing agents was examined. Indomethacin was used to inhibit conversion of arachidonate to prostaglandin endoperoxides and thromboxane A₂ and creatine phosphate/creatine phosphokinase (CP/CPK) was used in some experiments to convert released ADP to ATP. Synergism of collagen with ADP, arachidonate or thrombin was inhibited by indomethacin indicating that the arachidonate pathway plays a major role in the synergistic effects to which collagen contributes. Synergism of thrombin with collagen or arachidonate was inhibited by indomethacin but synergism of thrombin with ADP was only slightly affected. Indomethacin had little influence on the combined effects of these two agents on platelet aggregation. Thus it appears that the conversion of platelet arachidonate to prostaglandin endoperoxides and thromboxane A₂ plays a minor part in the synergistic effects in which thrombin or A23,187 are involved. Thus, the non-steroidal anti-inflammatory drugs may have only limited use in inhibiting the contribution of thrombin and ADP to the formation of platelet thrombi at sites of vessel injury.     

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.     

Analysis of platelet alpha2-adrenergic receptor activity in stable coronary artery disease patients on dual antiplatelet therapy

Combined antiplatelet therapy reduces recurrent atherothrombotic events in stable coronary disease patients; however, high residual platelet reactivity measured ex vivo still raises concerns as a condition related to treatment failure. Alpha-2 adrenoceptor enhances platelet reactivity and might contribute to this phenomenon. For the present study, 121 stable angina patients on standard dual antiplatelet therapy (75 mg clopidogrel and 100 mg acetylsalicylic acid) were recruited. Born aggregometry was performed with adenosine diphosphate (ADP), collagen and epinephrine. To verify platelet adrenergic activity, potentiation by low-dose epinephrine and inhibition by selective alpha-2 receptor blocker atipamezole were determined. To assess the P2Y(12)-specific residual activity, cangrelor was used. Plasma norepinephrine, soluble CD40-ligand, high-sensitivity-C-reactive protein (hsCRP) - and in 24 subjects platelet P-selectin positivity were measured. Epinephrine - at very low concentration (10(-9)g/ml) - significantly potentiates (1.25 microM ADP: 26.5% vs. 43%; 5 microM ADP: 53% vs. 64.5%; collagen: 17% vs 42%, p < 0.001) while atipamezole inhibits ADP- and collagen-induced platelet aggregations (1.25 microM ADP: 26.5% vs. 23%; 5 microM ADP: 53% vs. 47%; collagen: 17% vs. 11%, p < 0.001). Patients with high adrenergic activity have significantly increased baseline ADP- and collagen-induced platelet aggregation. Based on cangrelor's efficacy, these patients have significantly more residual P2Y(12) activity as well. HsCRP and soluble CD40-ligand levels were similar. In conclusion, stable coronary heart disease patients with prominent adrenoceptor activity in vitro have significantly increased platelet aggregability and more functional P2Y(12) receptor, indicating poor inhibitory response to thienopyridines. Therefore, platelet adrenergic receptor represents a considerable, dynamic factor of high residual platelet reactivity and might contribute to cardiovascular events indicating failure of antiplatelet therapy.     

Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of ³H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.