The effects of GPIIb-IIIa antagonists and a combination of three other antiplatelet agents on platelet-leukocyte interactions

The effects of the GPIIb-IIIa antagonists abciximab and MK-852 on platelet-leukocyte interactions in vitro were studied and the results compared with those obtained with a combination of aspirin, dipyridamole and AR-C69931 (Asp/Dip/AR-C). Platelet-monocyte (P/M) and platelet-neutrophil (P/N) conjugate formation increased when blood was stirred or a platelet agonist was added. Leukocyte activation also occurred as judged by expression of surface tissue factor antigen and CD11b. Abciximab and MK-852 potentiated P/M, especially when collagen was used. They also increased the amount of tissue factor on the monocytes, but not CD11b. The Asp/Dip/AR-C did not enhance P/M or tissue factor exposure. Augmented tissue factor expression on monocytes in the presence of a GPIIb-IIIa antagonist may be relevant to the increased mortality associated with trials of such antagonists when given orally in patients with vascular disease. The Asp/Dip/AR-C was superior to abciximab and MK-852 in inhibiting platelet and leukocyte function.     

Effects of aspirin,dipyridamole, nifedipine and cavinton which act on platelet aggregation induced by different aggregating agents alone and in combination

Summary The effects of aspirin, nifedipine, dipyridamole and cavinton on platelet aggregability in patients with atherosclerosis has been studied using various agents to induce aggregation.The drugs reduced platelet aggregability when aggregation was induced by ADP, adrenaline, or collagen alone. However, if platelet aggregation were induced by combinations of the agonists (including combinations of ADP with either adrenaline or platelet-activating factor (PAF), adrenaline with PAF, and collagen with ADP), the anti-aggregant effects of aspirin, dipyridamole, and cavinton were significantly reduced. The effect of nifedipine was less markedly reduced, especially by combinations which included adrenaline.The data suggest that positive agonist interactions may lead to a reduction in the therapeutic activity of antiplatelet drugs.     

Effects of plumbagin on platelet aggregation and platelet-neutrophil interactions

The effects of plumbagin were investigated on platelet aggregation in vitro and ex vivo, on the binding of thrombin-stimulated platelets to neutrophils, and platelet aggregation induced by intact neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF) activated neutrophils, by use of the methods of Hamburger, McEver and Born, respectively. The results showed that plumbagin in vitro significantly inhibited adenosine diphosphate (ADP)-, arachidonic acid (AA)-, or platelet activating factor (PAF)-induced platelet aggregation, in a concentration-dependent manner. The medium inhibitory concentrations (IC 50 ) were 39.4, 82.7 and 38.1 microM, respectively. Intragastric plumbagin at 10 mg/kg markedly suppressed platelet aggregation induced by ADP, AA, or PAF. Plumbagin decreased the binding between thrombin-stimulated platelets and neutrophils with an IC 50 of 62.9 microM. Plumbagin significantly inhibited washed platelet aggregation stimulated by fMLP- or PAF-activated neutrophils. The IC 50 values were 54.3 and 47.6 microM, respectively. On the other hand, plumbagin and aspirin increased the inhibition of intact neutrophils on AA-induced platelet aggregation. It is suggested that plumbagin inhibited platelet aggregation in vitro and ex vivo, suppressed the binding of activated platelets to neutrophils, inhibited platelet aggregation induced by activated neutrophils, and increased inhibition of intact neutrophils on platelet reactivity. Abbreviations. DMSO:dimethyl sulphoxide fMLP: N-formyl-methionyl-leucyl-phenylalanine ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet activating factor     

On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP

Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2-(methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor-selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner.     

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

三乙酰莽草酸对凝血酶和局灶性脑缺血再灌注诱导的血小板与白细胞粘附的作用及其机制

目的：研究三乙酰莽草酸（TSA）对凝血酶和局灶笥脑缺血再灌注诱导的血小板与中性粒细胞粘附及活化血小板膜P-选择素表达的作用。方法：观察玫瑰花环形成率作为血小板与中性粒细胞粘附率指标,并用流式细胞仪测定血小板表达P-选择素的表达。结果：TSA10-1000μmol/L可明显抑制凝血酶0.4kU/L诱导的血小板与中性粒细胞的粘附。TSA50-200mg/kg剂量依赖性抑制大脑中动脉阻断3h再灌注21h引起的血小板与中性粒细胞的粘附。TSA可明显抑制凝血酶诱导的血小板P-选择素的表达和ADP5μmol/L诱导的全血中血小板膜表面P-选择素的表达。结论：脑缺血再灌注和凝血酶引起血小板和中性粒细胞粘附,TSA对P-选择素表达的影响可能是其抑制血小板与中性粒细胞粘附的重要机制。     

Antiplatelet aggregation activity of diterpene alkaloids from Spiraea japonica

Six diterpene alkaloids with an atisine-type C(20)-skeleton isolated from the Chinese herbal medicines Spiraea japonica var. acuta and S. japonica var. ovalifolia, as well as eight derivatives of spiramine C and spiradine F were evaluated for the ability to inhibit aggregation of rabbit platelets induced by arachidonic acid, ADP, and platelet-activating factor (PAF) in vitro. The results showed that 12 of the 14 atisine-type diterpene alkaloids significantly inhibited PAF-induced platelet aggregation in a concentration-dependent manner, but had no effect on ADP- or arachidonic acid-induced aggregation, exhibiting a selective inhibition. It is the first report that C(20)-diterpene alkaloids inhibit PAF-induced platelet aggregation. However, spiramine C1 concentration-dependently inhibited platelet aggregation induced by PAF, ADP and arachidonic acid with IC(50) values of 30.5+/-2.7, 56.8+/-8.4 and 29.9+/-9.9 microM, respectively, suggesting a non-selective antiplatelet aggregation action. The inhibitory effect of spiramine C1 on arachidonic acid was as potent as that of aspirin. Primary studies of the structure-activity relationships for inhibition of PAF-induced aggregation showed that the oxygen substitution at the C-15 position and the presence of an oxazolidine ring in spiramine alkaloids were essential to their antiplatelet aggregation effects. These results suggest that the atisine-type alkaloids isolated from S. japonica are a class of novel antiplatelet aggregation agents.     

Effect of dipyridamole and aspirin on the platelet-neutrophil interaction via the nitric oxide pathway

This study was designed to determine the influence of the combination of aspirin and dipyridamole on the interaction in vitro between neutrophils and platelets through the nitric oxide (NO) pathway. Collagen-induced platelet aggregation (impedance method) was determined in platelet-rich plasma and in platelet-rich plasma+neutrophils, and cGMP (enzyme immunoanassay) and NO levels (electrochemical method, with a ISO-200 electrode) were also measured. The 50% inhibitory concentration (IC(50)) of aspirin was 139+/-11 microM in platelet-rich plasma, 367+/-21 microM in platelet-rich plasma+L-N(G)-nitro-arginine-methyl-ester (L-NAME), and 42+/-3 microM in platelet-rich plasma+L-arginine. The IC(50) for dipyridamole in platelet-rich plasma was not affected by L-NAME or L-arginine; the combination of aspirin with 20 microM dipyridamole (which has no effect per se) led to an IC(50) of 51+/-2 microM in platelet-rich plasma, 101+/-7 microM in platelet-rich plasma+L-NAME, and 13+/-2 microM in platelet-rich plasma+L-arginine. The cGMP levels showed the greatest increases in the aspirin plus dipyridamole group. Dipyridamole and aspirin increased the leukocyte production of NO: 50% increases were obtained at concentrations of 285+/-31 microM aspirin, 110+/-9 microM dipyridamole, and 16+/-2 microM aspirin+dipyridamole. Dipyridamole alone at a concentration of 20 microM had no significant effect on NO levels. We conclude that the combination of aspirin and dipyridamole significantly increases the antiplatelet effect of leukocytes, through an increase of NO, and that this effect is further evidence of the therapeutic benefits of this combination of drugs.     

Differential effect of platelet inhibitors in normal and in hypercholesterolaemic subjects.

Dibutyryl cyclic AMP, forskolin, dipyridamole and butyl imidazole inhibited platelet aggregation (induced by ADP or collagen) in washed platelets more than in platelet-rich plasma preparations. Aspirin, indomethacin and epoprostenol (prostacyclin, PGI2) showed no preferential inhibition of these platelet preparations. When platelet-rich plasma from either normal or familial hypercholesterolaemic (FH) subjects was used, aspirin, indomethacin and dipyridamole (but not forskolin) inhibited platelet aggregation in normal subjects more than in FH patients. When low doses of aspirin (75 mg daily for 7 days) or dipyridamole (250 mg, single dose) were administered in vivo, platelet aggregation was inhibited more in the normal subjects in comparison to the patient group.     

Antiplatelet drugs: clinical pharmacology and therapeutic use.

Because platelets are so important in thrombus formation, drugs which inhibit platelet function (the 'antiplatelet drugs') have considerable potential as antithrombotic agents. Among the antiplatelet drugs, only aspirin, sulphinpyrazone, dipyridamole, hydroxychloroquine, and clofibrate have had wide clinical trial. Their effects on platelet metabolism differ. Aspirin prevents platelet prostaglandin synthesis by acetylating and irreversibly inactivating platelet prostaglandin synthetase, while sulphinpyrazone is a reversible inhibitor of the same enzyme. Both aspirin and sulphinpyrazone impair the platelet release reaction and reduce platelet aggregation, but neither prevents platelet adhesion to the subendothelium or to foreign surfaces. On the other hand, dipyridamole reduces platelet adhesion as well as aggregation, probably by inhibiting phosphodiesterase and so raising platelet cyclic AMP levels. The effects of hydroxychloroquine and clofibrate have been less well defined. As the antiplatelet drugs form a diverse group of substances with differing effects on platelet function, it is hardly surprising that every potential clinical application of each antiplatelet drug or drug combination has had to be tested separately, and that these drugs have not proved to be equally effective. One or more antiplatelet drugs have now been evaluated in each of the following situations.     

In-vitro and Ex-vivo Inhibition of Blood Platelet Aggregation by Naftazone

Because of the considerable interest in the role of platelets and antiplatelet therapy in cardiovascular disease, including the aggregation of platelets to each other during arterial thrombosis and atherogenesis, we have studied the effect of naftazone (Etioven), an original vasculotropic drug on platelet aggregation. Rat and human platelets were prepared and incubated in-vitro with different concentrations of naftazone. We found that naftazone inhibited both platelet secretion and aggregation in platelet-rich plasma (PRP) and washed platelets after stimulation with thrombin or ADP. Rats were also treated intraperitoneally for five days with various naftazone doses (0.125-10 mg kg^?1) and ex-vivo platelet aggregation compared, at various times after the last injection, with that of control animals. Inhibition by naftazone was dose-dependent in both PRP and isolated platelets. The inhibition was transient, a maximum value (～ 50%) being obtained about 3–6 h after the last injection, with a return to near-control values after 24 h. Naftazone also facilitated platelet deaggregation after in-vitro stimulation with thrombin or ADP. In another series of experiments, rats were treated intraperitoneally for five days with 10 mg kg^?1 of aspirin, ticlopidine, dipyridamole or naftazone. Platelets were prepared and tested for aggregation 90 min after the last injection. Thrombin-induced aggregation in PRP and washed platelets was significantly reduced after in-vivo treatment with ticlopidine and naftazone. Except for dipyridamole, all the drugs inhibited ex-vivo ADP-induced aggregation in PRP. In isolated platelet preparation, only naftazone induced a significant inhibition of ADP-or thrombin-stimulated aggregation. We conclude that naftazone inhibits platelet aggregation in-vitro and ex-vivo.     

Effect of 2(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca⁺⁺, [¹²⁵I]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca⁺⁺ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.     

牛心内膜腺苷三磷酸双磷酸酶对血小板聚集的抑制作用

AIM: To study the anti-aggregatory effect of bovine endocardial endothelial cell (EEC)-associated apyrase. METHODS: Cultured bovine EEC was used. Adenosine diphosphate (ADP) was analyzed by reversed phase HPLC, and rabbit platelet aggregation was measured turbimetrically. RESULTS: Incubation of EEC with ADP 500 mumol.L-1 resulted in a progressive decrease in ADP concentration, which was paralleled by the decrease in platelet aggregating potential of the unmetabolized ADP. In the presence of aspirin (Asp 1 mmol.L-1)-treated EEC 1 x 10(9) cells.L-1, the aggregation of Asp (1 mmol.L-1) and methylene blue (10 mumol.L-1)-treated platelets in response to thrombin 500 U.L-1 and platelet activating factor (PAF 1 nmol.L-1) was markedly inhibited and was reversible, which was very similar to that in apyrase-treated platelets. The supernatants of EEC had no effect on platelet aggregation. EEC inhibited ADP (5 mumol.L-1)-induced platelet aggregation, but failed to inhibit adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S, an unmetabolizable structural analog of ADP, 15 mumol.L-1)-induced platelet aggregation. CONCLUSION: ADP hydrolysis by EEC-associated apyrase is a major anti-thrombotic mechanism of bovine EEC.     

Attenuation of the P2Y receptor-mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs

1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.     

Recent advances in antiplatelet agents

Platelet aggregation plays a key role in the pathogenesis of thromboembolic diseases such as myocardial infarction, stroke, unstable angina and peripheral artery disease. Until recently, aspirin was the only antiplatelet agent available to prevent or treat these events. Over the past several years, there has been a substantial expansion in the antiplatelet armamentarium as well as in the understanding of the clinical importance of antiplatelet therapy in limiting the complications of thrombosis. Aspirin was one of the first agents to be adopted and it remains as the standard therapy with the higher amount of available clinical information. Following aspirin, ADP receptor antagonists like ticlopidine and clopidogrel as well as phosphodiesterase inhibitors dipyridamole and cilostazol have been introduced. Glycoprotein (GP) IIb/IIIa receptor antagonists like eptifibatide, tirofiban and abciximab are the newer antiplatelet agents which act at the end of the common pathway of platelet aggregation. Although results of clinical studies with the first oral GPIIb/IIIa antagonists were disappointing, agents of the new generation might expand the potential application of GPIIb/IIIa targeted therapy. This review will highlight recent advances in the development of aspirin, phosphodiesterase inhibitors, ADP receptor antagonists and the platelet glycoprotein IIb/IIIa inhibitors. The emphasis of this paper has been placed on the chemical aspects of these agents.     

酸枣仁皂苷A的抗血小板聚集活性研究

目的 研究酸枣仁皂苷A的抗血小板聚集活性。方法 采用兔体外血小板聚集实验方法,结合酶联免疫吸附法测定血清血小板活化因子（platelet activating factors,PAF）、P-选择素和血小板因子-4（platelet factor-4,PF-4）的含量,探讨不同浓度的酸枣仁皂苷A对二磷酸腺苷（adenosine diphosphate,ADP）、花生四烯酸（arachidonic acid,AA）和PF-4诱导血小板聚集的抑制活性。结果 酸枣仁皂苷A能有效抑制AA、ADP和PF-4诱导的血小板聚集,能显著降低PAF、P-选择素和PF-4的含量。结论 酸枣仁皂苷A具有明显的抗血小板聚集活性。     

ADP receptors--targets for developing antithrombotic agents

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Inhibition of NAD(P)H oxidase attenuates aggregation of platelets from high-risk cardiac patients with aspirin resistance

Up to one-third of serious vascular events in high-risk patients is attributable to a failure of aspirin (ASA) to suppress platelet aggregation. We hypothesized that inhibition of NAD(P)H oxidase may inhibit aggregation of platelets from ASA-resistant (ASA-R) patients. Thus, platelet-rich plasma was isolated from ASA-sensitive (ASA-S) and ASA-R patients (aspirin resistance was defined as higher than expected aggregation to collagen and epinephrine [> or = 40%] after chronic oral treatment with 100 mg/day ASA). Aggregation to adenosine diphosphate (ADP) (5 and 10 micromol/l), collagen (2 microg/ml) and epinephrine (10 micromol/l) in the absence and presence of the NAD(P)H oxidase inhibitors: diphenylene iodonium (DPI) (1 micromol/l) and apocynin (3 x 10(-4) mol/l) was measured by optical aggregometry. Maximal aggregation of ASA-R platelets to collagen and epinephrine was significantly decreased by DPI and apocynin, whereas they had no effect in ASA-S platelets. Maximal aggregation to ADP was unaffected by NAD(P)H oxidase inhibition in either group. In ASA-R platelets both NADPH-driven O2(.-) production (lucigenin chemiluminescence assay) and expression of gp91phox and p67phox subunits of the NADPH oxidase (Western blotting) tended to increase. Collectively, inhibition of NAD(P)H oxidase effectively suppressed collagen and epinephrine-induced aggregation of platelets from ASA-R patients, which may represent a novel pharmacological target for cardioprotection in high-risk cardiac patients.     

Current issues in thrombosis prevention with antiplatelet drugs

In this review, the major current problems related to the pharmacology and clinical use of antiplatelet drugs are discussed in relation to the physiopathology of the platelet-vessel wall interaction and arterial thrombus formation. Although platelet adhesion to injured vessels is a crucial step in thrombogenesis, none of the currently used antiaggregating drugs prevents this phenomenon. Why the normal endothelium does not react with platelets is not known. Thus we are unable to pharmacologically restore endothelial 'non-thrombogenicity' when lost by single or repeated injury. In contrast, more information is available on the mechanisms controlling and amplifying platelet activation by physiological stimuli (such as collagen and thrombin), and on their pharmacological modulation. The 3 main amplification loops involve arachidonic acid metabolism, ADP release and possibly the availability of a phospholipid platelet activating factor. These pathways are in turn activated by the phosphatidylinositol cycle. The most widely used antiaggregating drug is aspirin. It prevents the formation of arachidonic acid metabolites both in platelets and in vascular cells. The use of low-dose aspirin, thromboxane-synthase inhibitors, thromboxane receptor antagonists, epoprostenol (prostacyclin) and its stable analogues, and ticlopidine all appear to be promising pharmacological approaches, but none has so far been tested in clinical trials for thrombosis prevention. On the other hand, aspirin (in relatively large doses of 300 to 1500 mg daily), sulphinpyrazone and dipyridamole have been tested alone or in combination in the secondary prevention of thromboembolic complications. Aspirin has significantly reduced both the occurrence of myocardial infarction and mortality rate in patients with unstable angina and/or previous myocardial infarction; it has also proved beneficial in cerebrovascular disease. The beneficial effect of aspirin was dose-independent. In some of these trials aspirin was combined with either dipyridamole or sulphinpyrazone. When used alone, the latter compound has reduced sudden death or thromboembolic complications in patients with myocardial infarction. It remains to be established whether antiplatelet therapy may prevent or stop the progression of atherosclerosis.