Aspirin resistance: definitions, mechanisms, prevalence, and clinical significance

Aspirin is the most commonly used therapeutic agent in prevention of vascular ischemic events. Aspirin exerts its antithrombotic effect primarily by interfering with the biosynthesis of thromboxane A2 (TXA2) and inhibition of TXA2 -dependent platelet aggregation. A meta-analysis of secondary prevention trials indicated that aspirin reduced major cardiovascular or cerebral events by 25%. This led to the widespread use of aspirin for prevention of cardiovascular events. However, it appears that aspirin antiplatelet effect is not uniform in all patients and previous studies estimated that 8-45% of the population were aspirin resistant. Furthermore, (i) the optimal dosage of aspirin for complete inhibition of platelet aggregation by physiological agonists (i.e arachidonic acid) is subject to great interindividual variability, (ii) the tests to detect aspirin resistance in vitro are subject to debate and (iii) the mechanisms by which some patients are resistant to aspirin in vitro remain to be determined. Despite these unresolved questions, recent clinical studies provide the reliable evidence that aspirin resistance correlates with confirmed clinical unresponsiveness, highlighting the clinical interest of determining the aspirin inhibitory effects on patients' platelets. In conclusion, discovery of aspirin resistance in individuals might be important in order to devise better anti-platelet strategies and improve our ability to prevent acute thrombotic complication.     

Sex difference in the effect of aspirin on intracellular Ca2+ mobilization and thromboxane A2 production in rat platelets

The intracellular Ca2+ mobilization in thrombin-stimulated platelets was greater in male rats than in female rats. Thromboxane (TX) B2 production in male platelets was greater than that in female platelets. Aspirin suppressed Ca2+ mobilization in rat platelets, but the inhibitory effect of aspirin was more efficient in males than that in females. Aspirin inhibited TXB2 production, and this inhibitory effect of aspirin was stronger in male platelets than in female platelets. Castration decreased Ca2+ mobilization and TXB2 production and weakened the effect of aspirin on them. It is suggested that the sex difference in the antiplatelet effect of aspirin results from the difference in the inhibition of Ca2+ mobilization via the inhibition of TXA2 production in thrombin-stimulated rat platelets.     

G-protein dependent platelet signaling--perspectives for therapy

Platelet activation and aggregation is an integral component of the pathophysiology that leads to thrombotic and ischemic diseases such as cerebral stroke, peripheral vascular disease and myocardial infarction. Anti-platelet agents (such as aspirin, ADP receptor antagonists, and GPIIb/IIIa antagonists), phosphodiesterase inhibitors and anti-coagulants are major part of the current treatment towards treating ischemic diseases. However, their limited efficacy in the setting of arterial thrombosis, unfavorable side effect profile and cost-to-benefit issues substantiate the need for the development of newer and more efficacious antithrombotic drugs. Various platelet agonists like adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2) activate platelets by acting via their respective surface receptors, which couple to one or more distinct G-proteins belonging to either the G(i), G(q), G(12/13) or G(s) families. Upon activation, each of these G-proteins trigger a series of intracellular signaling cascades, causing the platelets to undergo shape change, secrete their granular contents, generate positive feedback mediators and form stable platelet aggregates. In addition, various G-protein-mediated signaling cascades act in synergy with one another to amplify the magnitude of the platelet responses. The significance of G-proteins as key mediators of the platelet function and normal hemostasis is further corroborated by extensive gene knockout studies. In this review we will limit our discussion to understanding the role of G-proteins in the process of platelet activation and discuss some of the anti-thrombotic drugs that mediate their beneficial effects by interfering with or preventing the initiation of the G-protein signaling pathway.     

Acyclic analogues of adenosine bisphosphates as P2Y receptor antagonists: phosphate substitution leads to multiple pathways of inhibition of platelet aggregation

Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.     

Effect of thromboxane A2 synthetase inhibition, singly and combined with thromboxane A2/prostaglandin endoperoxide receptor antagonism, on inositol phospholipid turnover and on 5-HT release by washed human platelets

Differential effects on human platelet function of thromboxane A2 (TXA2) synthetase inhibition singly and of TXA2 synthetase inhibition combined with TXA2/prostaglandin endoperoxide receptor antagonism were revealed, using ridogrel as a probe. Ridogrel combines selective TXA2 synthetase inhibition with TXA2/prostaglandin receptor antagonism in one molecule: in washed human platelets, the compound reduces the production of TXB2 (IC50 = 1.3 X 10(-8) M) and increases that of PGF2 alpha, PGE2, PGD2 from [14C]arachidonic acid. Additionally, at higher concentrations (Ki = 0.52 X 10(-6) M), it selectively antagonizes the breakdown of inositol phospholipids, subsequent to stimulation of TXA2/prostaglandin endoperoxide receptors with U 46619. The latter happens in a competitive way with fast receptor association-dissociation characteristics. At low concentrations (1 X 10(-9)-1 X 10(-7) M) producing single TXA2 synthetase inhibition, ridogrel reduces the collagen-induced formation of TXB2 by washed platelets, but enhances [32P]phosphatidic acid (PA) accumulation and [3H]5-hydroxytryptamine (5-HT) release. At higher concentrations (1 X 10(-6)-1 X 10(-5) M) which additionally block U 46619-induced [32P]PA accumulation, ridogrel inhibits the [32P]PA accumulation and release of [3H]5-HT by human platelets stimulated with collagen. These observations, corroborated by results obtained with OKY 1581, sulotroban, indomethacin and human serum albumin, suggest a causal role for prostaglandin endoperoxides in the stimulation by TXA2 synthetase inhibition of platelet reactions to collagen. They reinforce the concept that TXA2 synthetase inhibition-induced reorientation of cyclic endoperoxide metabolism, away from TXA2 into inhibitory prostanoids, requires additional TXA2/prostaglandin endoperoxide receptor antagonism to achieve optimal anti-platelet effects.     

Anti-platelet therapy: ADP receptor antagonists

The P2Y(12) receptor on platelets with which ADP interacts has an important role in promoting platelet function and thereby platelet involvement in both haemostasis and thrombosis. Agents that act as antagonists at this receptor are thus likely to provide effective antithrombotic therapy, provided that there are no adverse effects on haemostasis. Here we describe the ADP receptor antagonists that are available and in development. We also consider their mode of action and ask whether there are additional mechanisms through which they exert their inhibitory effects on platelet function.     

PAR-1 Inhibitors: A Novel Class of Antiplatelet Agents for the Treatment of Patients with Atherothrombosis

Stroke and myocardial infarction are leading causes of death and disability worldwide. Typically, these events are triggered by the rupture or erosion of "vulnerable" atherosclerotic plaque, a phenomenon termed atherothrombosis.Three platelet activation pathways are presumed to be particularly important in the genesis of atherothrombosis and are triggered by 1) cyclo-oxygenase (COX)-1 mediated thromboxane A2 (TXA2) synthesis and activation via the TXA2 receptor, 2) adenosine diphosphate (ADP) via the P2Y12 receptor, and 3) thrombin via the protease activated receptor (PAR)-1.Despite the efficacy of aspirin and of a growing family of P2Y12 receptor antagonists on the first 2 pathways, major cardiovascular events continue to occur in patients with coronary and cerebrovascular disease, suggesting that thrombin-mediated platelet activation may contribute to these adverse events.Recently, a novel class of antiplatelet agents able to inhibit thrombin-mediated platelet activation has been developed, PAR-1 inhibitors. In this chapter, we will discuss the rationale underlying the development of this novel class of agents focus on the two drugs in the most advanced stages of development: vorapaxar (SCH530348) and atopaxar (E5555).     

Effects of enteric-coated,low-dose aspirin on parameters of platelet function

BACKGROUND: Aspirin is widely used as an anti-thrombotic drug; however, it has been suggested that enteric-coated formulations of aspirin may be less bioavailable and less effective as anti-thrombotic agents. AIM: To assess the effect of a formulation of enteric-coated, low-dose (81 mg) aspirin on serum generated thromboxane B2 and platelet aggregation in healthy subjects. METHODS: Twenty-four subjects participated in a double-blind, randomized, placebo-controlled, parallel-group, multiple-dose study. Twelve subjects in each of two groups received a daily oral dose of enteric-coated aspirin (81 mg) or matching placebo for 7 days. Serum thromboxane B2 and platelet aggregation (using 1 mm arachidonic acid and 1 microg/mL collagen as agonists) were measured 1-3 days prior to day 1, on day 1 (prior to therapy) and 4 h after the last dose on day 7. RESULTS: After seven daily doses of enteric-coated aspirin, the mean percentage inhibition from baseline of ex vivo generated serum thromboxane B2 was 97.4%, compared with a 7.8% increase after placebo treatment. The mean percentage inhibition of arachidonic acid- and collagen-induced platelet aggregation was 97.9% and 70.9%, respectively, following enteric-coated aspirin, compared with - 1.0% and 2.7%, respectively, after placebo. CONCLUSIONS: The anti-platelet effects of multiple, daily, low-dose aspirin (as assessed by inhibition of serum thromboxane B2 and platelet aggregation) are not adversely affected by enteric coating.     

Intracellular signaling as a potential target for antiplatelet therapy

Three classes of inhibitors of platelet aggregation have demonstrated substantial clinical benfits. Aspirin acts by irreversibly inhibiting COX-1 and therefore blocking the synthesis of proaggregatory thromboxane A (2) (TxA(2)). The indirect acting (ticlopidine, clopidogrel, prasugrel) and the direct acting (ticagrelor) antagonists of P2Y(12) block the thrombus stabilizing activity of ADP. Parenteral GP IIb-IIIa inhibitors directly block platelet-platelet interactions. Despite well-established benefits, all antiplatelet agents have important limitations: increased bleeding and gastrointestinal toxicities (aspirin), high incidence of thrombotic thrombocytopenic purpura (ticlopidine), potentially nonresponders (clopidogrel), severe bleeding (prasugrel, GP IIb-IIIa antagonists) and "complicated" relationships with aspirin ticagrelor). In this chapter, we present the genetic and pharmacological evidence that supports the development and expectations associated with novel antiplatelet strategies directed at intrasignaling pathways.     

Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor

Activation of the P2Y(1) nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y(1) receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC(50) value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC(50) values of 62.8 nM and 1.5 microM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y(1) receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca(2+). No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y(1) receptor antagonists with the proaggregatory P2Y(12) receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y(1) receptor, and MRS2500 is the most potent such antagonist yet reported.     

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.     

Decrease in agonist affinity for human platelet thromboxane A2/prostaglandin H2 receptors induced by a platelet-derived supernatant

Platelets possess membrane receptors which mediate the aggregatory response to thromboxane A2 (TXA2) and prostaglandin H2 (PGH2). It has been observed recently that the affinities for a series of TXA2/PGH2 mimetics are decreased in crude human platelet membranes and solubilized membranes compared to intact washed platelets. The present study investigated the notion that platelets contain a substance that is released during platelet lysis that reduces the affinity of the TXA2/PGH2 receptor for agonists. The displacement of 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13 - aza-15 alpha beta-omega-tetranor-TXA2 ([125I]PTA-OH), a TXA2/PGH2 receptor antagonist, from its binding site in intact washed platelets by TXA2/PGH2 mimetics and antagonists was characterized in the presence or absence of the supernatant (50,000 g) obtained from sonicated platelets. In the presence of the supernatant, there was a significant (P less than 0.025) increase in the IC50 values for the TXA2/PGH2 mimetics U46619, SQ26655, and ONO11113. The increase in the IC50 for U46619 induced by the supernatant was abolished by either boiling or treating the supernatant with trypsin. The supernatant did not affect the Kd or Bmax of [125I]PTA-OH or the IC50 of the TXA2/PGH2 antagonist, SQ29548. Pretreatment of the platelets with the supernatant resulted in a significant (P less than 0.02) reduction in the aggregation response induced by U46619. Gel filtration (Sephacryl S200) of the supernatant revealed a fraction (molecular weight approximately 100,000 daltons) which significantly increased the IC50 for U46619 to displace [125I]PTA-OH from its binding site. Thus, human platelets appear to possess a protein(s) that is released into the supernatant upon sonication and inhibits the binding of TXA2/PGH2 agonists but not antagonists to their receptor. This protein may play a role in the regulation of platelet responses to the aggregatory stimuli TXA2/PGH2.     

Future prospects in anti-platelet therapy: a review of potential P2Y12 and thrombin receptor antagonists

Atherothrombosis is an acute complication that develops on the surface of a ruptured atheromatous plaque or as a consequence of endothelial erosion that may cause myocardial infarction or ischemic stroke. Anti-platelet therapy has been highly effective at reducing atherothrombotic risk. However, patients continue to experience thrombotic events despite the use of agents such as aspirin and clopidogrel. Many of these events occur, in part, because of the inadequate response to these drugs. This has prompted the pursuit of novel agents with aspirations of optimizing anti-platelet therapy. New opportunities have emerged to address the deficiencies in current anti-platelet agents. Among these are thrombin receptor antagonists, such as SCH530348 and P2Y12 receptor antagonists, such as prasugrel, cangrelor, and AZD6140. The patents describe these novel compounds and compositions, their ability to inhibit platelet activation and/or aggregation and their use in the treatment of atherothrombotic diseases. These drugs have pharmacologic properties that translate into increased potency, more rapid onset of action, and less variability in response compared to standard therapy. In this review, we highlight the current data on these potential drugs and the role they could play in atherothrombotic disease.     

Anti-platelet and anti-thrombotic effects of triacetylshikimic acid in rats.

Because shikimic acid is the key intermediate in the shikimate pathway in plants and microorganisms, shikimic acid and its derivatives have been described as herbicides and anti-microbial agents. Triacetylshikimic acid (TSA) is an acetylate derivative of shikimic acid. The possible anti-platelet activity and anti-thrombotic efficacy of TSA were evaluated and its effect on arachidonic acid (AA) metabolism and second messengers including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) was evaluated. After oral pretreatment with TSA, adenosine diphosphate (ADP)-, collagen-, and AA-induced rat platelet aggregation was inhibited ex vivo in a dose-dependent manner. In an arteriovenous-shunt thrombosis model, oral administration of TSA resulted in a dose-dependent inhibition of thrombus growth. TSA markedly increased the cAMP level and showed no effect on the cGMP level in rat platelets. Also, no significant changes in ADP-induced thromboxane B2 formation in rat platelets or 6-keto-prostaglandin F 1alpha production from the abdominal aorta were observed after oral administration of low and medium doses of TSA (12.5 and 50 mg/kg). Additionally, prothrombin time, activated partial thromboplastin time, and thrombin time were unchanged at effective anti-platelet doses of TSA. These results demonstrate that TSA exerts oral anti-platelet and anti-thrombotic efficacy without perturbation of systemic hemostasis in rats, which was partially concerned with the elevation of cAMP in platelets.     

Thromboxane Receptors Antagonists and/or Synthase Inhibitors

Atherothrombosis is the major cause of mortality and morbidity in Western countries. Several clinical conditions are characterized by increased incidence of cardiovascular events and enhanced thromboxane (TX)-dependent platelet activation. Enhanced TX generation may be explained by mechanisms relatively insensitive to aspirin. More potent drugs possibly overcoming aspirin efficacy may be desirable. Thromboxane synthase inhibitors (TXSI) and thromboxane receptor antagonists (TXRA) have the potential to prove more effective than aspirin due to their different mechanism of action along the pathway of TXA(2). TXSI prevent the conversion of PGH(2) to TXA(2), reducing TXA(2) synthesis mainly in platelets, whereas TXRA block the downstream consequences of TXA(2) receptors (TP) activation.TXA(2) is a potent inducer of platelet activation through its interaction with TP on platelets. TP are activated not only by TXA(2), but also by prostaglandin (PG) D(2), PGE(2), PGF(2α), PGH(2), PG endoperoxides (i.e., 20-HETE), and isoprostanes, all representing aspirin-insensitive mechanisms of TP activation. Moreover, TP are also expressed on several cell types such as macrophages or monocytes, and vascular endothelial cells, and exert antiatherosclerotic, antivasoconstrictive, and antithrombotic effects, depending on the cellular target.Thus, targeting TP receptor, a common downstream pathway for both platelet and extraplatelet TXA(2) as well as for endoperoxides and isoprostanes, may be a useful antiatherosclerotic and a more powerful antithrombotic intervention in clinical settings, such as diabetes mellitus, characterized by persistently enhanced thromboxane (TX)-dependent platelet activation through isoprostane formation and low-grade inflammation, leading to extraplatelet sources of TXA(2). Among TXRA, terutroban is an orally active drug in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. Despite great expectations on this drug supported by a large body of preclinical and clinical evidence and pathophysiological rationale, the PERFORM trial failed to demonstrate the superiority of terutroban over aspirin in secondary prevention of cerebrovascular and cardiovascular events among ~20,000 patients with stroke. However, the clinical setting and the design of the study in which the drug has been challenged may explain, at least in part, this unexpected finding.Drugs with dual action, such as dual TXS inhibitors/TP antagonist and dual COXIB/TP antagonists are currently in clinical development. The theoretical rationale for their benefit and the ongoing clinical studies are herein discussed.     

Ticagrelor: a P2Y12 antagonist for use in acute coronary syndromes

Agents that inhibit platelet function are used routinely in the treatment and prevention of acute coronary syndromes. The main antiplatelet treatments used combine aspirin with one of the thienopyridine P2Y(12) antagonists, either clopidogrel or prasugrel. By blocking the synthesis of thromboxane A(2) in platelets and by blocking the effects of ADP, respectively, these agents reduce platelet activity, platelet aggregation and thrombus formation. Ticagrelor (marketed by AstraZeneca as Brilinta? in the USA, and as Brilique(?) or Possia(?) in Europe) is a cyclopentyl-triazolo-pyrimidine, a new chemical class of P2Y(12) antagonist that is now approved for use in the wide spectrum of acute coronary syndromes. In this article we provide an overview of ticagrelor. We discuss the differences in mode of action compared with other P2Y(12) antagonists, examine its pharmacodynamic, pharmacokinetic and safety profile, and summarize the various clinical trials that have provided information on its efficacy in combination with aspirin. Ticagrelor appears to overcome some of the difficulties that have been encountered with other antiplatelet treatments, clopidogrel in particular.     

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.     

Targeting von Willebrand factor as a novel anti-platelet therapy; Application of ARC1779, an Anti-vWF aptamer, against thrombotic risk

Excessive activation of platelets is a causative factor for thrombotic diseases such as acute coronary syndrome or stroke, and various anti-platelet drugs were developed. Aspirin and clopidogrel have been used as gold standards for anti-platelet therapies, however, their clinical limitations including bleeding problem have increased the demand driving development of novel anti-platelet drugs with new targets. Among several activating pathways leading to platelet aggregation, the interaction between von Willebrand factor (vWF) and glycoprotein Ib, which mainly occurs under high shear stress in arterioles, is recently suggested to be a new promising target. The anti-thrombotic efficacy of anti-vWF agents, such as ARC1779, has been proved in several preclinical and clinical studies. Here, we will discuss the potential benefits of targeting vWF as a novel antiplatelet therapy, providing an insight into the role of vWF in increased thrombotic risk.     

Effect of cyclooxygenase-2 inhibitor (celecoxib) on the infarcted heart in situ.

Several attempts have been made to replace aspirin with compounds without gastric toxicity; a cyclooxygenase-2 (COX-2) inhibitor, celecoxib, and a nitric oxide-aspirin, NCX-4016, have been developed for this purpose. This paper compares effects of celecoxib, NCX-4016 and aspirin on production of prostacyclin (PGI2) and thromboxane A2 (TXA2) and activation of the inducible form of nitric oxide synthase (iNOS) in infarcted heart in situ. Aspirin was most effective in reducing myocardial PGI2 synthesis and formation of TXA2. Myocardial effects of celecoxib resemble those of NCX-4016, although the two compounds have different modes of action.     

Safety and efficacy of targeting platelet proteinase-activated receptors in combination with existing anti-platelet drugs as antithrombotics in mice

BACKGROUND AND PURPOSE

Developing novel anti-platelet strategies is fundamental to reducing the impact of thrombotic diseases. Thrombin activates platelets via proteinase-activated receptors (PARs), and PAR antagonists are being evaluated in clinical trials for prevention of arterial thrombosis. However, one such trial was recently suspended due to increased bleeding in patients receiving a PAR₁ antagonist in addition to anti-platelet drugs that most often included both aspirin and clopidogrel. Therefore, it remains unclear how to best manipulate PARs for safe antithrombotic activity. To address this, we have examined potential interactions between existing anti-platelet drugs and strategies that target PARs.

EXPERIMENTAL APPROACH

We used in vivo mouse models in which interactions between various anti-platelet strategies could be evaluated. We examined the effects on thrombosis and haemostasis in PAR₄−/− mice (platelets unresponsive to thrombin) treated with therapeutic doses of either aspirin or clopidogrel.

KEY RESULTS

Using a model in which occlusive thrombosis occurred in PAR₄−/− mice or wild-type mice treated with aspirin or clopidogrel, PAR₄−/− mice treated with either anti-platelet agent showed marked protection against thrombosis. This antithrombotic effect occurred without any effect on haemostasis with aspirin, but not clopidogrel. Furthermore, specifically targeting thrombin-induced platelet activation (via PARs) improved the therapeutic window of non-specifically inhibiting thrombin functions (via anticoagulants).

CONCLUSIONS AND IMPLICATIONS

Our results indicate that PAR antagonists used in combination with aspirin provide a potent yet safe antithrombotic strategy in mice and provide insights into the safety and efficacy of using PAR antagonists for the prevention of acute coronary syndromes in humans.