Targeting thrombin long-term after an acute coronary syndrome: Opportunities and challenges

Patients after an acute coronary syndrome (ACS) are at increased risk of recurrent thrombotic events, justifying the search for additional antithrombotic treatments. The pathophysiology of ACS involves arterial thrombus formation, in turn occurring because of a combination of platelet activation and fibrin formation, with thrombin playing a key role in both. Antiplatelet therapy, targeting the thromboxane pathway and the ADP P2Y₁₂ receptor has been widely accepted for secondary prevention after an ACS. Now, data from recent clinical trials in such patients also encourage the pursuit of inhibiting thrombin formation or thrombin-mediated platelet activation in addition to antiplatelet therapy. This “triple pathway inhibition”, including inhibition of thrombin activity or thrombin receptor(s), is currently an option in pure ACS, but already a must in the setting of ACS accompanied by atrial fibrillation (AF), where anticoagulants have been shown to be much more effective than antiplatelet agents in preventing stroke. We here discuss the challenges of managing combined thrombin activity or receptor inhibition and antiplatelet therapy in all such patients. Translating this into practice still requires further studies and patient tailoring to fully exploit its potential.     

Combination oral antithrombotic therapy for the treatment of myocardial infarction: recent developments

Introduction: There have been significant new developments in the treatment of patients with myocardial infarction with respect to oral antithrombotic agents over the past decade. Recent studies have explored the potential utility of targeting the dual pathway inhibition of platelet function with single or dual antiplatelet agents and the thrombin pathway with direct thrombin inhibitors or factor Xa inhibitors.

Areas covered: In this review, the authors focus on the recent developments of oral antithrombotic agents including antiplatelet and antithrombin agents. It is based on literature covering: aspirin, P2Y₁₂ receptor blockers, PAR-1 inhibitors, direct thrombin inhibitors and factor Xa inhibitors from PubMed since 2008.

Expert opinion: Since thrombus formation involves multiple pathways including platelet activation and aggregation and coagulation, simultaneous and optimal blockade of these pathways is essential to prevent thrombotic complications and to avoid excessive bleeding in the myocardial infraction setting. Despite an improved anti-ischemic effect associated with potent P2Y₁₂ inhibitors plus aspirin, the degree of adverse event reduction compared to clopidogrel therapy in large scale trials is modest along with significantly greater bleeding. Recent studies suggest that targeting the thrombin pathway in addition to antiplatelet agents in high risk patients may further mitigate the risk of ischemic event occurrences with improved safety profiles.     

New perspectives in antiplatelet therapy

Platelet activation is a complex mechanism of response to vascular injury and atherothrombotic disease, leading to thrombus formation. A wide variety of surface receptors -integrins, leucine-rich family receptors, G protein coupled receptors, tyrosine kinase receptors- and intraplatelet molecules support and regulate platelet activation. They are potential targets of antiplatelet therapy for the prevention and treatment of arterial thrombosis. Despite the overall clinical benefit of established antiplatelet drugs targeting cyclooxigenase-1 (COX-1), glycoprotein integrin αIIbβ3, and the purinergic P2Y(12) receptor of adenosine diphosphate, a significant proportion of treated patients continue to experience recurrent ischaemic events. This may be in partly attributed to insufficient inhibition of platelet activation. In addition, it should not be underestimated that these drugs are not immune from bleeding complications. The substantial progress in understating the regulation of platelet activation has played a key role in the development of novel antiplatelet agents. Current examples of drug under development and evaluation include: novel P2Y(12) receptor inhibitors (prasugrel, ticagrelor, cangrelor, and elinogrel), thrombin receptor PAR-1 antagonists (vorapaxar, atopaxar), new integrin glycoprotein IIb/IIIa inhibitors, and inhibitors targeting the thromboxane receptor (TP), phosphodiesterases, the collagen receptor glycoprotein VI, and intraplatelet signalling molecules. This review summarizes the mechanisms of action and current clinical evaluation of these novel antiplatelet agents.     

Platelet P2Y<Subscript>12</Subscript> Receptor Inhibition

Antiplatelet therapy is the cornerstone of treatment for patients with coronary artery disease. Since adenosine diphosphate (ADP) represents one of the most important mediators of thrombosis, the inhibition of the platelet ADP receptor, in particular the P2Y₁₂ subtype, plays a pivotal role in secondary prevention of recurrent atherothrombotic events in high-risk settings. Numerous clinical trials have shown the efficacy of clopidogrel, an inhibitor of the ADP P2Y₁₂ receptor, in patients presenting with an acute coronary syndrome and undergoing percutaneous coronary intervention. However, laboratory and clinical experience with clopidogrel have led to understanding some of the limitations of this drug, the most important of which is its broad range in interindividual response variability, resulting in the development of novel ADP P2Y₁₂ receptor-inhibiting strategies. This article provides an overview of ADP P2Y₁₂ receptor-inhibiting strategies, including high clopidogrel dosing regimens and novel agents under advanced clinical development.     

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 P2Y₁₂ antagonists, either clopidogrel or prasugrel. By blocking the synthesis of thromboxane A₂ 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 P2Y₁₂ 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 P2Y₁₂ 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.     

Genetics of platelet inhibitor treatment

Dual antiplatelet therapy with aspirin and a P2Y₁₂ receptor antagonist is the standard of care in patients undergoing percutaneous coronary intervention (PCI) and in patients with acute coronary syndromes (ACS) because this regimen has markedly decreased the rate of cardiovascular events. The substantial variability in pharmacodynamic response as well as the moderate antiplatelet efficacy of clopidogrel has raised major concerns, since high on-clopidogrel platelet reactivity has consistently been associated with increased risk for ischaemic events in PCI patients. Baseline demographic and clinical variables contributing to the observed variability have been identified. Besides this, research within the past decade has focused on the impact of genetic polymorphisms encoding transport systems or enzymes involved in the absorption and metabolism of these drugs. Loss-of-function polymorphisms in CYP2C19 are the strongest individual variables affecting pharmacokinetics and antiplatelet response to clopidogrel, but explain no more than 5 to 12% of the variability in adenosine diphosphate-induced platelet aggregation on clopidogrel. No genetic variables contributing to clinical outcomes of patients treated with the newer P2Y₁₂ receptor antagonists, prasugrel or ticagrelor, have been identified so far. This review aims to provide an update on the current status of genotype-based personalized therapy with clopidogrel.     

P2Y12 polymorphisms and antiplatelet effects of aspirin in patients with coronary artery disease

Aims

Recently, two genetic polymorphisms of the platelet ADP receptor P2Y₁₂ (haplotypes H2 and 34T) have been implicated in increased platelet aggregation and atherothrombotic risk. It was suggested that these polymorphisms contribute to a diminished response to antiplatelet drugs. Therefore, we investigated the effects of these polymorphisms on platelet aggregation in aspirin-treated patients with coronary artery disease (CAD).

Methods

Platelet aggregation was studied in platelet-rich plasma from 124 patients with CAD treated with 100 mg aspirin day⁻¹. P2Y₁₂ ADP receptor polymorphisms were determined by PCR-RFLP. The 52G > T polymorphism was used as tag-SNP for the H2 haplotype. Aggregation was induced by 1 mg l⁻¹ collagen. In a subgroup (n = 72), a concentration-response curve to collagen (0.5–10 mg l⁻¹), aggregation at 2 μmol l⁻¹ ADP and 1 mmol l⁻¹ arachidonic acid were determined.

Results

Whereas arachidonic acid-induced aggregation was inhibited in all patients, collagen and ADP-induced aggregation were highly variable. However, aggregation did not differ significantly between carriers and noncarriers of the 52T-allele (1 mg l⁻¹ collagen: 32.7% (21.9–38.6%) vs. 32.5% (21.2–41.6%); P = 0.77; ADP: 33.1% (29.9–40.9%) vs. 39.1% (31.5–49.7%); P = 0.34), respectively. EC₅₀ values were 1.26 mg l⁻¹ (0.79–2.02) and 1.54 mg l⁻¹ (0.98–2.4) collagen in noncarriers and carriers of the H2 haplotype, respectively (P = 0.56). Moreover, the 34°C > T polymorphism did not significantly affect any of the aggregatory responses.

Conclusions

Low-dose aspirin inhibits platelet aggregation to the same extent in patients carrying or not carrying the P2Y₁₂ H2 haplotype and/or the 34T allele. Our data do not support the hypothesis that these polymorphisms contribute to an attenuated antiplatelet effect of aspirin.

What is already known about this subject

• Genetic polymorphisms of the P2Y₁₂ ADP receptor on platelets have been shown to contribute to variability in platelet aggregation in healthy humans.
• P2Y₁₂ ADP receptor polymorphisms are more frequently present in patients with vascular disease than in healthy people.
• The majority of patients with vascular disease receive acetylsalicylic acid as an anti-aggregatory agent, which has also been shown to induce a variable response; however, the role of P2Y₁₂ ADP receptor polymorphisms in the platelet response to acetylsalicylic acid in patients with vascular disease has not yet been studied.

What this study adds

• The present data show that the platelet response to acetylsalicylic acid is independent of the presence or absence of P2Y₁₂ ADP receptor polymorphisms in patients with stable coronary artery disease who have had their first myocardial infarction.
• This is important, as studies in healthy humans had suggested that carriers of P2Y₁₂ ADP receptor polymorphisms may be at increased risk of experiencing cardiovascular events.
• However, the observed variability of the platelet response to the cyclooxygenase inhibitor acetylsalicylic acid (in our study) and to the P2Y₁₂ ADP receptor blocker clopidogrel (in a study by Angiolillo et al.[18]) in patients with coronary artery disease is clearly not determined by common P2Y₁₂ ADP receptor polymorphisms.

     

Antiplatelet therapy in acute coronary syndromes

Introduction: Coronary thrombosis is a frequent cause of death and myocardial infarction most often explained by superimposition of a platelet-rich thrombus on existing coronary artery disease. Therefore, antiplatelet drugs are essential in the treatment and secondary prevention of acute coronary syndromes (ACS) and during percutaneous coronary intervention. Several novel antiplatelet drugs are now available.

Areas covered: For several years, aspirin and clopidogrel remained the cornerstone of treatment for ACS. However, prasugrel and ticagrelor have a more consistent, faster-acting and more potent antiplatelet effect than clopidogrel, which translates into improved clinical outcomes, although at the expense of an increased bleeding risk. Importantly, some patients experience cardiovascular events despite current antiplatelet treatment, because platelet activation may occur via pathways not inhibited by these agents. Therefore, improved antiplatelet strategies are warranted.

Expert opinion: Despite undisputable benefits of current antiplatelet strategies, a considerable number of patients continue to experience adverse thrombotic events, although clinical outcomes have been improved with new oral P2Y₁₂ antagonists. New drugs have been developed, including intravenous P2Y₁₂ antagonists and oral antagonist targeting the protease-activated receptor-1 platelet activation pathway stimulated by thrombin. This review provides an overview of current and novel antiplatelet strategies and also discusses unmet needs related to antiplatelet therapy for ACS.     

Cangrelor: a novel P2Y12 receptor antagonist

Antiplatelet therapy is critical in the prevention of thrombotic complications of acute coronary syndrome and percutaneous coronary interventions. Current antiplatelet agents (aspirin, clopidogrel and glycoprotein IIb/IIIa antagonists) have demonstrated the capacity to reduce major adverse cardiac events. However, these agents have limitations that compromise their clinical utility. The platelet P2Y₁₂ receptor plays a central role in platelet function and is a focus in the development of antiplatelet therapies. Cangrelor is a potent, competitive inhibitor of the P2Y₁₂ receptor that is administered by intravenous infusion and rapidly achieves near complete inhibition of ADP-induced platelet aggregation. This investigational drug has been studied for use during coronary procedures and the management of patients experiencing acute coronary syndrome and is undergoing evaluation for use in the prevention of perioperative stent thrombosis.     

Features of reversible P2Y<Subscript>12</Subscript> receptor antagonists based on piperazinyl-glutamate-pyridines

P2Y₁₂ receptor becomes a favorable target for therapeutic antiplatelet agents. Reversible P2Y₁₂ receptor antagonists have more benefits than the irreversible ones on rapid absorption, onset of action and higher inhibition of platelet aggregation. As potent reversible P2Y₁₂ receptor antagonists, piperazinyl-glutamate-pyridines exhibit a significant improvement in efficacy while maintaining a better safety profile than the irreversible ones. In this study, we developed a 3D pharmacophore model with r ² = 0.966 based on a set of piperazinyl-glutamate-pyridines showing the important pharmacophore features (one hydrogen bond acceptor, one hydrogen bond donor, one ring aromatic and one hydrophobic feature) which is necessary for reversible P2Y₁₂ receptor antagonists. The obtained pharmacophore models were validated by using well-known methodologies such as test molecules and Fischer’s randomization method. A docking study was also performed based on the recently released P2Y₁₂ receptor crystal structure (PDB ID: 4NTJ) which revealed key residues (Tyr105, His 187 and Lys280) in the receptor–ligand interaction. The docking results were well in agreement with the pharmacophore model that raised the model’s reliability. The pharmacophore model was further confirmed by estimating the activity of six known drugs which can distinguish the reversible P2Y₁₂ receptor antagonists from the irreversible ones. The results of our study provide confidence for the utility of the selected chemical features to retrieve further compounds as reversible P2Y₁₂ receptor antagonists.     

Novel approaches to the treatment of thrombosis.

Thromboembolic diseases remain the main cause of death in Western societies despite current antithrombotic treatments. Recent advances in the molecular bases of haemostasis have highlighted new targets for novel antiplatelet or anticoagulant agents. Considering antiplatelet agents, selective antagonists of specific receptors (von Willebrand factor, collagen or thrombin receptors) are effective in thrombosis models and direct ADP receptor antagonists and nitric-oxide-releasing aspirin are in Phase I-II clinical trials. Concerning anticoagulants, inhibitors of tissue-factor-induced clotting activation, selective inhibitors of thrombin and factor Xa, and components of the anticoagulant protein C system (recombinant activated human protein C or human soluble thrombomodulin) have been studied. Some of these agents have had promising results in Phase III studies. Several achievements are anticipated from the development of new antithrombotics, including a further reduction of cardiovascular mortality and unwanted bleeding, and easier patient management.     

Resistance to antiplatelet drugs: what progress has been made?

Introduction: Despite well-documented efficacy, recurrent thrombotic event occurrences, particularly stent thrombosis, have been repeatedly demonstrated in stented patients treated with aspirin and clopidogrel. The latter observation stimulated the close scrutiny of the pharmacodynamic effects of clopidogrel and revealed the ‘wide variability’ and the phenomenon of ‘antiplatelet resistance’. High on-treatment platelet reactivity to ADP (HPR) during clopidogrel therapy is an independent risk factor for ischemic event occurrences in post-percutaneous coronary intervention (post-PCI) patients. Recent observational studies demonstrated a link between low on-treatment platelet reactivity to bleeding. The concept of a ‘therapeutic window’ of P2Y₁₂ receptor reactivity associated with both ischemic event occurrence (upper threshold) and bleeding risk (lower threshold) has been proposed.

Areas covered: An update on and a brief review of the current knowledge on antiplatelet resistance were presented. Evidence available from studies evaluating aspirin resistance and high and low on-treatment platelet reactivity to ADP during P2Y₁₂ receptor blocker therapy was collected from a selective literature search.

Expert opinion: The available evidence indicates that HPR is an independent risk factor for post-PCI ischemic event occurrences. The therapeutic window concept for the P2Y₁₂ receptor blocker therapy may facilitate the balance between reducing ischemic events and avoiding bleeding events, thereby improving net clinical outcome.     

The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation

BACKGROUND AND PURPOSE

Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.

EXPERIMENTAL APPROACH

The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca²⁺ mobilization respectively.

KEY RESULTS

When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca²⁺ rises. Blockade of ADP P2Y₁₂ receptor with 2-methylthioadenosine 5′-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5′-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

These results suggest that PAR4 acts in parallel with the P2Y₁₂/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.     

Antiplatelet effects of piplartine,an alkamide isolated from Piper tuberculatum: possible involvement of cyclooxygenase blockade and antioxidant activity

Objectives Piplartine (piperlongumine; 5,6‐dihydro‐1‐[1‐oxo‐3‐(3,4,5‐trimethoxyphenyl]‐2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. Methods We evaluated the in‐vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet‐rich plasma by the agonists collagen, adenosine 5′‐diphosphate (ADP), arachidonic acid (AA) and thrombin. Key findings Piplartine (100μg/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 μg/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 μg/ml) inhibited thrombin‐ (37%), ADP‐ (71%) and collagen‐ (98%) induced aggregation. The inhibitory effect of piplartine on ADP‐induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), l ‐arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y₁₂ purinoceptor antagonist). However, aspirin, a well‐known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic‐acid‐induced platelet aggregation. Conclusions The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A₂ formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.     

Prasugrel

Clinical trials have demonstrated the superior clinical efficacy of dual antiplatelet therapy with a thienopyridine (a P2Y₁₂ receptor blocker) and aspirin (COX-1 inhibitor) in patients undergoing stenting as well as patients with acute coronary syndromes. However, clopidogrel treatment is associated with a wide response variability and non-responsiveness in selected patients. The latter phenomenon is linked to the occurrence of recurrent ischaemic events including stent thrombosis in the recent studies. Prasugrel is a new thienopyridine derivative that produces more potent platelet inhibition and a rapid onset of action that is associated with irreversible P2Y₁₂ receptor blockade. The latter properties of prasugrel may provide a superior alternative to clopidogrel, with less response variability and a decreased prevalence of non-responsiveness.     

Editorial Commentary

Thrombotic events of the arterial circulation continue to be the major cause of cardiovascular death in spite of great efforts to prevent and treat thrombosis. Current antithrombotic strategies in clinical use are primarily based on aspirin (antiplatelet), heparin (anticoagulant) and coumadins (chronic anticoagulants). Recently, ReoPro®, an antibody blocking the platelet fibrinogen receptor, GpIIb/IIIa, has been introduced for limited acute use for prevention of ischaemic complications of coronary balloon angioplasty. Clopidogrel is a novel antiplatelet agent that has demonstrated antithrombotic efficacy beyond aspirin in preclinical studies and is now in clinical (Phase II) development for secondary prevention of broad ischaemic cardiovascular events including death. Clopidogrel is an ADP receptor antagonist with additional properties that produce effective and long-lasting antithrombotic actions. Clopidogrel seems to be free of the adverse side-effects that flawed ticlopidine, a closely related thieno-derivative, and is at least six-fold more potent. It is expected that clopidogrel will add significantly to the antichrombotic arsenal and reduce cardiovascular morbidity and mortality above and beyond currently available therapy.     

Developments in antiplatelet therapy for acute coronary syndromes and considerations for long-term management

ABSTRACT

Objective: This article reviews the currently available antiplatelet therapies and emerging investigational drugs in the treatment of acutecoronary syndrome (ACS), and considerations for primary and secondary prevention in the long-term management of ACS patients undergoing percutaneous coronary intervention (PCI).

Research design and methods: Primary studies and reviews in the peer-reviewed, English-language literature were identified through searches of MEDLINE (1966–2008) using the terms ‘acute coronary syndrome’, ‘antiplatelet’, ‘aspirin’, ‘long-term management’, ‘P2Y₁₂ receptor’, and ‘thienopyridine’. Additional references were obtained by searching the reference lists of the identified articles. Articles were included if they were recently published and pertinent, patient-focused, and authors were recognized as leaders in the field. Current review is limited by literature search on single database.

Results: Platelets play a major role in atherogenesis and the formation of thrombi, the main events in the pathogenesis of ACS. Although aspirin is an effective antiplatelet agent, efficacy and safety data from a number of randomized clinical trials on atherothrombotic disease support the use of dual antiplatelet therapies such as aspirin and thienopyridines over single antiplatelet therapy for ACS and up to 1?year following ACS. Antiplatelet agents reduce, but do not eliminate, ischemic events after ACS due, in part, to variable individual response (or resistance) in antiplatelet agents, non-compliance, progression of atherosclerosis, modest inhibition of platelet aggregation (IPA) levels and other factors. Several antiplatelet agents, including novel P2Y₁₂-receptor antagonists and thrombin-receptor antagonists, are currently under investigation for ACS and primary and secondary prevention in the long-term management of patients undergoing PCI.

Conclusions: Current antiplatelet therapies have clinical benefits such as reducing immediate and long-term cardiovascular risk, but substantial residual risk remains indicating a need for new therapeutic agents. Additional large randomized trials are necessary to determine the most appropriate treatment regimens for ACS patients.     

AZD6140

Oral antiplatelet therapy with P2Y₁₂ receptor blockers (especially clopidogrel) is the current choice of treatment during acute coronary syndromes and percutaneous interventions. To address the various limitations of thienopyridine therapy (including response variability and non-responsiveness) a novel drug, AZD6140, is under clinical development. AZD6140 is an oral and reversible P2Y₁₂ receptor blocker that does not require hepatic conversion to an active metabolite and produces an overall superior ADP-induced platelet inhibition with less response variability than clopidogrel. It has fast onset and offset actions that may be advantageous in patients who may have to undergo immediate surgery.     

Antithrombotic and antiplatelet activities of 2-chloro-3-[4-(ethylcarboxy)-phenyl]-amino-1,4-naphthoquinone (NQ12), a newly synthesized 1,4-naphthoquinone derivative

The possibility of NQ12 (2-chloro-3-[4-(ethylcarboxy)-phenyl]-amino-1,4-naphthoquinone) as a novel antithrombotic agent and its mode of action were investigated. The effects of NQ12 on platelet aggregation in human platelet-rich plasma in vitro, in rats ex vivo, and on murine pulmonary thrombosis in vivo, as well as the mode of antithrombotic action were examined. NQ12 potently inhibited ADP-, collagen-, epinephrine-, and calcium ionophore-induced human platelet aggregations in vitro concentration-dependently. NQ12 significantly inhibited rat platelet aggregation in an ex vivo study. NQ12 prevented murine pulmonary thrombosis in a dose-dependent manner. However, NQ12 did not affect coagulation parameters such as activated partial thromboplastin time, prothrombin time, and thrombin time. NQ12 inhibited fibrinogen binding to the platelet surface GPIIb/IIIa receptor, but failed to inhibit binding to the purified GPIIb/IIIa receptor. Thromboxane B(2) formation caused by thrombin or collagen was inhibited significantly by NQ12. The phosphoinositide breakdown induced by thrombin or collagen was inhibited concentration-dependently by NQ12. These results suggest that NQ12 may be a promising antithrombotic agent, and its antithrombotic activity may be due to antiplatelet aggregation activity, which may result from the inhibition of phosphoinositide breakdown and thromboxane A(2) formation.     

Effect of 5-HT2 receptor antagonist ergolines and their desisopropyl metabolites on rabbit platelet aggregation in vitro and ex vivo

Amesergide and LY215840 are potent and long-lasting 5-HT₂ receptor antagonists after oral administration to animals. In animals, these ergolines are metabolized to their desisopropyl ergoline congeners which have lower affinity (40–60 nM) at 5-HT₂ receptors in the rat relative to higher 5-HT₂ receptor affinity (2–3 nM) at the cloned human 5-HT₂ receptor. Because amesergide and LY215840 are effective in rabbit models of thrombosis, we asked whether their efficacy in the rabbit was related in part to the activity of both the parent and desisopropyl metabolites at rabbit platelet 5-HT₂ receptors. Platelet aggregation responses were first optimized to ADP and the combination of ADP and serotonin with regard to platelet number (300,000 platelets/μl of plasma) and time (70 to 140 min after platelet harvest). In ex vivo studies, both amesergide and LY215840 (3.0 mg/kg p.o.) showed similar and marked antagonism of rabbit platelet 5-HT₂ receptors at 1 and 24 h after their oral administration to rabbits. Furthermore, the desisopropyl ergoline metabolites of both amesergide and LY215840 inhibited serotonin-amplified platelet aggregation responses in vitro as did amesergide and LY215840. Thus, these studies add support to the hypothesis that the desisopropyl metabolites of amesergide and LY215840 may contribute to the oral antithrombotic efficacy of the parent molecules in rabbits.