Update on the clinical development of cangrelor

Antiplatelet drugs represent the cornerstone of treatment for cardiovascular atherothrombotic disease. Dual oral antiplatelet therapy with aspirin and oral ADP-receptor antagonists, such as clopidogrel, has been the standard choice for prevention of ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention. However, due to the limitations of clopidogrel, such as interindividual-response variability, drug–drug interactions, slow onset of action and irreversible inhibitory effects, novel antiplatelet agents are under clinical development. Cangrelor is a reversible, potent, competitive inhibitor of the ADP P2Y₁₂ receptor that is administered intravenously and rapidly achieves near complete inhibition of ADP-induced platelet aggregation. These pharmacological properties make cangrelor a promising drug for clinical use. However, recent large-scale Phase III clinical investigations failed to show significant clinical benefit on the primary end point with cangrelor. This article provides an overview of the current status of knowledge on cangrelor, focusing on its pharmacologic properties and clinical development.     

Antiplatelet options for secondary prevention in acute coronary syndromes

Current guidelines recommend dual antiplatelet therapy, a combination of aspirin and a P2Y₁₂ inhibitor, for 6–12 months after percutaneous coronary intervention with drug-eluting stent implantation in all patients and for 1 year in all patients after an acute coronary syndrome (ACS), irrespective of revascularization strategy. Clopidogrel has a pharmacokinetic and pharmacodynamic profile that results in a delayed and/or subtherapeutic antiplatelet effect, and wide variability in antiplatelet response. New P2Y₁₂ inhibitors, such as prasugrel and ticagrelor, have favorable pharmacodynamics and clinical efficacy over clopidogrel and offer an alternative antiplatelet treatment strategy in specific patients. Prasugrel has more potent, rapid, and consistent effects on inhibiting ADP-induced platelet aggregation than clopidogrel. Ticagrelor also appears to have more rapid and consistent antiplatelet effects than clopidogrel. The higher levels of antiplatelet inhibition provided by prasugrel and ticagrelor compared with standard-dose clopidogrel result in improved ischemic outcomes in patients with ACS. Despite an increase in bleeding risk, prasugrel and ticagrelor appear to have a better net clinical benefit, especially in higher-risk patients with ACS.     

Dual antiplatelet therapy unmasks distinct platelet reactivity in patients with coronary artery disease

Summary. Background: Platelet‐induced thrombosis is a major risk factor for recurrent ischemic events, although platelet function in patients with cardiovascular disease taking aspirin and clopidogrel is very poorly characterized. The aim of this study was to assess platelet reactivity in patients with cardiovascular disease taking aspirin and clopidogrel. Methods: We developed a rapid assay to measure platelet aggregation in response to arachidonic acid, collagen, adenosine diphosphate (ADP), epinephrine and thrombin receptor activating peptide (TRAP) in 80 healthy volunteers. We then recruited 200 consecutive patients from outpatient clinics and the cardiac catheterization laboratory and tested platelet function. Platelet aggregation induced by epinephrine is a marker of global platelet reactivity. We tested platelet function in 146 patients compliant with antiplatelet therapy. Platelet aggregation to epinephrine was divided into quartiles. The platelet response to the other agonists was analysed based on the response to epinephrine. Results: Platelet reactivity increased significantly across the quartiles in response to epinephrine in healthy volunteers and patients (P < 0.0001). A significant increase in response across quartiles was seen with all agonists in healthy volunteers (P < 0.001). In contrast, a significant increase in response across quartiles was only seen with ADP in patients (P < 0.0001). Hypertension, smoking and diabetes were significantly associated with increasing platelet reactivity to epinephrine (P < 0.05). Conclusion: This study shows that platelet response differs between healthy volunteers and patients on dual antiplatelet therapy. In patients with cardiovascular disease, dual antiplatelet therapy unmasks a distinct type of platelet reactivity in response to epinephrine and ADP but not other agonists.     

Vorapaxar in the secondary prevention of atherothrombosis

Dual antiplatelet therapy with aspirin, a platelet cyclooxygenase-1 inhibitor and P2Y₁₂ receptor blockers, remains the major drug strategy to prevent ischemic event occurrence in patients with acute coronary syndromes and in patients undergoing coronary stenting, but there some limitations that can be overcome by targeting novel targets. Unlike direct thrombin inhibitors that bind directly to thrombin, targeting the platelet thrombin receptor, protease activated receptor (PAR)-1, may offer a better choice for the attenuation of atherosclerosis progression, thrombus-mediated ischemic events and restenosis without interfering with primary hemostasis. Vorapaxar – a synthetic analogue of himbacine, is a high affinity and highly selective PAR-1 antagonist that can effectively inhibit thrombin-induced platelet aggregation. In the TRACER trial, the addition of vorapaxar to standard therapy in patients with non-stent thrombosis-elevation- acute coronary syndromes did not significantly reduce the primary composite end point occurrence of cardiovascular (CV) death, myocardial infarction (MI), stroke, hospitalization for ischemia, or urgent revascularization, but significantly increased the GUSTO moderate and severe bleeding (p < 0.001) and intracranial hemorrhage (ICH). In the TRA 2°P-TIMI 50 trial, in patients with a history of MI and peripheral arterial disease (PAD) (67% of the total population), the end point of CV death, MI, or stroke was significantly (20%) reduced with vorapaxar whereas GUSTO moderate or severe bleeding was increased (1.5-fold), but not ICH or fatal bleeding and the net clinical outcome favoring the vorapaxar therapy. Based on these favorable results, the FDA approved vorapaxar for the reduction of thrombotic cardiovascular events in patients with prior MI or with PAD for long term therapy. A careful patient selection is needed to balance efficacy versus safety. At this time, patients with high risk for recurrent ischemic event occurrence such as patients with diabetes mellitus and previous MI can be safely treated with vorapaxar for long-term therapy.     

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y₁ receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm . The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A₂, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y₁₂ receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A₂ generation, ATP release, and P2Y₁₂ activation.     

Clopidogrel discontinuation and platelet reactivity following coronary stenting

See also Lordkipanidzé M, Harrison P. Beware of being caught on the rebound. This issue, pp 21–3. Summary. Aims: Antiplatelet therapy with aspirin and clopidogrel is recommended for 1 year after drug‐eluting stent (DES) implantation or myocardial infarction. However, the discontinuation of antiplatelet therapy has become an important issue as recent studies have suggested a clustering of ischemic events within 90 days of clopidogrel withdrawal. The objective of this investigation was to explore the hypothesis that there is a transient ‘rebound’ increase in platelet reactivity within 3 months of clopidogrel discontinuation. Methods and Results: In this prospective study, platelet function was assessed in patients taking aspirin and clopidogrel for at least 1 year following DES implantation. Platelet aggregation was measured using a modification of light transmission aggregometry in response to multiple concentrations of adenosine diphosphate (ADP), epinephrine, arachidonic acid, thrombin receptor activating peptide and collagen. Clopidogrel was stopped and platelet function was reassessed 1 week, 1 month and 3 months later. Thirty‐two patients on dual antiplatelet therapy were recruited. Discontinuation of clopidogrel increased platelet aggregation to all agonists, except arachidonic acid. Platelet aggregation in response to ADP (2.5, 5, 10, and 20 μm ) and epinephrine (5 and 20 μm ) was significantly increased at 1 month compared with 3 months following clopidogrel withdrawal. Thus, a transient period of increased platelet reactivity to both ADP and epinephrine was observed 1 month after clopidogrel discontinuation. Conclusions: This study demonstrates a transient increase in platelet reactivity 1 month after clopidogrel withdrawal. This phenomenon may, in part, explain the known clustering of thrombotic events observed after clopidogrel discontinuation. This observation requires confirmation in larger populations.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP‐induced platelet activation

Chang H, Yanachkov IB, Dix EJ, Li YF, Barnard MR, Wright GE, Michelson AD, Frelinger AL 3rd. Modified diadenosine tetraphosphates with dual specificity for P2Y₁ and P2Y₁₂ are potent antagonists of ADP‐induced platelet activation. J Thromb Haemost 2012; 10: 2573–80. Summary. Background: Diadenosine 5′,5′′′‐P¹,P⁴‐tetraphosphate (Ap₄A), a natural compound stored in platelet dense granules, inhibits ADP‐induced platelet aggregation. Ap₄A inhibits the platelet ADP receptors P2Y₁ and P2Y₁₂, is a partial agonist of P2Y₁₂, and is a full agonist of the platelet ATP‐gated ion channel P2X1. Modification of the Ap₄A tetraphosphate backbone enhances inhibition of ADP‐induced platelet aggregation. However, the effects of these Ap₄A analogs on human platelet P2Y₁, P2Y₁₂ and P2X1 are unclear. Objective: To determine the agonist and antagonist activities of diadenosine tetraphosphate analogs towards P2Y₁, P2Y₁₂, and P2X1. Methods: We synthesized the following Ap₄A analogs: P¹,P⁴‐dithiotetraphosphate; P²,P³‐chloromethylenetetraphosphate; P¹‐thio‐P²,P³‐chloromethylenetetraphosphate; and P¹,P⁴‐dithio‐P²,P³‐chloromethylenetetraphosphate. We then measured the effects of these analogs on: (i) ADP‐induced platelet aggregation; (ii) P2Y₁‐mediated changes in cytosolic Ca²⁺; (iii) P2Y₁₂‐mediated changes in vasodilator‐stimulated phosphoprotein phosphorylation; and (iv) P2X1‐mediated entry of extracellular Ca²⁺.Results: Ap₄A analogs with modifications in the phosphate backbone inhibited both P2Y₁ and P2Y₁₂, and showed no agonist activity towards these receptors. The dithio modification increased inhibition of P2Y₁, P2Y₁₂, and platelet aggregation, whereas the chloromethylene modification increased inhibition of P2Y₁₂ and platelet aggregation, but decreased P2Y₁ inhibition. Combining the dithio and chloromethylene modifications increased P2Y₁ and P2Y₁₂ inhibition. As compared with Ap₄A, each modification decreased agonist activity towards P2X1, and the dual modification completely eliminated P2X1 agonist activity. Conclusions: As compared with Ap₄A, tetraphosphate backbone analogs of Ap₄A have diminished activity towards P2X1 but inhibit both P2Y₁ and P2Y₁₂ and, with greater potency, inhibit ADP‐induced platelet aggregation. Thus, diadenosine tetraphosphate analogs with dual receptor selectivity may have potential as antiplatelet drugs.     

Potentiating role of Gas6 and Tyro3, Axl and Mer (TAM) receptors in human and murine platelet activation and thrombus stabilization

Summary. Background: Interaction of murine Gas6 with the platelet Gas6 receptors Tyro3, Axl and Mer (TAM) plays an important role in arterial thrombus formation. However, a role for Gas6 in human platelet activation has been questioned. Objective: To determine the role of Gas6 in human and murine platelet activation and thrombus formation. Methods and Results: Gas6 levels appeared to be 20‐fold higher in human plasma than in platelets, suggesting a predominant role of plasma‐derived Gas6. Human Gas6 synergizes with ADP–P2Y₁₂ by enhancing and prolonging the phosphorylation of Akt. Removal of Gas6 from plasma impaired ADP‐induced platelet aggregation. Under flow conditions, absence of human Gas6 provoked gradual platelet disaggregation and integrin α_IIbβ₃ inactivation. Recombinant human Gas6 reversed the effects of Gas6 removal. In mouse blood, deficiency in Gas6 or in one of the TAM receptors led to reduced thrombus formation and increased disaggregation, which was completely antagonized by external ADP. In contrast, collagen‐induced platelet responses were unchanged by the absence of Gas6 in both human and mouse systems. Conclusions: The ADP–P2Y₁₂ and Gas6–TAM activation pathways synergize to achieve persistent α_IIbβ₃ activation and platelet aggregation. We postulate a model of thrombus stabilization in which plasma Gas6, by signaling via the TAM receptors, extends and enhances the platelet‐stabilizing effect of autocrine ADP, particularly when secretion becomes limited.     

Earlier recovery of platelet function after discontinuation of treatment with ticagrelor compared with clopidogrel in patients with high antiplatelet responses

Summary. Background: The rate of recovery of platelet function after discontinuation of P2Y₁₂ inhibitors depends on the reversibility of the antiplatelet effect and the extent of the on‐treatment response. P2Y₁₂ inhibition increases the bleeding risk in patients requiring surgery. Objectives: To evaluate recovery of platelet function after discontinuation of ticagrelor vs. clopidogrel in stable coronary artery disease (CAD) patients with high levels of platelet inhibition (HPI) during the ONSET/OFFSET study. Methods: Patients received aspirin 75–100 mg per day and either ticagrelor 90 mg twice‐daily or clopidogrel 75 mg daily for 6 weeks. This subanalysis included patients with HPI after the last dose of maintenance therapy, defined as: inhibition of platelet aggregation (IPA) > 75% 4 h post‐dose (ADP 20 μm , final extent); < 120 P2Y₁₂ reaction units 8 h post‐dose (VerifyNow P2Y₁₂ assay); or platelet reactivity index < 50% 8 h post‐dose (VASP‐P assay). Results: IPA > 75% was observed in 39 out of 47 ticagrelor‐treated and 17 out of 44 clopidogrel‐treated patients. The rate of offset of IPA over 4–72 h was greater with ticagrelor (IPA %/hour slope: ?1.11 vs. ?0.67 for clopidogrel; P < 0.0001). Mean IPA was significantly lower with ticagrelor than clopidogrel between 48 and 168 h post‐dose (P < 0.01). Similar findings were observed with the other assays. The average time for IPA to decline from 30% to 10% was 50.8 h with ticagrelor vs. 110.4 h with clopidogrel. Conclusions: In patients with HPI, recovery of platelet function was more rapid after discontinuation of ticagrelor than clopidogrel leading to significantly greater platelet reactivity by 48 h after the last dose in the ticagrelor group.     

Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12

Summary. Background: In our previous in vitro study, we reported a constitutively active chimeric P2Y₁₂ (cP2Y₁₂) and found that AR‐C78511 is a potent inverse agonist at this receptor. The role of cP2Y₁₂ in platelet activation and thrombosis is not clear. Objectives: To investigate the physiologic implications of cP2Y₁₂ for platelet activation and thrombus formation, and to evaluate the antiplatelet activity of AR‐C78511 as an inverse agonist. Methods and Results:  We generated transgenic mice conditionally and platelet‐specifically expressing cP2Y₁₂. High‐level expression of cP2Y₁₂ in platelets increased platelet reactivity, as shown by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice showed a shortened bleeding time, and more rapid and stable thrombus formation in mesenteric artery injured with FeCl₃. The constitutive activity of cP2Y₁₂ in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR‐C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited a superior antiplatelet effect to that of AR‐C69931MX in transgenic mice. Conclusions:  These findings further emphasize the importance of P2Y₁₂ in platelet activation, hemostasis, and thrombosis, as well as the prothrombotic role of the constitutive activity of P2Y₁₂. Our data also validate the in vivo inverse agonist activity of AR‐C78511, and confirm its superior antiplatelet activity over neutral antagonists.     

Mechanism of action and clinical development of platelet thrombin receptor antagonists

Atherothrombotic disease is the leading cause of death worldwide. Currently, dual antiplatelet therapy with aspirin and ADP receptor antagonists has shown improved short- and long-term clinical outcomes but is associated with increased bleeding risk, and the rates of recurrent ischemic events still remain high. Selective inhibition of the principal protease-activated receptor (PAR)-1 for thrombin, the most potent platelet activator, represents a promising novel strategy to reduce ischemic events without increasing the risk of bleeding. Two PAR-1 antagonists are currently being tested in clinical trials: SCH 530348 and E5555. Both have demonstrated an antiplatelet effect without increasing bleeding time in preclinical trials. Results of Phase II trials showed that SCH 530348, in addition to standard antiplatelet therapy, was well tolerated and not associated with increased bleeding risk. The safety and tolerability of E5555 is being evaluated in patients with coronary artery disease and non-ST-segment elevation acute coronary syndrome in four Phase II clinical trials. Two large-scale Phase III trials assessing the efficacy of SCH 530348 in addition to the standard of care are currently ongoing. This article provides an overview of the current status of knowledge on platelet thrombin receptor antagonists, focusing on pharmacologic properties and clinical development.     

Dietary Fats and Platelet Function among Finnish Men

The aim of this study was to elucidate the role of platelet aggregation as a risk factor for ischemic heart disease (IHD) and the relationship between fatty acids and platelet function. Platelet aggregation upon adenosine diphosphate (ADP), adrenaline and thrombin were measured in middle aged men in east and west, two regions of Finland with a nearly twofold difference in IHD mortality. Platelet aggregation results were correlated with the fatty acid compositions of plasma lipid fractions, adipose tissue triglycerides and platelet phospholipids.

There was no significant east-west difference in platelet reactivity to ADP, adrenaline and thrombin. ADP-induced platelet aggregation showed significant negative correlations with all the platelet C20-C22 n-3 polyunsaturated fatty acids (PL)FA), but significant positive correlations with the percentage of 18:2n-6 in adipose tissue and plasma cholesterol esters (CE) and triglycerides (TG). Adrenaline-induced aggregation correlated negatively with the percentage of 20:5n-3 in plasma CE and TG, and positively with the total percentage of saturated fatty acids in platelets. Aggregation upon thrombin had a negative correlation with the 20:3n-6/20:4n-6 ratio in plasma CE and a positive correlation with 18: 2n-6 in adipose tissue.

The percentages of the major PUFA in platelets correlated sgnificantly with the same fatty acids in plasma CE and phospholipids PL. Platelet 20: 5n-3 had a highly significant negative correlation with the percentage of 18:2n-6 in plasma and adipose tissue lipids. Platelet 20: 4n-6 was unrelated to its precursors in plasma. The percentage of 18: 2n-6 in plasma and adipose tissue lipids was consistently lower in men from eastern than western Finland, whereas the percentage of platelet 20: 5n-3 was higher in the eastern men.

The results indicate, that (1) platelet function may not play a role in the regional gradient in IHD rate in Finland, (2) in a free-living population, relatively small changes in plasma and platelet fatty acids may have significant influence on platelet aggregation and on thrombosis tendency and (3) an increase in dietary PUFA content may not affect platelet function favourably, unless attention is paid to the maintaining of the n-3/n-6 ratio in the diet.     

Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel

Summary. Background: The thienopyridine P2Y₁₂ receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y₁₂ blockade. Methods: Healthy volunteers received a 600‐mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y₁₂ receptor number was assessed using a ³³P‐2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. Results: Rifampicin enhanced clopidogrel AM production [area‐under‐the‐curve (AUC): clopidogrel 89 ± 22 ng h mL^?1, clopidogrel + rifampicin 335 ± 86 ng h mL^?1, P < 0.0001], and P2Y₁₂ blockade (unblocked receptors: clopidogrel 48 ± 24, clopidogrel + rifampicin 4 ± 2, P < 0.0001) and reduced PA (5 μmol L^?1 ADP: clopidogrel 20 ± 4, clopidogrel + rifampicin 5 ± 2, P < 0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 μmol L^?1 was particularly sensitive to low levels of receptor blockade. Conclusion: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y₁₂ blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y₁₂ blockade.     

Inhibition of Primary ADP-Induced Platelet Aggregation in Normal Subjects after Administration of Nitrofurantoin (Furadantin)

The evidence indicating that platelets may play a role in the occurrence of certain thromboembolic phenomena has stimulated a search for inhibitors of platelet function. This report presents data to indicate that nitrofurantoin is a potent inhibitor of primary ADP-induced platelet aggregation. The addition of 10 muM nitrofurantoin to citrated platelet-rich plasma obtained from 12 normal subjects produced a 29+/-6% (2 SD) inhibition of the velocity of platelet aggregation induced by 2 muM ADP. The inhibitory effect of nitrofurantoin demonstrated competitive kinetics in respect to ADP. The intravenous (180 mg) or oral (200 mg) administration of nitrofurantoin produced a serum nitrofurantoin concentration ranging from 2.7 to 23 muM in 28 normal subjects. Platelet-rich plasma obtained from these subjects demonstrated inhibition of the velocity of ADP-induced platelet aggregation that correlated with the log of the serum nitrofurantoin concentration (P < 0.001). Collagen-induced platelet aggregation was also inhibited in a dose-related manner, and the bleeding time was significantly prolonged in the two subjects with the highest serum nitrofurantoin concentration. These studies indicate that nitrofurantoin in vivo inhibits platelet function to a degree that is proportional to the serum nitrofurantoin concentration.     

Platelet activation by ADP: the role of ADP antagonists

ADP plays a key role in haemostasis and thrombosis. Despite its early identification in 1961 as the first known aggregating agent, the molecular basis of ADP-induced platelet activation is only beginning to be understood. Two purinergic receptors contribute separately to the complex process of ADP-induced platelet aggregation: the P2Y1 metabotropic receptor responsible for mobilization of ionized calcium from internal stores, which initiates aggregation, and P2Y receptor coupled to adenylyl cyclase inhibition, which is essential for the full aggregation response to ADP and stabilization of platelet aggregates. The latter is the molecular target of the ADP-selective antiaggregating drugs ticlopidine and clopidogrel and the ATP analogues of the AR-C series. In addition, it is probably defective in patients with a bleeding diathesis characterized by selective impairment of platelet responses to ADP. Finally, the P2X1 ionotropic receptor is also present in platelets, but its role is not yet known. Studies with P2Y1 knock-out mice as well as the use of selective P2Y1 antagonists have shown that, in addition to the P2Y receptor, which is the target of clopidogrel, the P2Y1 receptor is an important potential target for new antithrombotic drugs.     

Novel assay demonstrates that coronary artery disease patients have heightened procoagulant platelet response

Essentials

• Procoagulant platelets can be detected using GSAO in human whole blood.
• Stable coronary artery disease is associated with a heightened procoagulant platelet response.
• Agonist‐induced procoagulant platelet response is not inhibited by aspirin alone.
• Collagen plus thrombin induced procoagulant platelet response is partially resistant to clopidogrel.

Summary

Background

Procoagulant platelets are a subset of highly activated platelets with a critical role in thrombin generation. Evaluation of their clinical utility in thrombotic disorders, such as coronary artery disease (CAD), has been thwarted by the lack of a sensitive and specific whole blood assay.

Objectives

We developed a novel assay, utilizing the cell death marker, GSAO [(4‐(N‐(S‐glutathionylacetyl)amino)phenylarsonous acid], and the platelet activation marker, P‐selectin, to identify procoagulant platelets in whole blood by flow cytometry.

Patients/Methods

Using this assay, we characterized the procoagulant platelet population in healthy controls and a cohort of patients undergoing elective coronary angiography.

Results

In patients with CAD, compared with patients without CAD, there was a heightened procoagulant platelet response to thrombin (25.2% vs. 12.2%), adenosine diphosphate (ADP) (7.8% vs. 2.7%) and thrombin plus collagen (27.2% vs. 18.3%). The heightened procoagulant platelet potential in CAD patients was not associated with other markers of platelet function, including aggregation, dense granule release and activation of α_2bβ₃ integrin. Although dual antiplatelet therapy (DAPT) was associated with partial suppression of procoagulant platelets, this inhibitory effect on a patient level could not be predicted by aggregation response to ADP and was not fully suppressed by clopidogrel.

Conclusions

We report for the first time that procoagulant platelets can be efficiently detected in a few microliters of whole blood using the cell death marker, GSAO, and the platelet activation marker, P‐selectin. A heightened procoagulant platelet response may provide insight into the thrombotic risk of CAD and help identify a novel target for antiplatelet therapies in CAD.     

Cangrelor: a review on its mechanism of action and clinical development

In patients with acute coronary syndromes and undergoing percutaneous coronary intervention, numerous large-scale clinical trials have shown that adjunctive treatment with the P2Y₁₂ receptor antagonist clopidogrel in addition to aspirin reduces ischemic events. These studies underscore the importance of blockade of the P2Y₁₂ signaling pathway in these settings. However, recent findings have shown that clopidogrel therapy may have some shortcomings. These include its broad range of interindividual-response profiles, where patients with low P2Y₁₂ inhibitory effects have an increased risk of recurrent ischemic events, including stent thrombosis, and its irreversible mechanism of action. These observations underscore the need for novel antiplatelet agents overcoming these limitations. Cangrelor (AR-C69931MX) is an intravenous, direct-acting and reversible P2Y₁₂ receptor antagonist. Cangrelor has a rapid onset and offset of action and achieves significantly greater degrees of platelet inhibition compared with clopidogrel. This article provides an overview of the current status of knowledge on cangrelor, focusing on its pharmacologic properties, clinical development and potential future applications.     

The Effect of Psychoactive Drugs on in vitro Platelet Function

BACKGROUND: Neuro-hormonal and hemostatic mechanisms are important in a wide range of psychological and cardiovascular diseases. The use of psychoactive drugs in mental illnesses is often involved with hematologic side effects including impaired platelet function. Subsequently, the risk for the development of cardiovascular diseases may be higher in these patients. Interestingly, platelets that play a key role in cardiovascular complications contain quite a number of neuronal receptors which are involved in psychotic disorders. It has been widely discussed whether psychoactive drugs used in the therapy of psychotic disorders have a direct effect on platelet function and whether the effects are transmitted through the corresponding receptors on the platelet surface. MATERIAL AND METHODS: In this study, we tested several psychoactive drugs regarding their impact on whole blood platelet aggregation. RESULTS: Antidopaminergics preferentially inhibited ADP-induced aggregation whereas anticholinergics mainly inhibited U46619-induced aggregation. Because platelets respond selectively to different psychoactive drugs we assume that corresponding receptors have a functional aspect on platelets and that receptor blockade affects platelet aggregation through different mechanisms. CONCLUSION: The knowledge about the effects of psychoactive drugs on platelet function may help to characterize neuronal receptors on platelets and may contribute to a better understanding of altered platelet function during therapy with psychoactive drugs.