Curcumin by activation of adenosine A2A receptor stimulates protein kinase a and potentiates inhibitory effect of cangrelor on platelets.

Curcumin is a natural polyphenol derived from the turmeric plant (Curcuma longa) which exhibits numerous beneficial effects on different cell types. Inhibition of platelet activation by curcumin is well known, however molecular mechanisms of its action on platelets are not fully defined. In this study, we used laser diffraction method for analysis of platelet aggregation and Western blot for analysis of intracellular signaling mechanisms of curcumin effects on platelets. We identified two new molecular mechanisms involved in the inhibitory effects of curcumin on platelet activation. Firstly, curcumin by activation of adenosine A2A receptor stimulated protein kinase A activation and phosphorylation of Vasodilator-stimulated phosphoprotein. Secondly, we demonstrated that curcumin even at low doses, which did not inhibit platelet aggregation, potentiated inhibitory effect of ADP receptor P2Y12 antagonist cangrelor which partly could be explained by activation of adenosine A2A receptor.

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti-platelet therapy.

BACKGROUND: Endothelium-derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO-sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. OBJECTIVE: We investigated using GC-activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti-thrombotic effect of both drugs. METHODS: In vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride-induced arterial thrombosis model were also performed. RESULTS: The GC-activator BAY-70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra-platelet cyclic nucleotide levels. Furthermore, mice administered sub-maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. CONCLUSIONS: Using in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti-platelet effects without altering blood flow. Therefore, modulation of intra-platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti-thrombotic regimen while minimizing vasodilator side effects.

Designing Natural Product Hybrids Bearing Triple Antiplatelet Profile and Evaluating Their Human Plasma Stability.

Cardiovascular diseases (CVDs) are becoming major contributors to the burden of disease due to genetic and environmental factors. Despite current standard oral care, cardiovascular risk remains relatively high. A triple antiplatelet therapy with a cyclooxygenase-1 (COX-1) inhibitor, a P2Y12 receptor antagonist, and a protease-activated receptor-1 (PAR-1) antagonist has been established in the secondary prevention of atherothrombosis in patients with acute myocardial infraction and in those with peripheral artery disease. However, due to the combinatorial use of three different drugs, patients receiving this triple therapy are exposed to enhanced risk of bleeding. Conforming to polypharmacology principles, the discovery of a single compound that can simultaneously block the three platelet activation pathways (PAR-1, P2Y12, and COX-1) is of importance. Natural products have served as an inexhaustible source of bioactive compounds presenting a diverse pharmaceutical profile, including anti-inflammatory, antioxidant, anticancer, and antithrombotic activity. Indeed, principal component analysis indicated that natural products have the potential to inhibit the three aforementioned pathways, though existed reports refer to single inhibition mechanism on specific receptor(s) implicated in platelet activation. We thus set out to explore possibilities that take advantage of this potential of natural products and shape the basis to produce novel compounds that could simultaneously target PAR-1, P2Y12, and COX-1 platelet activation pathways. Polyunsaturated fatty acids (PUFAs) have multiple effects leading to improvements in blood pressure and cardiac function and arterial compliance. A promising approach to achieve the desirable goal is the bioconjugation of natural products with PUFAs. Herein, we describe the principles that should be followed to develop molecular hybrids bearing triple antiplatelet activity profile.

Salvianolic acids from antithrombotic Traditional Chinese Medicine Danshen are antagonists of human P2Y1 and P2Y12 receptors.

Many hemorheologic Traditional Chinese Medicines (TCMs) that are widely-used clinically lack molecular mechanisms of action. We hypothesized that some of the active components of hemorheologic TCMs may function through targeting prothrombotic P2Y1 and/or P2Y12 receptors. The interactions between 253 antithrombotic compounds from TCM and these two G protein-coupled P2Y receptors were evaluated using virtual screening. Eleven highly ranked hits were further tested in radioligand binding and functional assays. Among these compounds, salvianolic acid A and C antagonized the activity of both P2Y1 and P2Y12 receptors in the low µM range, while salvianolic acid B antagonized the P2Y12 receptor. These three salvianolic acids are the major active components of the broadly-used hemorheologic TCM Danshen (Salvia militorrhiza), the antithrombotic molecular mechanisms of which were largely unknown. Thus, the combination of virtual screening and experimental validation identified potential mechanisms of action of multicomponent drugs that are already employed clinically.

Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis.

CONTEXT: Corydalis yanhusuo W.T. Wang (Papaveraceae) (Rhizoma Corydalis) showed inhibitory effects on rabbit platelet aggregation induced by ADP, thrombin (THR) or arachidonic acid (AA). OBJECTIVE: This study separates and identifies the possible target-related platelet proteins and suggests possible signal cascades of RC antiplatelet aggregation. MATERIALS AND METHODS: Based on comparative proteomics, the differentially expressed platelet proteins treated before and after with 50 mg/mL RC 90% ethanol extract (for 15 min at 37 °C) were analyzed and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS. To further verify the possible signalling pathways of RC antiplatelet aggregation function, the concentration of calcium (Ca2+) was measured by Fura-2/AM fluorescence (Ex 340/380 nm, Em 500 nm) (RC final concentrations of 0.0156-0.1563 mg/mL), the levels of P-selectin and cyclic guanosine monophosphate (cGMP) were quantified by ELISA (OD. 450 nm) (RC final concentrations of 0.0156-1.5625 mg/mL), and the 5-hydroxytryptamine (5-HT) level was measured using ortho-phthalaldehyde (OPT) fluorescence (Ex 340 nm, Em 470 nm) (RC final concentrations of 0.3125-1.5625 mg/mL). RESULTS: The expression of 52 proteins were altered in rabbit platelets after the treatment and the MALDI-TOF-MS analysis indicated that those proteins include 12 cytoskeleton proteins, 7 cell signalling proteins, 3 molecular chaperone proteins, 6 proteins related to platelet function, 16 enzymes and 7 other related proteins. Furthermore, RC extract could decrease the levels of 5-HT [inhibition rate of 96.80% (p < 0.05, vs. THR-activated group) treated with 0.7813 mg/mL of RC], Ca2+ [172.73 ± 5.07 to 113.56 ± 5.46 nM (p < 0.001, vs. THR-activated group) treated with 0.0313 mg/mL of RC] and P-selectin [13.48 ± 0.96 ng/3 × 108 to 11.64 ± 0.17 ng/3 × 108 (p < 0.05, vs. THR-activated group) treated with 0.0156 mg/mL of RC], and increase in cGMP level [38.93 ± 0.57 to 50.26 ± 4.05 ng/3 × 108 (p < 0.05, vs. THR-activated group) treated with 1.5165 mg/mL of RC] in ADP (10 µmol/L), THR (0.25 u/mL) or AA-(0.205 mmol/L) activated rabbit platelets. DISCUSSION AND CONCLUSION: The present study indicated that P2Y12 receptor might be one of the direct target proteins of RC in platelets. The signal cascades network of RC after binding with P2Y12 receptor is mediating Gαi proteins to activate downstream signalling pathways (AC and/or PI3K signalling pathways) for the inhibition of platelet aggregation.

New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors.

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5') tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.

Molecular mechanism of tanshinone IIA and cryptotanshinone in platelet anti-aggregating effects: an integrated study of pharmacology and computational analysis.

Tanshinone IIA and cryptotanshinone are two pharmacologically active diterpenoids extracted from the roots of Salvia milthiorriza Bunge, a plant used in Chinese traditional medicine for the treatment of some cardiovascular and cerebrovascular disease. Until now, the molecular mechanisms of action of these two diterpenoids on platelets are partially known. To clarify this aspect, here we utilized an integrated study of pharmacology and computational analysis. Our results demonstrate that cryptotanshinone is able to inhibit in a concentration dependent manner the rat platelet aggregation and also is endowed of Gi-coupled P2Y12 receptor antagonist as demonstrated by docking studies. This computational method was also performed for tanshinone IIA demonstrating even for this diterpenoid an interaction with the same receptor. The findings from our study enable a better understanding of tanshinone IIA and cryptotanshinone biological properties, which could ultimately lead to the development of novel pharmaceutical strategies for the treatment and/or prevention of some cardiovascular disease.

Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: evidence from clinic to laboratory.

Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use.

Platelet P2Y12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng.

BACKGROUND AND PURPOSE: Saponins isolated from Panax notoginseng (Burk.) F.H. Chen have been shown to relieve thrombogenesis and facilitate haemostasis. However, it is not known which saponin accounts for this haemostatic effect. Hence, in the present study we aimed to identify which saponins contribute to its haemostatic activity and to elucidate the possible underlying mechanisms. EXPERIMENTAL APPROACH: Platelet aggregation was analysed using a platelet aggregometer. Prothrombin time, activated partial thromboplastin time and thrombin time were measured using a blood coagulation analyser, which was further corroborated with bleeding time and thrombotic assays. The interaction of notoginsenoside Ft1 with the platelet P2Y12 receptor was determined by molecular docking analysis, cytosolic Ca(2+) and cAMP measurements, and phosphorylation of PI3K and Akt assays. KEY RESULTS: Among the saponins examined, Ft1 was the most potent procoagulant and induced dose-dependent platelet aggregation. Ft1 reduced plasma coagulation indexes, decreased tail bleeding time and increased thrombogenesis. Moreover, it potentiated ADP-induced platelet aggregation and increased cytosolic Ca(2+) accumulation, effects that were attenuated by clopidogrel. Molecular docking analysis suggested that Ft1 binds to platelet P2Y12 receptors. The increase in intracellular Ca(2+) evoked by Ft1 in HEK293 cells overexpressing P2Y12 receptors could be blocked by ticagrelor. Ft1 also affected the production of cAMP and increased phosphorylation of PI3K and Akt downstream of P2Y12 signalling pathways. CONCLUSION AND IMPLICATIONS: Ft1 enhanced platelet aggregation by activating a signalling network mediated through P2Y12 receptors. These novel findings may contribute to the effective utilization of this compound in the therapy of haematological disorders.

Permeation of fluorophore-conjugated phalloidin into live hair cells of the inner ear is modulated by P2Y receptors.

Phalloidin, a toxin isolated from the death cap mushroom, Amanita phalloides, binds to filamentous actin with high affinity, and this has made fluorophore-conjugated phalloidin a useful tool in cellular imaging. Hepatocytes take up phalloidin via the liver-specific organic anion transporting polypeptide 1b2, but phalloidin does not permeate most living cells. Rapid entry of styryl dyes into live hair cells has been used to evaluate function, but the usefulness of those fluorescence dyes is limited by broad and fixed absorption spectra. Since phalloidin can be conjugated to fluorophores with various spectra, we investigated whether it would permeate living hair cells. When we incubated mouse utricles in 66 nM phalloidin-CF488A and followed that by washes in phalloidin-free medium, we observed that it entered a subset of hair cells and labeled entire hair bundles fluorescently after 20 min. Incubations of 90 min labeled nearly all the hair bundles. When phalloidin-treated utricles were cultured for 24 h after washout, the label disappeared from the hair cells and progressively but heterogeneously labeled filamentous actin in the supporting cells. We investigated how phalloidin may enter hair cells and found that P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid and suramin, blocked phalloidin entry, while the P2Y receptor ligands, uridine-5'-diphosphate and uridine-5'-triphosphaste, stimulated uptake. Consistent with that, the P2Y6 receptor antagonist, MRS 2578, decreased phalloidin uptake. The results show that phalloidin permeates live hair cells through a pathway that requires metabotropic P2Y receptor signaling and suggest that phalloidin can be transferred from hair cells to supporting cells in culture.

Plant extracts inhibit ADP-induced platelet activation in humans: their potential therapeutic role as ADP antagonists.

Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. Platelet hyperactivity is associated with vascular disease and also has a key role in haemostasis and thrombosis. ADP activates platelets through three purinoceptor subtypes, the G(q)-coupled P2Y(1) receptor, G(i)-coupled P2Y(12) receptor and P2X(1) ligand-gated cation channel. Platelet ADP purinergic receptors are therefore suitable targets for antiplatelet drugs. Thienopyridines such as clopidogrel and ticlopidine, as well as other ADP receptor antagonists like prasugrel, ticagrelor, cangrelor and elinogrel have demonstrated clinical benefits via the inhibition of the selective purinergic ADP receptor, P2Y(12). However, they still have limitations in their mode of action and efficacy, like increased risk of bleeding. Thus, the ongoing pursuit to develop newer and more effective antiplatelet agents continues. There is a growing interest in the purinergic antiplatelet properties exhibited by plant extracts. This article considers the following: pomolic acid isolated from Licania pittieri, brazilin isolated from the heartwood of Caesalpinia sappan L, phylligenin isolated from the twigs of Muraltia vulpina, bark oil of Gonystylus velutinus, seed and bark extracts from Aesculus hippocastanum L. and red wine phenolics and catechins isolated from green tea. Moreover, the method used to investigate platelet purinergic receptors should be considered, since using a more sensitive, high-resolution platelet sizer can sometimes detect platelet variations when the light transmission method was not able to do so. The exact mechanisms by which these plant extracts work need further investigation. They all however inhibit ADP-induced activation in human platelets. This could explain, at least in part, the protective effect of plant extracts as antiplatelet agents.

[Synergistic action of Compound Danshen Dripping Pill (CDDP) on Clopidogrel Bisulfate (CPG) counteracting platelet aggregation].

OBJECTIVE: To study the synergistic action of Compound Danshen Dripping Pill (CDDP) on Clopidogrel Bisulfate (CPG) counteracting platelet aggregation. METHODS: 40 Sprague-Dawley (SD) rats were randomized into four groups: normal control group (CMC-Na), CPG alone group (30 mg/kg), CDDP alone group (324 mg/kg), co-administration group (CPG 30 mg/kg and CDDP 324 mg/kg). The rats received gastric infusion of corresponding drugs for 21 continuous days. Blood sample of SD rats were collected by puncture of the abdominal artery for the determination of coagulation parameters such as prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) concentration and thrombin time (TT) in each group. Another 40 SD rats were used for the observation of inhibitory effect on the thrombosis in arteriovenous shunt. Finally, homology modeling and molecular docking were employed to simulate their interaction between the P2Y purinoceptor 12 (P2Y12) target and bioactive compounds contained in CDDP. RESULTS: The bleeding time of coagulation parameters was prolonged, the thrombosis in arteriovenous shunt was inhibited in the medication group. The above effect was much better in the combination group. The molecular docking showed that bioactive compounds contained in CDDP was able to inhibit target P2Y12. CONCLUSION: CDDP can enhance the effect of CPG on inhibiting platelet aggregation.

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay.

BACKGROUND: Microfluidic devices recreate the hemodynamic conditions of thrombosis. METHODS: Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate=200 s(-1)) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y1 inhibitor (MRS 2179), P2Y12 inhibitor (2MeSAMP) or combined P2Y1 and P2Y12 inhibitors. RESULTS: High dose ASA (500 µM), 2MeSAMP (100 µM), MRS 2179 (10 µM), or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p<0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. R(COX) and R(P2Y) (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had R(COX)<1 or R(P2Y)<1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had R(P2Y)<1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of R(COX) and R(P2Y) to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively). CONCLUSIONS: Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self-normalized metric of platelet function for a single blood sample.

High-dose aspirin in dogs increases vascular resistance with limited additional anti-platelet effect when combined with potent P2Y12 inhibition.

INTRODUCTION: With the arrival of the potent P2Y12 antagonists, ticagrelor and prasugrel, the need for co-treatment with aspirin in acute coronary syndromes must be re-examined. This study assessed whether high-dose aspirin: a) provides additional anti-platelet efficacy, assessed in vivo and ex vivo, when combined with P2Y12 inhibition; and/or b) has a negative effect on vascular function. MATERIALS AND METHODS: Using an anaesthetized dog model of thrombosis, the effects of aspirin (50mg/kg) in addition to clopidogrel and ticagrelor were evaluated at two levels of P2Y12 inhibition, maximal (≥96%) and sub-maximal (~80%), as assessed by ex vivo ADP-induced whole blood impedence aggregometry. RESULTS: In the absence of aspirin, maximal and sub-maximal P2Y12 inhibition inhibited arachidonic acid-induced platelet aggregation by approximately 80% and 24%, respectively, without affecting platelet TXA2 formation. During maximal P2Y12 inhibition, aspirin provided less additional inhibition of ex vivo arachidonic acid- and collagen-induced platelet aggregation, as compared with sub-maximal P2Y12 inhibition, without additional anti-thrombotic effect in vivo. Aspirin significantly decreased in vivo PGI2 production (27%) and increased vascular resistance (16%), independently of P2Y12 antagonism. CONCLUSION: In the dog, P2Y12 antagonists inhibit TXA2-mediated platelet-aggregation independently of aspirin. Aspirin provides less additional anti-platelet effects during maximal compared with sub-maximal P2Y12 inhibition but increases vascular resistance.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling.

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbß3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin ß3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

Advances in the monitoring of anti-P2Y12 therapy.

Although there are many new and effective anti-P2Y(12) drugs available, clopidogrel in its original or generic forms will probably remain the most widely used and cheaper option. As clopidogrel is a pro-drug, there is marked heterogeneity in drug responsiveness between individuals and lack of responsiveness is associated with poorer clinical outcomes. Various platelet function and genetic tests are now available for potentially measuring whether clopidogrel effectively inhibits platelet function. Monitoring of P2Y(12) inhibition and/or identification of loss of function cytochrome P-450 genotypes could, therefore, offer the potential of tailoring therapy by identifying poor responders to clopidogrel and optimizing the levels of platelet inhibition using, for example, alternative drugs such as prasugrel or ticagrelor. The question remains whether any of these tests have prognostic utility with a defined therapeutic window to reliably identify hypo or hyper-responsive patients who may have an increased risk of thrombosis or bleeding, respectively? Once such patients are identified, can the tests then be subsequently used to demonstrate a change or improvement in platelet reactivity by using alternative therapies and equate this with improved clinical outcome? In this review, we describe an overview of the current platelet and genetic tests available and discuss whether these tests will ever become used routinely.

A randomized trial of prasugrel versus clopidogrel in patients with high platelet reactivity on clopidogrel after elective percutaneous coronary intervention with implantation of drug-eluting stents: results of the TRIGGER-PCI (Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy With Prasugrel) study.

OBJECTIVES: This study sought to investigate the efficacy, safety, and antiplatelet effect of prasugrel as compared with clopidogrel in patients with high on-treatment platelet reactivity (HTPR) after elective percutaneous coronary intervention (PCI). BACKGROUND: The extent to which prasugrel can correct HTPR and improve clinical outcomes in patients undergoing elective PCI is unknown. METHODS: Stable coronary artery disease (CAD) patients with HTPR (>208 P2Y(12) reaction units [PRU] by the VerifyNow test) after elective PCI with at least 1 drug-eluting stent (DES) were randomly assigned to either prasugrel 10 mg daily or clopidogrel 75 mg daily. Platelet reactivity of the patients on the study drug was reassessed at 3 and 6 months. The study was stopped prematurely for futility because of a lower than expected incidence of the primary endpoint. RESULTS: In 212 patients assigned to prasugrel, PRU decreased from 245 (225 to 273) (median [interquartile range]) at baseline to 80 (42 to 124) at 3 months, whereas in 211 patients assigned to clopidogrel, PRU decreased from 249 (225 to 277) to 241 (194 to 275) (p < 0.001 vs. prasugrel). The primary efficacy endpoint of cardiac death or myocardial infarction at 6 months occurred in no patient on prasugrel versus 1 on clopidogrel. The primary safety endpoint of non-coronary artery bypass graft Thrombolysis In Myocardial Infarction major bleeding at 6 months occurred in 3 patients (1.4%) on prasugrel versus 1 (0.5%) on clopidogrel. CONCLUSIONS: Switching from clopidogrel to prasugrel in patients with HTPR afforded effective platelet inhibition. However, given the low rate of adverse ischemic events after PCI with contemporary DES in stable CAD, the clinical utility of this strategy could not be demonstrated.

Docking-based virtual screening of potential human P2Y12 receptor antagonists.

Platelet plays essential roles in hemostasis and its dysregulation can lead to arterial thrombosis. P2Y12 is an important platelet membrane adenosine diphosphate receptor, and its antagonists have been widely developed as anti-coagulation agents. The current P2Y12 inhibitors available in clinical practice have not fully achieved satisfactory anti-thrombotic effects, leaving room for further improvement. To identify new chemical compounds as potential anti-coagulation inhibitors, we constructed a three-dimensional structure model of human P2Y12 by homology modeling based on the recently reported G-protein coupled receptor Meleagris gallopavo ß1 adrenergic receptor. Virtual screening of the modeled P2Y12 against three subsets of small molecules from the ZINC database, namely lead-like, fragment-like, and drug-like, identified a number of compounds that might have high binding affinity to P2Y12. Detailed analyses of the top three compounds from each subset with the highest scores indicated that all of these compounds beard a hydrophobic bulk supplemented with a few polar atoms which bound at the ligand binding site via largely hydrophobic interactions with the receptor. This study not only provides a structure model of P2Y12 for rational design of anti-platelet inhibitors, but also identifies some potential chemicals for further development.