Inhibited Adenosine Diphosphate Research Articles

Mechanistic Study of Hemoglobin Induced Platelet Activation

Abstract 5262Nitric oxide (NO) has been previously shown to inhibit adenosine diphosphate (ADP)- and thrombin- mediated platelet activation. This inhibitory effect of NO is blocked by cell-free oxyhemoglobin (Villagra, Shiva et al. 2007). Cell-free hemoglobin (Hb) also appears to directly activate platelets even in the absence of ADP. However, the precise mechanisms for cell-free Hb-induced platelet activation have not been fully delineated. We hypothesized that oxyhemoglobin can promote platelet activation through one of three potential mechanisms: 1) direct apoprotein interaction with platelet receptors, 2) reactive oxygen species (ROS) production, and/or 3) scavenging of endogenous NO.Platelets were isolated from whole blood obtained from healthy volunteers (n=15) using sodium citrate blood collection tubes. Platelet activation was measured by examining immunofluorescent Pac-1 labeling by flow cytometry using activated glycoprotein IIb/IIIa as a measure of activation. In the presence of 10 uM concentration of NO donor mahmaNONOate, ADP-induced platelet activation was inhibited by 44%. Incubation of 100 uM cell-free oxyhemoglobin eliminated the inhibitory effect of NO. In addition, cell-free oxyhemoglobin (100 uM concentration) induced platelet activation by 17% above unstimulated controls even in the absence of ADP.Methemoglobin was used to test if direct Hb protein interaction with platelets was sufficient for platelet activation, independent of heme redox state. To produce methemoglobin, the ferrous iron in oxyhemoglobin was oxidized to its ferric form, thus methemoglobin is likely to preserve all direct protein interactions. However, unlike oxyhemoglobin, incubation of 100 uM methemoglobin did not induce platelet activation.Next, we tested whether reactive oxygen species production was responsible for hemoglobin-induced platelet activation. In the absence of hemoglobin, superoxide dismutase (SOD) (100 U/ml) and catalase (100 U/ml) reduced baseline platelet activation, verifying a ROS (Pignatelli, Pulcinelli et al. 1998). However, in the presence of oxyhemoglobin, SOD and catalase did not significantly abrogate the effect of Hb on platelet activation, suggesting that the ROS superoxide and hydrogen peroxide are not involved in hemoglobin-induced platelet activation.To determine if scavenging of endogenous NO was responsible for hemoglobin-induced platelet activation, platelets were incubated in the presence or absence of NO scavengers, carboxy-PTIO (5mM) and Fe-DTCS (3 mM), or nitric oxide synthase (NOS) inhibitor, LNMMA (100 uM). Results from these investigations suggest scavenging of endogenous NO was not responsible for hemoglobin-induced platelet activation. Overall our data suggest a complex mechanism for oxyhemoglobin-mediated platelet activation that is likely to involve more than a single contributing pathway.These data aid in elucidating the mechanism of platelet activation by cell-free hemoglobin and may provide novel insights to designing potential treatments to reduce the platelet activation underlying hypercoagulable states associated with a variety of pathological conditions including hemolytic anemias, stroke, and diabetes.This work was supported by NIH grants HL058091 and HL098032 Disclosures:Gladwin:Patents filed related to treating hemolysis.: Patents & Royalties. Kim-Shapiro:Patents filed related to treating hemolysis. Honororia from Sangart Inc.: Honoraria, Patents & Royalties.

하고초 추출물의 항혈전 효능 및 혈소판 응집 억제작용

한방생약제의 항혈전 및 혈소판 응집 억제 효능을 탐색하기 위하여 하고초의 물 추출물로 혈전 용해능 활성과 혈액 응고시간 지연효과 즉 PT (prothrombin time), APTT (activated partial thromboplastin time)와 혈소판 응집억제 활성 등에 대해 항혈전 효능을 평가하였다. 혈전용해도를 측정하는 fibrin plate가 용해되어 형성된 투명환의 넓이를 측정하는 실험을 진행한 결과 혈전용해도가 농도의존적으로 효능을 나타내었다. 혈액 응고 cascade에 미치는 영향을 알아보기 위해 혈액 응고 시간 지연 및 단축 효과를 확인하고자 APTT와 PT에 미치는 영향을 조사한 결과 PT의 경우 10 mg/ml, 5 mg/ml의 경우에는 대조군보다 우수한 지연효과를 보였다. APTT의 경우에는 10 mg/ml, 5 mg/ml는 대조군과 비교하여 매우 탁월한 지연효과를 보이고, 2.5 mg/ml, 1.25 mg/ml에서도 높은 지연효과를 나타냈다. 혈소판의 응집에 따라 형성되는 두 전극 사이에 형성된 전기적 저항의 변화로 나타나는 실험을 시행한 결과 하고초의 ADP와 collagen에서 뛰어난 응집억제 활성을 보였다. 따라서 위의 항혈전 효능평가 실험결과를 볼 때 하고초를 향후에 혈전 질환의 치료제 개발에 효과적으로 이용될 수 있을 것으로 사료된다. This study was performed to develop effective antithrombotic agents from traditional herb extracts. Prunella vulgaris L. has been used traditionally as a medical resource in cancer therapy, as well as treatment of hypertension and inflammation, and as a diuretic. However, the effects of Prunella vulgaris on thrombosis and platelet activation have not been clearly understood. Antithrombotic and antiplatelet activities of oriental medicinal herbs were investigated by evaluating the effect of the aqueous extract from Prunella vulgaris on the blood coagulation, platelet aggregation and fibrinolysis. Prunella vulgaris extracts showed effective anticoagulant activity in coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT). Prunella vulgaris also inhibited adenosine diphosphate (ADP)- and collagen-induced platelet aggregation. In addition, evaluation of fibrinolytic activity showed that the Prunella vulgaris extracts have high solubility. From these results, it is suggested that Prunella vulgaris can be a potential candidate for anticoagulants and antiplatelets, as well as fibrinolytic agents.

Antiplatelet Drugs: Mechanisms and Risks of Bleeding Following Cardiac Operations

Preoperative antiplatelet drug use is common in patients undergoing coronary artery bypass grafting (CABG). The impact of these drugs on bleeding and blood transfusion varies. We hypothesize that review of available evidence regarding drug-related bleeding risk, underlying mechanisms of platelet dysfunction, and variations in patient response to antiplatelet drugs will aid surgeons as they assess preoperative risk and attempt to limit perioperative bleeding. The purpose of this review is to (1) examine the role that antiplatelet drugs play in excessive postoperative blood transfusion, (2) identify possible mechanisms to explain patient response to antiplatelet drugs, and (3) formulate a strategy to limit excessive blood product usage in these patients. We reviewed available published evidence regarding bleeding risk in patients taking preoperative antiplatelet drugs. In addition, we summarized our previous research into mechanisms of antiplatelet drug-related platelet dysfunction. Aspirin users have a slight but significant increase in blood product usage after CABG (0.5 U of nonautologous blood per treated patient). Platelet adenosine diphosphate (ADP) receptor inhibitors are more potent antiplatelet drugs than aspirin but have a half-life similar to aspirin, around 5 to 10 days. The American Heart Association/American College of Cardiology and the Society of Thoracic Surgeons guidelines recommend discontinuation, if possible, of ADP inhibitors 5 to 7 days before operation because of excessive bleeding risk, whereas aspirin should be continued during the entire perioperative period in most patients. Individual variability in response to aspirin and other antiplatelet drugs is common with both hyper- and hyporesponsiveness seen in 5 to 25% of patients. Use of preoperative antiplatelet drugs is a risk factor for increased perioperative bleeding and blood transfusion. Point-of-care tests can identify patients at high risk for perioperative bleeding and blood transfusion, although these tests have limitations. Available evidence suggests that multiple blood conservation techniques benefit high-risk patients taking antiplatelet drugs before operation. Guidelines for patients who take aspirin and/or thienopyridines before cardiac procedures include some or all of the following: (1) preoperative identification of high-risk patients using point-of-care testing; (2) withdrawal of aspirin or other antiplatelet drugs for a few days and delay of operation in patients at high risk for bleeding if clinical circumstances permit; (3) selective perioperative use of evidence-based blood conservation interventions (e.g., short-course erythropoietin, off-pump procedures, and use of intraoperative blood conservation techniques), especially in high-risk patients; and (4) platelet transfusions if clinical bleeding occurs.

Hydrocortisone inhibition of adenosine diphosphate (ADP)-induced platelet aggregation in horse

The aim of this study was to evaluate the effect of hydrocortisone on adenosine diphosphate (ADP)-induced platelet aggregation in horse. The study was carried out on 35 healthy horses of various breeds and gender. On all horses, citrated blood samples were collected by jugular venipuncture to assess platelet aggregation. On all samples, platelet-rich and platelet-poor plasma were prepared by centrifugation and divided into two different aliquots. On the first aliquot, platelet aggregation was determined after platelet activating with 1 and 0.5 μM ADP (group A). On the last one, the effect of 20-min preincubation with hydrocortisone in ADP-induced aggregation was determined at final ADP concentrations of 1 and 0.5 μM (group B). By means of an aggregometer, the platelet response was quantitated as maximum degree of platelet aggregation and initial velocity of platelet aggregation. All data were analyzed using unpaired Student's t test. A P ≤ 0.05 was considered statistically significant. The obtained results showed a statistically significant effect of hydrocortisone on both the maximum degree of platelet aggregation and the slope of platelet aggregation at the two final concentrations of ADP used. These results are compatible with the possibility that the vessel wall releases smooth muscle-relaxing prostaglandins when injured and that the inhibition of prostaglandin formation by hydrocortisone enhances hemostasis by allowing vasoconstriction to be maintained. Further investigations could be useful utilizing different concentrations of hydrocortisone in order to apply the knowledge of the effect of glucocorticoids on platelet aggregation.

Plasma sequestration: a good therapy to prevent the deleterious effect of clopidogrel when clopidogrel is used within one to four days of coronary bypass surgery

Clopidogrel (Plavix) is an inhibitor of ADP-induced platelet aggregation, acting by direct inhibition of adenosine diphosphate (ADP) binding to its receptor and of the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex. A common recommendation for coronary artery bypass surgery is to stop clopidogrel five to seven days before surgery.(1) Patients taking clopidogrel within five days of surgery are more likely to show an increase in post-operative bleeding, an increase need for blood products, and an increased chance for secondary operations to control bleeding. This paper presents an option which appears to decrease the adverse effects of clopidogrel for patients undergoing coronary artery bypass surgery within one to four days of stopping the use of clopidogrel. This option is autologous plasma sequestration. The process of plasma sequestration collection prior to the administration of heparin during surgery offers a significant therapy to minimize bleeding post bypass. Twelve consecutive patients are included in this study (one a REDO) each receiving clopidogrel within one to four days of surgery. Throughout their hospital stay, three patients required packed red blood cell (RBC) transfusions (two received 2 units, one received 1 unit). No other donor blood products were required. Post-operative bleeding was minimal, and the need for a secondary operation due to bleeding was eliminated. The average stay in the cardiovascular intensive care unit(CICU) was 2.8 days, with an average hospital stay of 7.25 days.

The pharmacokinetics and pharmacodynamics of prasugrel in healthy Chinese, Japanese, and Korean subjects compared with healthy Caucasian subjects

Prasugrel is a novel thienopyridine prodrug metabolised to an active metabolite that binds irreversibly to the platelet P2Y(12) receptor and inhibits adenosine diphosphate (ADP)-induced platelet aggregation. We compared prasugrel pharmacokinetics, pharmacodynamics, and tolerability in healthy Chinese, Japanese, Korean and Caucasian subjects. In an open-label, single-centre, parallel-design study, 89 healthy subjects (25 Chinese, 20 Japanese, 22 Korean and 22 Caucasian) aged 20-65 years were given a prasugrel 60-mg loading dose (LD) followed by daily 10-mg maintenance doses (MD) for 7 days and then 5-mg MD for 10 days. Plasma concentrations of prasugrel's active metabolite and inhibition of ADP-induced platelet aggregation (IPA) were determined. Mean exposure to prasugrel's active metabolite in all treatment regimens was higher in each of the Asian groups than in the Caucasian group, although there was considerable overlap between individual exposure estimates in Asians and Caucasians. The mean IPA was also higher in Asians than in Caucasians following a prasugrel 60-mg LD, although the difference did not consistently achieve statistical significance. Prasugrel 10-mg or 5-mg MD produced statistically significantly higher IPA in each Asian group compared with that in the Caucasians. Prasugrel was well tolerated during the LD and MD regimens by all groups. Mean exposure to the prasugrel active metabolite following prasugrel 60-mg LD and during daily 10-mg or 5-mg MD was higher in each of the Asian groups than in the Caucasian group, which resulted in greater platelet inhibition.

Association of Cytochrome P450 2C19 Genotype With the Antiplatelet Effect and Clinical Efficacy of Clopidogrel Therapy

Clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (ADP)-dependent platelet activation. However, nonresponsiveness is widely recognized and is related to recurrent ischemic events. To identify gene variants that influence clopidogrel response. In the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study (2006-2008), we administered clopidogrel for 7 days to 429 healthy Amish persons and measured response by ex vivo platelet aggregometry. A genome-wide association study was performed followed by genotyping the loss-of-function cytochrome P450 (CYP) 2C19*2 variant (rs4244285). Findings in the PAPI Study were extended by examining the relation of CYP2C19*2 genotype to platelet function and cardiovascular outcomes in an independent sample of 227 patients undergoing percutaneous coronary intervention. ADP-stimulated platelet aggregation in response to clopidogrel treatment and cardiovascular events. Platelet response to clopidogrel was highly heritable (h(2) = 0.73; P < .001). Thirteen single-nucleotide polymorphisms on chromosome 10q24 within the CYP2C18-CYP2C19-CYP2C9-CYP2C8 cluster were associated with diminished clopidogrel response, with a high degree of statistical significance (P = 1.5 x 10(-13) for rs12777823, additive model). The rs12777823 polymorphism was in strong linkage disequilibrium with the CYP2C19*2 variant, and was associated with diminished clopidogrel response, accounting for 12% of the variation in platelet aggregation to ADP (P = 4.3 x 10(-11)). The relation between CYP2C19*2 genotype and platelet aggregation was replicated in clopidogrel-treated patients undergoing coronary intervention (P = .02). Furthermore, patients with the CYP2C19*2 variant were more likely (20.9% vs 10.0%) to have a cardiovascular ischemic event or death during 1 year of follow-up (hazard ratio, 2.42; 95% confidence interval, 1.18-4.99; P = .02). CYP2C19*2 genotype was associated with diminished platelet response to clopidogrel treatment and poorer cardiovascular outcomes.

A class of novel N-(3 S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)- l-amino acid derivatives: their synthesis, anti-thrombotic activity evaluation, and 3D QSAR analysis

To find new anti-thrombotic agents, a natural amino acid was introduced into the 3-position of anti-platelet aggregation active 3 S-tetrahydroisoquinoline-3-carboxylic acid (THIQA), and 20 novel dipeptide derivatives, 3 S-tetrahydroisoquinoline-3-carboxyamino acids ( 6a– t), targeting the intestinal peptide transport system were provided. In vitro anti-platelet aggregation assay of 6a– t indicated that their potencies of inhibiting adenosine diphosphate (ADP), arachidonic acid (AA), platelet-activating factor (PAF), and thrombin (TH) induced platelet aggregations were higher than that of THIQA, and the in vivo anti-thrombotic assay of 6a– t indicated that their potencies of inhibiting thrombogenesis in rats were also higher than that of THIQA. According to MFA based Cerius2 QSAR module, using training/test set of 6a, b, d, g– p/ 6c, e, f, q and training/test set of 6a– p/ 6q– t, two equations ( r, 0.984 and 0.996) correlating the structures with in vitro or in vivo activity of 6a– t were established.

Prediction of Prasugrel Active Metabolite Concentrations From 2 Downstream Inactive Metabolite Concentrations Using Multilinear Regression Analysis

Prasugrel is a thienopyridine prodrug that is metabolized to an active metabolite (Pras-AM), which inhibits adenosine diphosphate (ADP)-induced platelet aggregation. The study objective was to describe a multilinear regression correlation model that was used to quantitatively predict concentrations of Pras-AM from downstream inactive metabolites, R-119251 and R-106583, for the purpose of estimating Pras-AM exposure in patients in the TRITON-TIMI 38 substudies. The model development included 1462 Pras-AM, 1345 R-119251, and 1456 R-106583 concentration data points from 103 healthy participants with a prasugrel dose range of 15 to 80 mg. The model was shown to provide good correlation between predicted and observed concentrations with only a minor deviation of approximately 6% from the unity line and described the variability within approximately 4.5%. Examination of the data indicated that regardless of ethnicity, age, weight, moderate hepatic impairment, or renal impairment, predictions were reliable. Predicted Pras-AM concentrations in TRITON-TIMI 38 were comparable with historical data.

The Effect of Milrinone on Platelet Activation as Determined by TEG® Platelet Mapping™

Milrinone is a phosphodiesterase III inhibitor that increases intracellular cyclic adenosine monophosphate resulting in improved ventricular function and vasodilation. Increased intracellular levels of cyclic adenosine monophosphate also inhibit adenosine diphosphate (ADP) and arachidonic acid (AA)-induced platelet aggregation. We hypothesized that inhibition of ADP and AA-induced platelet activation by therapeutic blood concentrations of milrinone could be quantified using TEG Platelet Mapping. Blood was taken from 15 healthy adults who had not been taking antiplatelet medications. Milrinone was added to whole blood in three clinically relevant concentrations (30, 100, and 300 ng/mL). Conventional thromboelastography (TEG) and TEG Platelet Mapping were performed on whole blood without milrinone and at each of these three concentrations. Increased blood concentrations of milrinone were associated with increased inhibition of ADP and AA-induced platelet activation (P < 0.0001). Milrinone at a blood concentration of 300 ng/mL markedly impaired the platelet activation response to ADP and AA. Therapeutic blood concentrations of milrinone exhibit a significant inhibitory effect on ADP and AA-induced platelet activation as determined by TEG Platelet Mapping, without affecting the conventional kaolin-activated TEG. We suggest that TEG Platelet Mapping results be interpreted with caution in patients being treated with milrinone, and other drugs that modify platelet cyclic nucleotide concentrations.

Novel antiplatelet strategies in acute coronary syndromes

Antiplatelet therapies for the treatment of acute coronary syndromes (ACS) act to interrupt various pathways of platelet activation. Clopidogrel, an established thienopyridine antiplatelet medication, inhibits adenosine diphosphate (ADP)-induced platelet aggregation to a modest degree and with wide variability in platelet response. Accumulating data suggest that a 600-mg loading dose of clopidogrel may help overcome the suboptimal response to the standard 300-mg dose seen in some patients. Prasugrel is a third-generation investigational thienopyridine that demonstrates more potent inhibition of platelet aggregation and more consistent platelet response compared with standard- and high-dose clopidogrel. A large clinical trial showed prasugrel to be superior to standard-dose clopidogrel in reducing ischemic events in patients with ACS scheduled for percutaneous coronary intervention, although prasugrel was associated with a significantly higher risk of major bleeding events. Other investigational antiplatelet agents also display more potent and consistent inhibition of platelet aggregation than is seen with clopidogrel. These include AZD6140, a reversible ADP receptor blocker; cangrelor, a rapidly acting intravenous ADP receptor blocker; and the thrombin receptor antagonist SCH 530348.

Perioperative Management of Patients on Adenosine Diphosphate Inhibitors in the Era of Drug-Eluting Stents: Review of the Literature and Clinical Implications

The current adenosine diphosphate inhibitors, ticlopidine and clopidogrel, are thienopyridine compounds that inhibit adenosine diphosphate mediated platelet aggregation. They interfere with platelet activation by selectively and irreversibly blocking P2Y12 sub-unit of the adenosine diphosphate receptor on the surface of platelets. This provides an antiplatelet effect that is additive to the inhibition of the thromboxane A2 pathway by aspirin. Dual antiplatelet therapy is extensively used in cardiovascular medicine. Randomized controlled trials have substantiated the fact that thrombotic complications after percutaneous coronary intervention procedures can be decreased by using dual antiplatelet therapy. However, there is a concern of bleeding due to enhanced and irreversible platelet inhibition in patients who will require any operation including coronary artery bypass grafting while on adenosine diphosphate inhibitors. This applies to a large population of patients requiring either coronary artery bypass grafting after angiographic definition of their coronary anatomy, or patients requiring semi-elective or urgent operation while under dual antiplatelet therapy. This concern is more present in era of drug-eluting stents, where long-term use of dual antiplatelet therapy is encouraged, and the incidence of late thrombosis after late cessation of adenosine diphosphate inhibitors is increasingly surfacing in the literature. The goal this review is to provide the medical chemistry of most commonly used adenosine diphosphate inhibitors, examine the literature on the effect of adenosine diphosphate inhibitors in hemorrhagic-related complications after surgical intervention, and provide the ramifications and alternatives in modern clinical practice.

Adenosine inhibition of adenosine diphosphate and thrombin-induced monocyte-platelet aggregates in cardiac syndrome X

Introduction We previously showed that platelet reactivity at rest is increased in patients with cardiac syndrome X (CSX), but that exercise reduces platelet reactivity in these patients. Adenosine was suggested to be involved in this phenomenon. In this study we investigated the effect of adenosine on adenosine diphosphate (ADP) and thrombin-induced platelet reactivity in CSX patients. Materials and methods We studied 15 CSX patients and a control group of 15 healthy subjects. Formation of monocyte-platelet (MONO-PLT) aggregates in vitro was assessed by flow cytometry: 1) at baseline; 2) after ADP (10 - 7 M) stimulation alone; 3) after ADP stimulation in presence of adenosine (10 - 5 M); 4) after thrombin (10 - 11 M) stimulation alone; 5) after thrombin stimulation in presence of adenosine. Results In non stimulated samples there were no relevant differences between the two groups in cytometry variables. Compared to controls, ADP induced a higher increase in MONO-PLT aggregates in CSX patients (P < 0.01), which was significantly inhibited by adenosine (P < 0.01). Thrombin also induced a greater increase in MONO-PLT aggregates in CSX patients (P < 0.001), which was also significantly blunted by adenosine. Similar trends were observed for platelet CD41 (glycoprotein IIb-IIIa) receptor and for monocyte receptors CD142 ad CD162 in MONO-PLT aggregates. Conclusions In CSX patients platelet reactivity is increased at rest, compared to healthy controls. Pre-incubation with adenosine reduces the agonist-induced platelet hyper-reactivity in these patients, suggesting that adenosine may be involved in the reduction of platelet reactivity observed in CSX patients after exercise in our previous study.

Thrombin as a common downstream target blocking both platelet and monocyte activation

Thromb Haemost 2009; 101: 220–221 Platelets play a pivotal role in the pathogenesis of thrombotic complications after percutaneous coronary interventions (PCI) (1). The use of optimal antiplatelet therapy is critical in reducing adverse events in this setting. Despite the efficacy of dual antiplatelet therapy in patients undergoing PCI, thrombo-ischaemic events and, in particular, stent thrombosis are frequent complications (2). High post-treatment platelet reactivity is associated with cardiovascular outcome after PCI (3) and coronary stent implantation (4). Different agents are currently in use for adjunctive anticoagulant treatment on top of adequate antiplatelet regimen during PCI. Traditionally, unfractionated heparin (UFH) has been the standard anticoagulant administered during coronary intervention. Enhanced platelet activation in vivo and ex vivo has been shown after UFH treatment (5, 6). Moreover, heparin does not suppress the transient increase in thromboxane biosynthesis associated with coronary angioplasty (7). Low-molecular-weight heparin (LMWH), in contrast, does not affect platelet activation in vivo and ex vivo after PCI (5, 8). Recently bivalirudin, a short-acting direct thrombin inhibitor, has been shown to be effective and safe in patients undergoing PCI (9), with reduced bleeding as compared with heparin. Reduced inhibition of adenosine diphosphate (ADP)-induced platelet aggregation at the time of PCI is associated with an increased risk for adverse thrombotic complications (10). Bivalirudin, given during PCI in patients pretreated with 600 mg of clopidogrel, is unlike UFH associated with further inhibition of platelet aggregation (11). A similar effect by bivalirudin was previously described on platelet P-selectin (12). Moreover, bivalirudin alone or coupled with clopidogrel may significantly down© 2009 Schattauer GmbH, Stuttgart

Another view on prasugrel

Thromb Haemost 2009; 101: 12–13 The combination of aspirin with clopidogrel is currently the mainstay of antiplatelet therapy for patients with acute coronary syndromes (ACS) (1). While aspirin inhibits platelet thromboxane A2 production and platelet activation, clopidogrel inhibits adenosine diphosphate (ADP)-induced platelet activation by blocking the P2Y12 platelet receptor. When added to aspirin therapy in patients with ACS, clopidogrel significantly reduces the risk of recurrent ischaemic events (1). Clopidogrel s onset of action is delayed and variable between individuals. A therapeutic level of inhibition is reached 4–6 hours (h) after a 300 mg loading dose, and 2 h after a 600 mg loading dose. Laboratory analysis reveals that a substantial proportion of patients do not show adequate inhibition of ADP-induced aggregation. Low responsiveness to clopidogrel has been shown to be significantly associated with adverse outcomes after percutaneous coronary intervention (PCI) (2). Prasugrel is a novel thienopyridine pro-drug with a greater rate, magnitude, and consistency of platelet ADP inhibition, as compared to clopidogrel. The therapeutic level of the inhibition of platelet aggregation is reached within 1 h after a 60 mg loading dose (3). TRITON-TIMI 38 (4) validated the hypothesis that more intensive antiplatelet blockade via the ADP receptor decreases the incidence of ischaemic events. Patients (13,608) with acute coronary syndromes scheduled for PCI and receiving aspirin were randomly assigned to receive either prasugrel or clopidogrel. After a median duration of 14.5 months, the primary efficacy outcome of cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke occurred in 12.1% of patients taking clopidogrel and in 9.9% of those taking prasugrel. Stent thrombosis was also reduced in the prasugrel group (2.4% clopidogrel vs. 1.1% prasugrel). However, major bleeding was increased (1.7% vs. 2.5%), as was fatal bleeding (0.1% vs. 0.4%) in the prasugrel group. Overall mortality did not differ significantly between treatment groups. In their viewpoint article, Serebruany et al. (5) discuss different aspects of the clinical development of prasugrel and, in particular, the pivotal trial TRITON-TIMI-38. Although their interpretations and evaluations are definitely debatable, this article highlights several points which are of outstanding interest for clinical and research approaches to platelet function inhibition: – Standardization: Serebruany et al. (5) highlight two aspects of standardization. First, the standardization of definitions of endpoints for clinical trials; in order to be able to compare event rates in different studies, a standardized definition of endpoints is desirable. Second, standardization of monitoring techniques for the assessment of anti-platelet therapy is crucial. The authors comment on discrepancies that occurred in the early phase of clinical development of prasugrel, which may be attributed to the use of different equipment for aggregation analysis in participating centers. Today, there is still a wide variation in the literature and clinical practice with respect to instruments, reagents, sample preparation, sample anticoagulants, as well as the analysis algorithms and clinical cut-offs applied for anti-platelet drug monitoring. – Patient compliance: An intensified anti-platelet treatment is usually equalized with a decreased rate of low patient responsiveness to anti-platelet treatment. Serebruany et al. (5) remind us that in a real-life scenario, patient compliance is crucial. Intensifying anti-platelet therapy might generate an increased rate of annoying minor bleeding complications during everyday activities, such as shaving or brushing teeth, which may again lead to patient non-compliance. For the physician, this includes the comforting fact that even with more modern and intensified anti-platelet regimens, the role of personal communication with the patient is still important for the appropriate adherence of the patient to his life-saving treatment. – Focus on safety: With the growing efficacy of antithrombotic treatments in ACS and in other areas such as prophylaxis of deep venous thrombosis, there is an increased focus on drug safety. Melagatran and ticlopidine are examples of antithrombotic drugs that were either withdrawn from the market, or experienced a sharp drop in use due to safety concerns. Referring to prasugrel, Bhatt (6) states that in a “realworld” setting, such as an elderly patient with multiple coexisting conditions, the risk of major bleeding and even fatal bleeding may increase to an even greater degree than was seen in TRITON–TIMI 38. This expectation is also expressed by Serebruany et al. in their viewpoint article (5). – Transformation of clinical trials into routine patient management: Clinical trials have inclusion and exclusion © 2009 Schattauer GmbH, Stuttgart

Acute Reversal of Clopidogrel-Related Platelet Inhibition Using Methyl Prednisolone in a Patient with Intracranial Hemorrhage

Clopidogrel and ticlopidine are agents that irreversibly inhibit adenosine diphosphate (ADP)–induced platelet aggregation.[1][1] Interaction of ADP with the P2Y1 receptor of the platelet induces platelet shape change, reversible aggregation, initial glycoprotein IIb/IIIa activation, phospholipase

(3 S)- N-( l-Aminoacyl)-1,2,3,4-tetrahydroisoquinolines, a class of novel antithrombotic agents: Synthesis, bioassay, 3D QSAR, and ADME analysis

To increase antithrombotic activity, 3 S-tetrahydroisoquinoline-3-carboxylic acid ( 1) was modified with natural amino acids to form 19 novel dipeptide analogs, 3 S-tetrahydroisoquinoline-3-carboxyamino acids ( 5a– s), targeting the intestinal peptide transport system. In vitro assay of 5a– s indicated that their potencies for inhibiting adenosine diphosphate (ADP), arachidonic acid (AA), platelet-activating factor (PAF), and thrombin (TH)-induced platelet aggregations were higher than that of 1. Additionally, in vivo assay of 5a– s indicated that their potencies for inhibiting thrombogenesis in rats were also higher than that of 1. Among the candidates, 5h with Ser attachment showed the most impressive features for further development. According to molecular field analysis based Cerius 2 QSAR module, two equations ( r, 0.961 and 0.988) correlating the structures with both in vitro and in vivo activities of 5a– s were established. ADMET calculations predict higher intestinal absorption for compounds 5a– s. Further investigation with 5h as a lead compound is underway.

Effect of CYP3A5*3 genotype on the pharmacokinetics and antiplatelet effect of clopidogrel in healthy subjects

Clopidogrel is activated by cytochrome P450 3A (CYP3A) to generate an active metabolite that inhibits adenosine diphosphate (ADP)-induced platelet aggregation through irreversible binding to the platelet P2Y12 receptor. The objective of this study was to assess the effect of the CYP3A5 genotype on the pharmacokinetics and antiplatelet effect of clopidogrel in healthy subjects. Twenty-two healthy subjects (CYP3A5*1/*1, n = 6; CYP3A5*1/*3, n = 8; CYP3A5*3/*3, n = 8) were recruited. After the administration of a loading dose of 300 mg of clopidogrel followed by 75 mg once daily for 6 days, plasma concentrations of clopidogrel and SR26334, an inactive metabolite, were measured for 24 h. The antiplatelet effect of clopidogrel was also measured, by determining the inhibition of ADP-induced platelet aggregation for 168 h, according to CYP3A5 genotype. Mean plasma concentration profiles of clopidogrel and SR26334 were comparable between CYP3A5 genotype groups. In addition, the CYP3A5 genotype did not affect the pharmacokinetics of either clopidogrel or SR26334. CYP3A5 genotype also did not modulate the inhibitory effect of clopidogrel on platelet aggregation. The CYP3A5*3 genotype plays a minor role in causing interindividual variability of the disposition of clopidogrel and its antiplatelet effect in humans.

The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities.

Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y(12) adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities. As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates. R-138727 reduced monocyte-platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 +/- 0.1 microM). In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte-platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.

The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities

Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y(12) adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities. As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates. R-138727 reduced monocyte-platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 +/- 0.1 microM). In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte-platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.