The in vitro effects of Xancor, a synthetic astaxanthine derivative, on hemostatic biomarkers in aspirin-naïve and aspirin-treated subjects with multiple risk factors for vascular disease.

Astaxanthine is a polar carotenoid metabolite derived from a proprietary prodrug, Xancor, which aligns parallel with the membrane phospholipids exhibiting potent antioxidant, anti-inflammatory, and cell protective properties, although the precise mechanism of action is unknown. This prodrug is currently under development for hepatic, neurologic, and vascular disease indications. Considering established links between heart disease and stroke with platelets, coagulation cascade, and fibrinolysis, the aim of the study was to assess the effect of asthaxantine on human biomarkers of hemostasis. The rationale was to test a hypothesis that the drug may diminish activation of hemostasis, making it a potentially attractive addition to treat patients with vascular disease. In vitro effects of whole blood preincubation with escalating concentrations of asthaxantine (0.3 microM, 1 microM, 3 microM, 10 microM, 30 microM, and 100 microM) were assessed from 12 aspirin-naïve and eight aspirin-treated volunteers with multiple risk factors for vascular disease. A total of 25 biomarkers were measured, of which 12 were related to platelet function, 10 to coagulation, and three to fibrinolysis. Platelet aggregation induced by ADP, collagen, and arachidonic acid and expression of CD31, CD41, GP IIb/IIIa, CD51/61, P-selectin, CD63, CD107a, CD151+CD14, and CD154 were not affected. Coagulation indices such as aPTT, prothrombin time, thrombin time, fibrinogen, antithrombin III (antigen and activity), Protein C, Protein S (free and activity), and von Willebrand factor remained unchanged after incubation with astaxanthine. Fibrinolytic activity biomarkers such as plasminogen, D-dimer, and FDP were also not affected after in vitro pretreatment of blood samples with astaxanthine. In the projected subclinical (less than 1 microM), therapeutic (3 microM to 30 microM), and supratherapeutic concentration (100 microM), astaxanthine in vitro does not affect platelet, coagulation, or fibrinolytic indices in either aspirin-naïve or aspirin-treated subjects. These results are important for the assessment of the safety profile, but remain to be confirmed preclinically, in vivo, and ultimately in the clinic.

Selective thromboxane inhibition after vascular protectant AGI-1067: results of assessment of lipoprotein profiles (ALPS) biomarkers in vitro and in vivo substudy.

BACKGROUND: Oxidative stress play an important role triggering platelet/endothelial activation. AGI-1067 is a novel, phenolic antioxidant, and vascular protectant which dose-dependently inhibits PEA biomarkers in vitro. Whether treatment with AGI-1067 alters platelets in vivo is not known. We serially assessed release of established PEA biomarkers in subjects treated with AGI-1067 versus placebo in the frame of Assessment of Lipoprotein Profiles Randomized Trial (ALPS). METHODS: Healthy subjects (18-65 years) with multiple risk factors for coronary artery disease were randomized 1:1 to receive 300 mg AGI-1067 (n = 112) or matching placebo (n = 117) daily for 12 weeks. Anticoagulants, aspirin, NSAIDS, and COX inhibitors were not permitted in this study. Plasma samples were collected at baseline, and at week 12 after randomization. Platelet factor 4 (PF4), beta-thromboglobulin (betaTG), P-selectin, thromboxane (TxB2), and prostacyclin (6-keto-PGF1a) were measured by ELISA. RESULTS: Treatment with AGI-1067 was associated with a highly significant reduction of TxB2 release (P < 0.0001) when compared to the placebo. There were no differences in PF4, betaTG, P-selectin, and 6-keto-PGF1a between and within groups. AGI-1067 also inhibits TxB2 release from calcium ionophore (A23187)-stimulated human platelets with the IC50 equals 1 microM; but does not interfere with 6-keto-PGF1alpha release in either A23187-, or TXA2-stimulated human aortic endothelial cells. CONCLUSION: AGI-1067 selectively reduces TxB(2 )production from stimulated platelets, and diminishes plasma TxB2 levels in ALPS participants. These data support earlier in vitro, and pilot ex vivo experiments suggesting antiplatelet properties of AGI-1067. Lack of 6-keto-PGF1a down regulation may represent an attractive advantage of AGI-1067 over currently available antiplatelet regimens.

Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in diabetic patients: the PLavix Use for Treatment Of Diabetes (PLUTO-Diabetes) trial.

BACKGROUND: Clopidogrel is widely used in diabetic patients after vascular events; however, the ability of this thienopyridine to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study with comprehensive assessment of platelet activity. The objective of this study was to compare the antiplatelet profiles of clopidogrel + aspirin in combination (C + ASA) versus aspirin alone (ASA) in patients with type 2 diabetes mellitus. METHODS: Seventy patients with documented diabetes already treated with antecedent aspirin were randomly assigned to receive C + ASA or ASA in the PLUTO-Diabetes trial. Platelet studies included adenosine diphosphate-, collagen-, and arachidonic acid-induced aggregometry; PFA-100 (Dade-Behring, Miami, FL) and Ultegra (Accumetrics, San Diego, CA) analyzers; and expression of 6 major receptors by flow cytometry at baseline and at day 30 after randomization. RESULTS: There were no differences in the baseline clinical and platelet characteristics between the C + ASA and ASA groups, or subsequent significant changes in platelet biomarkers in the ASA group, except for diminished collagen-induced aggregation (P = .02). In contrast, when compared with the ASA group, therapy with C + ASA resulted in significant inhibition of platelet activity assessed by adenosine diphosphate aggregation (P = .0001); closure time prolongation (P = .0003) and reduction of platelet activation units with Ultegra (P = .0001); and expression of platelet/endothelial cell adhesion molecule 1 (P = .002), glycoprotein IIb/IIIa antigen (P = .0002), and activity (P = .0001). CONCLUSION: Treatment with C + ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in diabetic patients in this small randomized trial. However, despite dual antiplatelet regimen, diabetic patients exhibit high residual activity of some platelet biomarkers, including unaffected protease-activated receptor 1 receptor expression.

Magnitude and time course of platelet inhibition with extended release dipyridamole with or without aspirin in healthy Japanese volunteers. The AGgrenox versus Aspirin Therapy Evaluation (AGATE-Japan).

Randomized trials showed greater stroke prevention with extended release dipyridamole in combination with low dose aspirin than with either aspirin or dipyridamole alone. However, most studies with this formulation (Aggrenox) were carried out in Europe and North America. Considering potential inter-racial differences in drug response, we conducted a small randomized study in healthy Japanese volunteers to compare antiplatelet regimens with regard to the changes in the platelet biomarkers. Thirty healthy volunteers (18-40 years old, 15 male and 15 female) of Japanese descent were randomized to Aggrenox (n = 17) or aspirin 81 mg (n = 13 volunteers) for 30 days. Platelet function was assessed at baseline, and on days 15, and 30 by conventional aggregometry, whole blood flow cytometry, and cartridge-based analyzer. Both Aggrenox and aspirin provided sustained platelet inhibition at Day 15 and Day 30. Therapy with Aggrenox, however, was associated with more prominent and significant inhibition of collagen-induced aggregation (p = 0.08, Day 15), as well as prolongation of the closure time (p = 0.001, Day 30); diminished expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) (p = 0.02, Day 30), glycoprotein IIb (GPIIb) antigen (p = 0.001 and 0.024 for Day 15 and Day 30), and GPIIb/IIIa activity by PAC-1 antibody (p = 0.014 and 0.03), CD62 (P-selectin) (p = 0.03 for Day 15 and Day 30), as well as inhibition of protease activated receptors (PAR-1) associated with intact WEDE-15 (p = 0.002 and 0.003) and SPAN-12 (p = 0.002 and 0.04) thrombin receptors when compared with aspirin. The magnitude and durability of platelet response after Aggrenox in healthy Japanese is similar to those effects observed in Caucasians and African-Americans. A larger study to assess drug efficacy and safety in the Japanese post-stroke patients is warranted.

Antiplatelet properties of escitalopram in patients with the metabolic syndrome: a dose-ranging in vitro study.

There is an increasing body of evidence suggesting that selective serotonin reuptake inhibitors exhibit clinical benefit beyond treating depression, by simultaneously inhibiting platelet activity. We recently demonstrated that escitalopram (ESC), but not its major metabolites, inhibits multiple platelet biomarkers in healthy volunteers. Considering that the metabolic syndrome represents one of the major risk factors for vascular disease, we here determined how ESC affects platelet activity in such patients. We assessed the in vitro effects of preincubation with escalating (50-200 nM/l) concentrations of ESC on platelet aggregation, expression of major surface receptors by flow cytometry, and quantitatively by platelet function analyzers. Blood samples were obtained from 20 aspirin-naïve patients with documented metabolic syndrome. Pretreatment of blood samples with medium (150 nM/l), or high (200 nM/l) doses of ESC resulted in a significant inhibition of platelet aggregation induced by ADP (p=0.007) and by collagen (p=0.004). Surface platelet expression of GPIb (CD42, p=0.03), LAMP-3 (CD63, p=0.04), and GP37 (CD165, p=0.03) was decreased in the ESC-pretreated samples. Closure time by the PFA-100 analyzer was prolonged after the 200 nM/l dose (p=0.02), indicating platelet inhibition under high shear conditions. On the other hand, the lowest tested concentration of ESC (50 nM/l) did not affect platelet activity in these patients. The in vitro antiplatelet characteristics of ESC in patients with the metabolic syndrome are similar to those in healthy volunteers. However, higher ESC doses are required to induce equally potent platelet inhibition. These data justify prospective ex vivo studies with the highest therapeutic dose to determine the potential clinical advantage of ESC in high-risk patients with vascular disease.

Consistent platelet inhibition during long-term maintenance-dose clopidogrel therapy among 359 compliant outpatients with documented vascular disease.

BACKGROUND: Numerous reports have dichotomized responses after clopidogrel therapy using varying definitions and platelet tests in patients immediately after acute vascular events; however, no large study has assessed platelet characteristics in outpatients receiving long-term treatment for more than 30 days with the maintenance dose (75 mg/d) of clopidogrel. The aim of this study was to describe the responses of ex vivo measures of platelet aggregation and activation to long-term clopidogrel therapy in a large population of outpatients after coronary stenting or ischemic stroke. METHODS: We conducted a secondary post hoc analysis of a data set represented by presumably compliant patients after coronary stenting (n = 237) or a documented ischemic stroke (n = 122) treated with clopidogrel-and-aspirin combination antiplatelet therapy. RESULTS: The mean duration of treatment was 5.8 months (range 1-21 months). Every patient exhibited a significant inhibition of adenosine diphosphate-induced platelet aggregation (mean 52.9%, range 36%-70%) as compared with the preclopidogrel measures. Inhibition of aggregation strongly correlated with a diminished expression of PECAM-1 (platelet/endothelial cell adhesion molecule 1, r = 0.75), glycoprotein IIb/IIIa (r = 0.62), and PAR-1 (protease-activated receptor 1, r = 0.71). None of the patients developed hyporesponsiveness (reduction from the baseline <15%) or profound inhibition (residual platelet activity <10%). CONCLUSIONS: In contrast to the wide variability of responses that exists in the acute setting, long-term therapy with clopidogrel leads to consistent and much less variable platelet inhibition. Lack of nonresponse and profound inhibition with clopidogrel allow for the maintenance of a delicate balance between proven efficacy and acceptable bleeding risks for long-term secondary prevention in outpatients after acute vascular events.

Effects of escalating doses of tirofiban on platelet aggregation and major receptor expression in diabetic patients: hitting the TARGET in the TENACITY trial?

INTRODUCTION: Ongoing search for the optimal dosing regimens, and valid concerns that some GPIIb/IIIa inhibitors may cause rebound platelet activation are limiting the use of these agents in patients with acute vascular events. MATERIALS AND METHODS: We assessed the in vitro effects of preincubation with escalating (12.5-200 ng/mL) concentrations of tirofiban on platelet biomarkers in 20 diabetic patients. Platelet activity was assessed by ADP-, and collagen-induced conventional plasma aggregometry, and by whole blood flow cytometry measuring expression of PECAM-1, GPIb, GP IIb/IIIa antigen and activity, vitronectin, P-selectin, LAMP-1, GP 37, LAMP-3, activated and intact PAR-1 thrombin receptors, GPIV, and platelet-monocyte formation. All patients were treated with aspirin (at least 81 mg daily for 1 month); other antiplatelet agents were not allowed. RESULTS: Significant decrease of ADP-induced platelet aggregation was observed starting at the low 12.5 ng/mL concentration (p=0.0001), with total inhibition occurring at 50 ng/mL of tirofiban dose. Inhibition of collagen-induced platelet aggregability requires 25 ng/ml of tirofiban (p=0.002), and was complete at 100 ng/mL. Dose-dependent blockade of GP IIb/IIIa activity was observed with tirofiban concentrations over 50 ng/mL (p=0.003). Other receptors were unaffected even with the high doses of tirofiban (100-200 ng/mL). CONCLUSION: Tirofiban completely inhibits ADP- and, with the higher dose, collagen-induced platelet aggregation. Higher loading dose of tirofiban used in the ongoing TENACITY trial (100 ng/mL) may be superior with regard to clinical outcomes to the regimens used in PRISM-PLUS (25 ng/mL), or TARGET (50 ng/mL). Selective inhibition of GPIIb/IIIa activity, and lack of alternative platelet activation beyond the GP IIb/IIIa blockade may represent the therapeutic advantage of tirofiban over other agents.

Monitoring platelet inhibition after clopidogrel with the VerifyNow-P2Y12(R) rapid analyzer: the VERIfy Thrombosis risk ASsessment (VERITAS) study.

INTRODUCTION: Clopidogrel inhibits platelet P2Y12 ADP receptors, while ADP, as an inductor of aggregation, stimulates both P2Y12 and P2Y1 platelet receptors. Despite a clinical loading dose routine with clopidogrel, some patients still experience coronary stent thrombosis suggesting persistent platelet activation. The VerifyNow-P2Y12 is a rapid assay that test platelet activity over 3 min and uses of the combination of ADP and prostaglandin E1 (PGE1) to directly measure the effects of clopidogrel on the P2Y12 receptor. ADP is used to maximally activate the platelets by binding to the P2Y1 and P2Y12 platelet receptors, while PGE1 is used to suppress the ADP-induced P2Y1-mediated increase in intracellular calcium levels. OBJECTIVE: The VERIfy Thrombosis risk ASsessment (VERITAS) was a prospective study designed to measure platelet response to clopidogrel therapy in subjects with multiple risk factors or history of vascular disease using this novel point-of-care assay. METHODS: 166 participants were enrolled in 4 participating sites. Data from 147 participants were analyzed after exclusion of 19 patients due to protocol violations. Platelets were assessed twice at baseline (before clopidogrel) and at 24 h post-loading 450 mg (110 participants) or 7 days after chronic clopidogrel treatment (75 mg/day) (37 patients). All participants received aspirin 81-325 mg for at least 2 days before the study enrollment. Results from the VerifyNow-P2Y12 assay are reported in P2Y12 reaction units (PRU). RESULTS: Clopidogrel therapy resulted in a mean 64.0+/-25.3% PRU reduction. No participant reached PRU inhibition below 10% of baseline. Distribution of PRU values for the VerifyNow-P2Y12 assay shows a separation from baseline to post-clopidogrel assay values with some overlap due to high inter-individual variations in response. CONCLUSIONS: VerifyNow-P2Y12 is a reliable, fast and sensitive device suitable for monitoring of platelet inhibition during clopidogrel therapy.

Combination antiplatelet therapy with aspirin and clopidogrel: the role of antecedent and concomitant doses of aspirin. An analysis of 711 patients.

BACKGROUND: Numerous randomized studies have shown that the combination of clopidogrel with aspirin yields better clinical outcomes than monotherapy in patients with acute vascular events. However, the impact of the aspirin dose on the antiplatelet potency of clopidogrel is unclear. We sought to compare the antiplatelet profile of aspirin 81 mg (n = 252) versus aspirin 325 mg (n = 459) before and during conventional clopidogrel loading (300 mg), and/or clopidogrel maintenance (75 mg/daily) therapy. METHODS: Secondary post hoc analysis of an existing dataset consisting of 711 patients after coronary stenting (n = 601) and ischemic stroke (n = 110) treated previously with aspirin for at least 1 month, and then with aspirin + clopidogrel for at least 7 days was performed. Platelet assessments include conventional and whole blood aggregometry, rapid cartridge-based analyzers, and expression of platelet/endothelial cell adhesion molecule-1, P-selectin, and GPIIb/IIIa activity by flow cytometry measured before and after addition of clopidogrel. RESULTS: There was a small but consistent yet non-significant trend towards more potent platelet inhibition with aspirin 325 mg compared to aspirin 81 mg for every platelet activation parameter before addition of clopidogrel. However, after loading and/or 1 week of chronic treatment with clopidogrel + aspirin, measured platelet parameters became very similar between the groups, and identical for collagen-induced aggregation and PFA-100 analyzer readings. CONCLUSIONS: Before addition of clopidogrel, aspirin 325 mg has a tendency to provide stronger platelet inhibition than aspirin 81 mg. However, when clopidogrel and aspirin are used in combination, the higher aspirin dose does not translate into superior antiplatelet action. Given that the existing body of evidence supports the comparable efficacy and, particularly, superior safety of lower versus higher doses of aspirin, aspirin 81 mg should be the dose used in combination with clopidogrel.

Does heart failure etiology, New York Heart Association class, or ejection fraction affect the ability of clopidogrel to inhibit heightened platelet activity?

The ability of clopidogrel to inhibit platelet function in patients with congestive heart failure (CHF) was proved by the PLUTO-CHF trial. We retrospectively analyzed platelet characteristics with respect to CHF etiology, class, and ejection fraction in patients enrolled in the PLUTO-CHF study. Twenty-five patients were divided by CHF etiology, severity, and ejection fraction. All patients received aspirin 325 mg for at least 1 month prior to screening. Platelet function studies were performed at baseline and after 30 days of therapy. There were no differences in platelet parameters dependent on clinical characteristics of CHF, except for a significant (P = 0.023) decrease in platelet/endothelial cell adhesion molecule 1 (PECAM-1) expression in the New York Heart Association class III-IV due to the higher baseline values. Therapy with clopidogrel resulted in a significant inhibition of platelet activity assessed by ADP-induced and epinephrine-induced aggregation, closure time, expression of PECAM-1, glycoprotein Ib, glycoprotein IIb/IIIa antigen, glycoprotein IIb/IIIa activity with PAC-1, CD151, and reduced formation of platelet-leukocyte conjugates when compared with baseline. Clopidogrel provides antiplatelet protection in the broad spectrum of patients with CHF independently of its etiology, severity, or myocardial contractility. This uniform platelet inhibition with clopidogrel may be an important consideration in designing future large-scale clinical trials.

Variability in platelet responsiveness to clopidogrel among 544 individuals.

OBJECTIVES: We sought to describe the responses of patients to clopidogrel using ex vivo measures of platelet aggregation and activation in a large, heterogeneous population. BACKGROUND: Recently, a number of reports, using various definitions, have dichotomized patients who are treated with clopidogrel into a minority of "non-responders" and a majority of "responders." Such classifications imply that treatment leads to an all-or-none response, with potentially important clinical implications. METHODS: We conducted secondary post-hoc analyses of a dataset consisting of volunteers (n = 94) and patients after coronary stenting (n = 405), with heart failure (n = 25), and after stroke (n = 20). RESULTS: The response of subjects to clopidogrel followed a normal, bell-shaped distribution, with a mean and standard deviation of 41.9 +/- 20.8% when aggregation was induced by 5 mumol/l of adenosine diphosphate. When hyporesponsiveness and hyper-responsiveness to clopidogrel were considered to be two standard deviations less than and greater than the mean, respectively, the prevalence of hyporesponsiveness and hyper-responsiveness in these patients was 4.2% and 4.8%, respectively. Pretreatment platelet activity and clinical characteristics were not associated with responsiveness to clopidogrel. CONCLUSIONS: Individuals receiving clopidogrel exhibit a wide variability in response that follows a normal distribution. The clinical implications of this variability are unknown but potentially are important. Clinical trials are needed to define whether hyporesponders to clopidogrel are at increased risk for thrombotic events and whether hyper-responders are at increased risk for bleeding. If so, the individualization of antiplatelet therapy, including clopidogrel dosing, may be possible in the future but will require the ability to easily and reproducibly measure responsiveness by a method that has been proven to be predictive of clinical events.

Valsartan inhibits platelet activity at different doses in mild to moderate hypertensives: Valsartan Inhibits Platelets (VIP) trial.

BACKGROUND: Previous in vitro studies have suggested that valsartan produces significant inhibition of human platelets, probably targeting angiotensin I platelet receptors. To test whether valsartan inhibits platelet activity in mild to moderate hypertensives we conducted the randomized Valsartan Inhibits Platelets (VIP) trial. METHODS AND RESULTS: Seventy-five patients with mild to moderate hypertension were randomized to valsartan 80 (n = 25), valsartan 160 (n = 29), or valsartan 320 mg/d (n = 21) for 9 weeks. Platelet function was assessed at baseline, week 5, and week 9 by aggregometry, flow cytometry, and cartridge-based analyzer. Independently of dose and duration, valsartan provided early sustained significant inhibition of adenosine diphosphate-induced platelet aggregation, decreased shear-induced activation measured with PFA-100 analyzer, and diminished expression of GP IIb/IIIa activity measured by PAC-1 antibody, GPIb (CD42b), vitronectin receptor (CD51/61), P-selectin (CD62p), lysosome-associated membrane protein (CD107a), and CD40-ligand (CD154). The antiplatelet properties of valsartan were more profound in patients with diabetes (n = 28) when compared with the nondiabetic group (n = 47). In subgroup analyses of patients with diabetes there appeared to be stronger inhibition of the platelet receptors, a significant decrease of adenosine diphosphate- and collagen-induced platelet aggregation, and more profound inhibition of GP IIb/IIIa activity. CONCLUSIONS: In the randomized VIP trial, valsartan produced sustained inhibition of platelet aggregation and major platelet receptors. The antiplatelet properties of valsartan were not dose or time dependent. In subgroup analyses patients with diabetes with mild to moderate hypertension tended to have greater platelet inhibition, a finding which, if confirmed in future studies suggests possible additional advantages for using valsartan in this high-risk population.

Effect of statins on platelet PAR-1 thrombin receptor in patients with the metabolic syndrome (from the PAR-1 inhibition by statins [PARIS] study).

We investigated whether, in primary prevention patients with metabolic syndrome, statins affect the platelet protease-activated receptor-1 (PAR-1) thrombin receptor by performing serial measurements of its activity and the antigen expression level by flow cytometry before and during treatment. Recent data from randomized trials of statins are compatible with the possibility of clinically relevant pleiotropic effects. The use of statins is associated with a reduced thrombosis burden and diminished platelet activity, as shown in animal models and in vitro studies. Seventy patients with the metabolic syndrome who were not taking antiplatelet agents were assigned consecutively at starting doses at the discretion of the responsible clinician to 1 of 6 statins (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin) or to a no-statin group for 6 weeks. Platelet expression of intact (SPAN12 antibody) and cleaved (WEDE15) PAR-1 thrombin receptors were assessed by flow cytometry at baseline and at weeks 4 and 6 of treatment. At baseline, no difference was found in receptor expression. However, after 4 weeks of treatment, all statins had significantly inhibited (46% to 55%) the activated epitope of PAR-1 expression. After 6 weeks, inhibition remained, despite a slight rebound (22% to 37%). Also, a delayed pattern of inhibition of the intact PAR-1 receptor epitope was found. In conclusion, all statins inhibited the activity and antigen level of the platelet PAR-1 thrombin receptor, which has a major role in regulating platelet activity and thrombin formation. These observational data offer a plausible mechanism for the recently demonstrated pleiotropic effects of statins that may contribute to early clinical benefit.

The in vitro effects of a novel vascular protectant, AGI-1067, on platelet aggregation and major receptor expression in subjects with multiple risk factors for vascular disease.

Oxidation-sensitive signals are important in platelet activation. The novel, phenolic, intracellular and extra-cellular antioxidant AGI-1067 inhibits the expression of a number of proinflammatory genes involved in atherosclerosis. The effect of AGI-1067 on human platelets was evaluated. Blood obtained from 20 aspirin-naïve volunteers with multiple risk factors for vascular disease was preincubated with escalating concentrations of AGI-1067 for the assessment of its ex vivo effects on platelet aggregation and expression of major surface receptors flow cytometry, evaluated by flow cytometry. AGI-1067 resulted in significant inhibition of a variety of activation-dependent platelet biomarkers in healthy volunteers, including adenosine diphosphate-induced platelet aggregation and decreased surface platelet expression of glycoprotein IIb/IIIa antigen, activity with PAC-1 antibody, and glycoprotein Ib (CD42b). The effect of AGI-1067 differs from other known antiplatelet agents, suggesting opportunities for therapeutic combination. These data need to be confirmed in subjects receiving orally dosed AGI-1067 to be clinically relevant.

Platelet/endothelial biomarkers in depressed patients treated with the selective serotonin reuptake inhibitor sertraline after acute coronary events: the Sertraline AntiDepressant Heart Attack Randomized Trial (SADHART) Platelet Substudy.

BACKGROUND: Depression after acute coronary syndromes (ACSs) has been identified as an independent risk factor for subsequent cardiac death. Enhanced platelet activation has been hypothesized to represent 1 of the mechanisms underlying this association. Selective serotonin reuptake inhibitors (SSRIs) are known to inhibit platelet activity. Whether treatment of depressed post-ACS patients with SSRIs alters platelet function was not known. Accordingly, we serially assessed the release of established platelet/endothelial biomarkers in patients treated with sertraline vs placebo in the Sertraline AntiDepressant Heart Attack Randomized Trial (SADHART). METHODS AND RESULTS: Plasma samples (baseline, week 6, and week 16) were collected from patients randomized to sertraline (n=28) or placebo (n=36). Anticoagulants, aspirin, and ADP-receptor inhibitors were permitted in this study. Platelet factor 4, beta-thromboglobulin (betaTG), platelet/endothelial cell adhesion molecule-1, P-selectin, thromboxane B2, 6-ketoprostaglandin F1a, vascular cell adhesion molecule-1, and E-selectin were measured by ELISA. Treatment with sertraline was associated with substantially less release of platelet/endothelial biomarkers than was treatment with placebo. These differences attained statistical significance for betaTG (P=0.03) at weeks 6 and 16 and for P-selectin (P=0.04) at week 16. Repeated-measures ANOVA revealed a significant advantage for sertraline vs placebo for diminishing E-selectin and betaTG concentrations across the entire treatment period. CONCLUSIONS: Treatment with sertraline in depressed post-ACS patients is associated with reductions in platelet/endothelial activation despite coadministration of widespread antiplatelet regimens including aspirin and clopidogrel. The antiplatelet and endothelium-protective properties of SSRIs might represent an attractive additional advantage in patients with depression and comorbid coronary artery and/or cerebrovascular disease.

Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in patients after recent ischemic stroke: for the Plavix Use for Treatment of Stroke (PLUTO-Stroke) trial.

BACKGROUND AND PURPOSE: Clopidogrel is widely used in patients after recent ischemic stroke; however, its ability to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study. To determine whether clopidogrel with aspirin (C+ASA) will produce more potent platelet inhibition than aspirin alone (ASA) in patients after ischemic stroke, we conducted the Plavix Use for Treatment of Stroke trial. METHODS: Seventy patients after ischemic stroke were randomly assigned to C+ASA or ASA groups. Platelet studies included aggregometry; cartridge-based analyzers; expression of PECAM-1, P-selectin, GP IIb/IIIa (antigen and activity), vitronectin receptor, and formation of platelet-leukocyte microparticles by flow cytometry. Platelet tests were performed at baseline and after 30 days after randomization. RESULTS: There were no deaths, hospitalizations, or serious adverse events. There were no differences in the baseline platelet characteristics between C+ASA and ASA groups, or significant changes in platelet parameters in the ASA group, except diminished collagen-induced aggregation (P=0.001). In contrast, therapy with C+ASA resulted in a significant inhibition of platelet activity assessed by ADP- (P=0.00001) and collagen-induced (P=0.02) aggregation; closure time prolongation (P=0.03), and reduction of platelet activation units with Ultegra (P=0.00001); expression of PECAM-1 (P=0.01), and GP IIb/IIIa activity with PAC-1 (P=0.02) when compared with ASA group. Therapy with C+ASA also resulted in the reduced formation of platelet-leukocyte microparticles (P=0.02). CONCLUSIONS: Treatment with C+ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in patients after recent ischemic stroke in the frame of the small randomized trial.

Relationship between release of platelet/endothelial biomarkers and plasma levels of sertraline and N-desmethylsertraline in acute coronary syndrome patients receiving SSRI treatment for depression.

OBJECTIVE: In a platelet/endothelial biomarker substudy of the Sertraline AntiDepressant Heart Attack Randomized Trial (SADHART), the authors sought to determine whether plasma levels of sertraline and its primary metabolite N-desmethylsertraline affect the release of platelet/endothelial biomarkers. METHOD: Fifty-five acute coronary syndrome patients with depression were randomly assigned to receive sertraline (N=23) or placebo (N=32). Twenty-six serial plasma samples collected at week 6 (N=12) and week 16 (N=14) were analyzed. Platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), platelet/endothelial cell adhesion molecule 1 (PECAM-1), P-selectin, thromboxane B(2) (TxB(2)), prostacyclin (6-keto-PGF1alpha), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were measured by enzyme-linked immunosorbent assay. Concentrations of sertraline and N-desmethylsertraline were determined by liquid chromatography with fluorescence detection in autologous samples. RESULTS: Strong, mostly time-dependent negative correlations were found for the plasma levels of sertraline and N-desmethylsertraline with PF4 (week 6: r=-0.69 and -0.33, respectively; week 16: r=-0.63 for both), beta-TG (week 6: r=-0.43 and -0.29; week 16: r=-0.66 and -0.57), PECAM-1 (week 6: r=-0.82 and -0.49; week 16: r=-0.60 for both), P-selectin (week 6: r=-0.82 and -0.49; week 16: r=-0.73 and -0.43), and TxB(2) (week 6: r=-0.66 and -0.59; and week 16: r=-0.64 and -0.41). Regression analysis revealed some borderline correlations for endothelial markers such as 6-keto- PGF1alpha and E-selectin and a positive correlation for VCAM-1. CONCLUSIONS: This is the first documented evidence that plasma release of platelet/endothelial biomarkers is directly related to the levels of sertraline and N-desmethylsertraline in acute coronary syndrome patients receiving SSRI treatment for depression. The clinical significance of these findings should be assessed in the setting of a randomized clinical trial.

Analysis of risk of bleeding complications after different doses of aspirin in 192,036 patients enrolled in 31 randomized controlled trials.

We sought to compare the risk of hemorrhage due to the low (<100 mg), moderate (100 to 200 mg), and high (>200 mg) doses of aspirin (acetylsalicylic acid [ASA]) in 192,036 patients enrolled in 31 clinical trials. Despite substantial differences in the reporting patterns of bleeding complications, low-dose ASA was associated with the lowest risk, and moderate doses caused a relatively high hemorrhagic event rate, especially with regard to minor, gastrointestinal, and total bleeding, and stroke. These findings should be considered when using combination antiplatelets, anticoagulant therapy, or both, with ASA, especially with the daily dose of >100 mg.

Absence of interaction between atorvastatin or other statins and clopidogrel: results from the interaction study.

BACKGROUND: Some, but not all, post hoc analyses have suggested that the antiplatelet effects of clopidogrel are inhibited by atorvastatin. We sought to address this issue prospectively by performing serial measurements of 19 platelet characteristics using conventional aggregometry, rapid analyzers, and flow cytometry. METHODS: The Interaction of Atorvastatin and Clopidogrel Study (Interaction Study) was designed for patients undergoing coronary stenting. All patients (n = 75) received 325 mg of aspirin daily for at least 1 week and 300 mg of clopidogrel immediately prior to stent implantation. They had been taking atorvastatin (n = 25), any other statin (n = 25), or no statin (n = 25) for at least 30 days prior to stenting. The main outcome measure was comparison of platelet biomarkers 4 and 24 hours after clopidogrel administration between study groups. RESULTS: At baseline, patients from both statin groups exhibited diminished platelet aggregation and reduced platelet expression of G-protein-coupled protease-activated thrombin receptor (PAR)-1. There were no significant differences in measured platelet characteristics among the study groups 4 and 24 hours after clopidogrel intake, with the exception of a lower collagen-induced aggregation at 24 hours and a constantly diminished expression of PAR-1 in patients treated with any statin. CONCLUSIONS: Statins in general, and atorvastatin in particular, do not affect the ability of clopidogrel to inhibit platelet function in patients undergoing coronary stenting. These prospective data also suggest that statins may inhibit platelets directly via yet unknown mechanism(s) possibly related to the regulation of the PAR-1 thrombin receptors.

Platelet activation in patients with congestive heart failure: do we have enough evidence to consider clopidogrel?

Our understanding of the pathogenesis of congestive heart failure (CHF) has improved remarkably in recent years. However, despite better knowledge and novel pharmaceutical strategies, this disease is still one of the most brutal killers in the Western world. The pathophysiology of CHF is complex, and much of our comprehension revolves strictly around the neurohormonal and mechanical mechanisms involved. It has been suggested that CHF is associated with altered hemostasis, but whether a prothrombotic state contributes to the pathogenesis and progression of the disease is still not well known. The purpose of this review article is to discuss our current knowledge of platelet activation in patients with CHF and the potential role of antiplatelet agents in preventing these hemostatic abnormalities. Clopidogrel is an established medication that reduces the incidence of stroke, myocardial ischemia, or vascular death. It is currently the drug of choice in the prophylaxis of subacute stent thrombosis and postischemic stroke treatment. Promising results of the most resent trials (Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events [CAPRIE] and Clopidogrel in Unstable angina to prevent Recurrent Events [CURE]) may expand future indications of this ADP receptor antagonist for prevention of thrombotic complications in the CHF population. Currently conducted clinical trials (Warfarin and Antiplatelet Therapy in Chronic Heart Failure [WATCH] and Plavix Use for Treatment of Congestive Heart Failure [PLUTO-CHF] should clarify the role of clopidogrel in these patients.