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.

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.

Effect of tenecteplase versus alteplase on platelets during the first 3 hours of treatment for acute myocardial infarction: the Assessment of the Safety and Efficacy of a New Thrombolytic Agent (ASSENT-2) platelet substudy.

BACKGROUND: Platelets play a pivotal role in the pathogenesis of acute myocardial infarction, as well as in the occurrence of coronary artery reocclusion and bleeding events. Therefore, the success of fibrinolytic therapy may be dependent on its direct effects on platelets. METHODS AND RESULTS: We sought to determine how tenecteplase (TNK) and alteplase (tPA) affect platelets in vitro in human volunteers and ex vivo by use of patient data from the Assessment of the Safety and Efficacy of a New Thrombolytic Agent (ASSENT-2) trial. For the in vitro studies, whole blood from 9 healthy volunteers was incubated with 30 mg TNK and 60 mg tPA. Platelet function was measured by conventional aggregometry, bedside point-of-care devices, and sensitive flow cytometry techniques. For the ex vivo study, 41 patients were selected from the ASSENT-2 trial: 21 had received TNK and 20 had received tPA. Each patient underwent 7 serial blood draws every 30 minutes for 3 hours. Levels of platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, beta-thromboglobulin, platelet factor 4, thromboxane, and prostacyclin were measured by enzyme-linked immunosorbent assay. Significant inhibition of conventional and whole blood aggregation and reduced platelet function by point-of-care analyzers were observed for both agents, but mostly in the TNK-treated samples. After incubation with TNK, flow cytometry revealed decreased expression of glycoprotein IIb/IIIa, PECAM-1, vitronectin receptor, CD151, and formation of the platelet-monocyte aggregates. Serial samples from patients in the ASSENT-2 trial showed a significant decrease of soluble platelet-endothelial biomarkers in the TNK group. There was a trend toward decreased platelet function characteristics with tPA; however, these differences were much smaller than those observed with TNK. CONCLUSIONS: Both tPA and TNK were shown to affect platelet function in human volunteers and in patients with AMI early after thrombolysis. The antiplatelet properties of TNK were shown to be more profound than those of tPA. These findings are relevant to ongoing investigations of combination therapy with fibrinolytic and antiplatelet agents in patients with AMI.