Warner TD. Aspirin and the in vitro linear relationship between thromboxane A 2 -mediated platelet aggregation and platelet production of thromboxane A 2 . J Thromb Haemost 2008; 6: 1933À43.Summary. Background: Currently, Ôaspirin resistanceÕ, the antiplatelet effects of non-steroid anti-inflammatory drugs (NSAIDs) and NSAID-aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A 2 formation is nonlinear and TXA 2 generation must be inhibited by at least 95% to inhibit TXA 2 -dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration-dependent relationships between TXA 2 generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA 2 production in response to arachidonic acid (0.03À1 mmol L ), in the presence of aspirin or vehicle, were determined in 96-well plates using blood taken from naı¨ve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA 2 production induced by either arachidonic acid or collagen were inhibited in concentrationdependent manners by aspirin, with logIC 50 values that did not differ. A linear relationship existed between aggregation and TXA 2 production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R 2 0.92; n = 224). The same relationships were seen in combinations of aspirin-treated and naı¨ve platelets, and in blood from individuals taking an antithrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA 2 generation and TXA 2 -mediated aggregation. This finding is important for our understanding of the anti-platelet effects of aspirin and NSAIDs, NSAIDÀaspirin interactions and Ôaspirin resistanceÕ.

Aspirin and clopidogrel are key anti-thrombotic therapies. Results from platelet reactivity testing during therapy, have been shown to correlate with future events and would allow for the optimisation of therapy. However, there is little agreement among current tests and there remains a clear clinical need for a universal standardised test. It was the objective of this study to explore the potential of 96-well plate aggregometry as a definitive clinical test of platelet reactivity with respect to aspirin and clopidogrel. A small non-blinded trial of 16 healthy male volunteers assigned to seven days of aspirin (75mg/day) or clopidogrel (75mg/day) therapy. Blood was collected before and on day 7 of treatment. Platelet aggregation was measured using a 96-well plate based aggregation method, and thrombi adhesion measured by colourimetric assay. Platelet agonists used were ADP (0.1-30microM), arachidonic acid (0.03-1.3mM), collagen (0.1-30microg/ml), adrenaline (0.001-100microM), ristocetin (0.2-3mg/ml), TRAP6 amide (0.130microM) and U46619 (0.130microM). Concentration response curves were constructed to each agonist under the various conditions and used to extract data such as log EC(50), Hill slope, and area under the curve. These demonstrated low intra- and inter-assay variability and strong discrimination of drug effects. This study demonstrates the ability of the 96-well plate based aggregation and adhesion method to detect and differentiate between stable aspirin and clopidogrel treatment in healthy volunteers. Moreover, this assay marries the ability to test subjects or patients using a range of platelet agonists with more rapidity and ease than the current gold standard platelet assay, traditional light transmission aggregometry, making it a serious alternative assay for use in clinical settings.

BackgroundClinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI2, formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice.Methodology/Principal FindingsA non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg−1, i.v.) and parecoxib (0.5 mg.kg−1, i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A2-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist.Conclusions/SignificanceInhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA2, but not that induced by U46619, which is independent of platelet TXA2. These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis.

Regular consumption of chocolate and cocoa products has been linked to reduced cardiovascular mortality. This study compared the effects of high flavanol dark chocolate (HFDC; 1064mg flavanols/day for 6 weeks) and low flavanol dark chocolate (LFDC; 88mg flavanols/day for 6 weeks) on blood pressure, heart rate, vascular function and platelet aggregation in men with pre-hypertension or mild hypertension. Vascular function was assessed by pulse wave analysis using radial artery applanation tonometry in combination with inhaled salbutamol (0.4 mg) to assess changes due to endothelium-dependent vasodilatation. HFDC did not significantly reduce blood pressure compared to baseline or LFDC. Heart rate was increased by LFDC compared to baseline, but not by HFDC. Vascular responses to salbutamol tended to be greater after HFDC. Platelet aggregation induced by collagen or the thromboxane analogue U46619 was unchanged after LFDC or HFDC, whereas both chocolates reduced responses to ADP and the thrombin receptor activator peptide, SFLLRNamide (TRAP6), relative to baseline. Pre-incubation of platelets with theobromine also attenuated platelet aggregation induced by ADP or TRAP6. We conclude that consumption of HFDC confers modest improvements in cardiovascular function. Platelet aggregation is modulated by a flavanol-independent mechanism that is likely due to theobromine.