Blood markers in remote ischaemic conditioning for acute ischaemic stroke: data from the REmote ischaemic Conditioning After Stroke Trial.

BACKGROUND AND PURPOSE: Remote ischaemic per-conditioning (RIC) is neuroprotective in experimental ischaemic stroke. Several neurohumoral, vascular and inflammatory mediators are implicated. The effect of RIC on plasma biomarkers was assessed using clinical data from the REmote ischaemic Conditioning After Stroke Trial (RECAST-1). METHODS: RECAST-1 was a pilot sham-controlled blinded trial in 26 patients with ischaemic stroke, randomized to receive four 5-min cycles of RIC within 24 h of ictus. Plasma taken pre-intervention, immediately post-intervention and on day 4 was analysed for nitric oxide (nitrate/nitrite) using chemiluminescence and all other biomarkers by multiplex analysis. Biomarkers were correlated with clinical outcome (day 90 National Institutes of Health Stroke Scale, modified Rankin Scale, Barthel index). RESULTS: Remote ischaemic per-conditioning reduced serum amyloid protein (SAP) and tissue necrosis factor α (TNF-α) levels from pre- to post-intervention (n = 13, two-way ANOVA, p < 0.05). Overall (n = 26), increases in SAP pre- to post-intervention and pre-intervention to day 4 were moderately correlated with worse day 90 clinical outcomes. No consistent significant changes over time, or by treatment, or correlations with outcome were seen for other biomarkers. CONCLUSIONS: Remote ischaemic per-conditioning reduced SAP and TNF-α levels from pre- to post-intervention. Increases in plasma levels of SAP were associated with worse clinical outcomes after ischaemic stroke. Larger studies assessing biomarkers and the safety and efficacy of RIC in acute ischaemic stroke are warranted to further understand these relationships.

How does measurement of platelet P-selectin compare with other methods of measuring platelet function as a means of determining the effectiveness of antiplatelet therapy?

Measurement of P-selectin on activated platelets as a means of measuring platelet function utilizing the technology described here has the advantage of not requiring immediate access to specialist equipment and expertise. Blood samples are activated, fixed, stored, and transported to a central laboratory for flow cytometric analysis. Here we have compared P-selectin with other more traditional approaches to measuring platelet function in blood and/or platelet-rich plasma (PRP) from patients with acute coronary syndromes on treatment for at least 1 month with either aspirin and clopidogrel or aspirin with prasugrel. The comparators were light transmission aggregometry (LTA), VerifyNow and Multiplate aggregometry (for determining the effects of aspirin) and LTA, VerifyNow and Multiplate together with the BioCytex VASP phosphorylation assay (for the P2Y12 antagonists). The P-selectin Aspirin Test revealed substantial inhibition of platelet function in all but three of 96 patients receiving aspirin with clopidogrel and in none of 51 patients receiving aspirin and prasugrel. The results were very similar to those obtained using LTA. There was only one patient with high residual platelet aggregation and low P-selectin expression. The same patients identified as "non-responders" to aspirin also presented with the highest residual platelet activity as measured using the VerifyNow system, although not quite as well separated from the other values. With the Multiplate test only one of these patients clearly stood out from the others. The results obtained using the P-selectin P2Y12 Test in 102 patients taking aspirin and clopidogrel were similar to the more traditional approaches in that a wide scatter of results was obtained. Generally, high values seen with the P-selectin P2Y12 Test were also high with the LTA, VerifyNow, Multiplate, and BioCytex VASP P2Y12 Tests. Similarly, low residual platelet function using the P2Y12 test was seen irrespective of the testing procedure used. However, there were differences in some patients. Prasugrel was always more effective than clopidogrel in inhibiting platelet function with none of 56 patients (P-selectin and VerifyNow), only 2 of 56 patients (Multiplate) and only 3 of 56 patients (Biocytex VASP) demonstrating high on-treatment residual platelet reactivity (HRPR) defined using previously published cut-off values. The exception was LTA where there were 11 of 56 patients with HRPR. It remains to be seen which experimental approach provides the most useful information regarding outcomes after adjusting therapies in treated patients.

A platelet P-selectin test predicts adverse cardiovascular events in patients with acute coronary syndromes treated with aspirin and clopidogrel.

There is wide variation in response to antiplatelet therapy and high on-treatment platelet reactivity is associated with adverse cardiovascular events. The objective here was to determine whether the results of a novel strategy for assessing platelet reactivity (based on P-selectin measurement) are associated with clinical outcomes in patients with acute coronary syndromes (ACS). This was a prospective cohort study of 100 ACS patients taking aspirin and clopidogrel. P-selectin tests designed to assess response to P2Y12 antagonists or aspirin were performed alongside light transmission aggregometry. For the P2Y12 P-selectin test, an optimal cutoff for high platelet reactivity was determined by receiver operating characteristic (ROC) curve analysis. Patients were divided into two cohorts based on this value: patients with (n = 42) or without (n = 58) high platelet reactivity. The primary endpoint was defined as the composite of cardiovascular death, myocardial infarction and stent thrombosis. After 12 months, the primary endpoint occurred in 12 patients. ROC curve analysis determined that the P2Y12 P-selectin test results were predictive of the primary endpoint (area under curve = 0.69, p = 0.046). The primary endpoint occurred more frequently in patients with high on-treatment platelet reactivity compared to those without (21.4% vs. 5.2%; hazard ratio (HR) 4.14; p = 0.026). The P2Y12 P-selectin test results correlated with light transmission aggregometry (Spearman p < 0.0001). Using the Aspirin P-selectin test, only two patients demonstrated high on-treatment platelet reactivity. This study suggests that a P2Y12 P-selectin test is capable of detecting high on-treatment platelet reactivity, which is associated with subsequent cardiovascular events.

DG-041 inhibits the EP3 prostanoid receptor--a new target for inhibition of platelet function in atherothrombotic disease.

Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E(2) (PGE(2)) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE(2), and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet-leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca(2+) mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y(12) antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE(2) on platelet aggregation depended on the nature of the agonist and the concentration of PGE(2) used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE(2) on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y(12) antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.

Potentiation of platelet aggregation by heparin in human whole blood is attenuated by P2Y12 and P2Y1 antagonists but not aspirin.

INTRODUCTION: Unfractionated heparin (UFH) potentiates platelet aggregation induced by some agonists. P2Y12 and P2Y1 receptors play a major role in amplifying platelet aggregation. We assessed the ability of cangrelor, a selective P2Y12 antagonist, A2P5P, a selective P2Y1 antagonist, and aspirin to block the potentiating effects of heparin. MATERIALS AND METHODS: Whole blood from healthy human volunteers was anticoagulated with either hirudin or UFH 10 IU/ml. Some tubes anticoagulated with hirudin also contained UFH 1 or 10 IU/ml. The low-molecular-weight heparin dalteparin was also assessed. Platelet aggregation was performed using whole blood single-platelet counting. Dense granule release was assessed using 14C-5HT-labelled platelets. RESULTS: UFH and, to a lesser extent, dalteparin potentiated platelet aggregation induced by ADP, PAF, 5HT, U46619, epinephrine and TRAP in a concentration-dependent manner but inhibited aggregation induced by collagen. Cangrelor effectively opposed the potentiating effects of heparins on sustained aggregation induced by ADP, PAF, 5HT, U46619 and TRAP but had less effect on epinephrine-induced aggregation, whereas A2P5P was more effective at blocking both the initial phase of ADP-induced aggregation and the aggregation response to epinephrine, reflecting the differences in G protein coupling between the agonist receptors. Aspirin had no effect on potentiation by heparin. Heparins did not increase ADP- or TRAP-induced 14C-5HT release. CONCLUSIONS: Heparins potentiate platelet responses to ADP and numerous other agonists. This potentiation is attenuated by cangrelor and A2P5P, and is not mediated by increased dense granule release. ADP receptor antagonists but not aspirin may have potential therapeutic benefits in counteracting the pro-thrombotic effects of heparins.