Novel strategies for assessing platelet reactivity

There are many approaches to assessing platelet reactivity and many uses for such measurements. Initially, measurements were based on the ability of platelets separated from other blood cells to aggregate together following activation with an appropriate ‘aggregating agent’. Later, measurements of platelet aggregation in blood itself were performed, and this led to a point-of-care approach to platelet function testing. Measurement of secretory activity through the appearance of the activation marker P-selectin on platelets now provides an alternative approach, which enables remote testing. Measurement of vasodilator-stimulated phosphoprotein phosphorylation is also moving toward application in situations remote from the testing laboratory. Here we provide an overview of the various approaches that are now available, assess their advantages and disadvantages, and describe some of the clinical situations in which they are being used

UDP-sugars activate P2Y 14 receptors to mediate vasoconstriction of the porcine coronary artery

Aims UDP-sugars can act as extracellular signalling molecules, but relatively little is known about their cardiovascular actions. The P2Y14 receptor is a Gi/o-coupled receptor which is activated by UDP-glucose and related sugar nucleotides. In this study we sought to investigate whether P2Y14 receptors are functionally expressed in the porcine coronary artery using a selective P2Y14 receptor agonist, MRS2690, and a novel selective P2Y14 receptor antagonist, PPTN (4,7-disubstituted naphthoic acid derivative). Methods and results Isometric tension recordings were used to evaluate the effects of UDP-sugars in porcine isolated coronary artery segments. The effects of the P2 receptor antagonists suramin and PPADS, the P2Y14 receptor antagonist PPTN, and the P2Y6 receptor antagonist MRS2578, were investigated. Measurement of vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry was used to assess changes in cAMP levels. UDP-glucose, UDP-glucuronic acid UDP-N-acetylglucosamine (P2Y14 receptor agonists), elicited concentration-dependent contractions in the porcine coronary artery. MRS2690 was a more potent vasoconstrictor than the UDP-sugars. Concentration dependent contractile responses to MRS2690 and UDP-sugars were enhanced in the presence of forskolin (activator of cAMP), where the level of basal tone was maintained by addition of U46619, a thromboxane A2 mimetic. Contractile responses to MRS2690 were blocked by PPTN, but not by MRS2578. Contractile responses to UDP-glucose were also attenuated by PPTN and suramin, but not by MRS2578. Forskolin-induced VASP-phosphorylation was reduced in porcine coronary arteries exposed to UDP-glucose and MRS2690, consistent with P2Y14 receptor coupling to Gi/o proteins and inhibition of adenylyl cyclase activity. Conclusions Our data support a role of UDP-sugars as extracellular signalling molecules and show for the first time that they mediate contraction of porcine coronary arteries via P2Y14 receptors

Safety and efficacy of intensive vs. guideline antiplatelet therapy in high-risk patients with recent ischemic stroke or transient ischemic attack: rationale and design of the Triple Antiplatelets for Reducing Dependency after Ischaemic Stroke (TARDIS) trial (ISRCTN47823388)

RATIONALE: The risk of recurrence following a stroke or transient ischemic attack is high, especially immediately after the event. HYPOTHESIS: Because two antiplatelet agents are superior to one in patients with non-cardioembolic events, more intensive treatment might be even more effective. SAMPLE SIZE ESTIMATES: The sample size of 4100 patients will allow a shift to less recurrence, and less severe recurrence, to be detected (odds ratio 0·68) with 90% power at 5% significance. METHODS AND DESIGN: Triple Antiplatelets for Reducing Dependency after Ischaemic Stroke (ISRCTN47823388) is comparing the safety and efficacy of intensive (combined aspirin, clopidogrel, and dipyridamole) vs. guideline antiplatelet therapy, both given for one-month. This international collaborative parallel-group prospective randomized open-label blinded-end-point phase III trial plans to recruit 4100 patients with acute ischemic stroke or transient ischemic attack. Randomization and data collection are performed over a secure Internet site with real-time data validation and concealment of allocation. Outcomes, serious adverse events, and neuroimaging are adjudicated centrally with blinding to treatment allocation. STUDY OUTCOME: The primary outcome is stroke recurrence and its severity ('ordinal recurrence' based on modified Rankin Scale) at 90 days, with masked assessment centrally by telephone. Secondary outcomes include vascular events, functional measures (disability, mood, cognition, quality of life), and safety (bleeding, death, serious adverse events). DISCUSSION: The trial has recruited more than 50% of its target sample size (latest number: 2399) and is running in 104 sites in 4 countries. One-third of patients presented with a transient ischemic attack

Clinical utility of remote platelet function measurement using P-selectin: assessment of aspirin, clopidogrel, and prasugrel and bleeding disorders

Vascular diseases such as myocardial infarction and ischemic stroke are associated with increased platelet function whilst the risk of recurrence is reduced by antiplatelet agents such as aspirin, clopidogrel, and prasugrel. However, some patients exhibit high platelet reactivity, especially with clopidogrel. Existing platelet function tests may not be ideal in that they can be expensive, are often time consuming, and measurements must be made near to the patient and within a few hours of blood collection. Platelet activation leads to translocation of P-selectin from alpha-granules to the cell surface. Following activation with arachidonic acid (which is blocked by aspirin) or adenosine diphosphate (inhibited by clopidogrel) and fixation, samples may be stored or posted to a laboratory performing flow cytometric quantification of platelet P-selectin expression. Acute myocardial infarction and ischemic stroke are associated with high platelet reactivity on clopidogrel in 6–58% of patients when assessed with P-selectin expression, and high reactivity was associated with an increased risk of recurrence after myocardial infarction. Use of P-selectin expression tests may also be of relevance to surgical and veterinary practice and the diagnosis of mild bleeding disorders. The present review explores this topic in further detail

Remote assessment of platelet function in patients with acute stroke or transient ischaemic attack

Background: Antiplatelets reduce recurrence after cerebral ischaemia. The international TARDIS trial assessed the safety and efficacy of intensive (combined aspirin, dipyridamole and clopidogrel) versus guideline (aspirin and dipyridamole, or clopidogrel alone) antiplatelet agents given for one month in patients with acute stroke or TIA. The aim of this substudy was to assess the effect of antiplatelet agents taken at baseline on platelet function reactivity and activation. Methods: In a substudy, platelet function, assessed by remotely measured surface expression of P-selectin (CD62P, Platelet Solutions Ltd), was assessed at baseline in patients who were and were not taking antiplatelet agents at the time of randomisation. Data are Median Fluorescence values (MF). Results: The aspirin P-selectin test demonstrated that platelet expression was lower in 485 patients taking aspirin than in 171 patients taking no aspirin: mean 209 (SD 188) vs. 552 (431), difference 343 (95% confidence intervals, CI 295.3, 390.7) (2p<0.001). Aspirin did not suppress P-selectin levels below 500 units in 22 (4.5%) patients. The clopidogrel P-selectin test showed that platelet reactivity was lower in 96 patients taking clopidogrel than in 586 patients taking no clopidogrel: 653 (297) vs. 969 (315), difference 316.1 (95% CI 248.6, 383.6) (2p<0.001). However, clopidogrel did not suppress P selectin level below 860 units in 24 (24.7%) patients. Conclusions: Aspirin and clopidogrel each suppress stimulated platelet P-selectin although one quarter of patients on clopidogrel have high on-treatment platelet reactivity. Platelet function testing, assessed as platelet P-selectin expression, may be performed remotely in the context of a large multicentre trial

Baseline characteristics of the 3,096 patients recruited into the 'Triple Antiplatelets for Reducing Dependency after Ischaemic Stroke' (TARDIS) trial

Background: The risk of recurrence following ischaemic stroke (IS) or transient ischaemic attack (TIA) is highest immediately after the event. Antiplatelet agents are effective in reducing the risk of recurrence and two agents are superior to one in the early phase after ictus. Design: The Triple Antiplatelets for Reducing Dependency after Ischaemic Stroke (TARDIS) trial was an international multicentre prospective randomised open-label blinded-endpoint trial that assessed the safety and efficacy of short-term intensive antiplatelet therapy with three agents (combined aspirin, clopidogrel and dipyridamole) as compared with guideline treatment in acute IS or TIA. The primary outcome was stroke recurrence and its severity, measured using the modified Rankin Scale at 90 days. Secondary outcomes included recurrent vascular events, functional measures (cognition, disability, mood, quality of life) and safety (bleeding, death, serious adverse events). Data are number (%) or mean (standard deviation, SD). Results: Recruitment ran from April 2009 to March 2016. 3,096 patients were recruited from 106 sites in 4 countries (Denmark 1.6%, Georgia 2.7%, New Zealand 0.2%, UK 95.4%). Randomisation characteristics included: age 69.0 (10.1) years; male 1945 (62.8%); time onset to randomisation 29.4 (11.9) hours; stroke severity (National Institutes for Health Stroke Scale) 2.8 (3.6); blood pressure 143.5 (18.2)/79.5 (11.4) mmHg; IS 2143 (69.2%), TIA 953 (30.8%). Conclusion: TARDIS was a large trial of intensive/triple antiplatelet therapy in acute IS and TIA, and included participants from four predominantly Caucasian countries who were representative of patients in many western stroke services

Clinical utility of remote platelet function measurement using P-selectin: assessment of aspirin, clopidogrel and prasugrel, and bleeding disorders

Vascular diseases such as myocardial infarction and ischaemic stroke are associated with increased platelet function whilst the risk of recurrence is reduced by antiplatelet agents such as aspirin, clopidogrel and prasugrel. However, some patients exhibit high platelet reactivity, especially with clopidogrel. Existing platelet function tests may not be ideal in that they can be expensive, are often time-consuming, and measurements must be made near to the patient and within a few hours of blood collection. Platelet activation leads to translocation of P-selectin from alpha-granules to the cell surface. Following activation with arachidonic acid (which is blocked by aspirin) or adenosine diphosphate (inhibited by clopidogrel) and fixation, samples may be stored or posted to a laboratory performing flow cytometric quantification of platelet P-selectin expression. Acute myocardial infarction and ischaemic stroke are associated with high platelet reactivity on clopidogrel in 6-58% of patients when assessed with P-selectin expression, and high reactivity was associated with an increased risk of recurrence after myocardial infarction. Use of P-selectin expression tests may also be of relevance to surgical and veterinary practice, and the diagnosis of mild bleeding disorders. The present review explores this topic in further detail