1. IntroductionThe optimal ‘non-monitored’ antiplatelet regimen for secondary prevention in individual patients following non-cardioembolic transient ischaemic attack (TIA) and ischaemic stroke remains contentious. Aspirin-dipyridamole combination therapy has been shown to be more effective than aspirin monotherapy at preventing recurrent stroke [[1]European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke.,[2]Antiplatelet therapy after noncardioembolic stroke.] or recurrent ischaemic vascular events overall in patients with TIA or ischaemic stroke [[3]Verro P. Gorelick P.B. Nguyen D. Aspirin plus dipyridamole versus aspirin for prevention of vascular events after stroke or TIA: a meta-analysis.,[4]Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial.], and can be safely started within 24 h of symptom onset [[5]Early treatment with aspirin plus extended-release dipyridamole for transient ischaemic attack or ischaemic stroke within 24 h of symptom onset (EARLY trial): a randomised, open-label, blinded-endpoint trial.]. However, aspirin-dipyridamole combination therapy has not been shown to be superior to clopidogrel monotherapy in an overall ischaemic cerebrovascular disease (CVD) population [[6]Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke.]. Dipyridamole has been shown to exert its antithrombotic effects via a variety of pathways, including inhibition of platelet activation [[7]Gurbel P. Jeong Y.h. Tantry U. ], platelet function [7Gurbel P. Jeong Y.h. Tantry U. , 8Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study., 9Platelet function testing in transient ischaemic attack and ischaemic stroke: a comprehensive systematic review of the literature.], the reuptake of adenosine by erythrocytes (which can in turn lead to vasodilation) [], expression of endothelial activation markers such as VWF antigen [[11]Longitudinal assessment of von Willebrand factor antigen and von Willebrand factor propeptide in response to alteration of antiplatelet therapy after TIA or ischaemic stroke.,[12]Effects of aspirin, clopidogrel and dipyridamole administered singly and in combination on platelet and leucocyte function in normal volunteers and patients with prior ischaemic stroke.], and may have indirect ‘anticoagulant effects’ by reducing peak and total thrombin generation ex-vivo following TIA or ischaemic stroke [[13]Longitudinal assessment of thrombin generation potential in response to alteration of antiplatelet therapy after TIA or ischaemic stroke.].More recent studies have shown that short-term treatment with aspirin-clopidogrel combination therapy can improve outcomes compared with aspirin monotherapy in selected patients with ‘high-risk’ TIA or minor ischaemic stroke [14Clopidogrel with aspirin in acute minor stroke or transient ischemic attack., 15Clopidogrel and aspirin in acute ischemic stroke and high-risk TIA., 16Clopidogrel plus aspirin versus aspirin alone for acute minor ischaemic stroke or high risk transient ischaemic attack: systematic review and meta-analysis., 17Dual antiplatelet therapy with aspirin and clopidogrel for acute high risk transient ischaemic attack and minor ischaemic stroke: a clinical practice guideline., 18Outcomes associated with clopidogrel-aspirin use in minor stroke or transient ischemic attack: a pooled analysis of clopidogrel in high-risk patients with acute non-disabling cerebrovascular events (CHANCE) and platelet-oriented inhibition in new TIA and minor ischemic stroke (POINT) trials.], whereas triple antiplatelet therapy (aspirin-dipyridamole-clopidogrel) has been shown to be harmful due to an increased risk of bleeding on same [[19]Antiplatelet therapy with aspirin, clopidogrel, and dipyridamole versus clopidogrel alone or aspirin and dipyridamole in patients with acute cerebral ischaemia (TARDIS): a randomised, open-label, phase 3 superiority trial.]. The bleeding risks on long-term aspirin-clopidogrel combination therapy outweigh the benefits of antiplatelet monotherapy in CVD patients [20Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events., 21Aspirin and clopidogrel compared with clopidogrel alone after recent ischaemic stroke or transient ischaemic attack in high-risk patients (MATCH): randomised, double-blind, placebo-controlled trial., 22SPS 3 Investigators et al.Effects of clopidogrel added to aspirin in patients with recent lacunar stroke.], unless required for cardiac or other vascular indications. Some patients may not benefit from antiplatelet combination vs. monotherapy due to a number of factors which may affect the efficacy of their prescribed antiplatelet regimen, with increasing interest in the phenomenon of ‘high on-treatment platelet reactivity’ (HTPR) ex vivo in CVD patients on antiplatelet therapy [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.,[9]Platelet function testing in transient ischaemic attack and ischaemic stroke: a comprehensive systematic review of the literature.,[23]Platelet function/reactivity testing and prediction of risk of recurrent vascular events and outcomes after TIA or ischaemic stroke: systematic review and meta-analysis.]. A randomized pilot study of 60 patients with type II diabetes and previous TIA suggested that the inhibition of ADP-induced aggregation in platelet rich plasma initially occurred more rapidly with clopidogrel than with aspirin-dipyridamole, but there was more marked inhibition of the expression of several markers of platelet activation over time with aspirin-dipyridamole vs. clopidogrel [[24]Antiplatelet profiles of the fixed-dose combination of extended-release dipyridamole and low-dose aspirin compared with clopidogrel with or without aspirin in patients with type 2 diabetes and a history of transient ischemic attack: a randomized, single-blind, 30-day trial.]. The pilot, longitudinal TRinity AntiPlatelet responsiveness (TRAP) study showed that 59% of CVD patients at approximately 14 days and 56% at ≥90 days following symptom onset did not have additional inhibition of platelet function on a ‘moderately-high shear stress’ test of platelet function (PFA-100®) in response to collagen and ADP when 200 mg of dipyridamole MR BD was added to aspirin; these patients were considered to have ‘dipyridamole-HTPR’ [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.]. As interest in the concept of personalised/precision medicine has emerged [[25]A new initiative on precision medicine.], these preliminary findings on dipyridamole-HTPR warranted re-evaluation in an independent cohort of patients on user-friendly tests of platelet function/reactivity under both high and low shear stress conditions because none of the aforementioned, large multicentre randomized controlled trials employed platelet function/reactivity testing during follow-up [[1]European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke.,[4]Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial.,[6]Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke.].1.1 Aims

The aims of this prospective, longitudinal, observational-analytical pilot study were:

1. To simultaneously compare the ability of established and relatively novel laboratory tests of platelet function/reactivity to identify patients with recent TIA or ischaemic stroke who do not have additional inhibition of platelet function/reactivity ex vivo after the addition of dipyridamole to aspirin using innovative longitudinal definitions of ‘dipyridamole-HTPR’.

2. To improve our understanding of the underlying platelet activation pathways and clinical, demographic and pharmacodynamic mechanisms influencing ex vivo on-treatment platelet reactivity in CVD patients when dipyridamole is added to aspirin.

3. To collect pilot data to assess the potential ability of this ‘novel longitudinal definition of dipyridamole-HTPR’ to predict the risk of recurrent vascular events during long-term follow-up.

We hypothesised that:

1a. Inhibition of platelet function/reactivity would be enhanced in an important minority of patients on aspirin-dipyridamole combination therapy compared with aspirin alone, and the PFA-100C-ADP cartridge would be confirmed to be able to detect such inhibition;

1b. Low shear stress ADP assays might be able to detect additional inhibition of platelet function/reactivity after the addition of dipyridamole to aspirin.

2. This study would provide further insights into the relationship between platelet activation and dipyridamole-HTPR status, and improve our understanding of the mechanisms influencing ex vivo on-treatment platelet reactivity on dipyridamole in CVD patients overall.

3. Pilot data from this study would inform the design a definitive multi-centre study assessing whether platelet reactivity testing improves our ability to predict the risk of recurrent vascular events in CVD patients treated with aspirin-dipyridamole combination therapy.

2. Methods2.1 Inclusion criteria

Consecutive eligible patients ≥18 years of age, who were on aspirin monotherapy and whose treating physician decided to change their antiplatelet treatment regimen to aspirin-dipyridamole combination therapy within 4 weeks of onset of a recent clinical diagnosis of TIA or ischaemic stroke were recruited to this component of our prospectively-planned, single centre, observational analytical study (the Optimal Antiplatelet Therapy in TIA and Ischaemic Stroke (OATS) study. Patients were intermittently recruited between October 2011 and January 2016 from the Rapid Access Stroke Prevention service, and from the inpatient population of the Neurology, Stroke, Age-Related Health Care and Vascular Surgery services at our secondary and tertiary referral university teaching hospital.

2.2 Exclusion criteria

Patients were excluded if they had a myocardial infarction, venous thrombo-embolism (DVT or PE) or recent surgery within the preceding three months; ongoing unstable angina or unstable symptomatic peripheral vascular disease; platelet count <100 × 109/L; known bleeding or clotting diathesis, including known platelet-related bleeding disorders; active proven vasculitis; active neoplasia; recent or prior history of intracranial haemorrhage, active infection and non-steroidal anti-inflammatory drug (NSAID) intake other than aspirin in the preceding 11 days; were unlikely to be able to attend for clinical follow-up and repeat testing at 14 +/− 7 days.

Dipyridamole was added to aspirin entirely at the discretion of the collaborating consultant staff; the supervising author (DJHM) and his coordinating research team (STL, SJXM) did not influence decisions regarding the choice of optimal antiplatelet therapy in any patients who were not directly under their care. If patients in this component of the OATS study had their index TIA or ischaemic stroke on aspirin monotherapy, or if the attending consultant felt that secondary prevention could be optimised in an evidence-based manner [[1]European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke.,[4]Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial.] in patients who were antiplatelet-naïve on presentation and had initially been prescribed aspirin monotherapy whilst they were undergoing their urgent neurovascular work up, then dipyridamole MR was subsequently added to establish them on this combination antiplatelet regimen. Typically, if these patients presented with their first TIA or ischaemic stroke, aspirin was initially prescribed at a dose of 300 mg daily for 2 weeks and then reduced to 75 mg daily. One week after commencing aspirin, 200 mg of dipyridamole MR once daily was typically added for one week, thereafter increasing to 200 mg BD of dipyridamole MR, and then combination treatment with 75 mg of aspirin daily and 200 mg BD of dipyridamole MR was continued thereafter. If a patient presented with a recurrent ischaemic cerebrovascular event on aspirin monotherapy, with no other obvious cause found that would warrant anticoagulation, and no history of ischaemic heart disease, aspirin therapy was usually continued and dipyridamole MR treatment was empirically added at a dose of 200 mg once daily for one week and then increased to 200 mg BD in combination with aspirin (usually 75 mg daily). If patients were deemed to be at ‘higher risk’ of recurrent vascular events whilst on aspirin monotherapy, e.g. in association with ≥50% extracranial internal carotid stenosis, some collaborators continued aspirin and added full dose dipyridamole MR 200 mg BD at the time of presentation (without a dose escalation phase) [[1]European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke.,[5]Early treatment with aspirin plus extended-release dipyridamole for transient ischaemic attack or ischaemic stroke within 24 h of symptom onset (EARLY trial): a randomised, open-label, blinded-endpoint trial.], once tolerated.2.3 Ethical approval

Written informed consent was obtained from all participants. This OATS study was approved by the St. James's Hospital/AMNCH Research Ethics Committee (REC Ref: 2011/35/03).

2.4 Clinical assessmentAll patients underwent detailed neurovascular assessment and were given a clinical diagnosis of a TIA or ischaemic stroke by their attending Consultant Neurologist or Stroke Physician after detailed investigations according to ESO recommendations [[26]European Stroke Organisation Executive Committee Guidelines for management of ischaemic stroke and transient ischaemic attack 2008.,[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.]. The diagnosis was also confirmed in all cases by a clinically-experienced Vascular Neurology Research SpR (STL/SJXM) or by the supervising Vascular Neurologist (DJHM). Information regarding vascular risk factors, including hypertension, prior TIA or stroke, ischaemic heart disease, atrial fibrillation, valvular heart disease, diabetes mellitus, hyperlipidaemia, peripheral vascular disease, migraine, family history of stroke, medication intake (including anti-thrombotic therapy), smoking status, alcohol intake, illicit substance intake, and the method of detection of carotid stenosis and timing of any carotid intervention in patients with large artery atherosclerosis was collected prospectively [[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.]. Details regarding antiplatelet regimens, dose and duration of therapy were recorded. Results of routine haematological (FBC), coagulation (PT/APTT), biochemical and blood glucose testing were collected prospectively. CT and/or MRI brain and colour Doppler ultrasound (CDUS) of neck vessels was performed in all patients, as well as magnetic resonance angiography (MRA) or CT angiography (CTA) to establish concordance between CDUS and another non-invasive imaging modality in recently symptomatic carotid stenosis patients. A chest radiograph, electrocardiograph (ECG), 24-h ECG recording and transthoracic or trans-oesophageal echocardiograph were obtained in all patients [[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.]. The underlying mechanism responsible for TIA or ischaemic stroke was categorised according to both the TOAST classification [[28]Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment.] and ASCOD classification systems [[29]The ASCOD phenotyping of ischemic stroke (Updated ASCO Phenotyping).].Patients underwent detailed clinical and laboratory assessment with venepuncture at baseline before changing treatment (baseline), with planned clinical and laboratory assessment at 14 +/− 7 days after (14d), and at least 90 days (90d) after their treating physician added dipyridamole to aspirin monotherapy. In patients with recently symptomatic ≥50% carotid artery stenosis, the 90d follow-up was performed at least 3 months following carotid surgery or endovascular treatment, unless intervention had been delayed for at least 3 months after symptom onset [[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.].Adherence to prescribed antiplatelet therapy in inpatients was confirmed by checking their inpatient prescription chart to ensure that medications had been dispensed and taken. Adherence in all outpatients was checked by history taking alone, but all patients were phoned to emphasise the importance of medication-adherence in the week prior to reassessment. Reassessment was deferred for 14 days in any patients deemed possibly non-adherent to their antiplatelet treatment, and any issues potentially affecting adherence were addressed [[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.].2.5 Clinical outcome eventsDetailed information regarding the occurrence of the pre-specified, primary composite clinical endpoint of non-fatal ischaemic stroke, non-fatal MI or vascular death during follow-up was collected in person at each clinical and/or laboratory follow-up visit. Data on other pre-specified secondary outcomes, including haemorrhagic complications [[6]Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke.], were recorded. Clinical follow-up to assess the longer-term risk of recurrent vascular outcome measures was performed using a validated in-person or telephone questionnaire at ≥1 year after symptom onset. We prospectively planned to confirm any outcome events determined by telephone interview alone by contacting the GP, reviewing relevant hospital consultant's letters or notes, and if necessary, by reviewing death certificates if the patient had unfortunately died before the 1-year follow-up visit.2.6 Laboratory assessmentsCareful, atraumatic venepuncture was performed from a free-flowing vein using a 21G butterly needle and a Vacutainer® system with a luer adapter after resting participants for ≥20 mins [[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.,[30]Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.] [[31]Increased platelet activation in early symptomatic vs. asymptomatic carotid stenosis and relationship with microembolic status: results from the platelets and carotid stenosis study.]. The first 3 ml citrate-anticoagulated blood sample was discarded. Two 2 ml sterile 3.2% buffered sodium citrate-anticoagulated samples were taken for analysis of platelet function/reactivity with the VerifyNow® system (Accriva Diagnostics, USA). Six further samples were taken into sterile 3 ml Vacutainers® containing 3.2% buffered sodium citrate. The first and second of these citrate-anticoagulated samples were used for whole blood flow cytometric analysis and for measurement of platelet function with the platelet function analyser (PFA-100®, Dade-Behring, Germany/Sysmex, UK), respectively, and the last sample was used to measure the platelet count, mean platelet volume (MPV) and platelet distribution width (PDW) in citrate between 2 and 4 h after venepuncture. Subsequently, one 3 ml double-walled Vacutainer® tube containing ‘recombinant hirudin anticoagulant’ was taken for platelet reactivity analysis on the Multiplate® system (Verum Diagnostica GmbH/Roche Inc., Germany). Three 3 ml sterile Vacutainer® tubes containing K2EDTA were obtained, the first of which was used to measure the full blood count (FBC), including measurement of the MPV and PDW in EDTA between 2 and 4 h after venepuncture.2.7 PFA-100® platelet function analyserThe degree of inhibition of platelet function in whole blood was assessed before and after adding dipyridamole to aspirin therapy at ‘moderately high shear stress’ following biochemical stimulation with collagen (2 μg) and 50 μg of ADP (Collagen-ADP cartridge), 10 μg epinephrine bitartrate (Collagen-EPI cartridge – sensitive to inhibition with aspirin), or 20 μg ADP/5 ng Prostaglandin E1/459μg CaCl (INNOVANCE PFA P2Y™ cartridge - sensitive to inhibition with clopidogrel/ P2Y12 antagonists), as previously described [[32]Assessment of the antiplatelet effects of low to medium dose aspirin in the early and late phases after ischaemic stroke and TIA.]. This device mimics the in vivo haemostatic process that one may see in a moderately stenosed artery. The time taken for activated platelets to occlude an aperture in the cartridge is called the ‘closure time’. The maximum closure time recorded by the device is 300 s, so we arbitrarily defined closure times >300 s as ‘301 s’ for statistical analyses and assumed that the data were not normally distributed (see statistical methodology section).2.7.1 Innovative longitudinal definition of dipyridamole-HTPR on the PFA-100In our laboratory, the intra-assay coefficient of variation (CV) was 7% for the C-ADP assay, 7.5% for the C-EPI assay and 7.8% for the INNOVANCE PFA P2Y assay. Although all 3 PFA-100 assays were performed, we only anticipated that the C-ADP cartridge would detect the antiplatelet effects of dipyridamole [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.]. For the purpose of this study, ‘dipyridamole-HTPR on the PFA-100’ was also defined as failure to prolong C-ADP closure times compared with the patient's baseline on aspirin monotherapy by more than twice the CV of the assay when dipyridamole was added to aspirin therapy i.e. failure to prolong C-ADP closure times by >14% of the patient's baseline C-ADP closure time [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.].2.8 VerifyNow® platelet function analyserThe VerifyNow® is a cartridge-based analyser which assesses ex vivo platelet reactivity at ‘low shear stress’ in response to stimulation with fixed doses of different platelet agonists in single-use cartridges containing fibrinogen-coated beads [[33]The use of the VerifyNow system to monitor antiplatelet therapy: a review of the current evidence.]. The reagents bound to the fibrinogen beads are arachidonic acid in the ‘Aspirin cartridge’, and adenosine diphosphate (ADP), iso- thrombin receptor activating peptide (iso-TRAP), and PAR-4 activating peptide in the ‘P2Y12 cartridge’. During the test, a 2 ml 3.2% sodium citrate-anticoagulated whole blood sample tube is inserted into the cartridge. The whole blood is mixed with the platelet agonists and the fibrinogen-coated beads by the movement of an electromagnetically-driven steel ball. The platelets become activated by the specific agonist in the cartridge to a degree that is dependent on the level of inhibition by the antiplatelet agent which the patient is taking. Activated platelets will then bind to the fibrinogen-coated beads, cause agglutination and will fall out of the solution. Within the instrument, the light absorbance through the solution is measured 16 times per second. Both the rate and extent of platelet-induced agglutination over a fixed period of time are measured and combined with a proprietary algorithm to report the values in ‘reaction units’ [[33]The use of the VerifyNow system to monitor antiplatelet therapy: a review of the current evidence.].2.8.1 Exploratory novel longitudinal definition of dipyridamole-HTPR on the VerifyNow P2Y12 assay

Intra-assay CVs were measured in our laboratory and found to be 0.1% for the Aspirin cartridge and 5.5% for the P2Y12 cartridge. Based on our experience with the PFA-100C-ADP assay, we provisionally defined ‘dipyridamole-HTPR on the VerifyNow’ as failure to decrease the P2Y12 Reaction Units (PRU) on the P2Y12 assay compared with the patient's baseline PRU on aspirin monotherapy by more than twice the CV of the assay when dipyridamole was added to aspirin i.e. failure to decrease the PRU by >11% of the patient's PRU at baseline.

2.9 Multiplate® assay

This whole blood platelet aggregation assay is based on measurement of impedance at ‘low shear stress’ as platelets adhere to 2 adjacent electrodes and aggregate to one another within a cuvette. The extent of platelet adhesion and aggregation is recorded as the Area Under the Curve (AUC) in ‘units (U)’ up to 6 min after the addition of either arachidonic acid (Aspirin test) or ADP (ADP test) to measure the antiplatelet effects of aspirin or clopidogrel/P2Y12 antagonists, respectively.

2.9.1 Exploratory novel longitudinal definition of dipyridamole-HTPR on the multiplate ADP assay

In our laboratory, the intra-assay CV for the Aspirin test was 7.3% and for the ADP test was 7.8% (N = 8 assays). We assessed the potential ability of the Multiplate ADP assay to detect additional inhibition of platelet function ex-vivo following stimulation with ADP when dipyridamole was added to aspirin. ‘Dipyridamole-HTPR on the Multiplate analyser’ was provisionally defined as failure to decrease the AUC of the Multiplate ADP test compared with the patient's baseline AUC on aspirin monotherapy by more than twice the CV of the assay when dipyridamole was added to aspirin therapy i.e. failure to decrease the AUC by >15.6% of the patient's AUC at baseline.

2.10 Whole blood flow cytometryPlatelets were distinguished from red and white blood cells, as described previously [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.,[27]Profile of reticulated platelets in the early, subacute and late phases after transient ischemic attack or ischemic stroke.,[30]Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.,[31]Increased platelet activation in early symptomatic vs. asymptomatic carotid stenosis and relationship with microembolic status: results from the platelets and carotid stenosis study.]. Platelet surface CD62P and CD63 expression [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.,[30]Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.,[31]Increased platelet activation in early symptomatic vs. asymptomatic carotid stenosis and relationship with microembolic status: results from the platelets and carotid stenosis study.], and the percentages of circulating neutrophil-platelet, monocyte-platelet and lymphocyte-platelet complexes were quantified as markers of platelet activation, using previously described and validated methods [[8]Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.,[30]Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemi