Role of platelet α2-adrenoreceptor in biological low response to Clopidogrel for patients with non cardioembolic ischemic stroke or transient ischemic attack

Abstract

Background and purpose: Low biological response to Clopidogrel prescribed after non cardioembolic ischemic stroke or transient ischemic attack (TIA) is a major clinical problem and could explain the recurrence of vascular events. Platelet α2-adrenoreceptors are involved in the high residual platelet reactivity in stable coronary artery disease patients on dual antiplatelet therapy. In the present study we investigated the impact of platelet α2-adrenoreceptors on ADP-induced platelet aggregation and on ADP-induced platelet membrane CD62P (P-selectin) expression, a marker of platelet activation on blood samples from patients hospitalized at the acute phase of a non cardioembolic ischemic stroke or TIA. Methods: 72 consecutive patients were prospectively recruited over the course of two years in a monocentric study. Patients received a daily 75 mg-dose of Clopidogrel. ADP-induced platelet aggregation was measured alone, with low dose epinephrine or with atipamezole, a selective α blocker of α2-adrenoreceptors, by Light Transmittance Aggregometry (LTA). Platelet membrane expression of P-selectin was measured by flow cytometry with either ADP alone or combined with epinephrine. Results: Epinephrine at low dose stimulated ADP-induced platelet membrane expression of CD62P whereas Atipamezole significantly inhibited 10 µM ADP-induced platelet aggregation. Conclusions: Our study showed the role of platelet α2-adrenoreceptors in biological low response to Clopidogrel for patients hospitalized for a non-cardioembolic ischemic stroke or TIA. Atipamezole could improve the status of biological response to Clopidogrel.

Introduction

Clopidogrel is one of the first-line treatments after non-cardioembolic ischemic stroke or transient ischemic attack (TIA) [1-3]. However, the recurrence rate of vascular events was 7.15% per year in CAPRIE trial (Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events), 16.7% at 18 months in MATCH trial (Management of ATherothrombosis with Clopidogrel in High-risk patients having experienced either transient ischemic attack or stroke), and 13.1% in PRoFESS trial (Prevention Regimen for Effectively avoiding Second Strokes) after a mean follow-up of 2.5 years [4-6]. In addition, Clopidogrel did not provide significant benefit in stroke patients when compared to aspirin treatment in CAPRIE trial [4]. In contrast, Clopidogrel was beneficial in patients with lower-limb occlusive arterial disease or diabetes, or with a history of stroke or myocardial infarction [4,7,8]. The well-known variability in biological response to Clopidogrel in coronary artery disease partly explains the recurrence of vascular events [9-11]. Indeed, in studies conducted specifically in ischemic stroke or TIA patients, the proportion of poor biological responders to Clopidogrel ranged from 8 to 55% according to the laboratory test and the cut-off value used [12-24].

The mechanisms underlying the variability in biological response to Clopidogrel have been studied. The involvement of platelet α2-adrenoreceptors in the high residual platelet reactivity has been investigated in stable coronary artery disease patients on dual antiplatelet therapy [25]. ADP-induced platelet aggregation was significantly potentiated by low-dose epinephrine (1.10^-9 g/ml) whereas it was inhibited by atipamezole (2 µM), a selective α2-adrenoreceptor blocker.

The aim of our study was to assess the influence of α2-adrenoreceptors on ADP-induced platelet aggregation and on ADP-induced platelet membrane P-selectin expression, in patients hospitalized at the acute phase of a non-cardioembolic ischemic stroke or a TIA and treated with a regular dose of Clopidogrel.

Materials and methods

Patients

Consecutive patients hospitalized in the Neurovascular Unit of Saint-Etienne University Hospital Centre following a non-cardioembolic ischemic stroke or TIA, and selected for treatment with Clopidogrel alone, were prospectively recruited between September 2013 and November 2015.

Patients were selected to receive Clopidogrel alone (Plavix 75 mg; Sanofi Pharma Bristol-Myers Squibb SNC, Paris, France) at the dose of 75 mg per day if this treatment was considered likely to confer greater benefit than aspirin.

Exclusion criteria comprised an abnormal laboratory test result (platelet count < 100 G/l, prothrombin time (PT) < 70% or > 130%, activated partial thromboplastin time (aPTT) < 27 s or > 39 s, aspartate aminotransferase or alanine aminotransferase > 2.5 N), need for continued treatment with aspirin, history of a stroke or TIA under Clopidogrel, or contraindication to Clopidogrel or to one of the excipients in the formulation. Ongoing treatment interacting with the α-adrenergic system was also an exclusion criterion.

Clinical and laboratory assessments

The following patients characteristics were recorded: age, sex, body mass index (BMI), medical history (hypertension, hypercholesterolemia, current smoking, diabetes, coronary artery disease, lower-limb occlusive arterial disease, ischemic stroke or TIA), ongoing treatment (β-blocker, calcium channel blocker, angiotensin converting enzyme inhibitor, angiotensin II receptor blocker, proton pump inhibitor, or statin). The definition of TIA established by the TIA Working group in 2002, the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) etiological classification, the National Institutes of Health Stroke Score (NIHSS) at inclusion, and the modified Rankin handicap Score (mRS) at discharge were used to characterize the index ischemic event [26-29]. The laboratory parameters analyzed were creatinine clearance (determined by the Cockcroft-Gault formula), glycated hemoglobin (HbA1c), low-density (LDL)-cholesterol, high-density (HDL)-cholesterol and triglycerides. Radiological parameters, evaluated by an independent radiologist, comprised the extent of white matter lesions of vascular origin according to the Fazekas classification, and the presence of microbleeds [30].

Evaluation of the biological response to Clopidogrel with Light Transmission Aggregometry (LTA)

Whole blood samples were drawn with a 19-gauge needle in citrated tubes (Sodium Citrate 0.105 M/3.2%, Becton Dickinson, Plymouth, UK) from the patients after 5 to 8 days of Clopidogrel treatment in order to ensure that the drug had reached a steady state dose [31]. Pre-analytical recommendations were followed [32]. Platelet rich plasma (PRP) was used for LTA.

The interval between blood sampling and LTA did not exceed 2 h. Platelet aggregation was measured using an aggregometer (TA4V; SD-Medical, Heillecourt, France) and according to LTA recommendations [33]. The percentage of maximal platelet aggregation (MPA) induced by ADP (Elitech) at final concentrations of 1.25, 5 and 10 µM was measured at 37°C [34].

Potentiation of ADP-induced aggregation by a low-dose of epinephrine (1.10^-9 g/l) was assessed with ADP 1.25 µM whereas the inhibition of ADP-induced aggregation was assessed with atipamezole (Antisedan^®, Atipamezole hydrochloride, Orion Pharma) and with ADP 10 µM. Preincubation of PRP was performed with 2 μM of atipamezole for 2 minutes.

Clopidogrel response according to LTA is usually considered as low if a maximal platelet aggregation value is > 70% [9].

Flow cytometry

10 µl of PRP was activated with ADP at a final concentration of 1.25, 5 and 10 µM for 10 minutes and this activation was stopped with thrombofix platelet stabilizer kit (Beckman Coulter, Villepinte, France). After 60 minutes, 10 µl of this mixture was incubated with 5 µl of anti P-selectin-PE antibody (Phycoerythrine, clone CLB-Thromb/6, Beckman Coulter) and 10 µL of anti CD61-PE-Cy7 antibody (phycoerythrin-cyanine 7, clone SZ21, Beckman Coulter) for 15 minutes in a dark room, at room temperature. Then 500 µl of phosphate buffer saline (PBS) were added and the acquisition of data was performed on 10000 platelets with a Navios (Beckman Coulter, Brea, USA) [35]. The Mean Fluorescence Intensity or MFI was determined on CD61-positive platelets and on P-selectin positive platelets by using the Kaluza 1.5a flow cytometry analysis software and the percentage of P-selectin positive platelets was determined.

Potentiation of ADP-induced platelet membrane P-selectin expression by low-dose of epinephrine (1.10^-9 g/l) was assessed with ADP 1.25 µM.

Clinical trials regulations

This study, registered at ClinicalTrials.gov (no. NCT01955642) was approved by the French health authorities and the local ethics committee. A signed consent form was provided by each patient included in the study.

Statistical analysis

Continuous data were expressed as mean and standard deviation or median and interquartile range (Q1-Q3) and categorical data as absolute and relative frequencies (expressed in percentages).

Maximal aggregation values induced by ADP 1.25 µM, 5 µM and 10 µM were compared using a Wilcoxon signed-rank test (paired data) as follows: i) ADP alone versus ADP in presence of low dose epinephrine; ii) ADP alone versus ADP combined with atipamezole.

All statistical tests were two-sided and P-values less than 0.05 were considered as statistical significant. Statistical analyses were performed with SAS (version 9.4).

Results

Seventy two patients were included in the study. The population characteristics are displayed in Table 1. The mean age was 68.7 +/- 9.6 years and 68.1% of the patients were males. The proportion of patients taking β-blocker, statin, PPI, ACE inhibitor/ARB and calcium channel blocker was respectively 15.3%, 69.4%, 36.1%, 58.3% and 33.3%. In our study, 38.9% of the patients were TOAST I. 25% of the patients were smokers.

Table 1.

Patients characteristics

Patients characteristics			Ongoing treatments
Age (y), mean (SD)		68.7 (9.6)	Beta-blocker	11 (15.3%)
Age ≥ 75 y		22 (30.6%)	Statin	50 (69.4%)
Male		49 (68.1%)	PPI	26 (36.1%)
BMI ≥ 30 kg/m2		14 (20.0%)	ACE inhibitor/ARB	42 (58.3%)
			Calcium channel blocker	24 (33.3%)
Medical history			Laboratory test parameters
Hypertension		48 (66.7%)	Clearance	12 (16.7%)
Hypercholesterolemia		39 (54.2%)	(Cockroft-Gault) < 60 ml/min
Diabetes		25 (34.7%)	Glycated hemoglobin ≥ 6%	37 (52.9%)
Current smokers		18 (25%)	LDL Cholesterol ≥ 2.6 mmol/l	41 (56.9%)
Coronary artery disease		7 (9.7%)	HDL Cholesterol ≤ 1 mmol/l	24 (35.3%)
Lower-limb arterial disease		5 (6.9%)	Triglycerides ≥ 1.70 mmol/l	27 (37.5%)
Ischemic stroke or TIA		14 (19.4%)
Characteristics of stroke/TIA			Radiological analysis
Type of event	Ischemic stroke	63 (87.5%)	Simplified Fazekas score ≥ 2	29 (40.3%)
	TIA	9 (12.5%)	Presence of microbleed	16 (22.2%)
TOAST classification	I	28 (38.9%)
	III	18 (25.0%)
	V	26 (36.1%)
NIHSS at admission	< 5	61 (84.7%)
	≥ 5	11 (15.3%)
mRS at discharge	< 3	63 (87.5%)
	≥ 3	9 (12.5%)

ACE inhibitor: angiotensin converting enzyme inhibitor; ARB: angiotensin II receptor blocker; BMI: body mass index; HDL: high-density lipoprotein; LDL: low-density lipoprotein; mRS: modified Rankin score; NIHSS: National Institutes of Health Stroke Score; NR patients: non-responder/low--responder/poor-responder patients; PPI: proton pump inhibitor; R patients: responder patients; SD: standard deviation; TIA: transient ischemic attack; TOAST: Trial of ORG 10172 in Acute Stroke Treatment.

Parameters of maximal aggregation induced by ADP 1.25, 5 and 10 µM are shown in Table 2. Median values of maximal aggregation values induced by ADP at 1.25 µM, 5 µM and 10 µM were 24.7%, 73.7% and 74.1% respectively.

Table 2.

Maximal platelet aggregation values (Aggmax) by ADP group

		ADP (N=72)	ADP + epinephrine (N=72)	ADP + atipamezole (N=72)
Aggmax: ADP 1.25 µM (%)	N	71	70	66
	Missing data	1	2	6
	Mean (SD)	30.4 (22.0)	31.3 (23.4)	29.8 (22.0)
	Median	24.7	22.7	21.0
	Min-Max	4.7-94.2	3.1-88.8	5.8-99.0
	Q1-Q3	16.5-33.2	14.6-34.9	15.7-32.3
Aggmax: ADP 5 µM (%)	N	72	72	68
	Missing data	0	0	4
	Mean (SD)	67.9 (20.1)	62.0 (18.2)	62.9 (21.5)
	Median	73.7	66.3	67.2
	Min-Max	16.0-101.2	15.4-90.8	1.2-97.2
	Q1-Q3	51.7-83.6	50.4-76.6	44.6-78.7
Aggmax: ADP 10 µM (%)	N	72	71	66
	Missing data	0	1	6
	Mean (SD)	70.9 (16.4)	65.5 (16.6)	66.6 (15.8)
	Median	74.1	70.5	70.5
	Min-Max	22.0-101.8	11.2-93.8	33.2-97.1
	Q1-Q3	64.2-80.9	55.7-76.6	54.3-78.7

All values are expressed as Mean (SD), Median and inter-quantile range (Q1-Q3).

Median values of maximal aggregation induced by 1.25 µM, 5 µM and 10 µM ADP in presence of low dose epinephrine were 22.7%, 66.3% and 70.5% (Table 2). A significant difference was found between values for ADP versus ADP in presence of low dose epinephrine with P values < 0.05 (Figure 1; P=0.01, n=70 for ADP 1.25 µM, P < 0.0001, n=72 for ADP 5 µM and P < 0.0001, n=71 for ADP 10 µM).

Figure 1.

Maximal platelet aggregation values (Aggmax) by ADP group. A. ADP 1.25 µM; B. ADP 5 µM; C. ADP 10 µM. *means a statistical Wilcoxon signed-rank test when comparing epinephrine group to control or atipamezole to control. The box-and-whisker plots show minimum and maximum values, 25^th and 75^th percentiles, and medians (horizontal bars) of Aggmax.

In representative dotplots (Figure 2A-F), low dose epinephrine potentiated ADP 1.25 µM induced platelet membrane P-selectin expression. On platelets from 67 patients, the median values of the percentage of P-selectin positive platelets induced by ADP and by ADP with low dose epinephrine (+ epi) were as follows: 16% with ADP 1.25 µM, 18.1% with ADP 1.25 µM + epi, 28.2% with ADP 5 µM, 31% with ADP 5 µM + epi, 30% with ADP 10 µM, 36.5% with ADP 10 µM + epi (Figure 2G).

Figure 2.

Effect of a low-dose of epinephrine on ADP-induced P-selectin expression assessed with flow cytometry. Representative density plots for ADP (A-C) versus ADP+low dose of epinephrine (1.10^-9 g/l) (D-F). Median values of the percentage of P-selectin positive platelets induced by ADP and by ADP with low dose epinephrine (G).

Median maximal aggregation values induced by 1.25 µM, 5 µM and 10 µM of ADP on PRP pretreated with atipamezole were: 21%, 67.2% and 70.5% respectively (Table 2). A significant difference was found between values for ADP versus ADP + atipamezole for ADP 5 µM and ADP 10 µM but not for ADP 1.25 µM (P=0.13, n=66 for ADP 1.25 µM, P=0.003, n=68 for ADP 5 µM and P=0.006, n=68 for ADP 10 µM). Furthermore, 33.3% of Clopidogrel low responder became responder in the “atipamezole” condition with an Aggmax value < 70% (n=14/42).

Discussion

Values of ADP-induced platelet aggregation for 5 and 10 µM ADP were close to the normal reference laboratory aggregation values previously published [36] from 100 healthy volunteers despite Clopidogrel treatment. Several hypotheses could explain this: a) the patients of our study were included at the acute phase of their ischemic stroke which favors the activation of platelets, especially for the patients with large-vessel cerebral infarction [37]; b) they received only one antiaggregant treatment without loading dose because of the risk of bleeding; c) a small number of patients received β-blocker, which is known to modulate the platelet reactivity. In addition, the conditions used in our study are significantly different from those previously published on patients hospitalized for a stable angor: all patients were treated by 100 mg aspirin, and 75 mg Clopidogrel and 92.6% were taking β-blocker [25].

Median values were taken into consideration because the number of patients included was low and because all the data were not normally distributed.

It is worth noting that epinephrine was applied at a dose falling within the range of catecholamines physiological concentrations. Indeed, at the nanomolar range, epinephrine cannot initiate platelet aggregation but is able to potentiate the effect of other agonist like ADP. A low concentration of ADP (1.25 µM) has been chosen to optimize the potentiating effect of low dose epinephrine on ADP-induced aggregation and ADP-induced platelet membrane P-selectin expression consecutively to translocation from alpha-granules following activation.

No potentiation of ADP induced aggregation by low-dose of epinephrine was observed. This is different from previous results likely because of the clinical settings [25]. A slight platelet inhibition has been described for patients with non-ST elevation acute coronary syndrome carrying the α2A-adrenoreceptor 6.3 kb variant and treated with 250 mg aspirin and 600 mg Clopidogrel [38]. This paradoxical result could be explained by a possible overstimulation of α2-adrenoreceptors in the context of an activated sympathetic nervous system and a desensitization of the α2-adrenoreceptors [38]. The sympathetic nervous system of our patients could be highly active because they were stressed at the acute phase of the ischemic stroke or of the TIA. During this phase like in non-ST elevation acute coronary syndrome a high level of blood catecholamines could induce an overstimulation and a desensitization of α2-adrenoreceptors [39].

However a potentiation by a low-dose of epinephrine of the ADP-induced P-selectin expression was observed. Thus the pathway linking α2-adrenoreceptor activation to the platelet membrane P-selectin expression is still active. This result confirms the hypothesis of Motulsky: the desensitization of α2-adrenoreceptors pathway occurs without change in the alpha 2-adrenoreceptors or in their coupling to an inhibition of adenylate cyclase [39].

A link has been suggested among smoking, serotonin and catecholamine signaling resulting in an increased tonic level of platelet activation in smokers [40]. We did not confirm these results (data not shown).

We found a significant inhibitory effect of atipamezole on 10 µM ADP-induced platelet maximal aggregation. Interestingly, after atipamezole incubation ADP-induced platelet maximal aggregation values were lowered. This could have a potential therapeutic interest for Clopidogrel low responders. This work could be further investigated by studying the impact of the autonomous nervous system on platelet function.

Conclusions

Our study showed the role of platelet α2-adrenoreceptors in biological low response to Clopidogrel for patients hospitalized for a non-cardioembolic ischemic stroke or TIA, and treated with Clopidogrel alone. Atipamezole could have a potential therapeutic effect for Clopidogrel low responder.

This transverse study involved clinical and research teams. This paper was written by NM and JV. English was improved by GL and FM. NM was in charge of the LTA and flow cytometry tests with AM. AM and CL provided the aggregometer and the flow cytometer. AG was in charge of the clinical research management. CC and SL were in charge of the statistical tests. CB was in charge of the radiological analysis. JV, SA and ME were in charge of patients recruitment; PG and PM supervised the clinical and pharmacological aspects of the study. The clinical team was also involved in the interpretation of the results.

Disclosure of conflict of interest

None.