Aspirin is the key treatment in the secondary prevention of atherothrombosis. Interindividual variability of response has been linked to a higher risk for ischemic events. The aim of this study was to identify clinical and biologic factors predicting high on-aspirin platelet reactivity (HPR) in a high-risk, “real-world” population of vascular patients. All platelet testing performed from 2011 to 2013 in consecutive patients receiving long-term treatment with aspirin for coronary or cerebrovascular disease was retrospectively analyzed. Indications for platelet testing were recurrent ischemic events or high-risk angioplasty. HPR was defined as aggregation intensity ≥20% using light-transmission aggregometry with arachidonic acid 0.5 mg/ml. Collagen-epinephrine platelet function analysis was also performed (threshold <165 seconds). Cardiovascular risk factors, usual biologic parameters, and antiplatelet treatment were recorded. A total of 1,508 patients were included (mean age 63 years, 71% men, 23% with diabetes). Antiplatelet treatment was aspirin alone in 333 patients and dual-antiplatelet therapy in 1,175 patients. HPR was found in 11.1% of patients. In multivariate analysis, independent predictive factors of HPR on light-transmission aggregometry with arachidonic acid were diabetes mellitus (odds ratio [OR] 2.10, 95% confidence interval [CI] 1.39 to 3.16), age (OR 1.25, 95% CI 1.06 to 1.47), fibrinogen level (OR 1.20, 95% CI 1.02 to 1.42), and von Willebrand factor level (OR 1.06, 95% CI 1.03 to 1.09). On light-transmission aggregometry with arachidonic acid and collagen-epinephrine platelet function analysis, fibrinogen remained the main factor associated with HPR (OR 1.33, 95% CI 1.19 to 1.61). Similar results were found in patients treated with aspirin alone or dual-antiplatelet therapy. A fibrinogen level >4.0 g/L was associated with a 3.9-fold increased risk for HPR in patients aged <75 years. In conclusion, fibrinogen level was the major predictor of HPR on aspirin in this large population of high-risk vascular patients.
Platelet function testing is not recommended in routine practice. However, assessment of platelet inhibition could be of interest in high-risk situations such as high-risk angioplasty (left main, residual artery, etc.) or in case of recurrent atherothrombotic events despite aspirin treatment. We hypothesized that finding factors predictive of high on-aspirin platelet reactivity (HPR) could help identify high-risk patients and to eventually modify antiplatelet therapy. Our main objective was to identify clinical and biologic predictive factors, routinely available, associated with HPR in a large real-life, high-risk population of patients receiving aspirin as monotherapy or dual-antiplatelet therapy (DAPT) in the setting of secondary prevention of coronary artery disease (CAD) or cerebrovascular disease (CVD).
Methods
This retrospective study was conducted at Lariboisiere Hospital (Paris, France) in 3 clinical departments (cardiology, neurology, and diabetology) from January 2011 to July 2013. We included all consecutive patients aged ≥18 years, treated with aspirin as monotherapy or DAPT (clopidogrel, prasugrel, or ticagrelor) in whom platelet function was assessed using light-transmission aggregometry with 0.5 mg/ml arachidonic acid (LTA-AA) and collagen-epinephrine platelet function analysis (PFA-EPI). Patients had to have received long-term treatment for ≥7 days with non-enteric-coated acetylsalicylic acid (lysine acetylsalicylate, Aspegic or Kardegic; Sanofi-Aventis, Paris, France) at the usual dose prescribed by their treating physicians, ranging from 75 to 300 mg/day without a change of dose within the previous week. Platelet reactivity was assessed in this highly selected population for the following reasons: (1) high-risk CAD, defined as angioplasty of the left main coronary artery, of the last patent coronary vessel, 3-vessel disease or complex proximal left anterior descending artery, recurrent acute coronary syndromes, stent thrombosis, and recurrent need for coronary angioplasty in patients with diabetes, and (2) high-risk CVD, defined as recurrent ischemic stroke or transient ischemic attack despite antiplatelet treatment and stenting of cerebrovascular arteries for atherosclerotic lesions. Platelet reactivity was assessed ≥7 days after an acute event. The following data were systematically recorded: age, gender, cardiovascular risk factors (diabetes mellitus, defined as treatment or glycated hemoglobin level >6.5%; treated hypertension; dyslipidemia, defined as statin treatment or low-density lipoprotein cholesterol level >1.60 g/L; active smoking; body mass index), antiplatelet therapy, and the usual biologic parameters (complete blood count, fibrinogen level, antigenic von Willebrand factor [vWF] level, factor VIII level, mean platelet volume, prothrombin time, and activated partial thromboplastin time). Patients were excluded if they had previously been treated for myeloproliferative neoplasms, had platelet counts outside the range of 100 to 500 G/L range, had hematocrit <25%, had prothrombin times <50%, or had prolonged activated partial thromboplastin times (>1.5). Before blood sampling, the exact time of the last aspirin intake was recorded, and blood sampling was delayed if there was doubt regarding adherence.
This study was conducted in accordance with the Declaration of Helsinki II and was approved by the local ethics committee. All patients gave informed consent for biologic testing.
Blood samples were obtained from venipuncture in the morning, just before the next aspirin intake (most often 24 hours after the last aspirin intake). Tests were performed <30 to 60 minutes after blood sampling. LTA-AA has been routinely performed for several years at our laboratory. Briefly, platelet-rich plasma is obtained by centrifugation of citrated whole blood at 156g for 12 minutes at 18°C and platelet-poor plasma by further centrifugation at 1,000g for 20 min. In vitro platelet aggregation is measured at 37°C in an aggregometer (model 490-4D; Chrono-Log Corporation, Kordia, The Netherlands) according to Born’s optical aggregometry method after the addition of 0.5 mg/ml of arachidonic acid. Aggregation is expressed as the maximum aggregation intensity (MAI) and maximal aggregation velocity from baseline using the autologous platelet-poor plasma as a reference. HPR is defined as MAI ≥20%. The PFA-100 system (Dade Behring International, Miami, Florida) is a whole-blood point-of-care assay that assesses platelet aggregation under high shear, measuring the time necessary to occlude an aperture in a membrane coated with collagen and epinephrine. HPR was defined by a closure time within the normal range (<165 seconds) on PFA-EPI in patients receiving aspirin therapy. PFA-EPI was used alone and in addition to LTA-AA to select a population with a high HPR profile.
The main objective of our study was to assess predictors of HPR defined by LTA-AA (MAI ≥20%). Continuous variables are presented as mean ± SD and were compared using Student’s unpaired t test if normally distributed or are presented as median (interquartile range) and were compared with the Mann-Whitney rank sum test if not. Usual clinical and biologic parameters (listed in Table 1 ) were selected for univariate analysis. Categorical variables are presented as counts and percentages and were compared by means of the chi-square test or Fisher’s exact test. Correlations between quantitative variables were assessed using Pearson’s or Spearman’s correlation coefficients. Predictive factors were determined using a stepwise multivariate logistic regression analysis. After univariate analysis, variables that presented significant associations with HPR (p <0.20) were entered in the multivariate model. A p value <0.05 was considered to indicate statistical significance. A similar analysis was performed using PFA-EPI and then both LTA-AA and PFA-EPI to determine HPR. A subgroup comparative analysis of stable patients (treated with aspirin alone) versus patients treated with DAPT was performed. Odds ratios (ORs) are presented with 95% confidence intervals (CIs). Statistical analyses were performed using MedCalc software (Mariakerke, Belgium).
| Overall population (n= 1508) | Presence of HPR (n= 168) | Absence of HPR (n= 1340) | p-value | |
|---|---|---|---|---|
| Clinical characteristics | ||||
| Age (years) | 63 [53-70] | 65 [57-78] | 62 [52-70] | p<0.0001 |
| Age > 75 years | 246 (16%) | 49 (29.2%) | 173 (12.9%) | p=0.0004 |
| Men | 1073 (71%) | 125 (74.4%) | 883 (65.9%) | NS |
| Diabetes mellitus | 345 (22.9%) | 57 (33.9%) | 288 (21.5%) | p=0.0004 |
| Hypertension ∗ | 538 (35.7%) | 73 (43.5%) | 465 (34.7%) | p=0.0309 |
| Dyslipidaemia ∗ | 693 (46.0%) | 95 (56.5%) | 598 (44.6%) | p=0.0046 |
| Smoker | 349 (23.1%) | 41 (24.4%) | 308 (23.0%) | NS |
| Body mass index (kg/m 2 ) | 26.2 [23.9-29.0] | 25.0 [23.9-29.4] | 26.2 [23.9-28.8] | NS |
| Indication of platelet function testing | ||||
| Coronary artery disease | 1277 (85%) | 153 (91.1%) | 1190 (88.8%) | NS |
| Cerebrovascular disease | 231 (15%) | 15 (8.9%) | 150 (11.2%) | NS |
| Medications | ||||
| Aspirin dose (mg) | 120 ±69 | 99 ±40 | 121 ±61 | p=0.0014 |
| Monotherapy | 333 (22%) | 29 (17.3%) | 304 (22.7%) | NS |
| Dual antiplatelet therapy | 1175 (78%) | 139 (82.7%) | 1036 (77.3%) | NS |
| Clopidogrel 75 or 150 mg/day | 728 (62%) | 88 (52.4%) | 699 (52.2%) | NS |
| Prasugrel 10 mg/day | 269 (23%) | 35 (20.8%) | 234 (17.5%) | NS |
| Ticagrelor 90 mg twice a day | 119 (10%) | 16 (9.5%) | 103 (8.7%) | NS |
| Biological characteristics | ||||
| Platelet count (G/L) | 241 [202-288] | 226 [179-272] | 242 [207-288] | p=0.0021 |
| Mean platelet volume (fL) | 7.8 [7.2-8.5] | 8.1 [7.1-8.4] | 7.8 [7.1-8.4] | p=0.0020 |
| Haemoglobin (g/dL) | 13.7 [12.6-14.6] | 13.0 [11.9-14.2] | 13.8 [12.8-14.7] | p<0.0001 |
| Haematocrit (%) | 40.8 [37.7-43.7] | 38.9 [36.0-42.7] | 41.0 [38.0-43.9] | p<0.0001 |
| Leukocytes count (G/L) | 7.1 [5.8-8.6] | 7.8 [6.0-9.6] | 7.0 [5.7-8.4] | p=0.0009 |
| Fibrinogen level (g/L) | 4.0 [3.4-4.9] | 4.6 [3.8-5.7] | 4.0 [3.4-4.8] | p<0.0001 |
| von Willebrand Factor >100 | 1206 (80%) | 142 (84.5%) | 942 (70.3%) | p=0.0002 |
| Prothrombin Time (%) | 104 [94-111] | 98 [88-107] | 104 [95-111] | p=0.0001 |
| activated Prothromboplastin Time (times the control time) | 1.0 [0.9-1.1] | 1.0 [1.0-1.2] | 1.0 [0.9-1.1] | p<0.0001 |
∗ Hypertension was defined as treated for hypertension, and dyslipidaemia as statin treatment or LDL cholesterol level >1.60g/L. Categorical variables are expressed as percentages and continuous variables as mean ± SD (if normally distributed) or median (interquartile range). P-values for comparison using Mann-Whitney test of patients with or without HPR, NS: non-significant (p>0.05).
Results
Among the 1,692 eligible patients treated from January 2011 to July 2013, 1,508 (89%) were included ( Figure 1 ). Clinical and biologic characteristics of the population are listed in Table 1 . The median dose of aspirin was 75 mg/day (range 75 to 300), and 62% of patients were treated with doses ≤100 mg/day.
