Thrombosis Research (2007) 120, 901–909
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Relationship between changes in platelet reactivity and changes in platelet receptor expression induced by physical exercise Cristina Aurigemma a , Andrea Fattorossi c , Alfonso Sestito a , Gregory A. Sgueglia a , Sara Farnetti b , Alexia Buzzonetti c , Fabio Infusino a , Raffaele Landolfi b , Giovanni Scambia c , Filippo Crea a , Gaetano A. Lanza a,⁎ a b c
Institute of Cardiology, Università Cattolica del Sacro Cuore, Rome, Italy Institute of Internal Medicine and Geriatrics, Università Cattolica del Sacro Cuore, Rome, Italy Laboratory of Immunology, Department of Oncology, Università Cattolica del Sacro Cuore Campobasso, Italy
Received 6 August 2006; received in revised form 9 January 2007; accepted 22 January 2007 Available online 6 March 2007
KEYWORDS Platelet; Atherosclerosis; Leukocyte–platelet aggregates; Exercise stress test; Platelet function analyser (PFA)-100
Abstract Introduction: In previous studies we have consistently shown a significant increase of platelet reactivity after exercise in patients with obstructive coronary artery disease (CAD). We also observed a significant individual variability in the response to exercise of platelet reactivity in these patients. Whether exercise-induced changes in platelet reactivity correlate with changes in platelet membrane receptors in patients with CAD is unknown. Methods: We studied 26 patients with stable CAD and 10 matched healthy controls who underwent a symptom-limited treadmill exercise stress test. Venous blood samples were collected at rest and within 5 min of peak exercise. Platelet reactivity was measured by the PFA-100 method as time to occlude (closure time, CT) a ring coated with collagen/adenosine diphosphate (C/ADP). Platelet expression of glycoprotein (GP) IIb/IIIa, in both global (CD41) and active form (PAC-1), and P-selectin (CD62P) and formation of leukocyte–platelet aggregates were assessed by flow cytometry. Results: After exercise CT did not change in controls (85.4 ± 12 to 84.0 ± 9 s, p = 0.37), whereas it decreased in CAD patients (98.8 ± 24 to 91.4 ± 25 s, p b 0.001). After
Abbreviations: CAD, coronary artery disease; PFA, platelet function analyzer; C/ADP, collagen/adenosine diphosphate; CT, closure time; ECG, electrocardiogram; PMN, polymorphonuclear cells; MONO, monocyte; GP, glycoprotein. ⁎ Corresponding author. Istituto di Cardiologia, Università Cattolica del Sacro Cuore, L.go A. Gemelli, 8, 00168 — Roma, Italy. Tel.: +39 06 3015 4187; fax: +39 06 3055 535. E-mail address:
[email protected] (G.A. Lanza). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.01.009
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C. Aurigemma et al. exercise, CD41 and PAC-1 platelet expression increased significantly in CAD patients (p = 0.04 for both), but not in controls (p = 0.39 and p = 0.98, respectively). To evaluate the relationship between the response to exercise of platelet reactivity and of platelet receptor expression, CAD patients were divided into two groups: CAD group 1 (16 patients, decrease in CT N 5 s after exercise) and CAD group 2 (10 patients no increase in platelet reactivity after exercise). CD41 and PAC-1 expression increased in CAD group 1 (p = 0.008 and p = 0.026, respectively) but not in CAD group 2 (p = 0.39 and p = 0.50, respectively). No significant differences were observed between the 2 groups for changes in CD62P and leukocyte–platelet aggregates. Conclusions: Our data show that, in patients with stable CAD, an increased platelet reactivity to C/ADP stimulation after exercise, as assessed by the PFA-100 method, is specifically associated with an increased expression of platelet GP IIb/IIIa receptor. © 2007 Elsevier Ltd. All rights reserved.
Introduction
Methods
Platelets are involved both in the mechanisms of atherogenesis [1] and in the thrombotic complications of atherosclerosis [2]. Several previous studies assessed the effects of lifestyle factors on platelet function [3–5]. The effects of exercise, in particular, have been extensively studied, but the results are contentious [4,6–11]. The reasons for that are likely multiple and include differences in patient selection, drug therapy and methods employed to assess study platelet function. Furthermore, techniques used to assess platelet reactivity, either in vitro or in vivo, are often associated with considerable methodologic difficulties, which might also account, at least in part, for the discrepancies reported in previous studies [12]. Recently, a new simple method has been proposed to measure platelet reactivity, the platelet function analyser (PFA)-100. By this method, platelet reactivity is measured as the time needed to occlude a ring in a cartridge coated with either collagen and adenosine diphosphate (C/ADP) or collagen and norepinephrine [13,14]. Using the PFA-100 method, in previous studies we have consistently shown a significant increase of platelet reactivity in response to C/ADP after exercise in patients with obstructive coronary artery disease (CAD) [9–11]. However, we also observed a significant individual variability in the response to exercise of platelet reactivity in these patients [10]. It is not known at present whether these individual differences are or not related to differences in changes of membrane platelet receptors in response to exercise. To address this issue, in this study we compared the exercise-induced changes of platelet reactivity (as assessed by the PFA-100 method) with the changes of platelet surface receptors and leukocyte–platelet aggregates, as assessed by flow cytometry, in a group of CAD patients.
Study groups We studied 26 patients (65 ± 9 years, 20 men) with clinically stable obstructive CAD, documented at coronary angiography (N 50% stenosis in ≥ 1 major epicardial coronary artery). All patients were studied while taking their usual medications. Patients receiving anticoagulant drugs or antiplatelet agents other than aspirin were excluded from the study. Patients taking aspirin were not excluded as previous reports showed that the platelet response to exercise, as assessed by the methods used in the present study (see below), is not significantly influenced by aspirin treatment [15,16]. Ten subjects without any evidence of CAD (60 ± 3 years, 4 men) were studied as a control group. These subjects were enrolled from the non-medical staff of our hospital and were selected to be comparable to CAD patients as to age and gender. Their clinical history excluded any potential cardiac symptom and all had normal standard 12 leads electrocardiogram (ECG), exercise stress test and two-dimensional and Doppler echocardiography. The study complies with the Declaration of Helsinki and was approved by our institutional review board; all subjects gave their written informed consent to participate in the study.
Exercise stress test After 30 min of rest in a sitting position, a symptom/ sign-limited exercise stress test was performed according to a standard Bruce protocol. Leads II, V2 and V5 were monitored continuously. A 12-lead ECG was printed at the end of each stage or when clinically indicated, and at 1-minute intervals in the recovery phase. Blood pressure was measured at
Relationship between changes in platelet reactivity and changes in platelet receptor expression baseline and during the last minute of each stage, unless otherwise indicated. The test was stopped in case of physical exhaustion, progressive angina (Borg scale N6), ST-segment depression N 3 mm, or clinically relevant events (e.g. dyspnoea, hypotension, arrhythmias). The test was considered “positive” for ischemia when horizontal or downsloping ST-segment depression ≥ 1 mm at 0.06 s from the J point occurred in at least one ECG lead.
Blood sampling Prior to exercise and within 5 min of peak exercise blood samples were drawn from an anticubital vein through clean venipunctures, using a 21G needle. Samples were drawn directly to plastic tubes containing 0.106 M trisodium citrate (blood:citrate = 9:1) after discarding the first 2 mL to minimize the formation of platelet aggregates. Beside immediate testings, anticoagulated blood was also centrifugated at 3000 g for 20 min and aliquots of plasma were stored at − 80 °C within 1 h of collection. Plasma von Willebrand factor antigen was quantitatively determined by automated latex particle enhanced immunoturbidometric assay (HemosIL, Instrumentation Laboratory, S.p.A, Milano, Italy) on a ACL TOP System (Instrumentation Laboratory, S.p.A, Milano, Italy). The assay was performed within 2 h of thawing the frozen samples. Results are reported as percentage of normality with a detection limit of 2.2%.
Platelet reactivity Details for the assessment of platelet reactivity by the PFA-100 method have been described in detail elsewhere [9–11]. Briefly, an aliquot of 800 μL was used within 1 h of collection to assess platelet reactivity by the PFA-100 method. The 800 μL of whole blood were added to a standardized disposable cartridge, containing a ring coated with 2 μg collagen (equine type 1) and 50 μg ADP. After incubation at 38.5 °C, blood was aspirated at arterial shear rates (5000 s− 1) through a C/ADP ring. Platelets within the sample adhere to the ring and are activated, forming an aggregate which occludes the opening. The time to occlusion (closure time, CT) is taken as a measure of platelet reactivity, with shorter CT indicating a faster aggregability. The measurements were performed only once by an experienced technician who was blinded to the clinical data and flow cytometry results. We previously found that the method shows good reproducibility [14] and is independent of sex,
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age, blood cell count, and most cardiological drugs, including aspirin [9–11,14].
Flow cytometry An aliquot of blood drawn as described before was used within 10 min of collection to prepare the samples for analyses by flow cytometry. Flow cytometry measurements were performed by an experienced technician who was blinded to the clinical data and PFA results. Platelet surface receptors Blood was diluted 1:10 in phosphate buffered saline (PBS) and labeled within 10 min of collection by incubation with specific antibodies. Briefly, blood aliquots (5 μL) were incubated for 15 min at room temperature with saturating concentrations of phycoerythrin (PE)-conjugated CD41 to study the platelet expression of glycoprotein (GP) IIb/IIIa receptor or PE-conjugated CD62P to study the platelet expression of P-selectin (Becton-Dickinson, Milan, Italy). In 18 CAD patients and 9 controls blood aliquots were also incubated with fluorescein isothiocynate (FITC)conjugated PAC-1 to study the platelet expression of the active form of the GP IIb/IIIa receptor. Following incubation, samples were diluted with 200 μL of PBS and immediately analysed by a flow cytometer Becton-Dickinson FACScan. An acquisition gate was first established on FSC/SSC signals. These were collected in logarithmic mode to improve discrimination between viable platelets and unwanted events (erythrocytes, white blood cells, debris and aggregates). The purity of the gate was always confirmed by backgating on CD41 staining. A low flow rate was used to minimize coincident events. A minimum of 10,000 platelets was counted for each test. Fluorescence data were evaluated as mean fluorescence intensity (MFI). Leukocyte–platelet aggregates Blood (100 μL) was labeled within 10 min of collection with a saturating concentration of FITCconjugated CD61 monoclonal antibody (Instrumentation Laboratory, Milan, Italy) for 15 min at room temperature, in order to specifically stain platelets by using one of its membrane receptors as an antibody target. Following incubation, erythrocytes were lysed with buffered ammonium chloride, and samples immediately analysed by FACScan. Polymorphonuclear cells (PMN) and monocytes (MONO) were selectively gated on FCS and SSC signals and aggregates measured as ratio between MFI of the population staining positive for CD61 and MFI of
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Table 1 Main clinical and exercise findings in patients with coronary artery disease and control subjects Controls p CAD patients (n = 10) (n = 26) Age (years) Sex (M/F) Cardiovascular risk factors Family history of CAD Hypertension Diabetes Hypercolesterolemia Smoking Drug therapy Diuretics ACE-inhibitors Nitrates Ca2+-antagonists Beta-blockers Statins Aspirin Exercise test data Rest Heart rate (bpm) Systolic blood pressure (mmHg) Rate–pressure product (bpm × mmHg) Peak Heart rate (bpm) Systolic blood pressure (mmHg) Rate–pressure product (bpm × mmHg) Duration of exercise (s) ≥ 1 mm ST-segment depression
65 ± 10 20/6
60 ± 3 6/4
0.11 0.41
9 (35%) 16 (62%) 3 (12%) 19 (73%) 4 (15%)
4 (40%) 4 (40%) 0 (0%) 5 (50%) 1 (10%)
1.00 0.29 0.54 0.25 1.00
7 (27%) 13 (50%) 6 (23%) 10 (39%) 16 (62%) 14 (54%) 22 (85%)
1 (10%) 2 (20%) 0 (0%) 1 (10%) 1 (10%) 3 (30%) 0 (0%)
0.40 0.14 0.16 0.13 0.01 0.27 b0.001
decrease in CT N5 s compared to the baseline CT value; 2) CAD group 2 included patients who did not show a significant increase in platelet reactivity with exercise (i.e., increase, no change or decrease ≤ 5 s in CT after exercise). The cut-off point of 5 s decrease in CT to divide CAD patients into 2 groups was chosen as this value corresponded to the top quartile of the changes in CT with exercise observed in a large group of CAD patients in a previous studied (i.e., 75% had a decrease in CT N 5 s after exercise) [10]. Correlations analysis between the changes in cytometry variables and CT were done by Spearman rho test. Data are shown as mean ± SD or median and interquartile intervals. A p value b0.05 was always required for statistical significance.
Results CAD patients vs. controls
67 ± 10 129 ± 19
77 ± 8 121 ± 14
0.006 0.29
9331 ± 4419
9324 ± 1501
0.69
120 ± 21 173 ± 23
153 ± 11 164 ± 23
20831 ± 25161 ± 5479 3978 445 ± 170 523 ± 119 15 (58%) 0 (0%)
b0.001 0.46 0.04 0.88 0.002
the population staining negative for CD61. A minimum of 30.000 PMN and MONO was counted for each test.
Statistics Between-group differences in continuous variables were compared by Mann–Whitney U test, whereas proportions were compared by Fisher exact test. Covariance analysis was applied to adjust differences in exercise-induced closure time changes between CAD patients and controls for basal differences of potentially confounding variables. To get insight into the relationship between the response to exercise of platelet reactivity and of platelet receptor expression, CAD patients were divided into two groups according to the changes in CT on the PFA-100 method with exercise: 1) CAD group 1 included subjects who showed an increase in platelet reactivity after exercise, defined as a
The main clinical characteristics of patients and controls are summarized in Table 1. Overall, there were no significant differences between the 2 groups in the main basal clinical data, including cardiovascular risk factors, but, as expected, CAD patients were taking more drugs, specifically betablockers and anti-platelet agents. Furthermore, peak heart rate and rate–pressure product were higher and exercise duration tended to be longer in controls, compared to CAD patients. Basal CT did not differ significantly between the 2 groups. Compared to baseline, after exercise CT did not change in controls (85.4 ± 12 vs. 84.0 ± 9 s, p= 0.37), whereas it decreased significantly in CAD patients (98.8 ± 24 vs. 91.4 ± 25 s, pb 0.001). The percentage change in CT in CAD patients (median −8.5%; interquartile interval −11% to −2.9%) was significantly different from that in controls (median −2.3%; interquartile interval −5.3% to +4%; p = 0.023). At rest, cytometry variables assessed in this study did not differ between the 2 groups. After exercise CD41 and PAC-1 platelet expression increased in CAD patients, but not in controls. On the other hand, PMN–platelet aggregates decreased in CAD patients whereas increased in controls. No other differences were observed in cytometry variables between the 2 groups (Table 2). No significant differences between the 2 groups were found in basal values and in the response to exercise of platelet count, hematocrit, plasma fibrinogen and von Willebrand factor (Table 2). In fact, hematocrit levels at rest tended to be higher in CAD patients than in controls (p = 0.08); however, the difference in the exercise closure time response
Relationship between changes in platelet reactivity and changes in platelet receptor expression Table 2
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Laboratory and cytometry results in patients with coronary artery disease and control subjects CAD patients (n = 26)
Closure time (s) Hematocrit (%) Platelet count (×103/μL) Fibrinogen (mg/dL) von Willebrand factor antigen (%) CD41 (MFI) CD62P (MFI) PAC-1 (MFI) PMN–PLT (MFI ratio) MONO–PLT (MFI ratio)
Controls (n = 10)
Rest
Peak exercise
p
Rest
Peak exercise
p
98.8 ± 24 47.8 ± 6 231 ± 57 314 ± 57 90.5 ± 27 287 ± 64 23.7 ± 5 149 ± 46 8.1 ± 1.9 9.0 ± 1.8
91.4 ± 25 48.9 ± 5 244 ± 58 316 ± 58 102.2 ± 24 318 ± 104 25.6 ± 6 160 ± 45 7.5 ± 1.5 8.7 ± 2.0
b0.001 0.015 0.01 0.75 0.008 0.04 0.01 0.04 0.02 0.44
85.4 ± 12 43.8 ± 6 228 ± 105 305 ± 38 90.2 ± 29 304 ± 36 23.2 ± 4 167 ± 68 7.4 ± 1.7 8.6 ± 2.5
84.0 ± 9 44.5 ± 7 237 ± 102 308 ± 45 102.7 ± 35 306 ± 35 26.6 ± 5 181 ± 70 8.1 ± 1.5 9.3 ± 2.2
0.37 0.038 0.022 0.64 0.036 0.58 0.09 0.26 0.05 0.169
between the 2 groups persisted unchanged (p b 0.001) after adjustment for hematocrit levels.
CAD patients Among CAD patients a significant increase in platelet reactivity after exercise (CT reduction N 5 s) was observed in 16 patients (62%; CAD group 1), whereas 10 patients (38%) did not show any significant variations in platelet reactivity (CAD group 2). The main clinical characteristics and exercise results of the two groups of CAD patients are summarized in Table 3. There were no differences in main clinical findings between the 2 CAD groups. There were also no significant differences in preexercise blood pressure, heart rate and rate–pressure product. Moreover, exercise duration and peak heart rate and rate–pressure product were also similar in the two groups. Significant ST-segment depression during exercise test was induced in 9 (56%) and in 6 (60%) patients in CAD group 1 and CAD group 2, respectively (p = 1.00). Laboratory variables potentially able to influence platelet reactivity (i.e. von Willebrand factor, fibrinogen, hematocrit and platelet count) also did not show any significant differences between the 2 CAD groups before and after exercise (Table 4).
Platelet reactivity and flow cytometry results in CAD groups Platelet reactivity results are shown in Table 4. According to grouping criteria, CT decreased significantly with exercise in CAD group 1 patients (from 98.9 ± 24 to 85.5 ± 23 s, p b 0.001), whereas there were no significant changes in CAD group 2 (98.6 ± 26 vs. 100.9 ± 25 s, p = 0.21). At baseline there were no differences between the two groups in flow cytometry variables. Compared to rest, the expression of platelet CD41 (Fig. 1) and PAC-1 increased significantly after
Table 3 Main clinical and exercise findings in the subgroups of CAD patients with (group 1) or without (group 2) exercise-induced increase in platelet reactivity CAD group 1 CAD group 2 p (n = 16) (n = 10) Age Sex (M/F) Cardiovascular risk factors Family history of CAD Hypertension Diabetes Hypercolesterolemia Smoking Coronary angiography 1-vessel 2-vessel 3-vessel Drug therapy Diuretics ACE-inhibitors Nitrates Ca2+-antagonists Beta-blockers Statins Aspirin Exercise test data Rest Heart rate (bpm) Systolic blood pressure (mmHg) Rate–pressure product (bpm × mmHg) Peak Heart rate (bpm) Systolic blood pressure (mmHg) Rate–pressure product (bpm × mmHg) Duration of exercise (s) ≥1 mm ST-segment depression
67 ± 1 12/4
61 ± 12 8/2
0.22 1.00
5 (31%) 10 (63%) 2 (13%) 12 (75%) 3 (19%)
4 6 1 7 1
(40%) (60%) (10%) (70%) (10%)
0.69 1.00 1.00 1.00 1.00
4 (25%) 7 (44%) 5 (31%)
3 (30%) 3 (30%) 4 (40%)
0.78
4 (25%) 7 (44%) 3 (18%) 5 (31%) 10 (63%) 8 (50%) 13 (81%)
3 6 3 5 6 6 9
(30%) (60%) (30%) (45%) (60%) (60%) (90%)
1.00 0.64 0.39 0.43 1.00 0.70 1.00
68 ± 8 130 ± 20
66 ± 13 128 ± 17
0.52 0.98
9875 ± 5459
8461 ± 1795
0.82
114 ± 18 169 ± 22
128 ± 25 179 ± 25
0.11 0.34
1949 ± 4967
2298 ± 5824
0.82
436 ± 166 9 (56%)
461 ± 185 6 (60%)
0.70 1.00
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Table 4 Laboratory and cytometry findings in the subgroups of CAD patients with (group 1) or without (group 2) exercise-induced increase in platelet reactivity CAD group 1 Closure time (s) Hematocrit (%) Platelet count (× 103/μL) Fibrinogen (mg/dL) von Willebrand factor antigen (%) CD41 (MFI) CD62P (MFI) PAC-1 (MFI) PMN–PLT (MFI ratio) MONO–PLT (MFI ratio)
CAD group 2
Rest
Peak exercise
p
Rest
Peak exercise
p
98.9 ± 24 47.5 ± 5 234 ± 57 314 ± 64 90.8 ± 26 292 ± 58 24.8 ± 5 146 ± 39 8.3 ± 2.2 9.1 ± 1.6
85.5 ± 23 49.1 ± 6 244 ± 60 315 ± 58 100.7 ± 27 345 ± 112 27.3 ± 6 166 ± 41 7.8 ± 1.7 8.9 ± 2.2
b0.001 0.009 0.035 0.86 0.029 0.008 0.023 0.026 0.18 0.47
98.6 ± 26 46.9 ± 7 228 ± 60 313 ± 59 90.2 ± 29 280 ± 75 21.9 ± 3 153 ± 58 7.7 ± 1.3 8.9 ± 2.3
100.9 ± 25 48.9 ± 7 242 ± 60 318 ± 58 103.7 ± 24 276 ± 78 23.0 ± 4 151 ± 54 7.0 ± 1.1 8.6 ± 1.9
0.21 0.021 0.018 0.21 0.011 0.39 0.033 0.50 0.09 0.65
exercise in CAD group 1 (p = 0.008 and p = 0.026, respectively) but not in CAD group 2 (p = 0.39 and p = 0.50, respectively). The percentage changes induced by exercise (median [interquartile interval]) in CD41 (+ 9.8% [+ 1.3%, + 29.7%] vs. − 3.4% [− 4.7%, + 1.1%]; p = 0.007) and in PAC-1 (+ 12.8% [+ 5.9%, + 26.6%] vs. − 1% [− 4.3%, + 2.1%]; p = 0.003) platelet expression were significantly different between the 2 CAD groups. Furthermore, a significant inverse correlation was found between CT changes and both CD41 and PAC-1 platelet expression in the whole population of CAD patients (r = − 0.40 and r = − 0.50, p = 0.037 and p = 0.034, Fig. 2). In contrast, platelet CD62P expression increased in both CAD groups (p = 0.023 and p = 0.033, respectively) without a significant between-group difference in the percent change with exercise (p = 0.18). PMN–platelet aggregates and MONO–platelet aggregates did not shown any significant change in both group of CAD patients without any significant between-group differences. No significant correlations were found between changes with exercise of CT and changes of CD62P or leukocyte–platelet aggregates.
Discussion In agreement with our previous data [9–11], this study shows that platelet reactivity in response to C/ ADP, as assessed by the PFA-100 method, increases with exercise in patients with clinically stable CAD whereas does not show any significant changes in controls. Our data, however, also confirm that the response to physical exercise of platelet reactivity in CAD patients is not homogeneous, showing a significant increase in most but not all patients. Most important, our results point out that the increased platelet reactivity induced by exercise in
CAD patients is associated with an increased expression of platelet GP IIb/IIIa receptors, in both global (CD41) and active (PAC-1) form, whereas no significant differences in the response to exercise were found between the patients with or without exercise-induced increased platelet reactivity on the PFA-100 method in platelet CD62P expression and platelet–leukocyte aggregates. The heterogenous platelet response to exercise in CAD patients was independent of the major clinical characteristics, platelet count, hematocrit, von Willebrand factor and fibrinogen, as well as exercise-induced myocardial ischemia and drug therapy, suggesting a different intrinsic response to exercise of platelets.
Exercise and platelet reactivity Patients with obstructive CAD have a slightly increased risk of acute coronary events during physical and emotional stress [17,18] and platelets may play an important role in triggering acute coronary events in stressful conditions. Despite that, there have been only scanty and conflicting data on the effect of exercise on platelet receptor expression. Thus in contrast with some studies demonstrating exercise-induced platelet reactivity [6,7,9–11], Lindmann et al. demonstrated an unexpected reduction of both CD41 and CD62P platelet expression, as well as of thrombin-induced platelet activation, after exercise in CAD patients, whereas no significant variations were observed in healthy controls [8]. Differences in selection and characteristics of patients and controls, and in procedures used to assess platelet function, as well as the small number of individuals usually included in the few available studies, might account, at least in part, for the conflicting results [12]. Moreover, platelet function is also difficult to assess with traditional techniques,
Relationship between changes in platelet reactivity and changes in platelet receptor expression
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Figure 1 Effect of exercise on platelet CD41 (GP IIb/IIIa) receptor expression in CAD patients with (CAD group 1) or without (CAD group 2) evidence of exercise-induced increased platelet reactivity by the PFA-100 method. Data in control subjects are also shown (●) Rest (○) Peak exercise.
which are usually time-consuming and require profound expertise. We recently used the PFA-100 method to assess platelet reactivity [5,9–11]. This technique is simple, quick, reproducible, largely operator independent and does not require any particular expertise. Using the PFA-100 method we recently consistently found increased platelet reactivity to C/ADP in CAD patients following both physical and mental stress, whereas no changes were observed in healthy controls [5,9–11], which is in accordance with the results of the present study. At the same time, however, we noticed a non-homogeneous response of platelet reactivity to exercise both in healthy subjects and, even more, in CAD patients [10]. The heterogeneous response in CAD patients was confirmed in this study, in which significant enhanced exercise-induced platelet reactivity was found in 62% of patients. In this study we show that the increase in platelet reactivity was specifically associated with an increased platelet expression of CD41 after exercise, thus suggesting that this change likely plays a major role in the exerciseinduced increase in platelet reactivity, at least as assessed by the PFA-100 method. Indeed, the increase of this platelet receptor was noticed in CAD patients but not in controls and, moreover, a concomitant increase of the expression of the PAC-1 was also observed in CAD patients only. In contrast to CD41, however, we observed a similar increase in platelet CD62P expression after exercise in controls and in patients with and in those without increased exercise-induced platelet reactivity. This finding was unexpected because both receptors are released by platelet alpha-granules in case of platelet activation [19]. The reasons for the different response to exercise of CD41 and CD62P in patients with increased platelet reactivity cannot be elucidated from our data. However, a possible explanation is that the difference might, at least in part, be related to a different mechanism of activation of platelets in
group 1 patients, resulting in a major translocation to platelet membranes of the superficial canalicular system, which is known to bring CD41 but not CD62P [19]. In the whole group of CAD patients we also observed a reduction after exercise of polymorphonuclear–platelet aggregates, without any significant differences between the two groups. This finding was unexpected because leukocyte–platelet aggregates are considered a sensitive marker of platelet activation [20]. A possible explanation for these results is that leukocytes–platelet aggregates might be seized from the circulating blood into the vessel wall, favoured by an increased expression of adhesion molecules (in particular of the platelet endothelial cell adhesion molecule 1 [PECAM-1]) on the surface of dysfunctional endothelium [21].
Clinical implications The heterogeneous response to exercise of platelet reactivity might have significant clinical implications. First, it might influence clinical outcome of CAD patients, as those with increased exercise-
Figure 2 Correlation between absolute changes in CT and percentage changes of CD41 (GP IIb/IIIa receptor) platelet expression induced by exercise, as compared to resting state, in patients with coronary artery disease.
908 induced platelet activation might be more exposed to stress-induced acute coronary events. Furthermore, it suggests that the assessment of the effectiveness of anti-platelet agents in clinical practice should also include their ability to prevent stress-induced platelet activation.
Limitations of the study Some limitations of the study should be acknowledged. Patients and controls differed for some drug therapy, in particular for aspirin assumption. However, we and others previously found that aspirin does not have any significant effect on the response to exercise of platelet reactivity as assessed by our methods, i.e., by PFA-100 method using C/ADP as platelet agonists and by cytometry [9,10,15,16]. Thus we were quite confident that including patients with or without aspirin would not have significantly biased our results. Moreover, it should be stressed that, as only most CAD patients, but not any controls, were taking aspirin, the different use of the drug should be expected to actually decrease, rather than increase, the differences in platelet response to exercise between the 2 groups. Finally, aspirin use did not differ between the subgroups of CAD patients with or without increased platelet reactivity after exercise according to PFA100 results, thus making unlikely any influence on the differences observed between these two groups. Similar comments apply to the use of betablockers and physical performance, as we would expect an increased platelet reactivity for higher levels of exercise. Moreover, we previously found that increase in platelet reactivity following exercise was largely independent of the level of exercise in stable CAD patients [9]. Some variables potentially able to influence exercise-induced platelet reactivity have not been taken into account in this study. In particular, physical fitness, which can improve the platelet response to exercise [24], was not measured. However, all subjects included in this study were sedentary, and it is unlikely that minor differences in physical activity among them did significantly influence the results. Finally, the results of our study cannot necessarily apply to platelet reactivity assessed by other methods or using other platelet stimuli. Further information might have, in fact, been derived from assessing platelet reactivity also in response to collagen/epinephrine with the PFA-100 method; however, data from previous studies [22,23] point out that the results obtained using these cartridges
C. Aurigemma et al. to stimulate platelets are significantly influenced by aspirin, thus precluding their use in our population.
Conclusions Our data show that the response of platelet reactivity to C/ADP after exercise in CAD patients is not homogeneous, as only a group shows a significant increase. The increased platelet reactivity after exercise seems to be specifically associated with an increase in platelet expression of GP IIb/IIIa. The mechanisms responsible for this heterogeneous response are worth to be investigated in future studies, due their possible pharmacological and clinical implications.
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