Inhibition by combined therapy with ticlopidine and aspirin of enhanced platelet aggregation during physical exercise in patients with coronary artery disease☆☆☆

Study subjects 

The trial was a single-center, open-label, randomized study. Of 60 screened Japanese patients with stable CAD, followed up in Mie University Hospital from February 1999 to January 2000, 48 patients (80%) were randomized according to the method of minimization for assigning patients to treatment and control groups. All patients had effort angina (Canadian Cardiovascular Society I or II) or a prior myocardial infarction (MI) and who had CAD determined by coronary angiography (>50% narrowing of luminal diameter). This study did not include patients with unstable angina, a recent MI, aortic aneurysm, malignant tumor, liver or renal failure, or steroid or warfarin users. Forty-eight patients (42 men and 6 women) between the age of 44 and 79 years were randomly distributed into 3 groups of 16 each. The respective groups were put on 3 different daily drug regimens: no antiplatelet drug (patient control group), 81 mg ASA only (ASA alone group) and 200 mg TIC plus 81 mg ASA (TIC + ASA group). No patient had taken other agents known to alter platelet function during the 2 weeks before the study. Antiplatelet therapy (ASA alone or TIC + ASA) was prescribed for 4 weeks before the ergometer exercise. There were no significant differences between the 3 groups regarding age, male/female ratio, stenotic vessel counts, left ventricular ejection fraction, coronary risk factors, or medication (β-blocker, calcium antagonist, and angiotensin-converting enzyme [ACE] inhibitor), as shown in Table I. All patients had been on antianginal drugs. The study protocol was approved by the institutional review committee at the Mie University Hospital. After the protocol was approved, all patients participating in this study gave informed written consent in accordance with the Declaration of Helsinki.

Table I. Clinical characteristics of study patients

		Patient control (n = 16)	ASA alone (n = 16)	TIC + ASA (n = 16)
	Mean age (y)	63.2 ± 9.2	62.2 ± 9.2	64.5 ± 8.2
	Male/female ratio	14:2	14:2	14:2
	No. of stenotic vessels*
	1	7	7	6
	2	2	6	7
	3	2	3	3
	Left ventricular ejection fraction (%)	62 ± 12	58 ± 13	63 ± 10
	Coronary risk factors
	Tobacco smokers	7	6	7
	Hypertension	7	8	8
	Diabetes	4	4	4
	Hypercholesterolemia†	7	7	7
	Obesity (body mass index >26.0)	2	3	2
	Medication
	β-Blockers	4	5	6
	Calcium antagonists	4	4	3
	ACE inhibitors	6	7	7
	*Coronary arteries narrowed >50%. †Total serum cholesterol >220 mg/dL, drug therapy for hypercholesterolemia, or both.

All P values are nonsignificant (Fisher exact test , χ2 test, or Kruskall-Wallis test).

Ergometer exercise test 

All ergometer exercise tests were performed between 11 AM and noon to avoid the enhancement of platelet aggregation in the morning between 6 and 9 am.19 Before start of the exercise, an indwelling 22-gauge intravenous line was inserted into a median antecubital vein of arm. Blood samples were drawn from this line before ergometer exercise (PRE) and immediately after exercise (POST). The exercise was carried out with the patient in a seated position on a cycle ergometer in which the work load could be regulated. The exercise work load increased steadily by 25 W every 3 minutes, beginning with 50 W up to 125 W of the last 3 minutes. During exercise, heart rate, blood pressure, and a 12-lead electrocardiogram (ECG) were continuously monitored. The exercise was terminated under the following conditions: ischemic symptoms such as chest pain, the target heart rate that was 85% of age-predicted maximal heart rate, impossible to continue exercise because of dyspnea or leg fatigue, or 2-mm ST depression at least 2 leads in the ECG.

Blood sampling 

At each time point, 21 mL of blood was drawn into a plastic syringe, without stasis. Nine milliliters of the sample was immediately mixed with 1 mL of 3.8% sodium citrate solution to study platelet aggregation; 5.4 mL of the sample was anticoagulated by adding 0.6 mL of 3.8% of trisodium citrate solution to assay thrombin-antithrombin complex (TAT) and plasmin-plasmin inhibitor complex (PIC); 5 mL of the sample was mixed with 5 mg of sodium ethylenediamine tetra-acetic acid to measure levels of catecholamines.

Measurement of platelet aggregation 

Platelet-rich plasma (PRP) was prepared by centrifugation at 120g for 15 minutes at room temperature, and the platelet count was adjusted to 3.0 × 105/μL to standardize the aggregation study, by adding homologous platelet-poor plasma obtained by centrifugation of the blood at 1500g for 5 minutes. Measurement of platelet aggregation was completed within 2 hours of blood sampling. Investigators measuring platelet aggregation were blinded to the treatment arm of each sample. Continuous monitoring of SIPA was done by use of a turbidimetric technique with a modified cone-plate viscometer, as described in detail elsewhere.12 To measure SIPA, 400 μL of PRP was applied to the chamber, and the cone was rotated with a computer-regulated rotor motor that generated constant shear stress. PRP was exposed to 6 dyne/cm2 shear stress for the first 20 seconds and then 108 dyne/cm2 shear stress for 5 minutes, and aggregation was monitored continuously by recording the intensity of the light transmitted through the PRP from the start of application of shear force. Agonist-induced platelet aggregation was monitored photometrically at 37°C with an AG10 aggregometer (Kowa, Tokyo, Japan), as described.20 A total of 2 μm of ADP (Sigma Chemical, St Louis, Mo) and 1.2 μg/mL collagen (Collagenreagent Horm, Nycomed Arzneimittel GmbH, München, Germany) were the agonists used. In either type of aggregation, responses were quantified as the maximum extent of aggregation.12, 16

Determination of plasma vWF 

Levels of plasma vWF were determined on the basis of the ristocetin cofactor activity of vWF (vWF/RCO) and by vWF antigen. vWF/RCO activity was measured with an aggregometer and a vWF reagent (Dade Boehring Marburg GmbH, Marburg, Germany).16 vWF antigen was assayed in enzyme-linked immunosorbent assays with use of a polyclonal anti-human vWF antibody (Dakopatts, Copenhagen, Denmark) as the primary antibody and a polyclonal peroxidase-conjugated immunoglobulin to human vWF as the secondary antibody. In both cases, the amount of plasma vWF (expressed as the percentage of the normal plasma activity level) was read as a calibration curve, constructed from serial dilutions of normal pooled plasma (100%) with isotonic saline solution to final vWF activities of 100%, 50%, 25%, 12.5%, and 6.3%.

Other hemostatic parameters 

Plasma levels of TAT and PIC were determined with use of the Enzygnost TAT (Behringwerke AG, Marburg, Germany) and PIC test (Teijin, Tokyo, Japan), respectively.

Assay of plasma catecholamines 

Plasma concentrations of epinephrine and norepinephrine were measured with an auto catecholamines analyzer (HLC-8030, Tosoh, Tokyo, Japan).

Statistics 

This study was designed to detect a 15% difference of SIPA between ASA alone and TIC + ASA groups on the basis of our previous report.16 To achieve a power of 80% with a 5% significance (2-sided), 16 patients would be required for each group.

Data were analyzed with use of the StatView statistical software package (version 5, SAS Institute, Cary, NC). Differences in the distributions of clinical characteristics among 3 groups were examined by Fisher exact test and χ2 test. Two-tailed nonparametric tests were used to analyze differences among the groups (Kruskal-Wallis test) and changes (data of postexercise and preexercise) within the group (Wilcoxon signed-ranks test). If statistical differences by Kruskal-Wallis test were identified among groups, positive pairwise comparisons (Dunn multiple comparison test) were calculated. Correlations were studied by linear regression analysis. All data are expressed as mean ± SD. Statistical significance was defined as a P value of less than .05.

Effect of exercise on cardiovascular variables, levels of catecholamines, and blood platelet counts 

The 3 groups were homogenous with respect to major baseline clinical and angiographic characteristics (Table I). As shown in Table II, the exercise tolerance time (in minutes) was 9.0 ± 2.3 in the patient control group, 7.9 ± 2.1 in ASA alone group, and 8.4 ± 2.4 in TIC + ASA group, respectively; there were no statistically significant differences among the 3 groups.

Table II. Exercise tolerance time and effect of exercise on double products, platelet counts, and plasma catecholamines

		Patient control (n = 16)	ASA alone (n = 16)	TIC + ASA (n = 16)	Statistical significance*
	Exercise tolerance time (min)	9.0 ± 2.3	7.9 ± 2.1	8.2 ± 2.4	NS
	Maximal work load (W)	108 ± 20	100 ± 19	102 ± 19	NS
	Double products (mm Hg)
	Preexercise	10,000 ± 2300]†	11,400 ± 2600]†	11,000 ± 3400]†	NS
	Postexercise	23,300 ± 4400	24,900 ± 5200	24,200 ± 7700	NS
	Platelet counts (×104/μL)
	Preexercise	39.1 ± 10.3]‡	37.7 ± 9.9 ]‡	35.4 ± 9.6 ]‡	NS
	Postexercise	43.6 ± 11.4	41.9 ± 10.7	39.8 ± 11.7	NS
	Epinephrine concentration (pg/mL)
	Preexercise	40 ± 20]†	49 ± 44]†	53 ± 22]†	NS
	Postexercise	115 ± 75	112 ± 72	142 ± 85	NS
	Norepinephrine concentration (pg/mL)
	Preexercise	561 ± 165]†	543 ± 272]†	632 ± 137]†	NS
	Postexericse	1352 ± 593	1324 ± 810	1382 ± 575	NS
	*By Kruskall-Wallis test. †P < .005. ‡P < .05.

NS, Not significant.

Effect of exercise on agonist-induced platelet aggregation 

As shown in Figure 1, ADP-induced platelet aggregation was significantly enhanced during exercise, in both the patient control group (35.4% ± 17.8 % to 40.9% ± 19.1%, n = 16, P < .05) and the ASA alone group (31.0% ± 9.3% to 33.7% ± 9.6%, n = 16, P < .05).

View Large Image Download to PowerPoint

Fig. 1. Effects of exercise test on ADP- and collagen-induced platelet aggregation in patients with stable CAD. Blood was obtained before (PRE) and immediately after (POST) ergometer exercise test in patient control (no medication) group, ASA-alone group and ASA + TIC group. Box and whisker plots show mean value (open circles), median (horizontal lines), 25th and 75th percentiles (boxes), and 90th percentiles (error bars). Asterisk, P < .05; two asterisks, P < .001.

Effect of exercise on SIPA 

Figure 2 shows the typical enhanced response of SIPA to exercise in a control group patient with CAD.

View Large Image Download to PowerPoint

Fig. 2. Typical curve of SIPA before (PRE) and after (POST) exercise test in a control patient with stable CAD. PRP was placed in a cone-plate viscometer and exposed to shear force, as described in Methods. The shear force applied to platelets is indicated by a broken line.

View Large Image Download to PowerPoint

Fig. 3. Effects of exercise test on SIPA in patients with stable CAD. Blood samples were taken before (PRE) and immediately after (POST) exercise from the patient control group, ASA-alone group and ASA + TIC group. Box and whisker plots are equivalent to those shown in Figure 1. Asterisk, P < .05; two asterisks, P < .001.

Effect of exercise on vWF/RCO activity and vWF antigen 

As shown in Figure 4, exercise led to a significant increase in vWF/RCO activity in all 3 groups (114% ± 36% to 137% ± 46%, P < .005 in the patient control group; 126% ± 35% to 140% ± 34%, P < .01 in the ASA-alone group; 120% ± 41% to 147% ± 43%, P < .005 in the TIC + ASA group) and also an increase in vWF antigen in these groups (126% ± 37% to 141% ± 36%, P < .005, in the patient control group; 130% ± 40% to 143% ± 40%, P < .01 in the ASA-alone group; 120% ± 37% to 138% ± 36%, P < .005, in the TIC + ASA group).

View Large Image Download to PowerPoint

Fig. 4. Effects of exercise test on VWF/RCO activity (upper panel) and vWF antigen (lower panel) in patients with CAD. Blood samples were taken before (PRE) and immediately after (POST) exercise in the patient control group, ASA-alone group, and ASA + TIC group. Box and whisker plots are equivalent to those shown in Figure 1. Asterisk, P < .05.

View Large Image Download to PowerPoint

Fig. 5. Correlation between the extent of SIPA and plasma vWF/RCO activity (upper panel) and vWF antigen (Ag) level (lower panel) before (closed circles) and after exercise (open circles) in the patient control group with CAD.

Effect of exercise on TAT and PIC levels 

As shown in Figure 6, TAT was significantly increased during exercise in all 3 groups (5.7 ± 6.9 ng/mL to 34.9 ± 35.9 ng/mL in the patient control group, 9.6 ± 7.6 ng/mL to 42.3 ± 37.7 ng/mL in the ASA-alone group, 7.8 ± 8.9 ng/mL to 29.4 ± 33.3 ng/mL in the TIC + ASA group, although increases in TAT during exercise test varied with each patient).

View Large Image Download to PowerPoint

Fig. 6. Effects of exercise test on levels of TAT (upper panel) and PIC (lower panel) in patients with stable CAD. Blood samples were taken before (PRE) and immediately after (POST) exercise in the patient control group, ASA-alone group, and ASA + TIC group. Asterisk, P < .05.

Study limitations 

This was a prospective randomized investigation but was not a double-blind study and all patients were enrolled in a single center. Nonetheless, no significant clinical and angiographic differences in demographics were apparent among the 3 groups. The study was small and hypothesis generating; therefore a large, double-blind, multicenter trial is required to confirm our results. Although the relationship between turbidimetric platelet aggregation and pathophysiologic events is not well established, turbidimetric aggregation remains the standard by which the pharmacologic effects of antiplatelet drugs are measured. Other measurements of platelet function (eg, fibrinogen binding, expressions of P-selectin or ligand-induced binding site, procoagulant activity) may refine the pharmacologic effects of the combination therapy with ASA and TIC. Clopidogrel, a newer thienopyridine derivative, is now being prescribed in Europe and the United States in lieu of ticlopidine because of its better tolerability and much fewer life-threatening side effects (eg, neutropenia and thrombotic thrombocytopenic purpura).32, 33 Clopidogrel is not licensed for use in Japan; hence we could not evaluate dual therapy with ASA.

Implications 

Pharmacologic intervention in platelet aggregation to prevent local thrombus formation is a prominent aspect of procedures used for patients with coronary atherosclerosis.15 The observations we report indicate that combined antiplatelet therapy with ASA and TIC effectively suppresses increases not only in vWF-dependent SIPA but also in agonist-induced aggregation during exercise in patients with CAD. Thus, in relatively high-risk CAD patients with diffuse atherosclerosis, dual therapy with low-dose ASA and a thienopyridine derivative may more effectively prevent thrombus formation and acute coronary events compared with the standard ASA monotherapy. Whether dual therapy reduces the rate of acute coronary events more effectively than ASA alone has to be tested in large clinical trials.