Exercise-Induced myocardial ischemia in patients with coronary artery disease: Lack of evidence for platelet activation or fibrin formation in peripheral venous blood

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CLINICAL STUDIES

Exercise-Induced Myocardial Ischemia in Patients With Coronary Artery Disease: Lack of Evidence for Platelet Activation or Fibrin Formation in Peripheral Venous Blood JOSEPH J. MARCELLA, MD, ALLEN B. NICHOLS, MD, FACC, LYNNE L. JOHNSON, MD, JOHN OWEN, MD, DENNIS S. REISON, MD, KAREN L. KAPLAN, MD, PAULJ.CANNON,MD,FACC New York, New York

The hypothesis that exercise-induced myocardial ischemia is associated with abnormal platelet activation and fibrin formation or dissolution was tested in patients with coronary artery disease undergoing upright bicycle stress testing. In vivo platelet activation was assessed by radioimmunoassay of platelet factor 4, beta-thrombo-globulin and thromboxane 8 2 , In vivo fibrin formation was assessed by radioimmunoassay of fibrinopeptide A, and fibrinolysis was assessed by radioimmunoassay ofthrombin-increasable fibrinopeptide 8 which reflects plasmin cleavage of fibrin I. Peripheral venous concentrations of these substances were measured in 10 normal subjects and 13 patients with coronary artery disease at rest and during symptom-limited peak exercise. Platelet factor 4, beta-thromboglobulin and thromboxane 8 2 concentrations were correlated with rest and exercise catecholamine concentrations to determine if exercise-induced elevation of norepinephrine and epinephrine enhances platelet activation. Left ventricular end-diastolicand endsystolic volumes, ejection fraction and segmental wall

Transient platelet activation could be related to exerciseinduced myocardial ischemia by the following mechanisms. First, flow-limiting platelet aggregates and fibrin strands could form at sites of atherosclerotic coronary artery lesions. If this occurs, thromboxane A z , released by aggregating From the Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York. This work was supported in part by U.S. Public Health Service Grant, HL-15486, HL-21006 and HL14148 from the National Insntutes of Health, Bethesda, Maryland. Dr. Nichols is a Senior Investigator of the New York Heart Association, and Dr. Kaplan was a Senior Investigator of the New York Heart Association dunng the course of this study. Manuscript received September 7, 1982; revised manuscript received December 7, 1982, accepted December 10, 1982. Address for reprints: Joseph J. Marcella, MD, Cardiovascular Laboratory, Presbyterian Hospital, 630 West 168 Street, New York, New York 10032. © 1983 by the Amencan College of Cardiology

motion were measured at rest and during peak exercise by first pass radionuclide angiography. All patients with coronary artery disease had documented exercise-induced myocardial ischemia manifested by angina pectoris, ischemic electrocardiographic changes, left ventricular segmental dyssynergy and a reduction in ejection fraction. Rest and peak exercise plasma concentrations were not significantly different for platelet factor 4, betathromboglobulin, thromboxane 8 2 , fibrinopeptide A and thrombin-increasable fibrinopeptide 8. Peripheral venous concentrations of norepinephrine and epinephrine increased significantly (p < 0.001) in both groups of patients. The elevated catecholamine levels did not lead to detectable platelet activation. This study demonstrates that enhanced platelet activation, thromboxane release and fibrin formation or dissolution are not detectable in peripheral venous blood of patients with coronary disease during exercise-induced myocardial ischemia.

platelets, could provoke coronary artery vasoconstnctIon (1-4). Second, platelet activation could occur in the microvasculature of ischemic or previously infarcted myocardial segments (5-8). Both platelet aggregation and thromboxane A z release can be potentiated by elevated catecholamine concentrations resulting from exercise (9-11). Platelet survival and platelet aggregation studies have been repeatedly performed in attempts to link platelet activation to clinical events such as angina pectoris and myocardial infarction (12-15). Many of these studies have suggested that platelets of patients with ischemic heart disease are abnormally reactive, but quantification of in vivo platelet activation has not been possible in these studies (16-18). Green et al. (19) first used radioimmunoassay of proteins released from platelets to study platelet activation in patients 0735-1097/83/0501185-9$0300

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with coronary artery disease at rest and during exercise. They reported that platelet factor 4 levels in peripheral venous blood were increased in patients with coronary artery disease during myocardial ischemia induced by exercise. However, other workers failed to confirm the finding that platelet protein levels were elevated during exercise-induced myocardial ischemia (20-23). In several of these conflicting studies, peripheral venous levels of only one platelet protein, either platelet factor 4 or beta-thromboglobulin, were measured (19-21,23). Determination of the levels of both platelet proteins in each blood sample has been suggested as a useful method for distinguishing platelet release that occurs in vivo from that which may occur in vitro during collection and processing of the blood samples (24,25). The objective of this investigation was to determine if in vivo platelet secretion, fibrinogen/fibrin proteolysis and thromboxane Az release are detectable in patients with coronary artery disease during exercise-induced myocardial ischemia, by measurement of platelet proteins, fibrinopeptide A, thrombin-increasable fibrinopeptide Band thromboxane Bz in peripheral venous blood. The study was designed to compare concentrations of these substances measured at rest, during exercise-induced myocardial ischemia and after recovery in patients with coronary artery disease and in healthy control subjects. The presence of myocardial ischemia was documented both by radionuclide angiography and exercise stress electrocardiography.

Methods Patient Selection Criteria for exclusion from the study were recent myocardial infarction « 3 months), diabetes mellitus, thrombophlebitis, valvular heart disease, congestive heart failure, atrial fibrillation, chronic lung, renal or liver disease, anemia or any bleeding disorder. Also excluded were patients who had undergone cardiac surgery and patients with an implanted pacemaker or other prosthetic device. Group 1 (control subjects) consisted of 10 healthy male volunteers (mean age 29 years, range 22 to 46). None of the control subjects had cardiac symptoms; all had normal cardiovascular examinations and none were taking medications. Group 2 (study patients) consisted of 13 men (mean age 55 years, range 36 to 69) with angina pectoris. Six of these patients had a prior myocardial infarction documented by electrocardiographic changes and a diagnostic increase in cardiac serum enzyme levels. Ten of the 13 patients underwent clinically indicated coronary arteriography and all had a documented stenosis of at least 70% of the luminal diameter of a major epicardial coronary artery. Of these 10, 1 patient had disease of the left anterior descending artery only, 4 patients had double vessel disease and 5 patients had triple vessel disease, including 1 with a left main lesion.

Three patients did not have cardiac catheterization. Two of these had a myocardial infarction occurring 5 and 60 months, respectively, before the exercise study. The other patient developed typical anginal pain, ischemic electrocardiographic changes and regional left ventricular wall motion abnormalities demonstrated by radionuclide angiography during stress testing. Five patients were receiving propranolol, one patient was receiving timolol and six patients were receiving long-acting nitrates. Beta-blockers were discontinued 14 hours (range 11 to 19) and nitrates were discontinued 13 hours (range I to 20) before exercise testing. Of the five patients receiving propranolol, four had a plasma level less than 2.5 ng/ml and one had a plasma level of 9.2 ng/ml-Ievels that have been shown to have a negligible effect on platelet aggregation in vitro (26). Three patients were receiving diuretic agents that were discontinued 24 hours before exercise testing. None of the patients with coronary artery disease had experienced an anginal episode during the 72 hours before exercise testing. None of the patients received anticoagulant agents, aspirin, dipyridamole, theophylline, hydralazine or nonsteroidal anti-inflammatory agents or any other drugs known to affect platelet function during the 2 weeks before exercise stress testing.

Exercise Protocol All subjects underwent graded upright exercise testing on a variable load bicycle ergometer. All patients were fasting and abstained from caffeinated beverages and smoking for 5 hours before testing. Radionuclide angiography was performed at rest and during peak exercise by the first pass technique using a computerized multicrystal scintillation camera (Baird Atomic System-77) as previously described (27). Left ventricular ejection fraction was calculated from background-corrected end-diastolicand end-systolic counts recorded for a representative cycle as previously described (27). Left ventricular end-diastolic volume was calculated by the area-length method of Dodge et al. (28). From the ejection fraction and end-diastolic volume measurements, the following data were derived: stroke volume = end-diastolic volume x ejection fraction, and cardiac output = stroke volume x heart rate. Ventricular volumes and cardiac output were normalized for body surface area. Regional wall motion of the anterior, apical and inferior segments of the left ventricle was evaluated from the regional ejection fraction image that is generated from stroke volume and end-diastolic analog images of the representative cardiac cycle. Electrocardiographic leads 1, 111 and V s were monitored continuously and recorded at I minute intervals during the exercise test and the 10 minute recovery period. Blood pressure was recorded with a cuff sphygmomanometer at rest, after each 3 minute stage of exercise and during the recovery

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period. Exercise was begun at 50 watts in group 1 and 25 watts in group 2. Work load was increased in 25 watt increments every 3 minutes. Pedaling was maintained at a constant rate of 50 rpm. For group 1 (control group), exercise resulted in leg fatigue after a heart rate of 85% of the predicted maximum was reached. For group 2 (men with angina pectoris), exercise was terminated when severe dyspnea, leg pain, fatigue or angina pectoris occurred. Work capacity, reported in kilojoules, was calculated by multiplying the exercise time (seconds) and watts for each stage and summing all stages. Patients were considered to have an ischemic response to exercise if two of the following three criteria were met: angina pectoris, ischemic electrocardiographic changes or development of left ventricular regional wall motion abnormalities. The electrocardiographic criterion for an ischemic response to exercise was junctional depression of at least 0.1 mY followed by a horizontal or downsloping ST segment of at least 80 ms duration (29).

Blood Sampling Protocol Samples of venous blood were drawn from a vein in the left antecubital fossa immediately after the rest radionuclide angiogram, immediately after completion of the exercise radionuclide angiogram and after a 10 minute recovery period while the patient remained in the upright position. A separate venipuncture with a 21 gauge scalp needle was used for each sample. Only direct venipunctures with smooth and rapid withdrawal of blood were considered acceptable; otherwise, the needle was removed and another venipuncture was performed with a new needle. For measurement of fibrinopeptide A and platelet protein concentrations, a multiple sample vacutainer Luer-adapter (Becton-Dickinson) was used to collect 9 ml blood directly into a chilled siliconized glass 10 ml vacutainer tube containing 1 ml anticoagulant of 1,000 U/ml Trasylol, 1,400 U/ml heparin, 0.10 M adenosine and 0.02 M theophylline in Hepes buffered saline solution (THAT). Blood was then collected into a 20 ml polypropylene syringe and 9 ml was transferred to a chilled siliconized 10 ml tube containing 0.1 mg indomethacin and 10 to 15 mg Na2EDTA (sodium ethylenediaminetetraacetic acid) for measurement of thromboxane B2. Additionally, 3 ml was transferred to a tube containing 5.4 mg EGTA and 3.6 mg glutathione for measurement of catecholamines; 3 ml was transferred to a heparinized tube for measurement of plasma renin activity and 4.5 ml blood was added to a chilled polystyrene tube containing 0.5 ml THAT for determination of thrombin-increasable fibrinopeptide B. The blood tubes were immediately placed on melting ice and the scalp needle was removed after obtaining samples. Blood samples were excluded if a hematoma developed at the venipuncture site during blood withdrawal, if blood flow stopped abruptly during withdrawal or if gross hemolysis was apparent in the plasma after centrifugation.

In patients receiving propranolol, 10 ml of blood was collected into a chilled heparinized 10 ml glass tube for determination of the plasma propranolol concentration. Radioimmunoassay of platelet factor 4, beta-thromboglobulin, fibrinopeptide A and thrombin-increasable fibrinopeptide B. Within 45 minutes of collection, each sample was centrifuged at 1,700 g for 15 minutes at 4°C. The supernatant plasma was pipetted into two polypropylene tubes with a siliconized pipette and centrifuged at 49,000 g for 10 minutes at 4°C. Then 0.5 ml of supernatant plasma from each tube was pipetted into a polypropylene tube and frozen at - 60°C. Platelet factor 4 and beta-thromboglobulin assays were performed according to previously described techniques (30,31). The remaining plasma was decanted into a polystyrene tube and frozen at - 60°C. After bentonite adsorption, fibrinopeptide A assay was performed as previously described (32). The supernatant plasma for thrombin-increasable fibrinopeptide B radioimmunoassay was precipitated with ethanol, dried, reconstituted and assayed as previously described (33). Radioimmunoassay of thromboxane B2 • The samples were initially centrifuged at 3,000 g for 15 minutes at 4°C; the plasma was then pipetted into a polycarbonate tube and centrifuged at 49,000 g for 15 minutes at 4°C, then pipetted into polypropylene tubes and frozen at - 20°C. The plasma was assayed directly for thromboxane B2 using a specific antibody donated by Dr. Lawrence Levine of Brandeis University. The cross-reactivity of various prostaglandins or prostaglandin metabolites was less than 0.1 % for prostaglandins E h E2, AI. F 2,,, 6-keto-prostaglandin F l a and 13,14 dihydro-15-keto prostaglandin E2. thromboxane B2, 100 to 125 Ci/mmol was obtained from New England Nuclear and the unlabeled thromboxane B2 from Upjohn Diagnostics. Each assay tube received 100 ILl of plasma or 100 ILl of standard (6 to 1,000 pg of unlabeled thromboxane 8 2 dissolved in Krebs-Ringer-phosphate bicarbonate buffer with I% bovine serum albumin), and approximately 6,000 countslmin of eH] thromboxane B2 dissolved in 100 ILl of Tris-buffered saline solution (10 mM, pH 7.4) with 0.1% gelatin, followed by 100 ILl of rabbit antithromboxane B2 antiserum diluted 1:20,000 in the Tris-buffer. The tubes were incubated in a shaking water bath at 37°C for 60 minutes and then placed on ice for 5 minutes. Antibody bound thromboxane B2 was separated from the unbound ligand by adding 100 ILl of normal rabbit plasma (diluted 1:200 with Tris buffer) followed by 50 ILl of sheep antirabbit gamma-G globulin. The tubes were incubated at 4°C for 18 hours and then centrifuged at 6,000 g for 30 minutes. The pellets were washed with 1 ml ice-cold Tris-buffer, centrifuged at 6,000 g for 15 minutes, dissolved in 200 ILl of O.lN NaOH and I ml of Tris buffer was added. The tubes were decanted into glass scintillation vials. After the addition of 15 ml of Hydrofluor, the vials were counted in a Packard Tri-Carb model 3330 liquid scintillationspectrometer.

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Standard curves were analyzed by the method of Rodbard and Hutt (34), a nonlinear transformation of the logit-log relation. The initial (zero point) binding was 56.9%; nonspecific binding was 1.2%. The amount of unlabeled thromboxane Bz required to displace zero point binding by 10% was 5 pg; the amount required to displace zero point binding by 50% was 40 pg. Recovery was determined by adding known amounts of unlabeled thromboxane Bz (25 to 800 pg) to plasma. Linear regression analysis of the recovery of standards revealed that y = 1.032x + 181 (r = 0.998, n = 3), where y is the amount of thromboxane Bz recovered and x is the amount added. The intraassay coefficient of variation was 4.7%; the interassay coefficient of variation was 7.9%. Assay of norepinephrine and epinephrine. The assay for norepinephrine and epinephrine was based on the modification of the procedure described by Passon and Peuler (35). The samples were centrifuged at 3,000 g for 15 minutes at 4°C. The plasma was then pipetted into 5 cc polypropylene tubes and frozen at - 20°C. Fifty JLl of plasma was assayed for norepinephrine and epinephrine; 100 pg of each catecholamine was added to 1 aliquot of each sample to serve as an internal standard. Enzymatic O-methylation was performed by adding the plasma sample and internal standard to 50 JLl of a reaction mix. The final concentration of reactants was as follows: 30 mM MgCIz'6H zO; 100 mM Tris; 0.05 mM benzylhydroxylamine; 10 mM EGTA; 5 JLCi S-adenosyl-L-methionine-WH] methyl) (9-11 Ci/mmol) and catechol-O-methyl transferase, 0.11 mg of a standardized enzyme preparation (22 mg/ml). The assay was then performed according to a previously described technique (36). Assay of plasma renin activity and propranolol. Plasma renin activity was measured by the method of Sealey and Laragh (37). A radioreceptor assay for propranolol was performed by the method of Bilezikian et al. (38). Statistical analysis. Logarithmic transformation was performed on those values not normally distributed: platelet factor 4, beta-thromboglobulin, fibrinopeptide A, thrombinincreasable fibrinopeptide Band thromboxane Bz. Rest hemodynamics and radionuclide volume data for the two groups were compared by the unpaired t test. Other comparisons between and among groups were performed by analysis of variance. Correlations were determined by linear regression analysis. Data are reported as mean values ±

standard deviation. A probability (p) value less than 0.05 was considered significant.

Results Exercise tests. None of the normal subjects (group 1) experienced angina pectoris or developed ischemic electrocardiographic changes or left ventricular regional wall motion abnormalities during exercise. All patients with coronary arterydisease(group 2) developed left ventricular regional wall motion abnormalities during exercise that were detected by radionuclide angiography. Eleven patients in group 2 experienced angina pectoris during stress testing, and nine patients developed ischemic electrocardiographic changes. Diagnostic ST segment depression could not be detected in three other patients who had left ventricular hypertrophy with strain pattern, left bundle branch block or nonsustained ventricular tachycardia with the onset of exercise-induced angina. Hemodynamic and ventriculographic changes during exercise (Table 1). Heart rate at rest and rate-pressure product (systolic blood pressure x heart rate) for the two patient groupsdid not differ significantly. The peak heart rate achieved during exercise was significantly (p < 0.001) greater for subjects in group 1. The rate-pressure product at peak exercise was significantly (p < 0.001) higher in subjects in group 1 and the mean work capacity was significantly (p < 0.001) lower for patients in group 2. Table 2 shows the changes in left ventricular volume and ejection fraction that occurred with exercise. Both groups of patients experienced a significant (p < 0.01) increase in end-diastolic volume index during exercise. Neither the mean values at rest nor the increase in end-diastolic volume index with exercise were significantly different for the two groups. End-systolic volume index decreased significantly (p < 0.01) for subjects in group 1 during exercise; in contrast, endsystolic volume index increased significantly (p < 0.01) for patients in group 2. End-systolic volume index was significantly (p < 0.01) higher at rest and during exercise in the patients in group 2. Rest ejection fraction values were higher (p < 0.01) for subjects in group 1 than for patients in group 2. During exercise, ejection fraction increased (p < 0.001) in the group 1 subjects, whereas ejection fraction in the

Table 1. Hemodynamics at Rest and Peak Exercise Heart Rate (beats/min) Group I Rest Exercise

80 ± 11 172 ± 8*

Work Capacity (kilojoules)

Rate-Pressure Product (mm Hg/min) Group 2

73 ± 18

117 ± 24*t

Group 2

Group I

Group 2

9.3 ± 2.2 x J03 18.0 ± 4.6 x 103 *t

88 ± 27

36 ± 14t

Group 1 3

9.4 ± 1.6 x 10 31.0 ± 4.3 x 103 *

*p < 0.001 compared with rest value; tp < 0.001 compared with group l. Values are expressed as mean values ± standard deviation. Group I = control subjects. Group 2

=

patients WIth coronary artery disease

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Table 2. Left Ventricular Volumes and Ejection Fraction at Rest and Peak Exercise EF (%)

ESVI (ml/rrr')

Rest Exercise

Group 1

Group 2

Group I

Group 2

Group 1

Group 2

64 ± 16

80 ± 25 97 ± 26*

27 ± 9 21 ± 6*

48 ± 28t 60 ± 27*t

59 ± 6 72 ± 5*

44 ± 13t 41 ± 13*t

72 ± 17*

* p < 0.05 compared with rest value, t p < 0.01 compared with group 1. EDVI = end-diastolic volume index, EF = ejection fraction, ESVI = end-systolic volume index: Group 1 disease. group 2 patients with coronary artery disease decreased (p < 0.05), The difference in the change of ejection fraction with exercise between groups I and 2 was highly significant (p < 0.001). Platelet protein and thromboxane B 2 concentrations. Platelet factor 4 and beta-thromboglobulin concentrations (Fig. I) did not change significantly during exercise or recovery in either group. The mean platelet factor 4 concentration at rest was higher (p < 0.02) in patients in group 2 compared with subjects in group I. There were no significant changes in the concentration of thromboxane B2 (Fig. 2) in peripheral venous blood during exercise or recovery in either group. Fibrinopeptide A and thrombin-increasable fibrinopeptide B (Fig. 3). There were no significant changes in fibrinopeptide A concentrations between rest and peak exercise in either group I or 2; however, values obtained during the recovery periods were elevated (p < 0.02) compared with values at rest. The mean fibrinopeptide A concentration at rest was higher (p < 0.01) in patients in group 2 compared with subjects in group I. In group 2, there were no significant changes in the concentration of thrombin-

Figure 1. Platelet protein concentration measured at rest, peak exercise and recovery (mean ± standard deviation). Open circles denote normal subjects (group I); closed circles denote patients with coronary artery disease (group 2). The mean platelet factor 4 concentration at rest was higher (p < 0.02) in patients in group 2 compared with subjects in group I. NS = no significant difference, comparing exercise with rest values and recovery with rest values within each group. • CORONARY ARTERY DISEASE GROUP

o NORMAL GROUP

= control subjects: Group 2 =

patients with coronary artery

increasable fibrinopeptide B during or after exercise-induced myocardial ischemia. Catecholamine concentrations and plasma renin activity (Table 3). Plasma norepinephrine concentrations at rest were not significantly different for groups I and 2. In both groups, plasma norepinephrine increased (p < 0.001) during exercise. The increase in plasma norepinephrine concentrations from rest to exercise was significantly (p < 0.003) smaller in the patients in group 2 with coronary artery disease. For both groups, concentrations of plasma norepinephrine measured during recovery were not significantly different from rest concentrations. Plasma epinephrine concentrations also increased significantly (p < 0.001) with exercise and returned to baseline (p = NS) during the recovery period. The normal subjects had a higher (p < 0.01) rest plasma epinephrine concentration than patients with coronary artery disease. The increase in plasma epinephrine concentration with exercise was significantly (p < 0.02) lower in patients with coronary artery disease. In both groups of patients, plasma renin activity (Table 3) increased (p < 0.001) during exercise. Neither the plasma renin activity values at rest nor the increase in plasma renin activity values with exercise was significantly different between the two groups of patients. Recovery plasma renin activity values remained elevated (p < 0.001) in both groups I and 2 compared with rest values. However, the difference in plasma renin activity levels between groups during the recovery period was not significant. In the normal group, Figure 2. Thromboxane B2 concentrations measured at rest, peak exercise and recovery (mean ± standard deviation). Symbols are identical to those in Figure I. NS = no significant difference comparing exercise with rest values and recovery with rest values within each group. • CORONARY ARTERY DISEASE GROUP

~=====::N:=IS.IS-THROMBOGLoeuLlN

~========NsI

I

...J

::l!

..,"z

NsI

PLATELET FACTOR 4

NS

4

NsI NS

o NORMAL GROUP 200

NS

NS

NS

NS

i"'-----=i

100 80

60 40 30

3

20

2

I

I

I

REST

EXERCISE

RECOVERY

10

I REST

I

I

EXERCISE

RECOVERY

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• CORONARY ARTERY DISEASE GROUP

o 40 30 20 -'

I 0

::;

08 06

~

~

04 03

I;

NORMAL GROUP

NS

D NS

~

INCREASABLE FIBRINOPEPTIDE B P
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NS NS

02 01

I REST

I EXERCISE

I RECOVERY

Figure 3. Fibrinopeptide A and thrombin-increasable fibrinopeptide B concentrations measuredat rest, peak exerciseand recovery (mean ± standard deviation). Symbols are identical to those in Figure I. P values refer to comparisons of recovery withrest values within each group. The mean fibrinopeptide A concentration at rest was higher (p < 0.01) in patients in group 2 compared with subjectsin group I. NS == no significant difference compared with rest value within each group. the increase in plasma renin activity during exercise correlated with the increase in norepinephrine concentrations (r == 0.7, P < 0.03); however, this relation was not observed in patients with coronary artery disease.

Discussion The present study was designed to determine whether exercise-induced myocardial ischemia in patients with coronary artery disease is associated with abnormal platelet activation or fibrin formation or dissolution detectable in peripheral venous blood by measurement of platelet factor 4, beta-thromboglobulin, thromboxane B 2 , fibrinopeptide A and thrombin-increasable fibrinopeptide B concentrations. Previous studies of exercise-induced myocardial ischemia using this approach have yielded conflicting results (1923,39-41). Widely discrepant results in other studies have been attributed to differences in patient selection, incomplete documentation of myocardial ischemia and differences in venipuncture technique and blood processing methods.

Indexes of Platelet Activation in Myocardial Ischemia In the present study, three indexes of platelet activation were used: platelet factor 4 and beta-thromboglobulin, which are specifically released platelet alpha-granule proteins (42), and thromboxane B2 , a stable metabolite of thromboxane A 2 , a compound formed from arachidonic acid during platelet activation (1,3). Each exercise test was performed with the patient seated upright on a bicycle ergometer with the left arm immobilized to facilitate direct venipuncture. Because previous studies have shown that repeated withdrawal of blood samples through indwelling venous catheters causes progressive increases in platelet protein and fibrinopeptide A concentrations

(22,43,44), separate venipunctures were used for the different time points. Platelet protein concentrations. In the present study, platelet protein concentrations were not significantly increased in either the patients with coronary artery disease during exercise-induced myocardial ischemia or in the normal subjects during maximal exercise. These results are similar to those reported recently by Mathis et al. (21), Tamay et al. (23), Stratton et al. (22) and Hughes et al. (20). Other groups, including Green et al. (19), Mehta and Mehta (40), Lindenfeld et al. (41) and Hyers et al. (39), have reported increases in platelet factor 4 or beta-thromboglobulin, or both, with exercise. The reasons for the differences between the two groups of studies may be related to differences in blood collection technique; for example, some of those who found increases with exercise had collected samples through indwelling venous catheters. Thromboxane B2 • Concentrations of thromboxane B2 , which provide a separate index of platelet activation, were also not increased with exercise in this study. In other studies, elevations in peripheral venous thromboxane B2 concentrations with exercise (45) or at rest (46) have been reported. In one of these (45), no inhibitor of cyclooxygenase (the enzyme that converts arachidonic acid to a cyclic endoperoxide which can then be converted to thromboxane A2 ) was included in the anticoagulant mixture. In the other study (46), samples may have been collected after the insertion of woven Dacron catheters, which have been reported to increase peripheral venous concentrations of thromboxane B 2 (44,47), Fibrinopeptide A and thrombin-increasable fibrinopeptide B. Proteolysis of fibrinogen during exercise was assessed by measurement of fibrinopeptide A, which is formed along with fibrin I when thrombin acts on fibrinogen (33), and of thrombin-increasing fibrinopeptide B, which is the product of plasmin action on fibrin 1 (48). In the present study, there was no significant increase in fibrinopeptide A during exercise in either the control group or the patient group. Thrombin-increasable fibrinopeptide B was measured only in patients with coronary artery disease and showed no significant change during exercise or recovery. This finding is interesting in view of extensive reports (49-52) on increased fibrinolytic activity during exercise, as determined by shortening of the euglobulin lysis time. Without measurements of thrombin-increasable fibrinopeptide B in the control group, it is impossible to know if the failure of thrombin-increasable fibrinopeptide B to increase in the patients is related to coronary artery disease (53), or if it indicates that thrombinincreasable fibrinopeptide B levels reflect a different phenomenon from that detected by the euglobulin lysis time. Slight, but significant increases were seen in fibrinopeptide A concentrations in both groups during the recovery period. These elevations may have been due to the indwelling Teflon

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Table 3. Mean Peripheral Venous Concentrations of Norepinephrine, Epinephrine and Plasma Renin Activity NE (pg/ml)

Rest Exercise Recovery

* p < O.llO]

EPI (pg/ml)

PRA (ng/ml per hour)

Group I

Group 2

Group I

Group 2

Group I

Group 2

504 :!: 155 1,844 :!: 537* 639 :!: 146

514 :!: 162 1,204 :!: 711 *t 524 :!: 215

112 :!: 46 214 :!: 77* 96 :!: 34

64 :!: 31t 115 :!: 72*t 61 :!: 33

1.3 :!: 0.7 2.8 :!: 1.8* 2.9 :!: 1.5*

1.4 :!: 1.2 2.8 :!: 2.7* 2.1 :!: 1.9*

compared with rest value; tp

<

0.02 compared with group I

EPI = epinephrine; Group I = control subjects; Group 2 = patients with coronary artery disease: NE = norepmephnne: PRA = plasma rerun acnvrty

intravenous catheter required for isotope injections for radionuclide angiography (44). The elevated mean peripheral venous concentrations of platelet factor 4 and fibrinopeptide A at rest in patients in group 2 may be due to the presence of a prior myocardial infarction in many of these patients (8). Catecholamine concentrations. Catecholamines, in supraphysiologic concentrations, promote platelet aggregation and secretion in vitro (9-11). Lower concentrations of catecholamines can potentiate the platelet response to agonists, and thus may have a role in modulating platelet responsiveness in vivo (9,11,54). In the present study, norepinephrine and epinephrine concentrations increased significantly with exercise in both groups; however, the increases in catecholamine concentrations were smaller in patients with coronary artery disease, compared with those in normal subjects, reflecting the reduced work load attained. Despite the significant increases in epinephrine and norepinephrine concentrations with exercise in both patient groups, no significant increases were found in the platelet protein concentrations during exercise. This is not surprising because it has been reported that 50 to 100 nM epinephrine is required for synergism with ADP-induced platelet activation in vitro (55), and the maximal epinephrine and norepinephrine concentrations reached with exercise were at least two orders of magnitude lower. Plasma renin activity. Plasma renin activity increased significantly in the normal subjects and patients with coronary artery disease, and remained elevated during the recovery period. Known stimuli for renin secretion during exercise include enhanced sympathetic neuron activity (56,57) and decreased renal blood flow (58), which may also be mediated by the sympathetic nervous system (57). Plasma renin activity in normal subjects may remain elevated up to 30 minutes after heavy exercise (59). In the present study, a significant correlation between the norepinephrine and plasma renin activity responses to exercise was observed in group 1 subjects, suggesting that the enhancement of sympathetic neuron activity by exercise led to increased renin section.

Implications This study shows that peripheral venous blood concentrations of platelet factor 4, beta-thromboglobulin, throm-

boxane B2 , fibrinopeptide A and thrombin-increasable fibrinopeptide B are not increased during exercise-induced myocardial ischemia in patients with coronary artery disease or in normal subjects during exercise. Thus, enhanced platelet activation, fibrin formation and dissolution are not detectable in peripheral venous blood during exercise-induced myocardial ischemia in patients with coronary artery disease. These results indicate either that abnormal platelet activation and fibrin formation do not occur in the coronary arteries or myocardial microvasculature during exercise-induced myocardial ischemia, or that the changes are quantitatively insufficient to cause significant increases in the peripheral venous concentrations of the substances reflecting these processes. Studies of arterial and coronary sinus levels of platelet proteins, thromboxane B2 , fibrinopeptide A and thrombin-increasable fibrinopeptide B will be required to resolve this issue. Currently available cardiac catheters cause artifactual increases of platelet protein and fibrinopeptide A concentrations, making such a study impractical. However, studies of heparin-bonded catheters suggest that such catheters may be suitable (47). We gratefully acknowledge the expert technical assistance of Robert Sciacca, Lloyd Fleisher, Betty Grossman, Thomas LIm, Carolme Rose and Leonard Norbert.

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