Studies of Platelet Factor 4 and Beta
Thromboglobulin Release During Exercise:
Lack of Relationship to Myocardial Ischemia
JOIN R. STRATTON, M.D., THOMAS W. MAtPASS, M.D., JAMES L. RITCHIE, M.D.,
MICHAEL A. PFEIFER, M.D., AND LAURENCE A. HARKER, M.D.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
SUMMARY Platelet activation, which results in release of the platelet-specific proteins platelet factor 4
(PF4) and BTG thromboglobulin (BTG), may participate in exercise-induced myocardial ischemia by the
formation of intravascular platelet aggregates or the generation of vasoactive substances such as thromboxane A2. We sought to determine if platelet release occurs during exercise-induced ntyocardial ischemia and
its relationship to exercise-induced catecholamine release in 10 normal males (mean age 29 + 6 years) and
25 males with proved coronary artery disease (mean age 60 ± 8 years) who performed maximal, symptomlimited treadmill exercise tests. None of the subjects took drugs known to modify platelet behavior; 17
coronary artery disease patients took B-blocking agents. Plasma and urine PF4, plasma BTG and plasma
catecholamines were measured before and immediately after exercise. Plasma PF4 and BTG were also
measured 30 minutes after exercise. Ischemia, defined as angina or 1 mm or more of horizontal or
downsloping ST depression, developed in 14 coronary artery disease patients.
In young normal subjects during exercise, plasma PF4 increased from 2.1 ± 1.2 at rest to 4.7 ± 2.6 ng/ml
at maximal exercise (p < 0.01 rest vs exercise) and plasma BTG increased from 11.7 ± 5.4 to 16.7 ± 7.7 ng/
ml (p < 0.01 rest vs exercise). Among the 25 coronary artery disease patients, plasma PF4 during exercise
increased slightly but significantly, from 2.1 1.4 at rest to 2.6 ± 2.2 ng/ml at exercise (p < 0.01); plasma
BTG did not change significantly, from 13.3 6.0 at rest to 14.1 ± 5.8 ng/ml during exercise. The small
exercise-induced elevations in plasma PF4 and BTG in all 25 coronary artery disease patients were significantly less than those in the 10 young normal subjects (both p < 0.01 vs normals). Among the subset of 14
coronary artery disease patients with exercise-induced ischemia, neither plasma PF4 nor BTG increased
significantly (PF4 from 2.6 1.7 at rest to 3.0 ± 2.9 ng/ml with exercise; plasma BTG from 13.6 ± 6.4 to
14.1 ± 7.1 ng/ml). The 14 coronary artery disease patients with ischemia had less exercise-induced
elevation in both plasma PF4 and BTG than normal subjects (both p < 0.05). Plasma PF4 and BTG levels 30
minutes after exercise were not significantly different from rest levels in any subject group. Mean rest, peak
exercise, and 30-minute postexercise plasma PF4 and BTG values did not differ significantly between the 17
coronary disease patients taking B-blocking agents and the eight coronary disease patients not taking
blocking drugs. Furthermore, urinary PF4 did not change significantly with exercise in either norrmal
subjects or in any of the coronary artery disease groups. Among all 35 subjects, exercise-induced increases
in plasma epinephrine correlated with increases in plasma PF4 (r = 0.35; p = 0.04) and BTG (r = 0.40; p
= 0.02). Increases in norepinephrine did not correlate with either increases in plasma PF4 (r = 0.24, p =
0.2) or BTG (r 0.23, p = 0.2).
We conclude that exercise-induced release of plasma PF4 and BTG, as measured by sampling peripheral
venous blood, is not a marker of myocardial ischemia. The small elevations in plasma PF4 and BTG induced
in normal subjects and in coronary artery disease patients by exercise are nonspecific for myocardial
ischemia and may be related to the magnitude of epinephrine release.
B-
PLATELET ACTIVATION, with the subsequent formation of platelet aggregates or the generation of
thromboxane A2, has been causally implicated in both
resting and exercise myocardial ischemia. 1-7 Both
platelet factor 4 (PF4) and B thromboglobulin (BTG)
are released from platelet a granules during platelet
activation. The development of radioimmunoassays
for these platelet-specific proteins permits their use as
indicators of platelet activation in man.i'7 The pres-
ence of these proteins in plasma has been proposed as a
measure of in vivo platelet release in a variety of disease states."'7 Studies of platelet a-granule release
during exercise have been inconclusive. In coronary
artery disease patients and in normal persons, no
or an increase in plasma PF4'9 20 or BTG21 22
during exercise have been reported. The divergent results in these and other studies of PF4 and BTG release
have been attributed to methodologic differences.'6'8
Specimen collection and processing are of primary
importance in ensuring that in vitro release of PF4 and
BTG is minimized. 16-18 23 In the current study, we used
a method for specimen handling and processing that
has been documented to minimize in vitro platelet a
granule release of PF4 and BTG. 7 Kaplan'6 suggested
that the simultaneous measurement of both plasma PF4
and BTG may be particularly useful in discriminating
between in vitro and in vivo release; release in vitro
would be expected to equally elevate plasma PF4 and
BTG, while release in vivo might disproportionately
elevate BTG because of its slower plasma clearance.
changet
From the Departments of Medicine, Veterans Administration Medical Center, Haborview Medical Center and the University of Washington, Seattle, Washington.
Supported in part by the Veterans Administration Medical Research
Service and by USPHS grants HL-18805-02, HL-18645, HL-l 1775,
HL-07093, and AM00738.
Dr. Pfeifer's current address: VA Medical Center, 800 Zorn Avenue,
Louisville, Kentucky 40202.
Address for correspondence: John R. Stratton, M.D., Division of
Cardiology (663/1 1 1), Veterans Administration Medical Center, 4435
Beacon Avenue South, Seattle, Washington 98108.
Received September 4, 1981; revision acccpted November 20, 198 1.
Circulation 66, No. 1, 1982.
33
34
CIRCULATION
The purpose of this study was to determine whether
PF4 or BTG release occurs during maximal upright
treadmill exercise in normal subjects or in patients with
proved coronary artery disease, and to relate any
changes to evidence of exercise-induced myocardial
ischemia or exercise-induced catecholamine release.
We also assessed the time course of PF4 release in
normal subjects during maximal supine bicycle exercise, since repeat venipunctures cannot be readily performed during upright treadmill testing. To explain the
exercise-related elevations of platelet-specific proteins
found in other studies, we also assessed whether repeated venipunctures from the same site or serial sampling from an indwelling venous catheter artifactually
elevated plasma PF4 levels.
Methods
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Subjects
Ten normal male volunteers (mean age 29 + 6
years, range 24-44 years) with no history or physical
findings of cardiovascular disease were studied. Nine
had normal resting ECGs; one had preexcitation. None
had taken any medications, including platelet-active
drugs (aspirin-containing compounds, dipyridamole,
sulfinpyrazone or other nonsteroidal antiinflammatory
drugs), within 1 week of the study. Young normal
volunteers were studied because they could be assumed to be free of coronary artery disease. No control
patients in the older age range were studied.
Twenty-five males (mean age 60 ± 8 years, range
46-74 years) with clinically stable coronary artery disease were selected for study from a cardiovascular
disease clinic. Sixteen had symptomatic angina pectoris and coronary artery disease (D 70% diameter reduction of at least one vessel) proved at catheterization; the other nine patients had sustained a remote
myocardial infarction. Overall, 19 of the 25 patients
had prior infarction, 14 transmural and five nontransmural. No patient had sustained a myocardial infarction within 3 months of the time of study. Myocardial
infarction was documented by new electrocardiographic Q waves, ST-T changes or both, as well as an
appropriate clinical history and elevation of creatine
kinase to more than twice the normal level. Nineteen
patients had a history of angina pectoris within 1
month before study. Five (patients 11, 12, 21, 33, 34
in table 1) had undergone coronary artery bypass grafting, four of whom had recurrent angina. No patient
had received platelet-active drugs for at least 1 week
before study. Sixteen patients were taking propranolol
(mean dose 221 ± 209 mg/day, range 40-800 mg/day)
and one patient was taking metoprolol (100 mg/day).
Patients 22 and 24 (table 1) were taking digoxin (0.25
mg/day). Ten patients were taking long-acting nitrates. All subjects were informed of the investigational nature of the study and gave informed consent.
Treadmill Exercise Testing
Symptom-limited maximal treadmill exercise testing was performed according to the standard Bruce
protocol24 using a 12-lead ECG after an overnight fast.
VOL 66, No 1, JULY 1982
Hard copies of leads II, aVF, and V5 were obtained
every minute and a full 12-lead ECG was obtained
during each stage and immediately after maximal exercise. Exercise testing was terminated because of
marked fatigue, severe chest pain, ventricular bigeminy, coupled premature ventricular complexes or exertional hypotension (> 10 mm Hg drop in systolic
blood pressure from baseline). Exercise testing was
not terminated at achievement of a target heart rate or
because of ST depression. The exercise test was considered positive for ischemia if the patient developed
typical angina pectoris (seven patients), more than 1
mm of horizontal or downsloping ST depression lasting at least 0.08 second after the J point in patients not
taking digoxin (one patient), or both angina and ST
depression (five patients). In addition, patient 24 (table
1), who was taking digoxin, developed 2 mm of horizontal ST depression and ventricular bigeminy, but not
angina, during exercise and was considered to have
had an ischemic response. Thus, 14 patients were classified as having exercise-induced ischemia.
Supine Bicycle Exercise Testing
For supine bicycle exercise testing, our previously
described protocol was performed in four additional
young normal subjects .25 The subject pedaled a bicycle
ergometer in the supine position for 4-minute stages,
beginning at 200 kilopond-meters (kpm)/min and increasing by 200 kpm/min until reaching a symptomlimited maximum.
Specimen Collection and Assay
For upright treadmill exercise tests, blood specimens for plasma PF4, BTG and catecholamine determinations were collected in all 35 subjects immediately before exercise with the subject sitting and within 1
minute of stopping exercise with the subject either
sitting or supine. Thirty minutes after exercise, specimens were also collected for plasma PF4 and BTG in
29 subjects, six normal and 23 with coronary disease.
Pre- and postexercise urine specimens for urine PF4
were collected in 33 subjects (10 normal and 23 with
coronary disease).
During the four supine bicycle exercise tests, all
subjects had an indwelling i.v. catheter in the right arm
for plasma catecholamine sampling. Saline without
heparin was used to keep this catheter open. Samples
for plasma PF4 and BTG measurement were drawn
from separate venipunctures in the opposite arm at
supine rest, during the tenth minute of exercise, immediately after exercise and at 30 minutes after exercise.
Our methods of specimen collection and processing
for plasma and urine PF4 and plasma ITG have been
previously described."7 At each sampling time, duplicate blood samples were collected by a single venipuncture with a 21-gauge butterfly needle into 5-ml
syringes that were precooled in ice and contained 1 ml
of acid-citrate-dextrose (ACD, NIH Formula A), 30
mM acetylsalicylic acid and 1 I! prostaglandin E,
(PGE,) final concentration. The samples were mixed
and placed immediately on ice for no longer than 30
35
PF4 AND BTG DURING EXERCISE/Stratton et al.
minutes before centrifugation at 48,000 g for 20 minutes at 4°C. One milliliter of plasma was immediately
collected from below the lipid interface and stored
frozen at - 20°C until assayed. Urine samples for PF4
were collected in a 10-ml plastic tube and kept on ice
until stored at - 20°C. A 1-ml aliquot of urine was
used for creatinine determination. The PF4 radioimmunoassay was performed in duplicate as previously
described. 17 Plasma BTG assays were performed using
a commercially available radioimmunoassay (Amer-
TABLE 1. Clinical, Exercise, Plasma Platelet Factor 4 and ,B Thromboglobulin Data from 35 Subjects
Treadmill Testing
Exercise tolerance test
Clinical data
Dura-tion
Ventriccular
depres- arrhythsion
mia*
ST
Plasma PF4 (ng/ml)
Exer30
Rest
cise
min
Plasma
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(secAge
p
Pt
(years) blocker onds)
Angina
Rest
Normal subjects (n = 10)
1
331
1085
1.3
3.1
ND
18
2
24
905
1.7
8.6
ND
6
3
44
815
1.4
4.4
ND
6
4
32
1020
2.3
6.6
ND
20
5
26
965
7
-1.4
4.8
1.6
6
25
802
1.2
1.8
5.1
10
7
31
730
4.6
4.8
1.8
16
8
26
820
3.9
8.6
3.5
17
9
27
1080
1.3
1.7
1.2
9
10
27
1005
1.7
2.4
1.7
8
±
Mean +SD 29 6
923 126
2.1 ± 1.2 4.7±2.6 2.5± 1.5
12±5
Coronary artery disease patients (n = 25)
11
+
+
+
74
230
1.6
1.7
1.3
11
+
+
+
12
56
500
2.3
5.0
3.3
22
+
+
+
2.5
13
55
460
1.6
7.8
6
14
+
+
1.6
71
435
5.8
8.6
10
+
+
15
+
1.6
1.9
16.0
50
520
12
+
+
+
16
63
445
1.9
2.6
2.9
10
17
+
2.4
65
+
2.6
1.7
255
24
18
+
2.5
3.7
60
450
19
5.4
19
+
+
1.1
1.0
46
375
11
1.0
20
+
+
1.4
1.9
1.3
50
285
13
-+
+
1.6
2.1
1.4
21
55
340
6
12.5
7.2
+
+
26
5.6
22
120 St'/2
63
1.9
1.9
1.9
10
+
66
+
360
23
+
11
1.5
1.8
1.9
+
24
67
465
2.7
16
1.7
1.5
25
52
555
14
1.1
1.2
1.1
+
26
71
305
25
2.8
4.0
2.7
+
27
495
59
11
2.1
3.0
2.1
+
28
63
360
ND
12
1.5
1.4
+
29
405
51
1.3
1.2
3
1.3
720
30
53
ND
17
1.7
1.3
70
60
- +
31
12
2.4
1.5
1.5
+
66
235
32
14
1.2
2.2
1.5
+
410
54
33
11
1.7
1.5
1.2
670
62
34
7
2.8
15.0
1.4
450
54
35
13+6
2.1 ± 1.4 2.6±2.2 3.8±4.3
391 ± 162
Mean SD 60 ± 8
ventricular
*Ventricular bigeminy or coupled premature
complexes.
Abbreviations: + = present; - = absent; ND = not done; 30 min = 30 minutes after completion of exercise; PF4
PTG = I thromboglobulin; St'/2 = stage '/2 of exercise tolerance test.
-
PTG (ng/ml)
Exercise
30
min
24
16
8
30
14
12
16
27
12
8
17±8
ND
ND
ND
ND
13
26
14
8
10
12
21
15
12
15
4
30
8
10
15
11
24
12
12
10
16
14
14
14
12
14±6
=
11
16
ND
18
10
9
13±4
8
24
22
16
23
14
22
22
10
14
14
28
8
11
12
12
20
10
ND
12
ND
10
14
15
28
16±6
platelet factor 4;
36
VOL 66, No 1, JULY 1982
CIRCULATION
TABLE 2. Mean Plasma Platelet Factor 4, ,B Thromboglobulin and Catecholamine Levels in Subgroups of Patients
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Normals (n = 10)
All CAD patients
(n = 25)
CAD with either
angina or ST
depression (n = 14)
CAD with neither
angina nor ST
depression (n = 11)
CAD with both
angina and ST
depression (n = 5)
CAD on 3
blockers (n = 17)
CAD on no P
blockers (n = 8)
CAD with CABG
(n 5 )
=
Rest
Plasma PTG (ng/ml)
Exercise
30 min
2.5 ± 1.5
11.7 ± 5.4
16.7 ± 7.7t'
12.8 ± 3.9
2.6±2.2t§
3.8+4.3
13.3±6.0
14.1±5.8
16.0+6.2
2.6+1.7
3.0±2.9
4.3±4.3
13.6±6.4
14.1+7.1
16.9±6.5
1.6±0.5
2.2±0.9§
3.1±4.5
12.9+5.6
14.0+3.8
14.8 5.8
1.8±0.3
2.7+1.3
6.3+6.0
12.2±5.9
15.0±6.3
18.2±6.9
2.0 ± 1.0
2.9 ± 2.7t
3.4 3.9
14.1 +6.3
14.8 ± 6.7
15.8 6.4
2.5±1.9
2.0+0.6§
4.8+5.2
11.8+5.4
12.5±2.9
16.6±6.3
1.6±0.4
2.5±1.4
1.7±0.9
12.8±5.9
14.2±7.8
15.0+5.7
Rest
Plasma PF4 (ng/ml)
Exercise
30 min
2.1 ± 1.2
4.7 + 2.6t
2.1
1.4
Values are mean ± SD.
*p < 0.05 compared with rest values.
p < 0.01 compared with rest values.
.p < 0.05 compared with normal subjects.
§.p < 0.01 compared with normal subjects.
Abbreviations: CAD = coronary artery disease; CABG
platelet factor 4; PTG =- thromboglobulin.
coronary artery
sham). Because problems of sample processing
than artifactually low levels,'6 the lower of the duplicate plasma values was selected to represent the in vivo
level of PF4 or 13TG. In 105 normal resting subjects,
the mean value in this laboratory for plasma PF4 was
1.8 + 0.9ng/ml (mean SD), forplasma,BTG6.0 +
3.6 ng/ml and for urine PF4 0.18 ± 0.16 ng PF4/mg
creatinine. 17
To determine if repetitive sampling through an indwelling catheter detectably elevated plasma PF4, we
16
NORMAL
*
13
CAD-EXERCISE
14
p < .01 vs. BASELINE
13
12
12
11
11/
e .
1O
I'
MIA
(N=14)
15
SEM
14
E
16B
CONTROLS
(N=10)
MAEAN+1
IMEAN+1 SEM
NS vs. CONTROL
t P
AND REST
FIGURE 1. Plasma platelet factor 4 (PF4)
levels at rest, immediately after maximal treadmill exercise, and 30 minutes after exercise in
10 normal control subjects and in all 14 coronary artery disease (CAD) patients who developed exercise-induced angina or ST depression. In normal subjects, peak exercise plasma
PF4 was significantly increasedfrom baseline.
In contrast, CAD patients with exercise-induced ischemia did not have a significant exercise-induced increase in plasma PF4.
10
C
E
9-
9-
8-
8
7
7
4-
4
3
2
02m
L
Rest
Maximum
Exercise
30 min.
Post Exercise
30 minutes after exercise; PF4
drew serial samples at the time of catheter placement
and 15 and 30 minutes later in six healthy males
through an 18-gauge Abbocath (Abbott Hospitals
Inc.). The first sample was drawn immediately upon
catheter placement. The catheter was kept open with
normal saline (50 ml/hour) without added heparin.
Also, to determine if repeated venipunctures in a single
vein at the same site elevate plasma PF4, we performed three repeated venipunctures every 15 minutes
in four normal subjects.
For plasma catecholamine determinations, 2.5 ml of
are
most likely to result in artifactually high levels rather
1S
bypass grafting; 30 min
REst
MPraximum
Exercie
30 min.
Post Exercise
PF4 AND I3TG DURING EXERCISE/Stratton et al.
37
TABLE 2. (Continued)
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Exercise
Rest
Exercise
115±64
653±367t
394± 122
3823± 1233t
0.26 ±0.29
82 ± 34
264 ±178t§
692 ±283§
2607 ±1740tt
0.19±0.23
0.28 ± 0.27
85 ± 33
272 ± 201t §
698 ±239§
2853 ±2051t*
0.27 ± 0.37
0.24± 0.32
78± 45
250 ±152t§
683 ±342t
2293 ±1268t§
0.22 ± 0.30
0.31 ± 0.12
87 ±47
404 ±276*
773 ±260§
3772 ±2682*
0.26±0.35
0.30±0.31
88±42
289±191t
762±279§
2972± 1858t
0.26±0.34
0.15±0.21t
69±26
211 ±144t§
543±241
1832± 1219*§
0.29 ± 0.31
0.27 ± 0.33
93 ±42
348 +207*
640 ±192t
3042 ±1357*
Rest
Exercise
Rest
0.24±0.15
0.31 ±0.12
0.23 ±0.30
venous blood were drawn at rest and at maximal exercise and placed into prechilled glass tubes containing
heparin and glutathione (5 mmol/l final concentration), which were immediately placed on ice. A singleisotope radioenzymatic assay was performed as previously described.26 The mean basal supine level in 95
normal subjects using this method was 252 + 138 pg/
ml for norepinephrine and 50 ± 22 pg/ml for epinephrine in this laboratory.
Statistical Analysis
The data were evaluated using Wilcoxon's signedrank test for paired data and the Mann-Whitney test for
unpaired data. Results are expressed as mean ± SD.
6.0
E
o5.0.
Cj
-t
U.
Plasma
norepinephrine
(pg/ml)
Plasma
epinephrine
(pg/ml)
Urine PF4
(ng PF4/mg
creatinine)
NORMALS(N 10)(tSEM)
§ NON-ISCHEMIC CAD(N = 11)
I SCHEMIC
CAD(N
=
14)
* p <.01 vs. REST
t p c .01 vs. NORMALS
4.0-
1.0
Rest
Maximal
Exercise
30 min.
Post Exercise
FIGURE 2. Mean plasma plateletfactor 4 (PF4) at rest, immediately after maximal treadmill exercise, and 30 minutes after
exercise in normal subjects and in coronary artery disease
(CAD) patients with or without exercise-induced myocardial
ischemia. Only the normal subjects had a significant elevation
in plasma PF4 during exercise.
Results
Plasma PF4 and BTG
-
Treadmill Exercise
Clinical data and plasma PF4 and BTG responses for
all 35 subjects undergoing upright treadmill testing are
presented in table 1. In normal subjects, mean plasma
PF4 was 2.1 ± 1.2 ng/ml at rest, increased to 4.7 +
2.6 ng/ml at peak exercise (p < 0.01 vs rest), and fell
to 2.5 ± 1.5 ng/ml at 30 minutes after exercise (NS vs
rest) (figs. 1 and 2). Among the 11 coronary artery
disease patients without exercise-induced ischemia,
plasma PF4 was 1.6 ± 0.5 ng/ml at rest, 2.2 ± 0.9 ng/
ml at peak exercise, and 3.1 ± 4.5 ng/ml at 30 minutes
after exercise (both NS vs rest) (fig. 2, table 2). Peak
exercise plasma PF4 was minimally but significantly
greater in normal subjects than in the 11 coronary
artery disease patients without exercise-induced ischemia (p < 0.01).
Among the 14 coronary artery disease patients who
developed evidence of myocardial ischemia during exercise (angina or ST depression), plasma PF4 was 2.6
± 1.7 ng/ml at rest, 3.0 + 2.9 ng/ml at peak exercise,
and 4.3 + 4.3 ng/ml at 30 minutes after exercise (both
NS vs rest) (figs. 1 and 2). The mean rest, exercise and
30-minute postexercise plasma PF4 values in the 14
patients with exercise-induced ischemia did not differ
significantly from the corresponding values in normal
subjects or from the values in the 11 coronary artery
disease patients without exercise-induced ischemia (table 2). The increase in plasma PF4 from rest to peak
exercise was significantly less in coronary artery disease patients with exercise-induced ischemia (0.4 +
2.5 ng/ml) than in normal subjects (2.6 ± 2.2 ng/ml;p
< 0.05), but not different from that in coronary artery
disease patients without exercise-induced ischemia
(0.6 ± 0.6 ng/ml; NS). The increase in plasma PF4
from rest to 30 minutes after exercise was not signifi-
38
CIRCULATION
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cantly different between coronary artery disease patients with ischemia (1.7 ± 4.6 ng/ml) and normal
subjects (0.4 ± 2.2 ng/ml) and coronary artery disease
patients without ischemia (1.5 ± 4.6 ng/ml) (both
NS).
When all 25 coronary artery disease patients are
considered together, plasma PF4 was 2.1 ± 1.4 ng/ml
at rest, 2.6 ± 2.2 ng/ml at peak exercise (p < 0.01 vs
rest) and 3.8 ± 4.3 ng/ml at 30 minutes after exercise
(NS vs rest). The exercise-induced increase in plasma
PF4 among all 25 coronary artery disease patients (0.5
± 1.9 ng/ml) was significantly less than that in normal
subjects (2.6 ± 2.2 ng/ml; p < 0.01).
Among normal subjects, mean plasma BTG was
11.7 ± 5.4 ng/ml at rest, 16.7 ± 7.7 ng/ml at peak
exercise (p < 0.01 vs rest) and 12.8 ± 3.9 ng/ml at 30
minutes after exercise (NS vs rest) (fig. 3). Among the
11 coronary artery disease patients who did not develop exercise-induced ischemia, plasma BTG was 12.9
± 5.6 ng/ml at rest, 14.0 ± 3.8 ng/ml at peak exercise, and 14.8 ± 5.8 ng/ml at 30 minutes after exercise
(both NS vs rest). Among the 14 coronary artery disease patients who developed exercise-induced ischemia, plasma BTG was 13.6 ± 6.4 ng/ml at rest, 14.1
± 7.1 ng/ml at peak exercise and 16.9 ± 6.5 ng/ml at
30 minutes after exercise (both NS vs rest). The increase in plasma BTG from rest to peak exercise in the
14 patients with exercise-induced ischemia (0.5 ± 3.4
ng/ml) and the 11 coronary disease patients without
exercise-induced ischemia (1.1 ± 2.9 ng/ml) was significantly less than the increase in normal subjects (5.0
± 4.1 ng/ml; both p < 0.05). When all 25 coronary
artery disease patients are considered together, plasma
BTG was 13.3 ± 6.0 ng/ml at rest, 14.1 ± 5.8 ng/ml
at peak exercise, and 16.0 ± 6.2 ng/ml 30 minutes
after exercise (both NS vs rest). The increase in plasma
BTG from rest to exercise in all patients with coronary
20 S NORMALS(n-10)(tSEM)
NON-ISCHEMIC CAD(n I 1)
18
E
*
ISCHEMIC CAD(n -14)
* p .01 vs. REST
t p a NS vs. NORMALS
16
;t16
14
S1
10
8j
Rest
Maximal
Exercise
30 min.
Post Exercise
FIGURE 3. Mean plasma fi thromboglobulin (PTG) at rest,
immediately after maximal treadmill exercise, and 30 minutes
after exercise in normal subjects and in coronary arterv disease
(CAD) patients with and without exercise-induced myocardial
ischemia. As with plasma platelet factor 4, only the normal
subjects had a significant elevation in plasma fiTG during
exercise.
7.0
VOL 66, No 1, JULY 1982
I
MEAN PF4 IN CAD PATIENTS ON BETA BLOCKERS
AND CAD PATIENTS NOT ON BETA BLOCKERS
6.0
E 5.0
CD
%-
U.
4.0
i ON BETA BLOCKERS (N=17)(iSEM)
+ ON NO BETA BLOCKERS (N-8)
* p < .01 vs. REST
p= NS ALL OTHER COMPARISONS
;r
Enl30
o(0 3.0
a-
c
0
2 2.0-
1.0-
Rest
Maximal
Exercise
30 mm.
Post Exercise
FIGURE 4. Mean plasma platelet factor 4 (PF4) in coronary
artery disease patients taking fl-blocking drugs and coronary
artery disease patients not takingJf-blocking drugs. There were
no significant differences between the two groups in plasma PF4
or plasma fl thromboglobulin (not shown) at rest, immediately
after maximal exercise, or at 30 minutes after exercise.
artery disease (0.8 ± 3.1 ng/ml) was significantly less
than that in normal subjects (5.0 ± 4.1 ng/ml; p <
0.01). Additionally, the five coronary artery disease
patients who developed two manifestations of ischemia during exercise (both angina and ST depression)
did not have significantly higher plasma PF4 or 1BTG
responses at peak exercise than either normal subjects
or the other 20 coronary artery disease patients (table 2).
Rest, exercise and postexercise plasma PF4 and
BTG levels were not significantly different between the
17 coronary artery disease patients taking 3 blockers
and the eight coronary artery disease patients not taking B blockers (table 2, fig. 4). At rest, plasma PF4 was
2.0 ± 1.0 ng/ml in the 17 coronary artery disease
patients taking B blockers and was 2.5 ± 1.9 ng/ml in
the eight coronary disease patients not taking B
blockers (NS). At peak exercise, plasma PF4 was 2.9
± 2.7 ng/ml in patients taking B blockers and was 2.0
± 0.6 ng/ml in patients not taking B blockers (NS).
Plasma BTG at rest was 14.1 + 6.3 ng/ml in patients
taking B blockers and 11.8 ± 5.4 ng/ml in patients not
taking B blockers (NS). At peak exercise, plasma BTG
was 14.8 + 6.7 in patients taking B blockers and 12.5
± 2.9 ng/ml in patients not taking B blockers (NS).
Plasma PF4 and BTG levels 30 minutes after exercise
in the two groups were also not significantly different
(table 2). The five patients with prior coronary artery
bypass grafting also did not have significantly different
PF4 or BTG responses from the 20 coronary artery
disease patients without prior bypass grafting or from
normal subjects (table 2).
Urinary PF4 did not change significantly from base-
PF4 AND BTG DURING EXERCISE/Stratton et al.
line to exercise in any subject group (all NS) (table 2).
Among all 35 patients, there was a highly significant
correlation between change in plasma PF4 and change
in plasma BTG from rest to peak exercise (r = 0.70, p
< 0.001). There was also a significant correlation between all rest and exercise PF4 and BTG values (r =
0.62, p < 0.001) in all 35 subjects.
Patient 2, the young normal subject who showed the
greatest change in plasma PF4 during treadmill exercise, underwent repeat maximal exercise testing on a
different day. On initial testing, plasma PF4 increased
from 1.7 to 8.6 ng/ml and on repeat testing plasma PF4
increased from 3.5 to 8.8 ng/ml, suggesting that exercise-induced increases in plasma PF4 were reproducible.
Plasma Epinephrine and Norepinephrine
During Treadmill Exercise
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Mean plasma epinephrine and norepinephrine levels
at rest and exercise among normal subjects and coronary artery disease subgroups are presented in table 2
and figure 5. Resting plasma norepinephrine was higher among all 25 coronary artery disease patients than
normal subjects (692 + 283 vs 394 ± 122 pg/ml, p <
0.001), which is probably related to differences in age
and in B-blockade therapy between the two groups.
Exercise norepinephrine was higher in normal subjects
(3823 ± 1233 vs 2607 ± 1740 pg/ml, p < 0.05).
Resting epinephrine was not significantly different between groups, but normal subjects had higher exercise
epinephrine levels than coronary artery disease patients (653 ± 367 vs 264 ± 178 pg/ml, p < 0.001).
The 14 coronary artery disease patients with exercise-induced ischemia did not have rest or exercise
catecholamine levels significantly different from those
in the 11 coronary artery disease patients without exercise-induced ischemia (table 2). Epinephrine increased
from 85 ± 33 to 272 ± 201 pg/ml in patients with
39
exercise-induced ischemia and from 78 ± 45 to 250 +
152 pg/ml in patients without ischemia (NS, patients
with ischemia vs patients without ischemia).
Norepinephrine increased from 698 ± 239 to 2853 ±
2051 pg/ml in patients with ischemia and from 683 ±
342 to 2293 ± 1268 pg/ml in patients without ischemia (NS).
Among all 35 subjects, there was a correlation between the increase in plasma epinephrine from rest to
exercise and the increase in plasma PF4 (r = 0.35, p
= 0.04) and BTG (r = 0.40 p = 0.02). There was no
correlation between exercise changes in plasma norepinephrine and PF4 (r = 0.24; p = 0.2) orBTG (r =
0. 23;p= 0.2).
Plasma PF4, BTG and Catecholamine Levels
During Supine Bicycle Exercise
Changes in plasma PF4, BTG and catecholamine
levels with moderate and maximal supine bicycle testing in four normal subjects are listed in table 3 and
figure 6. Mean duration was 19 ± 3 minutes (range
16-22 minutes). Mean plasma PF4 increased from 1.2
± 0.3 ng/ml at rest to 1.6 ± 0.3 ng/ml during the tenth
minute of exercise (NS vs rest) and to 2.1 ± 0.7 ng/ml
at maximal exercise (p < 0.05 vs rest) (fig. 6). Corresponding mean plasma BTG levels were 18.8 ± 9.6,
14.0 ± 4.9 and 15.8 ± 5.4 ng/ml (all NS, rest vs
exercise). Increases in plasma PF4 occurred at the time
of maximal increases in plasma catecholamines. Exercise plasma catecholamine levels were lower in the
supine protocol, as were maximal plasma PF4 and
BTG responses in this small group. There was no
change in urine PF4 during supine exercise.
Effects of Repeated Venipuncture and Sampling
Through an Indwelling Catheter
Mean serial plasma PF4 samples drawn through an
indwelling venous catheter in six normal subjects at the
MEAN PLASMA CATECHOLAMINE RESPONSES TO TREADMILL EXERCISE
IN NORMALS AND ALL CAD PATIENTS
I
EPINEPHRINE
NOREPINEPHRINE
I
800-
~~~~~~~~~~~*
4000-
700-
3500
E
n 600
a
0
3000-
0
cza 500-
=
2500
0)
I-O
0)
"
t
a
._x
10) 2000
400-
0
z
0
cL
0
o0 200
c 1000-
E
0 300
E 1500
0
4
0
100-
+ ALL CAD(N525)
500- i
*
t
Rest
Maximum
Exercise
NORMALS(N-10)
(tSEM)
Rest
p <.05 vs.
p
REST
<.05 vs. CAD
Maximum
Exercise
FIGURE 5. Mean plasma epinephrine and
norepinephrine at upright sitting rest and immediately after maximal treadmill exercise
among all 10 normal subjects and all 25 coronary artery disease (CAD) patients. At rest,
there was no significant difference between
mean plasma epinephrine levels, but CAD patients had a higher mean norepinephrine level
than normal subjects, possibly related to the
effects of age and fl-blockade drug therapy. At
exercise, normal subjects had significantly
greater plasma epinephrine and norepinephrine levels than CAD patients.
40
VOL 66, No 1, JULY 1982
CIRCULATION
TABLE 3. Platelet Factor 4 and Catecholamine Responses at Moderate and Maximal Supine Bicycle Exercise in Normal Subjects
Plasma ITG (ng/ml)
Plasma PF4 (ng/ml)
10 min
10 min
Max
30 min
Rest
Max
Rest
30 min
Pt
1.0
2.0
2.4
2.2
6
10
10
9
36
24
20
23
21
37
1.1
1.6
1.6
1.5
1.7
10
1.6
1.5
2.9
28
15
38
10
1.4
ND
17
16
1.1
1.2
15
ND
39
1.2
2.1
1.8
18.8
14.0
15.8
1.6
13.3
Mean
±0.7*
±0.4
4.9
±0.3
±0.3
5.4
±SD
+9.6
6.7
*p <0.05 compared to rest.
Abbreviations: Max = maximal exercise; 30 min = 30 minutes after completion of exercise; ND = not done; PF4 = platelet factor 4; fTG
=, thromboglobulin.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
time of catheter placement and 15 and 30 minutes later
were 2.0 + 0.4, 6.9 + 3.4, and 7.8 ± 3.6 ng/ml,
respectively (fig. 7). The 15- and 30-minute samples
were significantly elevated from baseline (both p <
0.05 vs baseline).
Mean plasma PF4 values obtained by repeated, separate venipunctures every 15 minutes from the same
venous site in four normal subjects showed no statistically significant elevations in this small sample (2.9 +
1.7, 1.8 ± 0.9, 3.8 ± 3.2 and 1.8 ± 0.7 ng/ml,
respectively). However, two of the four patients had
elevations of plasma PF4 greater than 1 ng/ml on repeated sampling, one from 2.5 to 3.6 ng/ml and one
from 5.4 to 8.5 ng/ml.
Discussion
Abnormal platelet activation, as evidenced by exercise-induced elevations in plasma PF4 or BTG, was not
detected during exercise-induced myocardial ischemia
in patients with proved coronary artery disease. In
contrast, the 10 normal control subjects had exerciseinduced increases in both plasma PF4 and BTG that
exceeded those in the 14 coronary artery disease patients with exercise-induced ischemia. The exerciseinduced increases in normal subjects were also greater
than the small increases seen in the entire group of 25
coronary artery disease patients. There were no differences in plasma PF4 or 13TG responses between the 17
coronary artery disease patients taking B blockers and
the eight coronary artery disease patients not taking B
blockers. Thus, it is unlikely that the lack of elevation
of plasma PF4 or BTG in coronary artery disease patients with exercise-induced myocardial ischemia was
due to B blocker therapy. In fact, the 17 patients taking
B blockers had a small but statistically significant increase in plasma PF4, but not in BTG from rest to peak
exercise. Among the three coronary artery disease subjects not taking B blockers who developed exerciseinduced ischemia, mean plasma PF4 actually fell from
rest to peak exercise. Lindenfeld et al.2U showed no
PLASMA PF4 DRAWN THROUGH AN INDWELLING VENOUS CATHETER
PLASMA PF4 DURING SUPINE EXERCISE IN NORMALS
(N=4)
3.0
14.0]
MEAN PF4tSEM
*
j MEAN PF4±1 SEM
p c .05 vs. O'
12.0
*
p=
0.01 vs. REST
10.0
NE
IL
tcLco
2.0
1'
U.a.
a
E
IL
Ee
8.0
6.0
4.0
1.
2.0
o0
Rest
FiGURE 6.
10 min.
Exercise
Plasma
Maximum
Exercise
platelet factor
4
(PF4) levels
15
30b
Minutes After Catheter Insertion
30 min.
Post Exercise
at rest, at
minutes of supine exercise, at maximal supine exercise, and 30
minutes after exercise in four young normal subjects. Plasma
PF4 progressively increased during supine exercise, as did
plasma catecholamines (table 3).
FIGURE 7. Plasma platelet factor 4 (PF4) levels drawn
through an 18-gauge indwelling venous catheter (Abbocath,
Abbott Hospitals, Inc.) at the time of catheter insertion and 15
and 30 minutes later. In all but one subject, plasma PF4 levels
increased at least threefold, presumably due to the thrombogenic stimulus of the catheter.
41
PF4 AND BTG DURING EXERCISE/Stratton et al.
TABLE 3. (Contittued)
Plasma
epinephrine (pg/mi)
Max
10 min
Rest
550
80
30
140
40
70
1240
50
30
180
100
60
528
68
33
+21
+28* +510*
Plasma
norepinephrine (pg/mi)
Max
10 min
Rest
1710
610
800
265
250
400
381
±167
430
420
670
580
600
4000
1060
1842
±187* ±1509:
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
difference in plasma PF4 response to exercise before
and after propranolol therapy in both patients with
angina and in normal subjects. Thus, propranolol therapy does not appear to significantly influence plasma
PF4 responses to exercise. The lack of an exercise
increase in plasma PF4 or BTG in patients with exercise-induced ischemia also cannot be attributed to
platelet-active drugs, since none of the subjects had
taken any platelet-active medications for at least 1
week before testing. Prior coronary artery bypass surgery did not appear to influence the results; the five
patients with prior surgery had similar results to the 20
coronary artery disease patients -without prior surgery.
However, four of these patients had recurrent postoperative angina and were, therefore, not representative
of all patients having coronary artery bypass grafting.
Thus, we cannot attribute the lack of an exercise'rise in
plasma PF4 and BTG in patients with proved coronary
artery disease and exercise-induced ischemia to either
B-blockade therapy, antiplatelet drug therapy, or prior
coronary artery bypass grafting. Our results support
the conclusion that exercise-induced myocardial ischemia does not produce platelet a' granule release that is
detectable in peripheral venous blood or in urine.
Our results are in agreement with the findings of
Mathis et al. , 18 who found exercise elevations of plasma PF4 above baseline levels in two of six normal
subjects but in none of 11 patients with proved coronary artery disease. In contrast, Green and co-workers'9 reported that 11 of 20 patients (55%) with exercise ST depression had a greater than 50% increase in
exercise plasma PF4, while only two of 20 patients
(10%) without ST depression had similar plasma PF4
elevations. Using their criteria of a 50% increase in
PF4 during exercise, 70% (seven of 10) of our normal
subjects had an abnormal exercise PF4 response, while
only 24% (six of 25) of our coronary artery disease
patients had an abnormal response. The difference between our results and those of Green et al. may be
because of differences in patient populations and differences in methods. Our normal subjects were younger than those studied by Green et al. (mean age 29 vs
48 years), and our coronary artery disease patients
were slightly older (mean age 60 vs 54 years). However, age differences alone cannot explain our finding of
no increase in plasma PF4 in patients with exerciseinduced myocardial ischemia. Fewer of our coronary
artery disease patients had ST depression during exercise than those of'Green et al.; however, the plasma
PF4 response in our coronary patients with ST depression was not different from that in our patients w'ithout
ST depression.
Differences in blood sample processing appear to be
a more likely explanation for the discrepant results.
Several investigators'6'8' 23 have noted the importance
of careful sample processing such that in vitro platelet
activation and release, which would spuriously elevate
PF4 and BTG levels, does not occur. Files et al. 17 found
lower resting plasma PF4 levels using the technique
used in this study than with the technique of either
Green et al. '9 or Lindenfeld et al.20 Differences in sample processing or in the radioimmunoassay may account for the differences in the normal ranges of plas0.9 ng/ml) and
ma PF4 between our laboratory (1.9
2.5 ng/ml).'9 However,
that of Green et al. (5.0
methodologic differences do not entirely explain the
differences in exercise results; we rarely found more
than a 50% increase in plasma PF4 in patients with
exercise induced ischemia, while Green et al. commonly did.'9
The use of separate venipunctures rather than an
indwelling venous catheter may be an additional important methodologic difference between this study
and others. Although Lindenfeld and colleagues20
found a four- to sixfold increase in plasma PF4 during
exercise in both normal subjects and in patients with a
history of angina, their samples were drawn through an
indwelling venous catheter instead of through separate
venipunctures. In our expenence, samples drawn
through an indwelling catheter showed progressively
increased levels of plasma PF4 over 30 minutes, presumably from platelet activation by the catheter (fig.
7). Knauer et al.27 showed a mean increase in plasma
PF4 of 55% over only 10 minutes when blood was
withdrawn from an indwelling Teflon catheter in five
normal subjects. Also, Hyers et al. ,21 using an indwelling catheter, found that plasma BTG increased from 24
6.5 to 42 + 12 ng/ml during maximal exercise in
six normal subjects. In addition, if heparin is used to
keep the catheter open, plasma PF4 may be artifactually elevated for reasons other than platelet release.
Thus, it seems likely that at least some reports of
dramatic elevations in plasma PF4 or BTG during exerc'ise in normal subjects and in patients with angina
might be explained by methodologic differences.
In the present study, BTG and PF4 assays were performed independently. The finding that exercise
changes in BTG paralleled exercise changes in PF4
further strengthens our conclusion that platelet-specific protein release, as measured in venous blood, is not
associated with myocardial ischemia. Urinary PF4 values appeared to offer even less discrimination between
patient groups than did plasma PF4 or BTG, as noted in
patients studied at rest.'7
Our results indicate that plasma PF4 and BTG increase minimally, but significantly, in normal subjects
duiring maximal exercise. In addition, if all 25 coronary artery disease patients are considered together,
plasma PF4, but not BTG, increased significantly, although t'o a lesser extent than in normal subjects The
-
42
VOL 66, No 1, JULY 1982
CIRCULATION
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
cause of the exercise-induced increase in plasma PF4
and 13TG in most of our normal subjects and in some
patients with coronary artery disease is uncertain. In
normal subjects, the increase in plasma BTG (5.0 ng/
ml) was greater than that in plasma PF4 (2.6 ng/ml);
these plasma levels parallel the relative amounts of
these proteins in platelets as measured in our laboratory. 17 Others have reported that because BTG and PF4
are cleared at different rates, a greater increase in plasma BTG might be helpful in discriminating between in
vivo release (BTG preferentially elevated due to slower
plasma clearance) and in vitro release (equivalent elevations of BTG and PF4). 13 16 In any event, the exercise
increases in plasma PF4 and BTG noted in the current
study are consistent with exercise-induced platelet activation and release in vivo. Part of the increases in
plasma PF4 and 3TG may be secondary to repeated
sampling from the same vein, as noted above. This
may explain the rise in plasma PF4 values at 30 minutes after exercise in four of the 25 coronary artery
disease patients and in one of the six normal subjects
sampled at that time. Therefore, a different venipuncture site was used, whenever possible, for each
sample.
Platelet activation during exercise may be related to
exercise duration, as suggested by the serial rises in
PF4 during supine bicycle exercise. However, among
all 35 subjects who underwent upright treadmill testing, exercise duration did not correlate with changes in
PF4 (r = 0.27, p = 0.1), but did correlate with
changes in plasma BTG (r = 0.48, p - 0.007). The
lack of plasma BTG elevation in patients with coronary
artery disease may be explained by this association,
since these patients exercised significantly less than
normal subjects.
Exercise changes in plasma PF4 and BTG may be
partially related to increases in circulating epinephrine, as suggested by the finding that among all 35
subjects, changes in plasma epinephrine during upright treadmill testing correlated with changes in plasmaPF4(r = 0.35,p = 0.04)andBTG(r= 0.45,p 0.01). Platelets possess a-adrenergic receptors28 29 and
the interaction of epinephrine with these receptors initiates platelet aggregation.28' 29 Alexander et al.28 postulated that these receptors are regulated by changes in
the circulating levels of a agonists. Kaplan et al.
showed that epinephrine at supraphysiologic levels in
vitro causes PF4 and BTG release. 15 Although epinephrine at physiologic levels does not cause platelet aggregation in vitro, it may enhance aggregation to other
stimuli.30 In the current study, there were no significant differences in catecholamine, PF4 or BTG levels
during exercise in coronary artery disease patients with exercise-induced ischemia compared with
coronary patients without exercise-induced ischemia.
Thus, increases in plasma PF4 and BTG during exercise may be related more to increases in epinephrine
than to the presence of myocardial ischemia.
Regional sampling of PF4 or BTG across the coronary vascular bed using specialized nonthrombogenic
arterial and coronary sinus catheters, as has been done
for circulating platelet aggregates3' and thromboxane
B232, 33 may reveal evidence of localized PF4 and BTG
release during myocardial ischemia. However, the
finding that increases in plasma PF4 and BTG in peripheral venous blood during exercise did not correlate
with the development of myocardial ischemia makes
these measurements of platelet ax-granule release of
limited clinical value, either in individual patients or in
groups, despite the suggestion that PF4 and BTG levels
might offer noninvasive assessment of therapy.
Acknowledgment
The authors thank Kathy W. McFadden, Esther K. Yee and David
Flatness for their expert technical help. They also thank Pat Jenkins,
Maxine Cormier, Lynda Taylor and Kathy Jelsing for their skillful
assistance in preparation of the manuscript.
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Studies of platelet factor 4 and beta thromboglobulin release during exercise: lack of
relationship to myocardial ischemia.
J R Stratton, T W Malpass, J L Ritchie, M A Pfeifer and L A Harker
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Circulation. 1982;66:33-43
doi: 10.1161/01.CIR.66.1.33
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