Plasma Beta-Thromboglobulin as a Measure of Platelet Activity Effect of Risk Factors and Findings in lschemic Heart Disease And After Acute Myocardial Infarction
CHARLES W. PUMPHREY, MD, MRCP and JOAN DAWES, DPh
The plasma concentration of beta-thromboglobulin (BTG), a platelet-specific protein released during platelet aggregation, is considered a sensitive marker of in vivo platelet activity. The mean plasma level in 133 asymptomatic individuals was 32.3 f 1.1 ng/ml, and there was no difference between those with no risk factors (32.2 f 1.2 ng/ml, n = 56) those who smoked (31.8 f 1.8 ng/ml, n = 45), those with hyperlipidemia (32.8 f 1.7 ng/ml, n = 15), and those exposed to both of these risk factors (34.1 f 2.7 ng/ml, n = 17). The mean plasma BTG level in 104 patients with symptomatic ischemic heart disease was significantly elevated (40.9 f 1.4 ng/ml, p
(37.0 f 1.8 ng/ml, n = 44), patients with 2 or more risk factors had a significantly elevated plasma BTG level (45.2 f 2.2 ng/ml, n = 47, p
Recent studies have highlighted the importance of coronary arterial thrombosis in both acute myocardial infarction and possibly unstable angina pectoris.lm” Occlusive arterial thrombosis results from a focus of platelet aggregation, and altered platelet behavior has been well documented in patients with ischemic heart disease.4-6 However, it remains uncertain whether this alteration in platelet activity causes atherosclerosis and is exacerbated by the presence of known coronary risk factors, or whether it is due to the presence of estab-
The plasma level of BTG, a platelet-specific protein released from platelet alpha granules during platelet aggregation, provides an accurate measure of in vivo platelet activity.7 We report herein the results of measurements of plasma BTG in patients with symptomatic ischemic heart disease and in normal asymptomatic individuals and compare the effect of known coronary risk factors on platelet activity in the 2 groups. We also report the results of studies on changes in plasma BTG in patients after their admission to the Coronary Care Unit with chest pain to identify changes in platelet activity after acute myocardial infarction.
lished darily.
arterial
disease
activating
the platelets
secon-
Methods From the Thrombosis Laboratory, Royal Infirmary, Edinburgh, Scotland, and the M.R.C. Immunoassay Team, Forrest Road, Edinburgh,Scotland. Manuscript received February 17, 1982; revised manuscript received June 1, 1982, accepted June 16. 1982. Address for reprints: Charles W. Pumphrey, MD, the London Hospital, Whitechapel, London El lBB, England.
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We studied 104 patients (76 male and 28 female) ranging in age from 27 to 65 years (mean age 51 years). All had a history of current exercise-induced angina pectoris and a resting or exercise electrocardiogram compatible with the diagnosis of ischemic heart disease. Patients with any recent change in
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symptoms and those with an elevated creatinine level or additional valve disease, which might affect the plasma BTG level, were excluded. Fifty-five had previously documented myocardial infarction. Sixty-three patients were on betablocking drugs and 30 of these were on additional long-acting nitrates or calcium antagonist drugs. Twenty had undergone coronary arteriography and all demonstrated occlusive coronary atherosclerosis. The smoking habits, blood lipid levels, blood pressure, and family history of ischemic heart disease were documented for each patient. A family history was considered positive for any patient who had a first-degree relative presenting with clinical ischemic heart disease under the age of 60 years. A smoker was defined as a patient who was still smoking at least 10 cigarettes a day. Hyperlipidemia was diagnosed when the serum cholesterol level was >7.0 mmol/liter or the serum triglyceride level was >1.8 mmol/liter in a strict fasting blood specimen despite treatment with diet, drugs, or both. A diastolic pressure >90 mm Hg despite antihypertensive treatment on at least 2 occasions was taken t.o indicate significant hypertension. No patient had taken aspirin or other anti-inflammatory drugs during the 10 days before sampling. Blood was taken to determine plasma BTG, platelet count, and serum cholesterol and triglyceride levels during fasting. To prevent ex vivo platelet release and spurious elevation of plasma BTG, all blood samples were carefully collected by 1 of us (CWP), placed in prechilled platelet inhibitor solution, and processed within 1 hour.7 Samples were assayed for BTG using the double-antibody technique described by Bolton et al,s which has an interassay coefficient of variation of 8%. The results were compared with those of a control group of 101 blood donors (52 male and 49 female subjects) ranging in age from 18 to 64 years (mean 39) who had no history of vascular disease, renal disease, or hypertension and no family history of premature coronary disease. Blood lipid levels were checked at the time of sampling and only those with normal values were included. Their smoking habits were documented. An additional 32 subjects (22 male and 10 female) ranging in age from 22 to 63 years (mean 41) who had no clinical vascular disease but who were attending the Lipid Clinic because of hyperlipidemia were also examined. In a separate study the platelet activity was measured in 30 patients admitted to the Coronary Care Unit with a diagnosis of possible acute myocardial infarction. Blood for BTG and platelet count was taken usually within 12 hours of the onset
AND DAWES
of symptoms (minimum 3 hours, maximum 20 hours) and then daily for 10 days or until the patient was discharged. Myocardial infarction was diagnosed on the basis of the conventional criteria of electrocardiographic changes and elevation in cardiac enzyme levels. Medical complications, particularly clinical evidence of deep venous thrombosis, elevated plasma creatinine levels, additional chest pain, pericarditis, or heart failure, were documented. The patients’ records were then reviewed at 8 months after discharge from the hospital. Results in asymptomatic individuals and patients with stable ischemic heart disease are expressed as mean f standard error. Statistical analysis was carried out using the unpaired Student’s t test. Results in patients followed up serially are expressed as a median value, and analysis was carried out using the Wilcoxon rank sum test.
Results Plasma BTG in clinically asymptomatic subjects: Mean plasma BTG concentrations in relation to the presence of identified risk factors for normal asymptomatic subjects are shown in Figure 1. There was no significant difference between smokers (31.8 f 1.6 ng/ml, n = 45) and nonsmokers (32.2 f 1.2 ng/ml, n = 56). Hyperlipidemic subjects with no clinical evidence of vascular disease had normal BTG levels (32.8 f 1.7 ng/ml, n = 15) and an additional 17 of these individuals who also smoked had a similar mean BTG level (34.1 f 2.7 ng/ml). No correlation was found between serum cholesterol or triglycerides and plasma BTG in hyperlipidemic subjects, nor was there a difference in BTG concentration among the different classes of hyperlipidemia.
Plasma BTG in patients with stable symptomatic ischemic heart disease: Patients with coronary artery disease,
however,
had
a significantly
elevated
mean
.. .. ::
Plasma BTG ng/ml
. ..
e” 3
1.‘.
&
50
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40
::.
t
:.: :. . ...
+
30
+
.I. .. . . .: .::
-I-
--J-
t
.;;
20 t 10 t N=
NO Risk FBCtOrS
Smokers
56
45
Hyparlipidaemia
Hyperlipidaemia and Smokers
15
Patimtn N.
17
IC4
0
FIGURE 1. Plasma BTG in factors.
asymptomatic
subjects:
relation
to risk
FIGURE 2. Mean and distributionof plasma BTG among control subjects and patients with ischemic
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plasma BTG level (40.9 f 1.4 ng/ml, n = 104, p
Plasma BTG ng/ml 60
.
50
-
t 40-
+
t
30
-
20
-
10
-
-I+
N=
NO Risk Factors
Smokers
13
23
iiyperllpldaemia
Positive Family History
High Blood Pressure
6
4
11
0
FIGURE 3. Plasma BTG in patients with ischemic heart disease: relation to risk factors.
failure developed with, again, no change in BTG level. In no patient did clinical signs of venous thrombosis, arterial embolism, or an increase in plasma creatinine level develop. After 8 months, 2 of the 29 patients had died suddenly (median BTG level on hospital discharge 34.0 ng/ml). One had had another infarct (BTG 37.4 ng/ml) and 8 continued to have angina pectoris (median BTG level 37.9 ng/ml). The other 18 patients were alive with no symptoms (median BTG level 45.1 ng/ml). Discussion These results support the concept that platelets function abnormally in patients with ischemic heart disease, evidence for which includes both increased in vitro platelet aggregation and accelerated in vivo Although the mean age of platelet consumption. 4-6,g~10 the control subjects was lower than that of the study group, age is not an independent determinant of plasma BTG levels.7 The increased platelet activity in our patients was not detectably augmented by single risk
Plasma BTG ng/ml 60
50
,
I
c
Plasma BTG ng/ml
30 *-*
Unstable
O-0
InfarCtlO”
Angum
N=l
N=22
20 NII or One Rsk Factor
Two or More Risk Factors
57
47
N: 0
t 10 t
FIGURE 4. Plasma BTG in patients with ischemic heart disease. Comparison of those with 1 or no risk factors to those with 2 or more risk factors.
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I
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0
I
I
1
’
’
’
I
1
1
I
1
2
3
4
5
6
7
8
9
10
1
Days
FIGURE 5. Changes in plasma BTG after admission with chest pain (median values).
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factors, although in patients with 2 or more risk factors the plasma BTG level was significantly increased. Such risk factors may operate synergistically, and each may have an effect that is not detectable unless these effects are combined. Alternatively, vascular disease may activate platelets which then contribute to further progression of the atherosclerotic process. The increased platelet activation seen in hypertension has been attributed to the existence of established atherosclerosis caused by hypertension rather than to a direct effect on the plate1ets.l’ Although not all patients who present with cardiac pain have fixed coronary artery stenosis, it was found in the 20 patients who underwent coronary arteriography. Therefore, the higher plasma BTG levels found in our patients with more risk factors may indicate more extensive disease throughout the arterial system. Our data on normal asymptomatic individuals with the identified coronary risk factors of smoking and hyperlipidemia reinforce this finding. Thus, although our definition of a risk factor was arbitrary, we did not find that the presence of risk factors per se in otherwise healthy subjects significantly affected platelet activity. Although others 12,13showed altered platelet activity in various types of hyperlipidemia, we found normal plasma BTG levels in asymptomatic patients with hyperlipidemia. In addition, although reduced platelet survival has been reported in apparently healthy smokers,14 our control subjects who smoked had normal plasma BTG levels and, furthermore, the 17 asymptomatic subjects with both risk factors had no exacerbation of platelet activity. It is likely that platelet survival and plasma BTG do not reflect similar aspects of platelet activity. Our failure to demonstrate a change in plasma BTG levels after infarction is surprising in view of recent evidence that coronary thrombosis occurs early in the evolution of infarction and may even initiate it.1,2 Although we were unable to measure plasma BTG levels before infarction, samples were taken as early as 3 hours after the event. If considerable BTG release had been associated with infarction, a subsequent decline in plasma BTG levels from an initial high level should have been observed. Our negative findings suggest that either BTG, with its short half-life, was cleared from the blood before the samples were taken or that the amount of BTG released from a platelet thrombus occluding a coronary artery is not sufficient to cause a measurable increase in the systemic circulation. Furthermore, our findings differ from those of Smitherman et al,‘” who ‘found in some cases very high levels of plasma BTG, although the temporal relation between peak plasma level and infarction was inexact. No reference is made to factors other than ischemia, such as pericarditis, altered renal function, venous thrombosis, or intercurrent infection, which might have contributed to these elevated plasma values. Similarly, in contrast to another report,l” we found no increase in plasma BTG levels after episodes of unstable angina. In the few patients who had continuing
IN ISCHEMIC HEART DISEASE-PUMPHREY
AND DAWES
symptoms after admission, blood samples were taken very shortly after the symptoms were relieved and still no alteration in plasma BTG was observed. Platelet thrombosis is probably not involved in the pathogenesis of unstable angina; alternatively, as already mentioned, systemic venous sampling is not sensitive enough to detect BTG released from platelets in the coronary circulation. The months after infarction are the period of increased risk of death or reinfarction. We found no correlation between plasma BTG and clinical history during the 8 months after discharge from the hospital. Plasma BTG did not distinguish the 2 patients who died suddenly or those who continued to have angina pectoris, so there is no suggestion that plasma BTG will identify patients with a poor prognosis. We conclude that elevated plasma BTG levels in patients with ischemic heart disease probably reflect platelet activation by established vascular disease. However, measurement of plasma BTG unable to detect platelet involvement in myocardial infarction or unstable angina pectoris. Acknowledgment: We are grateful to Professor M. Oliver and the staff of the Cardiac Department of the Royal Infirmary, Edinburgh, Scotland, for permission to study their patients.
References 1. DeWood MA, Spores J, Notske R, Mouser LT, Burroughs f?, Golden MS, Lang HT. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med 1980:303:897-902. 2. Rentrop P, Bfanke H, Karach KR, Kaiser H, Kostering H, Leitz K. Selective intracoronary thrombolysis in acute myocardial infarction and unstable angina pectoris. Circulation 1981:63:307-317. 3. Holmes DR, Harizfer GO, Smith HC, Fuster V. Coronary artery thrombosis in patients with unstable angina. Br Heart J 1981;45:411-416. 4. Hampton JR, Gorlin R. Platelet studies in patients with coronary artery disease and their relatives. Br Heart J 1972;34:465-471. 5. Steele PP, Weily HS, Davies H, Genton E. Platelet function studies in coronary artery disease. Circulation 1973;43: 1194-1200. 6. Szczekllk A, Grygfewskl RJ, Musiaf J, Grodrinska L, Serwonska M, Marcinkiewicz c.Thromboxane generation and platelet aggregation in survivors of mvocardial infarction. Thromb Haemost (Stutta) _. 1978:40: 66-74. . 7. Ludfam CA. Evidence for platelet-specificity of &thromboglobulin and studies on its plasma concentration in healthy individuals. Br J Haematol 1979;41:271-278. 8. Bofton AE, Ludfam CA, Moore S, Pepper DS, Cash JD. Three approaches to the radioimmunoassay of human fi-thromboglobulin. Br J Haematof 1976;32:233-238. 9. Dreyfuss F, Zahavi J. Adenosine diphosphate induced platelet aggregation in myocardiaf infarction and ischemic heart disease. Atherosclerosis 1973;17:107-120. 10. Kutti J, Weinfield A. Platelet survival and platelet production in acute myocardial infarction. Acta Med Stand 1979;205:501-504. 11. Vfachakis ND, Afedorl L. Platelet aggregation in relationship to plasma catechofamines in patients with hypertension. Atheroslcerosis 1979;32: 451-460. 12. Cavarfho ACA, Colman RW, Lees RS. Platelet function in hyperfipoproteinemia. N Engl J Med 1974;290:434-438. 13. Tremofi E, Maderna P, Sirfori M, Sirtori CR. Platelet aggregation and malondialdehyde formation in Type Ila hypercholesterolemia patients. Haemostasis 1979:8:47-53. 14. Fuster V, Chesebro JH, Frye RL, Efveback LR. Platelet survival and the development of coronary artery disease in the young adult: effects of cigarette smoking, strong family history, and medical therapy. Circulation 1981;63:546-551. 15. Smitherman TC, Mifam M, Woo J, Wilferson JT, Frenkel EP. Elevated (%thromboglobulin in peripheral venous blood of patients with acute myocardial ischemia. Direct evidence for enhanced platelet reactivity in viva. Am J Cardiol 1981;48:395-402. 16. Sobef M, Salzman EW, Davies GC, Handin RI, Sweeney J, Pfoetz J, Kurland G. Circulating platelet products in unstable angina pectoris. Circulation 1981;63:300-306.
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