Elevated beta thromboglobulin in peripheral venous blood of patients with acute myocardial ischemia: Direct evidence for enhanced platelet reactivity in vivo

SEPTEMBER

The American

Journal

1981

of CARDIOLOGY@ VOLUME NUMBER

48 3

CLINICAL STUDIES

Elevated Beta Thromboglobulin in Peripheral Venous Blood of Patients With Acute Myocardial Ischemia: Direct Evidence for Enhanced Platelet Reactivity in Vivo 1

THOMAS MARY

C.

SMITHERMAN,

MILAM,

MD

JANNIE

WOO,

PhD

JAMES

T. WILLERSON,

EUGENE

P. FRENKEL,

MD,

MD,

FACC

FACC

MD

Dallas and Houston, Texas

From tfm Veterans Administration Medical Center, Parkland Memorial Hospital, and the Cardiology Division of the Departnwnt of Medicine of the University of Texas Health Sctence Center, Daltas. Texas, and the University of Texas Medical School, Houston, Texas. This study was supported in part by the Medical Research Service of the Veterans Administration, Washington, D.C.. tfw Eugene B. McDermott Foundation, Dallas, Texas, Gsants CA231 15 and CA18132 from the National Cancer Institute. Washington. DC. and Grant fit_-17669 from the lschemic Heart Center Specialized Center of Besearch, National InstiMes of Health, Bethesda. Maryland. Manuscript received June 30.1980; revised manuscript received March 19. 1981. accepted March 31, 1981. Address for reprints: Thomas C. Smitherman, MD. Veterans Administration Medical Center (11 lA6). 4500 South Lancaster Road, Dallas, Texas 75216.

Levels of beta thromboglobulin, a platelet-specific protein, In platelet-Poor plasma from peripheral venous samples of patients wlth acute myocardlal Ischemia were measured in order to obtain direct evidence for enhanced platelet reactlvlty in vivo in these patients and to determine the value of beta thromboglobulln assay in studying platelet reactivity in patients with ischemlc heart disease. The normal beta thromboglobulln concentration in peripheral venous plasma, determined from normal volunteers and patients without known ischemic heart disease or disorders associated with enhanced platelet destruction (control group without platelet destruction), was 30.0 f 12.6 ng/ml (mean f standard deviation) (range 10 to 59). The mean beta thromboglobulin level In hospltallzed patients wlth chest pain judged not to be due to acute myocardlal lschemia was 33.7 f 12.0 (range 11 to 65). Patients with deep venous thrombosis, disseminated intravascular coagulation, thrombotlc thrombocytopenk purpura and idiopathic thrombocytopenic purpura provided a control group of patients with enhanced platelet destruction; the beta thromboglobulln level in this group was 131.0 f 12.6 ng/ml (range 36 to 250). Elevated beta thromboglobulin levels were observed In 4 (16 percent) of 25 patients wtth stable angina pectorls (mean 45.0 f 22.6 ng/ml, range 22 to 125), in 23 (59 percent) of 39 patients wlth unstable angina pectoris (mean 63.2 f 73.7 ng/ml, range 14 to 910) and in 12 (60 percent) of 15 patients wlth acute myocardlal lnfarcti~(mean 118.5 f 66.2 ng/ml, range 30 to 420) on one or more occasions during hospitalization. The mean valm of patients wlth platelet destructton in the control group (p
September 1961

The American Journal of CARDIOLOGY

Volume 48

395

BETA THROMBOGLOBULIN

WITH ACUTE MYOCARDIAL

ISCHEMIA-SMITHERMAN

The potential role of blood platelets in the origin and complications of coronary atherosclerotic disease has been given much interest and study in the last 20 years. Many studies have examined platelet reactivity in pa-

tients with ischemic heart disease using a variety of in vitro tests or analyses of the in vivo survival of in vitro radionuclide-labeled autologous platelets. Others have examined the effect of drugs that inhibit platelet reactivity on the course of patients with ischemic heart disease. The results of many of these studies have recently been reviewed. 1-4Many, but not all, of the in vitro studies of platelet reactivity have suggested that the platelets of patients with ischemic heart disease are abnormally reactive. The results of platelet-active drug intervention trials have been mixed; both a beneficial effect in decreasing morbidity and mortality from ischemic heart disease5 and only a trend toward a beneficial effect or negative results have been reported.s,7 The relation of in vitro tests of reactivity to platelet reactivity in vivo is uncertain. Even when drug intervention trials with platelet-active agents demonstrate positive results in reducing cardiac deaths, the possibility remains that the beneficial effect is mediated by actions independent of or in addition to drug effects on platelet reactivity.5 Consequently, a method that provides a direct measurement of platelet reactivity and destruction in vivo has obvious attraction. Recent development of a highly sensitive radioimmunoassay for a platelet-specific protein, beta thromboglobulin, which is secreted during the platelet release reaction,s,g appears to provide such a method for investigation of platelet function in vivo.l”J1 A platelet-specific beta globulin was first described 2 decades ago12J” and was later found to be located in the granules.14-l6 It has been identified, purified and characterized recently and given the name beta thromboglobulin by Moore et a1.17J8 They found that it is a basic protein with molecular weight of about 36,000 daltons, is relatively inert chemically and constitutes a major fraction of the total protein of platelet alpha granules. Although the function of beta thromboglobulin remains obscure, Moore et al.ls suggested that it may be a matrix or packing protein, helping to stabilize the active constituents of the alpha granules. Ludlam et al.lOJ’ developed a sensitive radioimmunoassay for beta thromboglobulin. Use of the radioimmunoassay has confirmed the specificity of beta thromboglobulin to platelets.lg Increased platelet-poor plasma levels of beta thromboglobulin were recently documented during circumstances of platelet utilization20-24 and in disease states thought to result in platelet activation.25-27 In this investigation, we measured the level of beta thromboglobulin in the peripheral venous blood of patients with acute myocardial infarction and of patients with stable and with unstable angina pectoris in order to determine directly if states of acute myocardial ischemia are associated with enhanced platelet reactivity in vivo.

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Methods Beta Thromboglobulin Assay Radioimmunoassay of beta thromboglobulin was performed with a kit from Amersham (Amersham, Arlington Heights, Illinois). Assays were performed according to the manufacturer’s instructions. Aliquots were assayed independently by a nonkit radioimmunoassay utilizing beta thromboglobulinspecific antibody raised in rabbits in the laboratory by one of us (J. Woo). Results by these two methods were virtually identical in spite of storage and transport under the conditions to be described. Blood was drawn t,hrough a 20 gauge needle from a vein in the antecubital fossa occluded by a lightly applied t,ourniquet. Three ml of blood was drawn into a 10 ml plastic syringe, the needle removed and exactly 2.5 ml expressed into a plastic tube containing ethylenediaminetetra-acetic acid (EDTA) and theophylline. The tubes were immediately inverted once and placed in ice. Within 30 minutes of venipuncture, the specimens of blood were centrifuged at 1,500 g for 30 minutes at 0’ C. The top 0.5 ml of the platelet-poor plasma was removed from each specimen with a Pasteur pipette, transferred to a clear plastic tube and frozen at -20’ C. The samples were kept frozen until just before beta thromboglobulin assay was performed. To avoid artifactual elevation of plasma beta thromboglobulin levels owing to in vitro platelet destruction, all blood samples were drawn by the same two persons and all of the platelet-poor plasma samples were prepared by the same person. Normal volunteers were tested repetitively throughout the study period.

Patient Selection Patients were evaluated who were admitted to the clinical services of the Department of Internal Medicine at either Parkland Memorial Hospital or the Veterans Administration Medical Center, Dallas, Texas. The study group included 54 patients with acute myocardial infarction or unstable angina pectoris who were admitted to the coronary care units and 25 in-hospital and ambulatory patients who were undergoing evaluation for chest pain and were considered to have stable angina pectoris. Three control groups were studied. These included (1) 11 healthy laboratory workers and 10 patients in the hospital or outpatient clinics who were not known to have ischemic heart disease, thrombotic disorders or hematologic disorders associated with increased platelet destruction; (2) 7 patients who were admitted to the hospital with acute chest pain that was ultimately judged definitely not to be due to myocardial ischemia; and (3) 26 patients with deep venous thrombosis or hematologic diseases associated with increased platelet destruction.

Patients with acute myocardial infarction or unstable angina: Patients considered to have acute myocardial ischemia were those admitted to the hospital with acute myocardial infarction (15 patients) or unstable angina pectoris (39 patients). The criteria for the diagnosis of acute myocardial infarction or unstable angina pectoris and differentiation between the two syndromes were similar to those that we have used before.2Ss2 Both groups of patients complained of chest discomfort typical for myocardial ischemia. Patients with unstable angina had been admitted to the hospital with worsening of previous angina or the new onset of progressive angina within 4 weeks of admission and did not manifest progression to acute myocardial infarction by the criteria outlined subsequently.

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THROMBOGLOBULIN

Supporting evidence for the presence of ischemic heart disease was also required for the diagnosis of unstable angina and was established by one or more of the following criteria:

(1) documented previous myocardial infarction; (2) transient or persistent electrocardiographic evidence of myocardial ischemia (greater than 1 mm of horizontal or downsloping S-T segment depression for 0.08 second beyond the J point in the absence of digitalis glycoside ingestion or left ventricular hypertrophy); (3) previous positive exercise test; (4) coronary arterial luminal narrowing of 50 percent or greater documented by coronary arteriography; and (5) a previous history of typical angina pectoris. For the diagnosis of unstable angina pectoris, we excluded patients with possible precipitating events (congestive heart failure, anemia, hypoxia, arrhythmia, fever, hypotension, hypertension or thyrotoxicosis), concurrent disease (renal failure, hepatic failure, malignant neoplasm, severe valvular heart disease or diabetes mellitus out of control or with major complications) or myocardial infarction in the 3 months before admission. Acute myocardial infarction was documented by electrocardiographic QHS changes characteristic of transmural infarction or S-T segment and T wave abnormalities consistent with myocardial ischemia, or both, associated with elevation of any two of the three serum enzymes (creatine kinase, glutamic oxaloacetic transaminase or lactic dehydrogenase) to at least twice the upper limits of normal. Most of the patients with acute myocardial infarction also underwent technetium-%m pyrophosphate scintigraphy to document an acute infarction. All of these patients had “3-4 +” myocardial uptake of radionuclide-labeled pyrophosphate.:):’ Blood was drawn for beta thromboglobulin measurements from all but one of the patients with acute myocardial ischemia within 24 hours of hospital admission. Samples in ad-

dition to the initial one were obtained from 11 patients with acute myocardial infarction and 17 patients with unstable angina pectoris on one or more occasions during the first 7 hospital days and during hospital days 10 to 14. Patients with acute myocardial &hernia were excluded from this study if there was any reason to suspect deep venous

thrombosis or if they had received aspirin in the week before admission or had received sulfinpyrazone, nonsteroidal anti-inflammatory drugs or dipyridamole in the 3 days before admission. Patients with stable angina pectoris: All 25 patients with stable angina pectoris had typical angina pectoris and no unstable angina pectoris or myocardial infarction in the preceding 3 months. Twenty-two patients also had additional evidence for ischemic heart disease by meeting at least one of the previously mentioned criteria numbered 1 to 4. Blood was drawn during a pain-free interval at least 12 hours after the last episode of angina. Control groups: Normal volunteers were 11 healthy hospital workers who had taken no medicines in the week before the blood sample was drawn. Six in-hospital and four ambulatory clinic patients were chosen at random from patients who had no known ischemic heart disease, venous thrombotic disease or any hematologic disorder associated with increased platelet destruction. The group of patients with acute chest pain not due to myocardial ischemia consisted of seven patients who were

hospitalized to evaluate their pain and who, after complete study, were judged not to have acute myocardial ischemia. This categorization was based on a careful review of the medical history, physical examination, electrocardiogram and all other relevant data before the results of the beta thromboglobulin assays were known. All of these patients underwent

WITH

ACUTE

MYOCARDIAL

ISCHEMA-SMITHERMAN

ET AL.

further evaluation for objective evidence of ischemic heart disease. The results were positive for only one patient, who was found to have significant three vessel coronary atherosclerosis on coronary arteriography. Two to four plasma samples were obtained, the first within 24 hours of hospital admission and subsequent samples at a rate of approximately two samples each week during the remainder of their hospitalization. The control group of patients with disorders associated with accelerated platelet destruction included 10 patients

with acute deep venous thrombosis, 3 with disseminated intravascular coagulation, 2 with thrombotic thromhocytopenic purpura and 11 with idiopathic thrombocytopenic purpura. Statistical analysis: The significance of any differences among the mean beta thromboglobulin values in the patients with stable angina, unstable angina and acute myocardial infarction and the two control groups with and without evidence of enhanced platelet reactivity was determined by one way analysis of variance. The significance of within-group differences was determined with Student-Newman-Keuls tests.:l” All reported values are mean f standard deviation.

Results Normal control group: Beta thromboglobulin values in peripheral venous plasma from the 11 normal volunteers ranged from 10 to 39 ng/ml (mean 26.4 f 9.3). Values were similar in the 10 in-hospital and ambulatory patients who had neither ischemic heart disease nor disorders associated with enhanced platelet destruction (range 12 to 59 ng/ml, mean 34.1 f 15.0). The mean value of all of these persons, combined to give a single control group without platelet destruction, was 30.0 f 12.6 ng/ml. Because the range of beta thromboglobulin values for healthy persons and patients without enhanced platelet destruction has not been firmly established, we arbitrarily chose 59 ng/ml, the highest value recorded from our control group without platelet destruction, as a conservatively high value to distinguish abnormal from normal, a level in agreement with the previous experience of others (Table I). More than 95 percent of the apparently healthy persons in these studies from various laboratories have beta thromboglobulin values of 59 ng/ml or less. We observed little variation in beta thromboglobulin levels on repetitive testing of normal subjects. None of the values exceeded 59 ng/ml. In one normal volunteer tested 17 times over a 2 year period during the conduct of this and related studies values ranged ng/ml (mean 32.1 f 11.1). Control patients with nonischemic

from 13 to 51 chest

pain:

The beta thromboglobulin values of the patients with acute chest pain judged definitely not to be due to myocardial ischemia are shown in Table II. Only one of these patients (Case 1) had objective evidence of ischemit heart disease. The highest value in this group was only 65 ng/ml, and this was the single elevated value in the group. The mean value in these patients, 33.7 f 12.0 ng/ml, was almost identical to that (34.1 f 15.0 ng/ml) in the control group of in-hospital and ambulatory patients with neither ischemic heart disease nor enhanced platelet destruction. The marked variations in beta thromboglobulin values demonstrated in patients with

September 1981

The American Journal of CARDIOLOGY

Volume 48

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BETA THROMBOGLOBULIN

WITH ACUTE MYOCARDIAL

ISCHEMIA-SMITHERMAN

ET AL.

TABLE I Reported Values (ng/ml) of Beta Thromboglobulin in Platelet-Poor Plasma of Apparently Healthy Persons Values Reference (first author) Ludlam20 O’Brien” Burrowsz5 Ludlam’O Dawesg Preston*’ Campbell26 Boughton Ludlamlg

Range

Mean

95 % of All Values (mean f 2 SD)

30-58 4-50’ 5-64 3-34’ 14.5-80.9 8-66’ 12-90

38.7

20.5-56.9

l

z2.9

27.8 12.5 30.8

3-185

Extrapolated from figure. SD = standard deviation; -

4-34 3.3-58.1

31047

3-52.6 6-32 lo-65

Fraction of Persons With Value >59 ng/ml 0135’ o/33* l/35’ o/13* -I35 3163’ 3135’ -I18 71180’

l

acute myocardial group. Control

= not specified.

&hernia

patients

were not observed

with platelet

in this

destruction:

Beta

thromboglobulin values ranged from 19 to 500 ng/ml (mean 136.3 f 130.1) in the 11 patients with idiopathic thrombocytopenic purpura; values were 68 and 200 ng/ml, respectively, in the two patients with thrombotic thrombocytopenic purpura and 73,160 and 500 ng/ml, respectively, in the three patients with disseminated intravascular coagulation and ranged from 38 to 250 ng/ml (mean 90.5 f 61.9) in the patients with deep venous thrombosis. The mean value in all of these patients, combined to give a single control group with platelet destruction, was 131.0 f 121.6 ng/ml. All but five of these patients had elevated beta thromboglobulin values. These five (and their respective beta thromboglobulin levels) included one patient with idiopathic thrombocytopenic purpura (19 ng/ml) and four patients with deep venous thrombosis (38, 50, 56 and 56 ngl ml). Unstable angina pectoris: Beta thromboglobulin values in the 39 patients with unstable angina pectoris ranged from 14 to 910 ng/ml (mean 83.2 f 73.7) (Table III, Fig. 1). Twenty-three (59 percent) had elevated beta thromboglobulin values on one or more tests, and 17 (74 percent) of the 23 had an initially elevated value. Sixteen of the 39 patients were tested on more than 1 day of their hospitalization. Three patients (Cases 4,7 and 8) had very high beta thromboglobulin levels (910,520

TABLE II Beta Thromboglobulin Values (ng/ml) in Seven Patients With Acute Chest Pain Judged Not to Be Due to Myocardial lschemia Hospital Day

398

Case

1

3-4

6-7

1

21 18

29 25

39

3’ 4

z

.”

65

ii

7

42

:z

1::

September 1981

8-9

39

36 47

J3” ,..

is’

:::

35

13-14

3.2’

and 600 ng/ml, respectively) just before hospital discharge at a time when their angina was well controlled. There was no apparent temporal relation in these patients between in-hospital elevation of beta thromboglobulin level and recurrence of unstable angina pectoris, electrocardiographic changes or drug administration. Acute myocardial infarction: Beta thromboglobulin values in the 15 patients with acute myocardial infarct.ion ranged from 30 to 420 ng/ml (mean 118.5 f 66.2) (Table III, Fig. 1). Twelve (80 percent) of these 15 patients had one or more elevated values, and 11 (92 percent) of the 12 had an initially elevated value. Eleven of the 15 patients had multiple beta thromboglobulin determinations. Beta thromboglobulin values generally remained elevated for the first 3 to 4 hospital days. As with the patients with unstable angina pectoris, there was no apparent temporal relation between changes in beta thromboglobulin values and the frequency or severity of angina, electrocardiographic or serum enzyme changes or drug administration during the in-hospital phase of recuperation. Role of heparin therapy: We found no apparent relation between elevations in beta thromboglobulin and the administration of heparin to patients with acute myocardial infarction or unstable angina pectoris. Fourteen of these 54 patients with acute myocardial ischemia received small dose heparin by subcutaneous administration at intermittent times during hospitalization. Eighteen beta thromboglobulin values obtained from 11 patients who received heparin on the same day ranged from 24 to 350 ng/ml. Nine of these 18 values were in the normal range. Four patients whose beta thromboglobulin level was measured more than once received heparin intermittently; we could discern no apparent heparin-induced alterations of beta thromboglobulin levels in these patients. Stable angina pectoris: Beta thromboglobulin levels in the 25 patients with stable angina pectoris, obtained during pain-free intervals, ranged from 22 to 125 ng/ml (mean 45.0 f 22.6) (Fig. 1). Four (16 percent) of the 25 patients had elevated beta thromboglobulin values. Comparison among the five patient groups: There were significant differences (F = 8.02, p <0.0005) in the

The American Journal of CARDIOLOGY Volume 48

BETA THROMBOBLOBULIN WITH ACUTE MYOCARDIAL ISCHEMIA-SMITHERMAN ET AL.

mean beta thromboglobulin levels in the five groups (Fig. 1). These groups are arranged in rank order according to ascending beta thromboglobulin values as follows: (1) control patients without enhanced platelet reactivity, (2) patients with stable angina pectoris, (3) patients with unstable angina pectoris, (4) patients with acute myocardial infarction, and (5) control patients with enhanced platelet reactivity. The mean values of the patients in the control group with platelet destruction (p
TABLE Ill Beta Thromboglobulln Levels (ng/ml) in 15 Patients Wlth Acute Myocardlal lnfarctlon and 39 Patients Wlth Unstable Angina Pectoris Hospital Day 1

Case

4

3

5-7

10-14

15 Patients With Myocardial Infarction

.

1 3 4 5

92 si

86

50

iib

‘ii

:::

1::

1;:

:::

ii

:::

:::

:::

6

139(3)’ 104 132(6)* 87(21)’ 300

ibi

1::

1::

1::

1::

:

1::(6,*

117 196

260 141

426

:::

: ::

9

149(24)’ 240

94

iSi

1::

1::

1::

.

.

..I

... .

:::

1::a9

.

..

.

:‘: ::

145 5: 40

.

::

1;:

ii% ...

Discussion This study provides direct evidence for enhanced in vivo platelet reactivity and platelet product release, as demonstrated by elevated beta thromboglobulin levels, in most patients (80 percent) with acute myocardial infarction and the majority of patients (59 percent) with unstable angina pectoris. The mean beta thromboglobulin values in peripheral venous plasma of patients with acute myocardial ischemia were higher than those in normal control subjects and patients with chronic stable angina; these values were almost as high as those in patients with deep venous thrombosis and hematologic disorders associated with increased platelet destruction. Beta thromboglobulin levels in the last group, control subjects with enhanced platelet destruction, were similar to those reported by others for patients with deep venous thrombosis,20,21,2s thromboembolism,‘O thrombocytosis secondary to myeloproliferative disorders,24 preeclampsia of pregnancy22 and diabetes mellitus.2”-27 It appears unlikely that the elevations in beta thromboglobulin in patients with acute myocardial ischemia were simply a result of an acute illness and hospitalization and their attendant stresses because similar elevations were not observed in patients admitted to the hospital with acute chest pain judged not to be due to myocardial ischemia. The results of this study also demonstrate, not unexpectedly, elevation of beta thromboglobulin in patients with disseminated intravascular coagulation, thrombotic thrombocytopenic purpura and idiopathic thrombocytopenic purpura. Role of abnormal platelet reactivity in ischemic heart disease: Platelets could participate in complications of ischemic heart disease in several ways. Aggregation of platelets at an atherosclerotic plaque

2

.

iii

‘34 120

39 Patients With Unstable Angina Pectoris

ii

. 53

62 z; I:

.

132 64 820 107 65

54

zi

::

39

t: a2

:B

bi :: ii .55 .56 42

44 .

44

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...

32 60 350 17 30 15 14

ii

12 53 24 1% 54 70

:1

48

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kil 600

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Two patients with infarction (Cases 5 and 8) had several samples drawn during the 1st hospital day. The number in parentheses indicates the number of hours that had elapsed since the apparent onset of the infarction at the time the sample was obtained.

September 1981

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The American Journal of CARDIOLOGY

Volume 48

399

BETA

THROMBOGLOBULIN

1

WITH

ACUTE

MYOCARDIAL

ISCHEMIA-SMITHERMAN

ET AL.

PC 0001 I

I

P< 0.001

I

P<

I 0.05

I

I

NS I

I

PC 0.01 r

I

140

NS

P
1zo-

,120 100

looPC0025

= 5 -c

80-

80

d

60-

60

40-

40

20-

20

5

O-

CONTROLS WITHOUT FNHANCED PLATELET DESTRUCTION

STABLE ANGINA PfCTORIS

UNSTABLE ANGINA PECTORIS

may form an obstructing platelet plugs” or initiate a coronary arterial thrombus. Platelet aggregates in the myocardial microcirculation may cause some cases of myocardial infarction and sudden cardiac death.:‘” Platelet aggregation releases thromboxane As, a powerful vasoconstrictor.s7 The higher mean values of beta thromboglobulin in patients with unstable angina and myocardial infarction compared with values in patients with chronic stable angina tend to support the concept that abnormal platelet reactivity is involved in syndromes of acute myocardial ischemia. Nevertheless, our results do not provide definite evidence that the enhanced platelet reactivity demonstrated in patients with acute myocardial infarction and unstable angina was a cause of their clinical deterioration. The temporal relation of elevated beta thromboglobulin values to clinical deterioration was not precise. Of the patients with acute myocardial ischemia and at least one elevated beta thromboglobulin value, the initial value was elevated in 92 percent of the patients with acute myocardial infarction but in only 74 percent of the patients with unstable angina pectoris. Furthermore, beta thromboglobulin was elevated in five of seven patients with either acute myocardial infarction or unstable angina pectoris 10 to 14 days after admission, a time

400

September 1991

The American Journal of CARDIOLOGY

CONTROLS WITH ENHANCED PLATELET DESTRUCTION

0

FIGURE 1. Beta thromboglobulin levels in plateletpoor plasma in the five study groups. The insert in each bar shows the mean f standard deviation and the number (n) of persons in each group. The probability (P) values for the differences among the mean values of the groups are shown on the appropriate connecting lines. NS = not significant.

when the patients were in clinically improved and stable condition (Table III). Significance of fluctuations in beta thromboglobulin levels: The considerable fluctuations in beta thromboglobulin levels we observed in patients with acute myocardial ischemia (Table III) surprised us. It appears that some of the patients in our study had recurring episodes of enhanced platelet reactivity in spite of apparent clinical improvement, especially the patients (Cases 4,7 and 8, Table III) with unstable angina who had very high beta thromboglobulin levels just before hospital discharge when their angina was controlled and their activity level was nearly normal. It appears unlikely that these variations in beta thromboglobulin values were artifactual or fortuitous, because we did not observe similar variations in normal subjects or in patients admitted to the hospital with acute chest pain judged not to be due to myocardial ischemia. In the 3 month follow-up period after discharge from the hospital, two of these three patients (Cases 4 and 7) experienced severe (New York Heart Association3s functional class IV) angina pectoris. The third patient (Case 8) complained of only minimal discomfort. Nevertheless, further work will be necessary to determine if continued or recurrent increases in platelet reactivity

Volume 48

BETA THROMBOGLOBULINWITH ACUTE MYDCARDIAL ISCHEMIA-SMITHERMAN ET AL.

as manifested by elevated beta thromboglobulin values are a prognostic marker for an increased risk of morbidity and mortality. From the results of this study, it is not possible to conclude that any particular degree of peripheral venous beta thromboglobulin elevation is associated with the immediate development of worsening angina. Comparison with previous studies: Our results are in agreement with the few previously reported data on beta thromboglobulin measurements in patients with ischemic heart disease. Preliminary results in patients with acute myocardial infarction were reported in two concurrently published letters to the editor.21,3g O’Brien et a1.21 measured beta thromboglobulin levels from a single sample in 17 patients 1 to 12 days after acute myocardial infarction. Two of these patients had values (80 and 120 ng/ml, respectively) that were well above their control values of 5 to 50 ng/ml. Denham et a1.3g measured beta thromboglobulin in four elderly patients with transmural acute myocardial infarction and mural thrombosis demonstrated at autopsy. From 11 measurements in these four patients, they determined a log mean value of 108.6 ng/ml and a 95 percent range of 47.4 to 248.8 ng/ml, which was significantly higher than the values of their elderly control patients. Han et aL40 and Steele41 recently reported in abstract form preliminary results demonstrating elevated beta thromboglobulin levels in patients with chronic angina. Our results are also in agreement with the limited data available on platelet factor 4, another plateletspecific protein, in patients with ischemic heart disease. Handin et a1.42reported elevations of platelet factor 4 in about 50 percent of their patients with acute myocardial infarction. About one third of their patients with unstable angina had some elevation of platelet factor 4. They did not find elevations in patients with chronic stable angina. Ellis et a1.43recently reported preliminary results of platelet factor 4 measurements in patients with chronic stable angina, unstable angina and acute myocardial infarction; the mean values in all three groups were significantly elevated. Clinical applications: Our results, which show substantial differences between the patients in the control groups with and without enhanced platelet reactivity, provide strong evidence that the beta thromboglobulin levels reflect an in vivo product of platelet activation and are not the result of residual platelets in the plasma sample. 44 Whereas the usefulness of beta thromboglobulin assay in patients with acute myocardial ischemia cannot be established until there are more data to indicate a cause-effect relation for enhanced

platelet reactivity and episodes of acute myocardial ischemia, our results suggest that assay of beta thromboglobulin is helpful in studying the state of platelet reactivity in patients with ischemic heart disease. The assay of such platelet-specific products that are secreted during the platelet-release reaction should obviate many of the uncertainties inherent in studies of platelet function that rely solely on in vitro platelet tests or platelet survival techniques. Use of beta thromboglobulin assays in studies should be complementary to use of platelet factor 4 assays. The very short half-life of platelet factor 4 in the blood9 recommends its use when examining short-term changes in platelet activation in vivo. The longer half-life of beta thromboglobulin in the blood (about 100 minutes)g recommends its use in gaining an estimate of platelet activity over longer periods of time and in situations in which phlebotomy immediately after an acute clinical event is impossible. Beta thromboglobulin assay may be the more useful method in patients receiving heparin anticoagulation. Administration of heparin has been reported to lead to in vivo release of platelet factor 4 but not beta thromboglobuling Although we did not study this issue systematically, the lack of any consistent effect of heparin administration on the beta thromboglobulin values of our patients

supports

that observation.

Implications: Future studies using beta thromboglobulin as a direct indicator of in vivo platelet reactivity should be of value in answering several important questions in patients with ischemic heart disease. These include: (1) What is the temporal relation of alterations in platelet reactivity to clinical deterioration? (2) Is enhanced platelet reactivity a prognostic marker for subsequent severe angina pectoris, acute myocardial infarct and cardiac death? (3) What drug treatment is most efficacious in diminishing platelet reactivity? Answers to the first two questions will aid in determining whether enhanced platelet reactivity in these patients is a cause or an effect of the various complications of the disease. Acknowledgment We are grateful to Joel L. Moake, MD and John D. Olson, MD, PhD for their assistance in developing and refining the human beta thromboglobulin radioimmunoassay used in these studies and for their frequent and thoughtful advice, Larry L. Burden for obtaining blood samples, Michael J. Newton for performing the beta thromboglobulin assays, the nursing staff and medical house staff in the intensive care.units for their cooperation and Betty Bibus for assistance in preparing the manuscript.

References Weiss HJ. Antiplatelet drugs: a new pharmacologic approach to the prevention of thrombosis. Am Heart J 1976;86:86-102. Packham MA, Mustard JF. Clinical pharmacology of platelets. Blood 1977;50:555-73. Haff JI. Role of blood platelets in coronary artery disease. Am J Cardiol 1979;43:1197-206. Schafer Al, Handin RI. The role of platelets in thrombotic and

vascular disease. Prog Cardiovasc Dis 1979;22:31-52. 5. The Anturane Reinfarction Trial Research Group. Sulfinpyrazone in the prevention of sudden death after myocardial infarction. N Engl J Med 1980;302:250-6. 6. The Persantine-Aspirin Reinfarction Study Research Group. Persantine and aspirin in coronary heart disease. Circulation 1980; 62:449-61.

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