451
Atherosclerosis, 32 (1979) 451-460 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
PLATELET AGGREGATION IN RELATIONSHIP TO PLASMA CATECHOLAMINES IN PATIENTS WITH HYPERTENSION
NICOLAS
D. VLACHAKIS
and LOUIS ALEDORT
Hypertension and Hematology Divisions of the Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, NY (U.S.A.) (Received 16 August, 1978) (Revised, received 15 December, (Accepted 29 December, 1978)
1978)
Summary Plasma catecholamine concentration and platelet aggregation were studied in 22 patients with uncomplicated primary hypertension and 13 age-matched normotensive, healthy subjects at rest and in some during isometric handgrip exercise. The effect of norepinephrine (NE) infusion upon platelet aggregation was also examined. Plasma catecholamine concentration was slightly higher in the hypertensive than the normotensive group, but the difference was not significant. However, platelet aggregation to ADP was significantly greater in the hypertensive than the normotensive subjects. Exercise increased significantly both catecholamines and aggregation in both groups. Platelet aggregation was correlated with age (r = 0.62, P< 0.01) and plasma NE (r = -0.34, P< 0.05 for the total group of subjects). The infusion of NE increased significantly plasma NE and platelet aggregation and there was an inverse correlation between NE increase and threshold decrease (r = -0.69, P < 0.05). Thus, plasma catecholamines are important determinants of platelet aggregation. However, in our study, uncomplicated primary hypertension was not associated with abnormal plasma catecholamine concentration. It is likely that the observed abnormal platelet aggregability to ADP represents a secondary phenomenon, possibly related to more advanced atherosclerotic vascular changes in hypertensive than normotensive subjects. Key words:
Exercise -Hypertension
-Plasma
catecholamines
-Platelet
aggregation
This work was supported by the Heart Research Foundation, Inc. and the Ma%ie Boas Memorial Fund. Part of the work was presented in the 6th International Congress on Haemostasis and Tbromboti. Philadelphia, June 1977. Reprint requests to: Nbolas D. Vlachakfs. M.D., Hypertension Division, University of Southern Callfornia, 2025 Zonal Avenue. Los Angeles, CA 90033. U.S.A.
452
Introduction Individuals with hypertension have more atherosclerosis than do normotensive subjects [ 11. There is strong evidence that the rate of development of degenerative arterial disease is related to the blood pressure [ 21. Several investigators have suggested that platelet abnormalities may be a factor in the development and progression of atherosclerosis [ 31. Platelet dysfunction has been observed in patients with ischemic heart disease, and cerebrovascular disease [4-61. Age [7], sex, smoking [ 81, hyperlipidemia [9],, diabetes mellitus [lo], stress [ 111, and hypertension [ 12-141, all suggested risk factors for atherosclerosis, have been found to affect platelet function. Both epineand pophrine and norepinephrine bring about platelet aggregation [l&--17], tentiate the aggregation of platelets by adenosine diphosphate [ 181. Increased platelet adhesiveness has been found in patients with pheochromocytoma and elevated plasma catecholamines, which returned toward normal after successful operation [ 191. Many laboratories have reported that patients with essential hypertension may have increased levels of plasma catecholamines compared to normotensive subjects [20,21]. Also it has been suggested that patients with labile hypertension have a higher blood pressure and catecholamine response to stress than normotensive controls [ 221. In the present study we report the results of platelet aggregation and plasma catecholamine studies in patients with uncomplicated primary hypertension and healthy subjects, at rest and in some during isometric handgrip exercise. The effect of norepinephrine infusion upon platelet aggregation also has been examined. Material and Methods Plasma catecholamines and platelet aggregation studies were carried out in 22 patients with uncomplicated primary hypertension and 13 normotensive, healthy subjects at rest. In 12 subjects from each group, similar studies were performed during exercise. Subjects with cigarette smoking habits, diabetes mellitus, lipid abnormalities, manifest atherosclerotic vascular disease, kidney or heart failure and thyroid dysfunction were excluded from the study. Drugs of any sort were discontinued for at least two weeks prior to the study. Routine testing in the patients with hypertension consisted of physical examination and laboratory procedures including electrocardiogram, chest X-ray, rapid sequence pyelogram, complete blood count, routine urinalysis, multi-channel automated blood chemistries and urinary catecholamine metabolite test. Blood was withdrawn for complete blood count and multichannel automated blood chemistries from the normal controls. Following a 12-h fasting period platelet aggregation studies were carried out during the morning hours, in a quiet room. Patients were either at rest or, in some cases, exercising. Before testing, each subject was instructed in the procedure and the technique of handgrip exercise, while during the procedure no discussion with the subject was permitted. Each subject was asked to lie comfortably on a bed and a 19-gauge siliconized needle was inserted, without the use of a tourniquet, into an antecubital vein. A slow infusion of physiologic saline was
453
used to keep the needle open. The catheter remained in place throughout the study. Following a 20-min period of relaxation, blood was withdrawn. Subsequently the subject (12 each of the normotensive and hypertensive groups) was asked to stand for 10 min and then subjected for 3 min to the physical stress of handgrip, using 2/3 of their maximal voluntary contraction, measured on a hand ergometer. After exercise blood was again obtained. Platelet aggregation studies were performed using the turbidimetric method of Born [23] as modified by Mustard et al. [24] with adenosine diphosphate (ADP) as the aggregating agent. Blood was sampled by free flow into plastic tubes containing l/10 volume acid citrate 3.2% of concentration and plateletrich plasma (PRP) was separated by immediate centrifugation at 150 X g for 15 min at room temperature. After removal of the upper two-thirds of the PRP, the remaining specimen was recentrifuged at 600 X g for 10 min to yield platelet-poor plasma (PPP). The platelet count of the PRP was adjusted to 300,000/mm3, when necessary, with the autologous PPP. Platelet aggregation was completed within 2 h of sampling. Samples of 0.45 ml of PRP in siliconized cuvettes were stirred while prewarming for 1 min, then placed in the aggregometer. The light transmission obtained with the tested PRP sample at 1 PM final concentration of ADP was compared to the light transmission of the blank. Autologous PPP was used as blank and set for 100% transmission. The degree of aggregation is related to the increase in light transmission since clumping reduces light absorption. All experiments were started with 1 PM ADP concentration and the percent light transmission recorded. Subsequently the minimal concentration of ADP (threshold) to induce the typical biphasic curve (irreversible aggregation) was determined (Fig. 1). ADP was maintained as a 10e3 M frozen stock solution and was diluted just before use with saline buffer. We determined two indices of platelet aggregability: (a) the percent of light transmission at 1 PM ADP and (b) the threshold for biphasic aggregation, i.e. the minimal concentration of ADP to induce a typical biphasic curve (Fig. 1). The intra- and interassay variability of both indices was determined in 5 subjects by testing multiple samples of similar platelet count in 3 consecutive days. There was a 1% variability for the light transmission, whereas the same concentration of ADP has produced the typical biphasic curve in all samples.
ADP
Fig. 1. Platelet aggregation indices. a: percent of light transmission during first phase of aggregation 1 PM ADP. This refers to the aggregation which occurs during first phase. b: typical biphasic curve.
at
454
For measurement of plasma catecholamines, the blood was withdrawn in a heparinized syringe and quickly transferred in a tube containing reduced glutathione and ethylene bis-(oxyethylene nitrilo) tetraacetic acid (EGTA). The tube was inverted gently and placed on ice until the end of the testing, then the plasma was separated in a cold centrifuge and kept in a freezer at -20°C until assayed (within two weeks of collection). A modification of the radioenzymatic assay of Passon and Peuler [25] and Peuler and Johnson [ 261 was used for measurement of norepinephrine and epinephrine. The assay is based on the conversion of catecholamines to their 0-methylated derivatives in the presence of catechol-O-methyl transferase and S-adenosyl methionine-3H. The total incubation volume was 70 ~1 containing 40 ~1 of plasma, 10 ~1 of buffer (1 mM tris, 100 mM EGTA, 600 mM MgCl*), 10 ~1 of the enzyme and 10 ~1 of the isotope activity 8.8-11.3 Ci/mmol). The (New England Nuclear Corp., specific decrease of the total incubation volume to 70 ~1, as compared to the Peuler and Johnson assay, produced an increase in sensitivity of assay by 25%. Also the increase in the concentration of MgCl?, in comparison to the assay of Peuler and Johnson, was associated with a further 25% increase in the sensitivity of epinephrine. For development of the chromatographic plates methyl alcohol and NH40H 1.4 M, 2 : 1, was used as a solvent, which required 15 min only for perfect separation of the normetanephrine and metanephrine bands. The bands containing the 0-methylated analogs were scraped directly into vials and counted by liquid scintillation. Approximately one picogram of catecholamine could be measured with an interassay coefficient of variation of 1.2% and 2.3% for norepinephrine and epinephrine, respectively. In each experiment aliquots of the same pooled plasma were assayed. To determine the effect of levels of NE found in the blood during real life stressful conditions upon platelet aggregation, we infused 10 subjects in the recumbent position with NE at a rate of 0.1 Erg/kg body weight/min for 15 min. Blood pressure was recorded every 2 min with an automatically inflated device (Arteriosonde 1216). Blood was withdrawn before and at the end of infusion for measurement of plasma catecholamines and platelet aggregation. For statistical analysis the Student t-test was used for comparison between groups and the paired t-test for changes within the same group. Correlation coefficients were computed. Results are expressed as mean f standard error of mean. Results Plasma catecholamines and platelet aggregation studies at rest and duringexercise Included in the normotensive group were 10 male and 3 female subjects with a mean age of 44 f 2.1 years, while in the hypertensive group were 15 male and 7 female patients with a mean age of 50 + 2.3 years (NS). The norepinephrine and epinephrine levels at rest were 234 f 25 pg/ml and 35 f 2.7, respectively, in the normotensive group and 277 i: 19 and 45 * 6.8, respectively, in the hypertensive group (NS). During standing isometric handgrip exercise both NE and E increased significantly in both groups (P < 0.01 for both) (Figs. 2 and 3),
455 Norepinephrme at Rest and During Exercise
Plasma Epinephrine Before and During Exercise
200180160140-i \ 120a” IOOW 80Q 6040200
F 1000 900a” 800w 700$ 600500400 300200 IOO-
0
n=l2 Rest Ex NORMOTENSIVE
n=ll Rest Ex HYPERTENSIVE
Fig. 2. Plasma norepinephrine in 12 norm&n&e (Ex).
n =I2 Rest Ex NORMOTENSIVE
n I2 Rest Ex HYPERTENSIVE ??
and 11 hypertensive subjects at rest and during exercise
Fig. 3. Plasma epinephrine in 12 normotensive and 12 hypertensive subjects at rest and during exercise.
but neither the attained levels nor the extent of the change was significantly different between the two groups. At rest, the percent of light transmission at 1 PM concentration of ADP was 28.0 f 2.0 for the normotensive and 36 + 2.3 for the hypertensive group (I’< 0.02). During exercise it increased significantly in both hypertensive (P < 0.025) and normotensive (P < 0.01) subjects. The threshold for biphasic aggregation was 3.5 f 0.33 FM in the normotensive and 2.14 k 0.30 ,uM in the hypertensive group (P < 0.01). During exercise the concentration of ADP
TABLE 1 CLINICAL DATA, PLASMA CATECHOLAMINES BOTH GROUPS OF SUBJECTS
Age (yr.9 Sex NorepinepMne (pg/ml) at rest Epinephrine @g/ml) at rest % of light transmission at 1 PM ADP at rest Threshold of biphasic aggregation at rest
AND PLATELET
AGGREGATION
RESULTS
Normotensives (N) n= 13
Hypertensives (H) n=22
FvaIues (H) vs. (N)
44 f 2.1 10 M, 3 234 + 35 f 26.0 f 3.5 *
60 f 2.3 15M.7F 277 f 45 f 36 f 2.14 +
NS NS NS NS (0.02
Atherosclerosis, 32 (1979) 451-460 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
PLATELET AGGREGATION IN RELATIONSHIP TO PLASMA CATECHOLAMINES IN PATIENTS WITH HYPERTENSION
NICOLAS
D. VLACHAKIS
and LOUIS ALEDORT
Hypertension and Hematology Divisions of the Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, NY (U.S.A.) (Received 16 August, 1978) (Revised, received 15 December, (Accepted 29 December, 1978)
1978)
Summary Plasma catecholamine concentration and platelet aggregation were studied in 22 patients with uncomplicated primary hypertension and 13 age-matched normotensive, healthy subjects at rest and in some during isometric handgrip exercise. The effect of norepinephrine (NE) infusion upon platelet aggregation was also examined. Plasma catecholamine concentration was slightly higher in the hypertensive than the normotensive group, but the difference was not significant. However, platelet aggregation to ADP was significantly greater in the hypertensive than the normotensive subjects. Exercise increased significantly both catecholamines and aggregation in both groups. Platelet aggregation was correlated with age (r = 0.62, P< 0.01) and plasma NE (r = -0.34, P< 0.05 for the total group of subjects). The infusion of NE increased significantly plasma NE and platelet aggregation and there was an inverse correlation between NE increase and threshold decrease (r = -0.69, P < 0.05). Thus, plasma catecholamines are important determinants of platelet aggregation. However, in our study, uncomplicated primary hypertension was not associated with abnormal plasma catecholamine concentration. It is likely that the observed abnormal platelet aggregability to ADP represents a secondary phenomenon, possibly related to more advanced atherosclerotic vascular changes in hypertensive than normotensive subjects. Key words:
Exercise -Hypertension
-Plasma
catecholamines
-Platelet
aggregation
This work was supported by the Heart Research Foundation, Inc. and the Ma%ie Boas Memorial Fund. Part of the work was presented in the 6th International Congress on Haemostasis and Tbromboti. Philadelphia, June 1977. Reprint requests to: Nbolas D. Vlachakfs. M.D., Hypertension Division, University of Southern Callfornia, 2025 Zonal Avenue. Los Angeles, CA 90033. U.S.A.
452
Introduction Individuals with hypertension have more atherosclerosis than do normotensive subjects [ 11. There is strong evidence that the rate of development of degenerative arterial disease is related to the blood pressure [ 21. Several investigators have suggested that platelet abnormalities may be a factor in the development and progression of atherosclerosis [ 31. Platelet dysfunction has been observed in patients with ischemic heart disease, and cerebrovascular disease [4-61. Age [7], sex, smoking [ 81, hyperlipidemia [9],, diabetes mellitus [lo], stress [ 111, and hypertension [ 12-141, all suggested risk factors for atherosclerosis, have been found to affect platelet function. Both epineand pophrine and norepinephrine bring about platelet aggregation [l&--17], tentiate the aggregation of platelets by adenosine diphosphate [ 181. Increased platelet adhesiveness has been found in patients with pheochromocytoma and elevated plasma catecholamines, which returned toward normal after successful operation [ 191. Many laboratories have reported that patients with essential hypertension may have increased levels of plasma catecholamines compared to normotensive subjects [20,21]. Also it has been suggested that patients with labile hypertension have a higher blood pressure and catecholamine response to stress than normotensive controls [ 221. In the present study we report the results of platelet aggregation and plasma catecholamine studies in patients with uncomplicated primary hypertension and healthy subjects, at rest and in some during isometric handgrip exercise. The effect of norepinephrine infusion upon platelet aggregation also has been examined. Material and Methods Plasma catecholamines and platelet aggregation studies were carried out in 22 patients with uncomplicated primary hypertension and 13 normotensive, healthy subjects at rest. In 12 subjects from each group, similar studies were performed during exercise. Subjects with cigarette smoking habits, diabetes mellitus, lipid abnormalities, manifest atherosclerotic vascular disease, kidney or heart failure and thyroid dysfunction were excluded from the study. Drugs of any sort were discontinued for at least two weeks prior to the study. Routine testing in the patients with hypertension consisted of physical examination and laboratory procedures including electrocardiogram, chest X-ray, rapid sequence pyelogram, complete blood count, routine urinalysis, multi-channel automated blood chemistries and urinary catecholamine metabolite test. Blood was withdrawn for complete blood count and multichannel automated blood chemistries from the normal controls. Following a 12-h fasting period platelet aggregation studies were carried out during the morning hours, in a quiet room. Patients were either at rest or, in some cases, exercising. Before testing, each subject was instructed in the procedure and the technique of handgrip exercise, while during the procedure no discussion with the subject was permitted. Each subject was asked to lie comfortably on a bed and a 19-gauge siliconized needle was inserted, without the use of a tourniquet, into an antecubital vein. A slow infusion of physiologic saline was
453
used to keep the needle open. The catheter remained in place throughout the study. Following a 20-min period of relaxation, blood was withdrawn. Subsequently the subject (12 each of the normotensive and hypertensive groups) was asked to stand for 10 min and then subjected for 3 min to the physical stress of handgrip, using 2/3 of their maximal voluntary contraction, measured on a hand ergometer. After exercise blood was again obtained. Platelet aggregation studies were performed using the turbidimetric method of Born [23] as modified by Mustard et al. [24] with adenosine diphosphate (ADP) as the aggregating agent. Blood was sampled by free flow into plastic tubes containing l/10 volume acid citrate 3.2% of concentration and plateletrich plasma (PRP) was separated by immediate centrifugation at 150 X g for 15 min at room temperature. After removal of the upper two-thirds of the PRP, the remaining specimen was recentrifuged at 600 X g for 10 min to yield platelet-poor plasma (PPP). The platelet count of the PRP was adjusted to 300,000/mm3, when necessary, with the autologous PPP. Platelet aggregation was completed within 2 h of sampling. Samples of 0.45 ml of PRP in siliconized cuvettes were stirred while prewarming for 1 min, then placed in the aggregometer. The light transmission obtained with the tested PRP sample at 1 PM final concentration of ADP was compared to the light transmission of the blank. Autologous PPP was used as blank and set for 100% transmission. The degree of aggregation is related to the increase in light transmission since clumping reduces light absorption. All experiments were started with 1 PM ADP concentration and the percent light transmission recorded. Subsequently the minimal concentration of ADP (threshold) to induce the typical biphasic curve (irreversible aggregation) was determined (Fig. 1). ADP was maintained as a 10e3 M frozen stock solution and was diluted just before use with saline buffer. We determined two indices of platelet aggregability: (a) the percent of light transmission at 1 PM ADP and (b) the threshold for biphasic aggregation, i.e. the minimal concentration of ADP to induce a typical biphasic curve (Fig. 1). The intra- and interassay variability of both indices was determined in 5 subjects by testing multiple samples of similar platelet count in 3 consecutive days. There was a 1% variability for the light transmission, whereas the same concentration of ADP has produced the typical biphasic curve in all samples.
ADP
Fig. 1. Platelet aggregation indices. a: percent of light transmission during first phase of aggregation 1 PM ADP. This refers to the aggregation which occurs during first phase. b: typical biphasic curve.
at
454
For measurement of plasma catecholamines, the blood was withdrawn in a heparinized syringe and quickly transferred in a tube containing reduced glutathione and ethylene bis-(oxyethylene nitrilo) tetraacetic acid (EGTA). The tube was inverted gently and placed on ice until the end of the testing, then the plasma was separated in a cold centrifuge and kept in a freezer at -20°C until assayed (within two weeks of collection). A modification of the radioenzymatic assay of Passon and Peuler [25] and Peuler and Johnson [ 261 was used for measurement of norepinephrine and epinephrine. The assay is based on the conversion of catecholamines to their 0-methylated derivatives in the presence of catechol-O-methyl transferase and S-adenosyl methionine-3H. The total incubation volume was 70 ~1 containing 40 ~1 of plasma, 10 ~1 of buffer (1 mM tris, 100 mM EGTA, 600 mM MgCl*), 10 ~1 of the enzyme and 10 ~1 of the isotope activity 8.8-11.3 Ci/mmol). The (New England Nuclear Corp., specific decrease of the total incubation volume to 70 ~1, as compared to the Peuler and Johnson assay, produced an increase in sensitivity of assay by 25%. Also the increase in the concentration of MgCl?, in comparison to the assay of Peuler and Johnson, was associated with a further 25% increase in the sensitivity of epinephrine. For development of the chromatographic plates methyl alcohol and NH40H 1.4 M, 2 : 1, was used as a solvent, which required 15 min only for perfect separation of the normetanephrine and metanephrine bands. The bands containing the 0-methylated analogs were scraped directly into vials and counted by liquid scintillation. Approximately one picogram of catecholamine could be measured with an interassay coefficient of variation of 1.2% and 2.3% for norepinephrine and epinephrine, respectively. In each experiment aliquots of the same pooled plasma were assayed. To determine the effect of levels of NE found in the blood during real life stressful conditions upon platelet aggregation, we infused 10 subjects in the recumbent position with NE at a rate of 0.1 Erg/kg body weight/min for 15 min. Blood pressure was recorded every 2 min with an automatically inflated device (Arteriosonde 1216). Blood was withdrawn before and at the end of infusion for measurement of plasma catecholamines and platelet aggregation. For statistical analysis the Student t-test was used for comparison between groups and the paired t-test for changes within the same group. Correlation coefficients were computed. Results are expressed as mean f standard error of mean. Results Plasma catecholamines and platelet aggregation studies at rest and duringexercise Included in the normotensive group were 10 male and 3 female subjects with a mean age of 44 f 2.1 years, while in the hypertensive group were 15 male and 7 female patients with a mean age of 50 + 2.3 years (NS). The norepinephrine and epinephrine levels at rest were 234 f 25 pg/ml and 35 f 2.7, respectively, in the normotensive group and 277 i: 19 and 45 * 6.8, respectively, in the hypertensive group (NS). During standing isometric handgrip exercise both NE and E increased significantly in both groups (P < 0.01 for both) (Figs. 2 and 3),
455 Norepinephrme at Rest and During Exercise
Plasma Epinephrine Before and During Exercise
200180160140-i \ 120a” IOOW 80Q 6040200
F 1000 900a” 800w 700$ 600500400 300200 IOO-
0
n=l2 Rest Ex NORMOTENSIVE
n=ll Rest Ex HYPERTENSIVE
Fig. 2. Plasma norepinephrine in 12 norm&n&e (Ex).
n =I2 Rest Ex NORMOTENSIVE
n I2 Rest Ex HYPERTENSIVE ??
and 11 hypertensive subjects at rest and during exercise
Fig. 3. Plasma epinephrine in 12 normotensive and 12 hypertensive subjects at rest and during exercise.
but neither the attained levels nor the extent of the change was significantly different between the two groups. At rest, the percent of light transmission at 1 PM concentration of ADP was 28.0 f 2.0 for the normotensive and 36 + 2.3 for the hypertensive group (I’< 0.02). During exercise it increased significantly in both hypertensive (P < 0.025) and normotensive (P < 0.01) subjects. The threshold for biphasic aggregation was 3.5 f 0.33 FM in the normotensive and 2.14 k 0.30 ,uM in the hypertensive group (P < 0.01). During exercise the concentration of ADP
TABLE 1 CLINICAL DATA, PLASMA CATECHOLAMINES BOTH GROUPS OF SUBJECTS
Age (yr.9 Sex NorepinepMne (pg/ml) at rest Epinephrine @g/ml) at rest % of light transmission at 1 PM ADP at rest Threshold of biphasic aggregation at rest
AND PLATELET
AGGREGATION
RESULTS
Normotensives (N) n= 13
Hypertensives (H) n=22
FvaIues (H) vs. (N)
44 f 2.1 10 M, 3 234 + 35 f 26.0 f 3.5 *
60 f 2.3 15M.7F 277 f 45 f 36 f 2.14 +
NS NS NS NS (0.02
