Age and Ageing (1975), 4, 105
PLATELET 5-HYDROXY-TRYPTAMINE IN THROMBOTIC AND NON-THROMBOTIC DISEASES D. P. MISRA, G. STADDON, P. JACKSON, N . POWELL AND J. MISRA
Summary
It is generally accepted that platelets form the main constituent of a thrombus. Experimental work has shown that 5-hydroxy-tryptamine (5 HT) in a concentration of 0.06 mg/ml produces clumping of the platelets. In view of this it was decided to estimate platelet 5 HT in thrombotic and non-thrombotic diseases. Thirty-five patients were investigated. Estimations of 5 HT were made on washed platelets rather than platelet-rich plasma which has been used in previous studies. Three groups were recognized on the basis of platelet 5 HT values. The first group consisted of five subjects who were clinically asymptomatic at the time of investigation but died 24 to 48 hours later and were shown at autopsy to have deep-vein thrombosis and pulmonary embolism; they had the highest level of platelet 5 HT. The second group comprised eight patients with cerebral thrombosis whose platelet 5 HT ranged between 1529 and 2183 ng/109. There were 22 subjects in the third group (11 apparently normal and 11 with different pathologies). Their platelet 5 HT ranged from 611 to 877 ng/109 and did not vary in the different pathological conditions studied. Since the highest level of platelet 5 H T was observed in patients prior to the formation or embolization of a thrombus, it is suggested that this may have some role in initiating thrombus formation. INTRODUCTION
Platelets form the main constituent of a thrombus (Levene & Levene, 1957; Mitchell & Schwartz, 1965; Woolf & Carstairs, 1967) and consequently much work has been done to find out the factors which produce changes in the platelets during experimental and clinical thrombus formation. The role of adenosine diphosphate (ADP) in experimental thrombus formation has been confirmed on several occasions (Gaarder et al, 1961; Mitchell & Sharp, 1964; O'Brien, 1964; Begent & Bown, 1970). It has been shown that platelets became spherical before they became sticky (O'Brien & Heywood, 1966; Begent & Bown, 1970). This is followed by adhesion of platelets as loose aggregates and lastly they become closely packed. During the intermediary phase of the reaction, fibrinogen and calcium are also required. It has been found that platelet clumping is produced by various agents and all seem to act by formation or release of ADP. Since the platelets form the main part of a thrombus and are rich in 5 HT, it was decided to measure the platelet and plasma 5 HT in patients with cerebral thrombosis of less than 24-hours duration and compare it with patients suffering from other diseases of the same age-group.
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
United Bristol Hospitals, Bristol
D. P. Misra, G. Staddon, P. Jackson, N. Powell andj. Misra
106
Material and Method
RESULTS
On the basis of the results of platelet 5 HT, three groups were recognized (Table I). Table I. Platelet 5 HT (ng/109 platelets) Apparently Rheumatoid normal arthritis
Osteoarthritis
Deep vein Uraemia Anaemia Cerebral thrombosis with thrombosis pulm. embolism
3000
o°o°°
2500 2000
°o o o o o
1500
"o
1000 500
D o 0 0 o o OoOo°
°0°
°o o °
°o
°o
Group 1 There were five cases in this group who, at the time of investigation, had no clinical evidence of venous thrombosis or pulmonary embolism. Their platelet 5 HT ranged
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
Thirty-five cases (15 men and 20 women) between the ages of 60 and 70 years were studied. Eight had hemiplegia or hemiparesis of less than 24-hours duration. Four of them died and were found at autopsy to have stenosis of the extracranial portion of one internal carotid artery; one had angiography which showed occlusion of one internal carotid artery, and the remaining three patients were having recurrent ischaemic attacks suggestive of internal carotid artery stenosis but no angiogram was done to demonstrate the obstruction; two had developed monoplegia at the time of investigation. Of the remaining 27 patients, 14 were admitted for social reasons and had no active pathology, six were immobile on account of painful osteoarthritis, three had rheumatoid arthritis, two had uraemia due to chronic pyelonephritis and two had iron-deficiency anaemia. Venous blood was collected from each patient between 9.00 a.m. and 10.00 a.m. The sample for 5 H T estimation was obtained in bottles containing 3 per cent EDTA in 0.7 per cent NaCl. The whole blood was spun in a MSE Junior centrifuge at 1000 rev./min for 20 minutes to separate red cells. The plasma was removed and spun at 2300 rev./min for 30 minutes to separate platelets. The supernatant was kept for chemical analysis. A solution of 3 per cent EDTA in 0.7 per cent NaCl was added to the platelets. The platelets and solution were agitated with a Whirlimixer for 5 to 10 minutes to suspend the platelets in solution. The platelet suspension was spun at 2300 rev./min for 30 minutes to separate platelets from solution. The washings were withdrawn and the platelets suspended in the EDTA solution. 5 H T was estimated by the spectrofluorimetric method of Crawford & Rudd (1962). The proteins were precipitated with trichloracetic acid; excess trichloracetic acid and interfering fluorochromes were removed with ether and the 5 H T was extracted with salt-saturated butanol after pH adjustment with borate buffer. Iso-octane was added to an aliquot of the butanol extract and the 5 H T was then extracted into 3 N - H C I for fluorescence measurement. An Aminco-Borman spectrophotofluorimeter was used with an activating wavelength of 295 nm and a fluorescent wavelength of 540 nm.
Platelet 5-Hydroxy-tryptamine
in Thrombotic and Non-thrombotic Diseases
107
from 3101 to 3382 ng/109. They died 24 to 48 hours after the blood was taken for estimation, and autopsy revealed deep-vein thrombosis with pulmonary embolism. The deep veins affected were femoral in two cases and calf veins in three cases. Group 2 This group comprised eight cases of cerebral thrombosis. Their platelet 5 HT ranged from 1529 to 2183 ng/10».
The plasma 5 HT ranged from 19 to 87 ng/ml and did not show any variation in the different pathological groups studied (Table II). There was no correlation between the results of plasma and platelet 5 HT. Table II. Plasma 5 HT (ng/ml) Apparently normal subjects
Deep vein Rheumatoid Uraemia Anaemia Cerebral thrombosis with thrombosis pulm. embolism arthritis
Osteoarthritis
o
80 0
o o
70 o
o
0
50
0
40
o
o
O O
0
°
o
o
o o°
o
0
o o
O o
O o
O
30 20
o
10
DISCUSSION
The formation of platelet thrombi at the site of injured vessels was demonstrated over a century ago (Wharton-Jones, 1851). Since then much work has been done on different aspects of the problem, but the mechanism of thrombus formation still remains obscure. It was suggested that a chemical substance, adenosine diphosphate (ADP), which has been found to initiate experimental thrombus formation (Honour & Mitchell, 1964; Spaet & Zucker, 1964) may be responsible. Later thrombus formation was produced by the iontophoresis of ADP through intact vessel wall, which further confirmed the role of ADP in thrombus formation (Begent & Bown 1970). The second theory stressed the importance of damaged endothelium in causing thrombus formation, by exposing the basement membrane collagen to the blood (Lyman et al, 1971). It was also shown that washed platelets suspended in human ringer solution exhibit negligible adhesion in contrast to washed platelets suspended in plasma. Addition of
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
Group 3 This group consisted of various patients including those with osteoarthritis, rheumatoid arthritis, uraemia, anaemia, and those with no active pathology. The platelet 5 HT ranged from 611 to 877 ng/109.
108
D. P. Misra, G. Staddon, P. Jackson, N. Powell and J. Misra
ACKNOWLEDGEMENTS
We wish to thank Drs W. H. Lloyd and A. C. M. Windsor for permission to study their cases and Dr D. D. Gibbs for his comments and advice on the paper. We are very grateful to Mr D. A. Lothian for his help in estimating 5 HT. REFERENCES
N. & BOWN, G. V. R. (1970). Growth rate in vivo of platelet thrombi produced by iontophoresis of ADP as a function of mean blood flow velocity. Nature, Lond. 227, 926. CRAWFORD, N. & RUDD, B. T. (1962). A spectrophotofluorimetric method for the determination of serotonin (5-hydroxytryptamine) in plasma. Clin. Chim. Acta 7, 114. GAARDER, A., JONSEN, J., LALAND, S., HELLEM, A. J. & OWREN, P. A. (1961). Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature, Lond. 192, 531. HONOUR, A. J. & MITCHELL, J. R. A. (1964). Platelet clumping in injured vessels. Br. J. exp. Pathol. 45, 75. KAGAN, A., DAWBER, T. R., KANNEL, W. B. & REVOTSKIE, N. (1962). The Framingham study: a prospective study of coronary heart disease. Fed. Proc. 21, suppl. 11, 52. LEVENE, M. & LEVENE, C. I. (1957). Pulmonary platelet thromboembolism. J. clin. Pathol. 10, 200. L/YMAN, B., ROSENBERG, L. & KARPATKIN, S. (1971). Biochemical and biophysical aspects of human platelet adhesion to collagen fibres. J. Clin. Invest. 50, 1854. MITCHELL, J. R. A. & SCHWARTZ, C. J. (1965). Arterial Disease, pp. 248, 199. Oxford: Blackwell. MITCHELL, J. R. A. & SHARP, A. A. (1964). Platelet clumping in vitro. Br. J. Haemotol. 10, 78. MORRELL, M. T. & DUNHILL, M. S. (1968). The post-mortem incidence of pulmonary embolism in a hospital population. Br. J. Surg. 55, 347. BEGENT,
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
human fibrinogen to the ringer solution completely restored the ability of washed platelets to adhere to collagen fibres, thus suggesting that fibrinogen was the factor in the plasma, which was required for platelet adhesion to purified collagen. It is obvious that experimental thrombus formation is not analogous to clinical thrombosis. The relevance of experimental findings in the clinical situation is also doubtful. Clinical studies have concentrated on the value of predisposing and environmental factors in the pathogenesis of thrombus formation. In the Framingham survey (Kagan et ah, 1962), the risk factors in coronary artery thrombosis included elevated serum lipids, elevated blood pressure, cigarette smoking and advancing age. On the other hand, leg vein thrombosis has been linked with operation, injury, childbirth, immobility and malignant diseases (Morrell & Dunhill, 1968), with the recent addition of oestrogencontaining contraceptives (Vessey & Doll, 1969). Investigations so far carried out do not provide any evidence as to what initiates a thrombus and how it is propagated. Although the importance of ADP is greatly emphasized and its value in experimental thrombus formation is undisputed, less attention has been paid to 5 HT, adrenaline and noradrenaline. Comparing the results of Group 1, 2 and 3 cases, it is apparent that the platelet 5 HT rises prior to the formation or embolization of a thrombus and may, therefore, influence thrombus formation in man. On the evidence obtained in the present work it is difficult to explain the discrepancy in the findings of plasma and platelet 5 HT in thrombotic diseases, but it is suggested that the turnover of platelet 5 HT is increased, i.e. the production and degradation of 5 HT in the platelets is enhanced.
Platelet 5-Hydroxy-tryptamine
in thrombotic and Non-thrombotic Diseases
109
J. R. (1964). Platelet stickiness: some methods and some theory. J. din. Pathol. 17, 472. J. R. & HEYWOOD, J. B. (1966). Effect of aggregating agents and their inhibitors on the mean platelet shape. J. Clin. Pathol. 19, 148. SPAET, T. H. & ZUCKER, M. B. (1964). Mechanism of platelet plug formation and the role of adenosine diphosphate. Am. J. Physiol. 206, 1267. VESSEY, M. P. & DOLL, R. (1969). Investigation of relation between use of oral contraceptives and thrombo-embolic disease: a further report. Br. Med. J. 2, 651. WHARTON-JONES, T. (1851). On the state of the blood and blood vessels in inflammation. Guy's Hospital Reports. Series 2, 7, 1. WOOLF, N. & CARSTAIRS, K. C. (1967). Infiltration and thrombosis in atherogenisis. Am. J. Pathol. 51, 373. O'BRIEN, O'BRIEN,
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
Present address: D. P. Misra, Good Hope Hospital, Sutton Coldfield, West Midlands, BIS 1RR
PLATELET 5-HYDROXY-TRYPTAMINE IN THROMBOTIC AND NON-THROMBOTIC DISEASES D. P. MISRA, G. STADDON, P. JACKSON, N . POWELL AND J. MISRA
Summary
It is generally accepted that platelets form the main constituent of a thrombus. Experimental work has shown that 5-hydroxy-tryptamine (5 HT) in a concentration of 0.06 mg/ml produces clumping of the platelets. In view of this it was decided to estimate platelet 5 HT in thrombotic and non-thrombotic diseases. Thirty-five patients were investigated. Estimations of 5 HT were made on washed platelets rather than platelet-rich plasma which has been used in previous studies. Three groups were recognized on the basis of platelet 5 HT values. The first group consisted of five subjects who were clinically asymptomatic at the time of investigation but died 24 to 48 hours later and were shown at autopsy to have deep-vein thrombosis and pulmonary embolism; they had the highest level of platelet 5 HT. The second group comprised eight patients with cerebral thrombosis whose platelet 5 HT ranged between 1529 and 2183 ng/109. There were 22 subjects in the third group (11 apparently normal and 11 with different pathologies). Their platelet 5 HT ranged from 611 to 877 ng/109 and did not vary in the different pathological conditions studied. Since the highest level of platelet 5 H T was observed in patients prior to the formation or embolization of a thrombus, it is suggested that this may have some role in initiating thrombus formation. INTRODUCTION
Platelets form the main constituent of a thrombus (Levene & Levene, 1957; Mitchell & Schwartz, 1965; Woolf & Carstairs, 1967) and consequently much work has been done to find out the factors which produce changes in the platelets during experimental and clinical thrombus formation. The role of adenosine diphosphate (ADP) in experimental thrombus formation has been confirmed on several occasions (Gaarder et al, 1961; Mitchell & Sharp, 1964; O'Brien, 1964; Begent & Bown, 1970). It has been shown that platelets became spherical before they became sticky (O'Brien & Heywood, 1966; Begent & Bown, 1970). This is followed by adhesion of platelets as loose aggregates and lastly they become closely packed. During the intermediary phase of the reaction, fibrinogen and calcium are also required. It has been found that platelet clumping is produced by various agents and all seem to act by formation or release of ADP. Since the platelets form the main part of a thrombus and are rich in 5 HT, it was decided to measure the platelet and plasma 5 HT in patients with cerebral thrombosis of less than 24-hours duration and compare it with patients suffering from other diseases of the same age-group.
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
United Bristol Hospitals, Bristol
D. P. Misra, G. Staddon, P. Jackson, N. Powell andj. Misra
106
Material and Method
RESULTS
On the basis of the results of platelet 5 HT, three groups were recognized (Table I). Table I. Platelet 5 HT (ng/109 platelets) Apparently Rheumatoid normal arthritis
Osteoarthritis
Deep vein Uraemia Anaemia Cerebral thrombosis with thrombosis pulm. embolism
3000
o°o°°
2500 2000
°o o o o o
1500
"o
1000 500
D o 0 0 o o OoOo°
°0°
°o o °
°o
°o
Group 1 There were five cases in this group who, at the time of investigation, had no clinical evidence of venous thrombosis or pulmonary embolism. Their platelet 5 HT ranged
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
Thirty-five cases (15 men and 20 women) between the ages of 60 and 70 years were studied. Eight had hemiplegia or hemiparesis of less than 24-hours duration. Four of them died and were found at autopsy to have stenosis of the extracranial portion of one internal carotid artery; one had angiography which showed occlusion of one internal carotid artery, and the remaining three patients were having recurrent ischaemic attacks suggestive of internal carotid artery stenosis but no angiogram was done to demonstrate the obstruction; two had developed monoplegia at the time of investigation. Of the remaining 27 patients, 14 were admitted for social reasons and had no active pathology, six were immobile on account of painful osteoarthritis, three had rheumatoid arthritis, two had uraemia due to chronic pyelonephritis and two had iron-deficiency anaemia. Venous blood was collected from each patient between 9.00 a.m. and 10.00 a.m. The sample for 5 H T estimation was obtained in bottles containing 3 per cent EDTA in 0.7 per cent NaCl. The whole blood was spun in a MSE Junior centrifuge at 1000 rev./min for 20 minutes to separate red cells. The plasma was removed and spun at 2300 rev./min for 30 minutes to separate platelets. The supernatant was kept for chemical analysis. A solution of 3 per cent EDTA in 0.7 per cent NaCl was added to the platelets. The platelets and solution were agitated with a Whirlimixer for 5 to 10 minutes to suspend the platelets in solution. The platelet suspension was spun at 2300 rev./min for 30 minutes to separate platelets from solution. The washings were withdrawn and the platelets suspended in the EDTA solution. 5 H T was estimated by the spectrofluorimetric method of Crawford & Rudd (1962). The proteins were precipitated with trichloracetic acid; excess trichloracetic acid and interfering fluorochromes were removed with ether and the 5 H T was extracted with salt-saturated butanol after pH adjustment with borate buffer. Iso-octane was added to an aliquot of the butanol extract and the 5 H T was then extracted into 3 N - H C I for fluorescence measurement. An Aminco-Borman spectrophotofluorimeter was used with an activating wavelength of 295 nm and a fluorescent wavelength of 540 nm.
Platelet 5-Hydroxy-tryptamine
in Thrombotic and Non-thrombotic Diseases
107
from 3101 to 3382 ng/109. They died 24 to 48 hours after the blood was taken for estimation, and autopsy revealed deep-vein thrombosis with pulmonary embolism. The deep veins affected were femoral in two cases and calf veins in three cases. Group 2 This group comprised eight cases of cerebral thrombosis. Their platelet 5 HT ranged from 1529 to 2183 ng/10».
The plasma 5 HT ranged from 19 to 87 ng/ml and did not show any variation in the different pathological groups studied (Table II). There was no correlation between the results of plasma and platelet 5 HT. Table II. Plasma 5 HT (ng/ml) Apparently normal subjects
Deep vein Rheumatoid Uraemia Anaemia Cerebral thrombosis with thrombosis pulm. embolism arthritis
Osteoarthritis
o
80 0
o o
70 o
o
0
50
0
40
o
o
O O
0
°
o
o
o o°
o
0
o o
O o
O o
O
30 20
o
10
DISCUSSION
The formation of platelet thrombi at the site of injured vessels was demonstrated over a century ago (Wharton-Jones, 1851). Since then much work has been done on different aspects of the problem, but the mechanism of thrombus formation still remains obscure. It was suggested that a chemical substance, adenosine diphosphate (ADP), which has been found to initiate experimental thrombus formation (Honour & Mitchell, 1964; Spaet & Zucker, 1964) may be responsible. Later thrombus formation was produced by the iontophoresis of ADP through intact vessel wall, which further confirmed the role of ADP in thrombus formation (Begent & Bown 1970). The second theory stressed the importance of damaged endothelium in causing thrombus formation, by exposing the basement membrane collagen to the blood (Lyman et al, 1971). It was also shown that washed platelets suspended in human ringer solution exhibit negligible adhesion in contrast to washed platelets suspended in plasma. Addition of
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
Group 3 This group consisted of various patients including those with osteoarthritis, rheumatoid arthritis, uraemia, anaemia, and those with no active pathology. The platelet 5 HT ranged from 611 to 877 ng/109.
108
D. P. Misra, G. Staddon, P. Jackson, N. Powell and J. Misra
ACKNOWLEDGEMENTS
We wish to thank Drs W. H. Lloyd and A. C. M. Windsor for permission to study their cases and Dr D. D. Gibbs for his comments and advice on the paper. We are very grateful to Mr D. A. Lothian for his help in estimating 5 HT. REFERENCES
N. & BOWN, G. V. R. (1970). Growth rate in vivo of platelet thrombi produced by iontophoresis of ADP as a function of mean blood flow velocity. Nature, Lond. 227, 926. CRAWFORD, N. & RUDD, B. T. (1962). A spectrophotofluorimetric method for the determination of serotonin (5-hydroxytryptamine) in plasma. Clin. Chim. Acta 7, 114. GAARDER, A., JONSEN, J., LALAND, S., HELLEM, A. J. & OWREN, P. A. (1961). Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature, Lond. 192, 531. HONOUR, A. J. & MITCHELL, J. R. A. (1964). Platelet clumping in injured vessels. Br. J. exp. Pathol. 45, 75. KAGAN, A., DAWBER, T. R., KANNEL, W. B. & REVOTSKIE, N. (1962). The Framingham study: a prospective study of coronary heart disease. Fed. Proc. 21, suppl. 11, 52. LEVENE, M. & LEVENE, C. I. (1957). Pulmonary platelet thromboembolism. J. clin. Pathol. 10, 200. L/YMAN, B., ROSENBERG, L. & KARPATKIN, S. (1971). Biochemical and biophysical aspects of human platelet adhesion to collagen fibres. J. Clin. Invest. 50, 1854. MITCHELL, J. R. A. & SCHWARTZ, C. J. (1965). Arterial Disease, pp. 248, 199. Oxford: Blackwell. MITCHELL, J. R. A. & SHARP, A. A. (1964). Platelet clumping in vitro. Br. J. Haemotol. 10, 78. MORRELL, M. T. & DUNHILL, M. S. (1968). The post-mortem incidence of pulmonary embolism in a hospital population. Br. J. Surg. 55, 347. BEGENT,
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
human fibrinogen to the ringer solution completely restored the ability of washed platelets to adhere to collagen fibres, thus suggesting that fibrinogen was the factor in the plasma, which was required for platelet adhesion to purified collagen. It is obvious that experimental thrombus formation is not analogous to clinical thrombosis. The relevance of experimental findings in the clinical situation is also doubtful. Clinical studies have concentrated on the value of predisposing and environmental factors in the pathogenesis of thrombus formation. In the Framingham survey (Kagan et ah, 1962), the risk factors in coronary artery thrombosis included elevated serum lipids, elevated blood pressure, cigarette smoking and advancing age. On the other hand, leg vein thrombosis has been linked with operation, injury, childbirth, immobility and malignant diseases (Morrell & Dunhill, 1968), with the recent addition of oestrogencontaining contraceptives (Vessey & Doll, 1969). Investigations so far carried out do not provide any evidence as to what initiates a thrombus and how it is propagated. Although the importance of ADP is greatly emphasized and its value in experimental thrombus formation is undisputed, less attention has been paid to 5 HT, adrenaline and noradrenaline. Comparing the results of Group 1, 2 and 3 cases, it is apparent that the platelet 5 HT rises prior to the formation or embolization of a thrombus and may, therefore, influence thrombus formation in man. On the evidence obtained in the present work it is difficult to explain the discrepancy in the findings of plasma and platelet 5 HT in thrombotic diseases, but it is suggested that the turnover of platelet 5 HT is increased, i.e. the production and degradation of 5 HT in the platelets is enhanced.
Platelet 5-Hydroxy-tryptamine
in thrombotic and Non-thrombotic Diseases
109
J. R. (1964). Platelet stickiness: some methods and some theory. J. din. Pathol. 17, 472. J. R. & HEYWOOD, J. B. (1966). Effect of aggregating agents and their inhibitors on the mean platelet shape. J. Clin. Pathol. 19, 148. SPAET, T. H. & ZUCKER, M. B. (1964). Mechanism of platelet plug formation and the role of adenosine diphosphate. Am. J. Physiol. 206, 1267. VESSEY, M. P. & DOLL, R. (1969). Investigation of relation between use of oral contraceptives and thrombo-embolic disease: a further report. Br. Med. J. 2, 651. WHARTON-JONES, T. (1851). On the state of the blood and blood vessels in inflammation. Guy's Hospital Reports. Series 2, 7, 1. WOOLF, N. & CARSTAIRS, K. C. (1967). Infiltration and thrombosis in atherogenisis. Am. J. Pathol. 51, 373. O'BRIEN, O'BRIEN,
Downloaded from http://ageing.oxfordjournals.org/ at University of Sydney on March 23, 2016
Present address: D. P. Misra, Good Hope Hospital, Sutton Coldfield, West Midlands, BIS 1RR
