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SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD: DEPENDENCE ON AGE AND HAEMATOCRIT
1213 months. One patient dropped out because of "dizziness" and another required surgery. The remaining seven experienced no subjective improvement in their claudication. The ankle/arm pressure ratio (doppler ultrasound) did not change, confirming De Cree’s findings. However, De Cree et al did report a significant improvement in thigh/arm pressure ratios, without stating whether thigh blood pressure rose or whether it was the fall in arm pressure that they observed that contributed to the rise in thigh/arm ratio. We also found that ketanserin did not alter platelet aggregation or thromboxane A2 release after the addition in vitro of adrenaline, adenosine diphosphate, or collagen.Thus ketanserin seems to inhibit only serotinin-induced aggregation (which is reversible in human platelets) and serotonin-enhanced aggregation and the clinical significance of such properties remains to be established. Ketanserin did not affect diabetic control or insulin or
glibenclamide dose requirements. However, we have noted two important side-effects of ketanserin. Of the nine diabetic patients and six others with Raynaud’s disease whom we have treated with this drug for intermittent claudication three have reported increased appetite and weight gain of 3 kg or more over 8 weeks. Cyproheptidine, another serotonin receptor antagonist, also stimulates appetite. Four patients have had symptoms of severe depression, which resolved when the drug was withdrawn. Ketanserin has al-adrenergic blocking effects,2 and other al blockers, such as prazosin and indoramin, can cause depression.3,4 In view of these al-blocking properties the terms "specific" and "selective" 5- HT antagonist (used by De Cree et al) are misleading. Departments of Chemical Pathology and Human Metabolism and of Pharmacology, Royal Free Hospital and School of Medicine, London NW3 2QG
V. FONSECA A. G. RAMAGE D. P. MIKHAILIDIS M. A. BARRADAS J. Y. JEREMY P. DANDONA
Jeremy JY, Barradas MA, Green N, Dandona P. Effect of ethanol on vascular prostacyclin (prostaglandin I2) synthesis, platelet aggregation and platelet thromboxane release. Br Med J 1983; 287: 1495-98. 2. Reimann IW, Frolich JC. Mechanism of antihypertensive action of ketanserin in man Br Med J 1983; 287: 381-83. 3. ABPI data sheet compendium. London: Datapharm Publications, 1984: 1588-89. 4. ABPI data sheet compendium. London: Datapharm Publications, 1984: 1084-87. 1.
SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD: DEPENDENCE ON AGE AND HAEMATOCRIT on the effect of haematocrit spontaneous platelet aggregation (SPA) in whole blood. Saniabadi et all and now Dr Harrison and his colleagues (Oct 27, p 991) manipulated Hct in vitro and showed that SPA, as measured by the fall in number of single platelets in stirred citrated blood, increases with increasing Hct. We have measured SPA in citrated blood and in heparinised blood obtained from different subjects and compared the values obtained with the Hct of the samples. We also looked for relationships between SPA and age, plasma fibrinogen (a co-factor in the aggregation process), and several other haematological indices and found that SPA correlates with age, Hct, fibrinogen, and platelet count, the major independent determinants being age and Hct. Blood was taken from 67 healthy subjects aged 18-88 years, and collected into citrate, heparin, and EDTA.2 Citrated and heparinised samples were used to determine SPA; 400 pl volumes were stirred at 370C for 8 min and the number of single platelets that remained were determined in an ’Ultra Flo 100’ whole-blood platelet counter.2The fall in the number of single platelets was expressed as a percentage of the number of platelets in the EDTA blood. Plasma fibrinogen was determined3in 31 of the citrated samples, and Hct and the other haematological indices were measured in 54 of the EDTA samples. SPA in whole blood taken from different individuals ranged from 0 to 96% (citrated) and 8 to 82% (heparinised). After the samples had been stirred for 8 min the values obtained in citrated blood and heparinised blood for the different individuals were closely
SIR,-Two groups have reported
(Hct)
on
CORRELATIONS BETWEEN SPA IN CITRATED BLOOD AND OTHER INDICES
correlated (r = 0 - 87, p<0 001), so SPA does not vary greatly with the type of anticoagulant. Significant positive correlations were found between SPA (citrated blood) and the individual’s age, fibrinogen, haematocrit and other red-cell related indices, and platelet count (table). Multiple regression analysis showed that the major contributors to the variation in SPA in citrated blood are age (p<0 01) and Hct (p<0 01), the contributions from fibrinogen and
platelet
count
being non-significant.
The clear relation between SPA and Hct that we and others have demonstrated may be a consequence of ADP being liberated from red cells. In 1960, Hellemshowed that the number of platelets that are lost when whols blood is passed through a column of glass beads is proportional to the Hct of the sample, and this S relation was attributed to ADP liberated from the red cells. Using an enzyme system that degrades ADP, Harrison and Mitchell6 provided evidence that ADP liberated from red cells is a major contributor to the loss of single platelets from blood held in a rotating glass flask. Both Fox et al2 and Saniabadi et al7 have shown that enzymes that degrade ADP reduce SPA in stirred whole blood, and Harrison and colleagues have now shown that increased SPA at high Hct is associated with increased red cell damage. SPA may be merely an in-vitro artefact of red cell damage and thus irrelevant to the in-vivo situation. However, the number of single platelets in whole blood falls even when the blood is simply allowed to stand on the bench2 and it is possible that red cells are continually liberating small quantities of ADP and that this happens in vivo as well as in vitro. This could explain why there is an increased risk of thrombosis when Hct is high.8 Our results also show a clear relation between SPA in whole-blood and the subjects’ age that is independent of Hct, and there is no doubt that the risk of an individual experiencing coronary heart disease, stroke, or peripheral arterial disease increases with age.9 SPA may itself be a risk marker for thrombosis. Large-scale prospective studies in which this rapid and simple test of platelet behaviour is measured are now required to test this hypothesis. We thank Miss J. Crawford and Miss M. Hawkins for technical assistance and staff in the haematology department, University Hospital, Nottingham, for the full blood counts. The work was supported by CORDA.
Department of Medicine, University Hospital, Queen’s Medical Centre, Nottingham NG72UH
M. E. BURGESS-WILSON S. GREEN S. HEPTINSTALL J. R. A. MITCHELL
AR, Lowe GDO, Barbenel JC, Forbes CD. Haematocrit, bleeding time, and platelet aggregation. Lancet 1984; i 1409-10. Fox S, Burgess-Wilson ME, Heptinstall S, Mitchell JRA Platelet aggregation in whole blood determined using the Ultra-Flo 100 platelet counter. Thromb Haemostas
1. Saniabadi
2
1982; 48: 327-29. 3. Clauss Von A. Gerinnungsphysiologische schellmethode fibrinogens. Acta Haematol 1957; 17: 237. 4
zur
bestimmung
des
Hellem AJ The adhesiveness of human blood platelets in vitro. Scand J Clin Lab Invest 1960; 12 (suppl 51) A, Jonsen J, Laland S, Hellem A, Owren PA. Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature 1961, 192:
5. Gaarder
531-32
6. Harnson MJG, Mitchell JRA. The influence of red blood-cells on platelet adhesiveness. Lancet 1966; ii: 1163-64. 7. Saniabadi AR, Lowe GDO, Barbenel JC, Forbes CD A comparison of spontaneous platelet aggregation in whole blood with platelet rich plasma Additional evidence for the role of ADP Thromb Haemostas 1984; 51: 115-18. 8. Dormandy JA. Haemorheology and thrombosis In Bloom AL, Thomas DP, eds Haemostasis and thrombosis. Edinburgh Churchill Livingstone, 1981; 610-25 9. Dawber TR Incidence of coronary heart disease, stroke, and peripheral arterial disease In: The Framingham study Cambridge, Mass Harvard University Press, 1980; 59-75.
glibenclamide dose requirements. However, we have noted two important side-effects of ketanserin. Of the nine diabetic patients and six others with Raynaud’s disease whom we have treated with this drug for intermittent claudication three have reported increased appetite and weight gain of 3 kg or more over 8 weeks. Cyproheptidine, another serotonin receptor antagonist, also stimulates appetite. Four patients have had symptoms of severe depression, which resolved when the drug was withdrawn. Ketanserin has al-adrenergic blocking effects,2 and other al blockers, such as prazosin and indoramin, can cause depression.3,4 In view of these al-blocking properties the terms "specific" and "selective" 5- HT antagonist (used by De Cree et al) are misleading. Departments of Chemical Pathology and Human Metabolism and of Pharmacology, Royal Free Hospital and School of Medicine, London NW3 2QG
V. FONSECA A. G. RAMAGE D. P. MIKHAILIDIS M. A. BARRADAS J. Y. JEREMY P. DANDONA
Jeremy JY, Barradas MA, Green N, Dandona P. Effect of ethanol on vascular prostacyclin (prostaglandin I2) synthesis, platelet aggregation and platelet thromboxane release. Br Med J 1983; 287: 1495-98. 2. Reimann IW, Frolich JC. Mechanism of antihypertensive action of ketanserin in man Br Med J 1983; 287: 381-83. 3. ABPI data sheet compendium. London: Datapharm Publications, 1984: 1588-89. 4. ABPI data sheet compendium. London: Datapharm Publications, 1984: 1084-87. 1.
SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD: DEPENDENCE ON AGE AND HAEMATOCRIT on the effect of haematocrit spontaneous platelet aggregation (SPA) in whole blood. Saniabadi et all and now Dr Harrison and his colleagues (Oct 27, p 991) manipulated Hct in vitro and showed that SPA, as measured by the fall in number of single platelets in stirred citrated blood, increases with increasing Hct. We have measured SPA in citrated blood and in heparinised blood obtained from different subjects and compared the values obtained with the Hct of the samples. We also looked for relationships between SPA and age, plasma fibrinogen (a co-factor in the aggregation process), and several other haematological indices and found that SPA correlates with age, Hct, fibrinogen, and platelet count, the major independent determinants being age and Hct. Blood was taken from 67 healthy subjects aged 18-88 years, and collected into citrate, heparin, and EDTA.2 Citrated and heparinised samples were used to determine SPA; 400 pl volumes were stirred at 370C for 8 min and the number of single platelets that remained were determined in an ’Ultra Flo 100’ whole-blood platelet counter.2The fall in the number of single platelets was expressed as a percentage of the number of platelets in the EDTA blood. Plasma fibrinogen was determined3in 31 of the citrated samples, and Hct and the other haematological indices were measured in 54 of the EDTA samples. SPA in whole blood taken from different individuals ranged from 0 to 96% (citrated) and 8 to 82% (heparinised). After the samples had been stirred for 8 min the values obtained in citrated blood and heparinised blood for the different individuals were closely
SIR,-Two groups have reported
(Hct)
on
CORRELATIONS BETWEEN SPA IN CITRATED BLOOD AND OTHER INDICES
correlated (r = 0 - 87, p<0 001), so SPA does not vary greatly with the type of anticoagulant. Significant positive correlations were found between SPA (citrated blood) and the individual’s age, fibrinogen, haematocrit and other red-cell related indices, and platelet count (table). Multiple regression analysis showed that the major contributors to the variation in SPA in citrated blood are age (p<0 01) and Hct (p<0 01), the contributions from fibrinogen and
platelet
count
being non-significant.
The clear relation between SPA and Hct that we and others have demonstrated may be a consequence of ADP being liberated from red cells. In 1960, Hellemshowed that the number of platelets that are lost when whols blood is passed through a column of glass beads is proportional to the Hct of the sample, and this S relation was attributed to ADP liberated from the red cells. Using an enzyme system that degrades ADP, Harrison and Mitchell6 provided evidence that ADP liberated from red cells is a major contributor to the loss of single platelets from blood held in a rotating glass flask. Both Fox et al2 and Saniabadi et al7 have shown that enzymes that degrade ADP reduce SPA in stirred whole blood, and Harrison and colleagues have now shown that increased SPA at high Hct is associated with increased red cell damage. SPA may be merely an in-vitro artefact of red cell damage and thus irrelevant to the in-vivo situation. However, the number of single platelets in whole blood falls even when the blood is simply allowed to stand on the bench2 and it is possible that red cells are continually liberating small quantities of ADP and that this happens in vivo as well as in vitro. This could explain why there is an increased risk of thrombosis when Hct is high.8 Our results also show a clear relation between SPA in whole-blood and the subjects’ age that is independent of Hct, and there is no doubt that the risk of an individual experiencing coronary heart disease, stroke, or peripheral arterial disease increases with age.9 SPA may itself be a risk marker for thrombosis. Large-scale prospective studies in which this rapid and simple test of platelet behaviour is measured are now required to test this hypothesis. We thank Miss J. Crawford and Miss M. Hawkins for technical assistance and staff in the haematology department, University Hospital, Nottingham, for the full blood counts. The work was supported by CORDA.
Department of Medicine, University Hospital, Queen’s Medical Centre, Nottingham NG72UH
M. E. BURGESS-WILSON S. GREEN S. HEPTINSTALL J. R. A. MITCHELL
AR, Lowe GDO, Barbenel JC, Forbes CD. Haematocrit, bleeding time, and platelet aggregation. Lancet 1984; i 1409-10. Fox S, Burgess-Wilson ME, Heptinstall S, Mitchell JRA Platelet aggregation in whole blood determined using the Ultra-Flo 100 platelet counter. Thromb Haemostas
1. Saniabadi
2
1982; 48: 327-29. 3. Clauss Von A. Gerinnungsphysiologische schellmethode fibrinogens. Acta Haematol 1957; 17: 237. 4
zur
bestimmung
des
Hellem AJ The adhesiveness of human blood platelets in vitro. Scand J Clin Lab Invest 1960; 12 (suppl 51) A, Jonsen J, Laland S, Hellem A, Owren PA. Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature 1961, 192:
5. Gaarder
531-32
6. Harnson MJG, Mitchell JRA. The influence of red blood-cells on platelet adhesiveness. Lancet 1966; ii: 1163-64. 7. Saniabadi AR, Lowe GDO, Barbenel JC, Forbes CD A comparison of spontaneous platelet aggregation in whole blood with platelet rich plasma Additional evidence for the role of ADP Thromb Haemostas 1984; 51: 115-18. 8. Dormandy JA. Haemorheology and thrombosis In Bloom AL, Thomas DP, eds Haemostasis and thrombosis. Edinburgh Churchill Livingstone, 1981; 610-25 9. Dawber TR Incidence of coronary heart disease, stroke, and peripheral arterial disease In: The Framingham study Cambridge, Mass Harvard University Press, 1980; 59-75.