Scand J Haematol (1976) 16, 266-270
Serotonin Uptake by Various Human Blood Platelet Populations P.KAMOUN, M.D., G . LAFOURCADE & H. JEROME, M.D. Laboratoire de biochimie ge'nktique (Chief, H . Jerome), HGpital Necker, Paris, France
Human blood platelets were separated into 4 populations by centrifugation on a sucrose gradient. Serotonin uptake expressed against cell protein content is the greater as platelets become lighter. The relation between these facts and ATP content in the various platelet populations is discussed. Ke,y words: blood platelets - serotonin uptake
Accepted for publication January 24, 1976 Correspondence to: Dr. P. Kamoun, Laboratoire de Biochimie Ginttique, HGpital Necker, 75730 Pans Cedex 15, France
It is possible to separate platelets into 4 more homogeneous populations on the basis of their size and density. Such a separation was successfully achieved by centrifugation on a discontinuous sucrose gradient (Booyse et a1 1968). The heaviest platelets have tentatively been characterized as the newly formed or young platelets (Booyse et a1 1968, Bmyse & Rafelson 1969). Abnormalities were described for serotonin uptake by blood platelets in renal chronic failure (Kamoun et a1 1970), Down's syndrome (Jerome & Kamoun 1970) and some haematological disorders (Hardisty & Stacey 1957), such as myeloid leukaemia (Polli et a1 1958). In all these diseases except the former an abnormal distribution of platelets between the 4 populations was described (Vainer et al 1971, Kamoun & Jerome 1976). It is therefore of interest
to determine in normal subjects whether differences in platelet size and density correlate with differences in platelet functions such as serotonin uptake. MATERIALS AND METHODS Blood (30 ml) from human volunteers was collected by venous punction in siliconed glassware (9 volumes of blood for 1 volume of isotonic anticoagulant solution disodium EDTA 27 mM, NaCl 154 mM). The method described by Dillard et al (1951) was used for the preparation of platelet rich plasma. After centrifugation at 50 x g for 45 min the plasma was discarded. Freshly isolated platelets were suspended in 5 ml of chilled 0.15 M NaCl and centrifuged at 500 x g for 15 min. The supernatant was removed from the pellet and the procedure repeated. The recovery of platelets from blood was about 25 %. At this stage no erythrocyte or leucocyte contamination could be detected by microscopic examination. The platelet pellet was resuspended in 2.2 ml of 0.15 M
SEROTONIN UPTAKE BY PLATELETS NaCl and 0.2 ml was removed for protein determination according to Lowry et al (1951). Then 1.9 ml of this suspension (ranging from 2.6 to 6.9 mg of platelet proteins) was layered on a 11.2 ml discontinuous sucrose gradient (50 %, 55 %, 59 %, 65 %, w/v) containing 2 mM disodium EDTA, 0.15 M NaCl and 10 mM TrisHCI buffered at pH 7.4. Centrifugation was carried out at 22,500 x g for 150 min in a SW 40 rotor of the Spinco Model centrifuge (Booyse et a1 1968). The gradient was collected from the bottom of the tube by fractions of 7 drops. In preliminary experiments protein assays were made on each fraction and the results summed for recovery determination. For the serotonin uptake measurements the platelets from each of the 4 populations obtained (1st population in tubes nos. 10 t o 20, 2nd in nos. 21 t o 32, 3rd in nos. 33 to 44, 4th in nos. 45 t o 58) were pelleted by centrifugation at 500 x g for 15 min and washed twice with chilled 0.15 M NaCI. Then the platelets were incubated with mechanical stirring at 37OC for 30 min in an isotonic medium buffered at pH 7.4: disodium phosphate 16.24 m M , monosodium phosphate 3.76 mM, disodium EDTA 2.69 mM, glucose 5.55 mM, sodium acetate 11.03 mM, sodium chloride 104.54 mM, potassium chloride 4.02 mM and 2 14C serotonin creatinine sulphate 2.32 p M (spec act 48 mCVmmo1). Then the platelets were pelleted by centrifugation at 4 O C 500 x g for 15 min and twice washed with chilled isotonic saline. The sedimented platelets were broken with 1 ml of 0.02 N HCI. An aliquot was used for protein assay (Lowry et a1 1951) and 0.5 ml was deproteinized by addition of 50 pl Zn so4 (347 mM) and 25 p1 of 1 N NaOH. After centrifugation at 2700 x g for 30 min, 0.5 ml of the supernatant was mixed with 10 ml of scintillation liquid (Instagel Packard) and counted in a liquid scintillation spectrometer (Packard 3380). These counts were never less than twice the background. Finally the results could be expressed as jcmoVg protein.
267
as per cent of amount of protein applied on the top of gradient. In preliminary assays it reached (mean jz SEM) 91.3 k 2.4 % (sum of the results found for each fraction of 7 drops). The recovery sinks to 24.8 k 0.8 % for 15 assays made on platelets pelleted after incubation for serotonin uptake determinations (sum of the results found for each of the 4 populations). The decrease of recovery was not uniformly distributed: it was particularly important for the 4th population which is the lightest (Table 1). Serotonin uptake is the highest in the lightest population (the 4th) and the decreasing order is 4th, 3rd, lst, 2nd (Table 2). According to Booyse et a1 (1968) and Booyse & Rafelson (1969) this corresponds to the platelet age. Thus the platelets take up serotonin the better the older 0pti:al density
0 .l
0.2
RESULTS
The separation of blood platelets in 4 populations was very reproducible and a typical pattern of one of the 12 preliminary assays is shown in Figure 1. Platelet recovery was measured by protein assays and expressed
Figure 1. Collection of sucrose gradient: from the bottom of the tube 7 drops were collected in each tube. In ordinate: optical density measured in protein assay as indicates by Lowry et a1 (1951).
P. KAMOUN, G . LAFOURCADE & H. JEROME
268
TABLE 1 Distribution o f human blood platelets between four populations
A
Mean SEM
28.7 1.3
37.7 1.3
17.3 0.8
16.4 0.6
B
Mean SEM
27.1 3.4
49.5 2.8
16.0 2.0
7.3 0.8
A - Preliminary assays: percentage of the sum of proteins measured on each tube collected from the gradient. B - Incubation assays: percentage of the sum of recovered proteins in the lysats prepared after platelet incubation for 30 min with serotonin. TABLE 2 Net uptake of serotonin f o r 30 min by 4 various human blood platelet populations
Age
I
Sex M M F F M M M F F M M M M F M
37 26
54 18 38 51 23 52 25 25 22 36 23 40 39
Mean SEM
1
(umol/g protein) 1st
1
2.9 3.3 2.1 2.3 2.1 2.3 2.3 2.5 2.9 2.5 3.4 2.1 2.3 2.8 2.0
1.7 2.6 2.2 1.9 1.5 1.7 2.1 1.9 2.2 1.4 1.o 2.7 1.9 1.7 1.3
2.5 0.11
1.9 0.12
they are. The individual values vary greatly but in regard to serotonin uptake the order of populations is almost constant among all subjects. A variance analysis perfomied on individual results shows no difference between subjects but a very significant difference between the four populations (p < 0.001). DISCUSSION
Blood platelets are separated by a sucrose
1
2nd
.
3rd
1
4th
2.9 3.7 3.9 4.1 4.9 4.1 3.1 1.9 3.2 2.1 1.5 2.5 1.7 2.0 1.1
4.8 6.7 3.7 4.5 3.9 3.0 5.0 2.6 4.4 3.8 2.9 3.7 3.2 4.2 3.1
2.8 0.29
4.0 0.27
gradient which is hyperosmolar and it may be possible that the platelets were obviously harmed by the isolation procedure to different extents along the gradient. That hypothesis may account for the variations observed in platelet recovery and serotonin uptake between the 4 populations. But we have to point out that the 4th population (the lightest) is isolated in sucrose 50 % (the smaller concentration) and even despite that the loss during incubation is particularly heavy for this population.
SEROTONIN UPTAKE BY PLATELETS
Blood platelets take up serotonin by an active energy-dependent mechanism (Hardisty & Stacey 1955), which is inhibited by metabolic inhibitors such as cyanide (Born & Gillson 1959) and fluoride (Weissbach et a1 1960). The serotonin taken up by platelets from the surrounding medium is localized at least partly in dense osmiophilic organelles (Tranzer et a1 1968) where it forms a complex with ATP (Da Prada et a1 1972). According to Born et a1 (1958) there is a direct relationship between ATP and serotonin concentrations. ATP concentrations in platelet populations have been investigated by Karpatkin (1969). His separation method enabled the separation of only two populations: a large heavy population with a density greater than 1.055 and a light small platelet population with a density below 1.046. The quantity of ATP contained in loll platelets came to 5.90 pmol for the heavy platelets and 2.08 pmol for the light platelets. The difference was less if the concentrations were expressed by ml of platelet volume (5.0 and 4.1 respectively), and cancelled out altogether if the ATP concentrations were related to the protein concentrations (25.6 and 23.3 pmoVg protein respectively, which scarcely differs from concentrations observed in whole blood platelets: 25.7 pmol/g protein). In our investigation the serotonin uptake was expressed against protein. It is conceivable therefore that the observed variations are independent of ATP concentrations. But since the protein content per platelet was distinctly less in the light populations [lightheavy ratio 0.39 for Karpatkin (1969)], conceivably if the serotonin uptake had been expressed in pmol per platelet it would have eliminated the differences observed between populations.
269
However, this does not explain the discordance observed between our results and those of Rendu & Caen (1973). They stated that serotonin uptake (expressed as pmoVg protein) is greater for the heavy platelets than for the light ones. The technique used by Rendu & Caen (1973) differs considerably from ours, because they separated the platelets into 4 populations only after serotonin uptake and without elimination of sucrose. Moreover, the platelets were incubated in plasma together with citric acid and without EDTA. Despite the serotonin concentration (5.4 1 c 6 M) used by these workers uptake for 30 min incubation was about 5 times less than that observed by us. Finally the authors do not account for the interference due to sucrose in determining protein content by the method of Lowry et a1 (1951). Our study is in agreement with the results of Hardeman (1974): platelet conservation at 37O C for 4 days with dialysis renewal of the plasma conservation medium does not modify serotonin uptake (expressed as pM/109 platelets). Since the life span of platelets is 7 days the absence of any overall variation in the serotonin uptake expressed by platelet would seem to be in favour of better serotonin uptake by the older (lighter) platelets than by the younger (heavier) ones when this uptake had been expressed against protein. ACKNOWLEDGEMENT These investigations were supported by a Grant from INSERM no. 874-24. REFERENCES Booyse F M, Hoveke T & Rafelson M E Jr (1968) Studies on human platelets. 11. Protein synthetic activity of various platelet populations. Biochim Biophys Acta 157, 660-63.
270
P. KAMOUN, G. LAFOURCADE & H. JEROME
Booyse F M & Rafelson M E J r (1969) Protein synthesis and platelet function. In S Johnson & M M Guest (eds) Dynamics o f thrombus formation and dissolution, pp 149-71. J B Lippincot, Philadelphia. Born G V R & Gillson R E (1959) Studies on the uptake of 5-hydroxytryptamine by blood platelets. J Physiol 146, 472-91. Born G V R, Ingram G I C & Stacey P S (1958) The relationship between 5-hydroxytryptamine and adenosine triphosphate in blood platelets. Brit J Pharmacol 13, 62-64. D a Prada M, Tranzer J P & Pletscher A (1972) Storage of 5-hydroxytryptamine in human blood platelets. Experientia 28, 1328-29. Dillard G H L, Brecher G & Cronkite E P (1951) Separation, concentration and transfusion of platelets. Proc Soc Exp Biol Med 78, 796-99. Hardeman M R (1974) The effect of continuous dialysis on platelet preservation. In C F Hogman, H W Krijnen & C R Valeri (eds) Symposium on platelet preservation and transfusion. Uppsala Offset Center, Uppsala, Sweden. Hardisty R M & Stacey R S (1955) 5-hydroxytryptamine in normal human platelets. 1 Physiol 130, 711-20. Hardisty R M & Stacey R S (1957) Platelet 5hydroxytryptamine (HT) in disorders of the blood. Br J Haematol 3, 292-98. Jerome H & Kamoun P (1970) Platelet binding of serotonin. Ann N Y Acad Sci 171, 543-50. Kamoun P & Jerome H (1976) Blood serotonin in
mental disorders. In D J Boullin (ed) Serotonin in mental disorders. John Wiley, London (in press). Karnoun P, Kleinknecht D, Ducrot H & Jerome H (1970) Platelet serotonin in uraemia. Lancet 1, 782. Karpatkin S (1969) Heterogeneity of human platelets. I. Metabolic and kinetic evidence suggestive of young and old platelets. J CIin Invest 48, 1073-82. Lowry 0 H, Rosebrough N J, Fan- A L & Randall R J (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-75. Polli E E, Bianchi P A & Crosti P F (1958) La serotonina piastrinica nelle leucemie. Haematologica Latina 1, 2 5 M 4 . Rendu F & Caen J P (1973) Effect of reserpine on 14C serotonin uptake by human platelet populations. Haemostasis 1, 161-68. Tranzer J P, Da Prada M & Pletscher A (1968) Electron microscopic study of the storage site of 5-hydroxytryptamine in blood platelets. Adv Pharmacol Chemother 6 A , 125-28. Vainer H, Besson P & Caen J (1971) La glycoghe synthktase dans les plaquettes et les populations plaquettaires anormales de syndromes myelo prolifkratifs. Nouv Rev Fr Hematol 11, 769-80. Weissbach H, Redfield B G & Titus E (1960) Effect of cardiac glycosides and inorganic ions on binding of serotonin by platelets. Nature 185, 99-100.
Serotonin Uptake by Various Human Blood Platelet Populations P.KAMOUN, M.D., G . LAFOURCADE & H. JEROME, M.D. Laboratoire de biochimie ge'nktique (Chief, H . Jerome), HGpital Necker, Paris, France
Human blood platelets were separated into 4 populations by centrifugation on a sucrose gradient. Serotonin uptake expressed against cell protein content is the greater as platelets become lighter. The relation between these facts and ATP content in the various platelet populations is discussed. Ke,y words: blood platelets - serotonin uptake
Accepted for publication January 24, 1976 Correspondence to: Dr. P. Kamoun, Laboratoire de Biochimie Ginttique, HGpital Necker, 75730 Pans Cedex 15, France
It is possible to separate platelets into 4 more homogeneous populations on the basis of their size and density. Such a separation was successfully achieved by centrifugation on a discontinuous sucrose gradient (Booyse et a1 1968). The heaviest platelets have tentatively been characterized as the newly formed or young platelets (Booyse et a1 1968, Bmyse & Rafelson 1969). Abnormalities were described for serotonin uptake by blood platelets in renal chronic failure (Kamoun et a1 1970), Down's syndrome (Jerome & Kamoun 1970) and some haematological disorders (Hardisty & Stacey 1957), such as myeloid leukaemia (Polli et a1 1958). In all these diseases except the former an abnormal distribution of platelets between the 4 populations was described (Vainer et al 1971, Kamoun & Jerome 1976). It is therefore of interest
to determine in normal subjects whether differences in platelet size and density correlate with differences in platelet functions such as serotonin uptake. MATERIALS AND METHODS Blood (30 ml) from human volunteers was collected by venous punction in siliconed glassware (9 volumes of blood for 1 volume of isotonic anticoagulant solution disodium EDTA 27 mM, NaCl 154 mM). The method described by Dillard et al (1951) was used for the preparation of platelet rich plasma. After centrifugation at 50 x g for 45 min the plasma was discarded. Freshly isolated platelets were suspended in 5 ml of chilled 0.15 M NaCl and centrifuged at 500 x g for 15 min. The supernatant was removed from the pellet and the procedure repeated. The recovery of platelets from blood was about 25 %. At this stage no erythrocyte or leucocyte contamination could be detected by microscopic examination. The platelet pellet was resuspended in 2.2 ml of 0.15 M
SEROTONIN UPTAKE BY PLATELETS NaCl and 0.2 ml was removed for protein determination according to Lowry et al (1951). Then 1.9 ml of this suspension (ranging from 2.6 to 6.9 mg of platelet proteins) was layered on a 11.2 ml discontinuous sucrose gradient (50 %, 55 %, 59 %, 65 %, w/v) containing 2 mM disodium EDTA, 0.15 M NaCl and 10 mM TrisHCI buffered at pH 7.4. Centrifugation was carried out at 22,500 x g for 150 min in a SW 40 rotor of the Spinco Model centrifuge (Booyse et a1 1968). The gradient was collected from the bottom of the tube by fractions of 7 drops. In preliminary experiments protein assays were made on each fraction and the results summed for recovery determination. For the serotonin uptake measurements the platelets from each of the 4 populations obtained (1st population in tubes nos. 10 t o 20, 2nd in nos. 21 t o 32, 3rd in nos. 33 to 44, 4th in nos. 45 t o 58) were pelleted by centrifugation at 500 x g for 15 min and washed twice with chilled 0.15 M NaCI. Then the platelets were incubated with mechanical stirring at 37OC for 30 min in an isotonic medium buffered at pH 7.4: disodium phosphate 16.24 m M , monosodium phosphate 3.76 mM, disodium EDTA 2.69 mM, glucose 5.55 mM, sodium acetate 11.03 mM, sodium chloride 104.54 mM, potassium chloride 4.02 mM and 2 14C serotonin creatinine sulphate 2.32 p M (spec act 48 mCVmmo1). Then the platelets were pelleted by centrifugation at 4 O C 500 x g for 15 min and twice washed with chilled isotonic saline. The sedimented platelets were broken with 1 ml of 0.02 N HCI. An aliquot was used for protein assay (Lowry et a1 1951) and 0.5 ml was deproteinized by addition of 50 pl Zn so4 (347 mM) and 25 p1 of 1 N NaOH. After centrifugation at 2700 x g for 30 min, 0.5 ml of the supernatant was mixed with 10 ml of scintillation liquid (Instagel Packard) and counted in a liquid scintillation spectrometer (Packard 3380). These counts were never less than twice the background. Finally the results could be expressed as jcmoVg protein.
267
as per cent of amount of protein applied on the top of gradient. In preliminary assays it reached (mean jz SEM) 91.3 k 2.4 % (sum of the results found for each fraction of 7 drops). The recovery sinks to 24.8 k 0.8 % for 15 assays made on platelets pelleted after incubation for serotonin uptake determinations (sum of the results found for each of the 4 populations). The decrease of recovery was not uniformly distributed: it was particularly important for the 4th population which is the lightest (Table 1). Serotonin uptake is the highest in the lightest population (the 4th) and the decreasing order is 4th, 3rd, lst, 2nd (Table 2). According to Booyse et a1 (1968) and Booyse & Rafelson (1969) this corresponds to the platelet age. Thus the platelets take up serotonin the better the older 0pti:al density
0 .l
0.2
RESULTS
The separation of blood platelets in 4 populations was very reproducible and a typical pattern of one of the 12 preliminary assays is shown in Figure 1. Platelet recovery was measured by protein assays and expressed
Figure 1. Collection of sucrose gradient: from the bottom of the tube 7 drops were collected in each tube. In ordinate: optical density measured in protein assay as indicates by Lowry et a1 (1951).
P. KAMOUN, G . LAFOURCADE & H. JEROME
268
TABLE 1 Distribution o f human blood platelets between four populations
A
Mean SEM
28.7 1.3
37.7 1.3
17.3 0.8
16.4 0.6
B
Mean SEM
27.1 3.4
49.5 2.8
16.0 2.0
7.3 0.8
A - Preliminary assays: percentage of the sum of proteins measured on each tube collected from the gradient. B - Incubation assays: percentage of the sum of recovered proteins in the lysats prepared after platelet incubation for 30 min with serotonin. TABLE 2 Net uptake of serotonin f o r 30 min by 4 various human blood platelet populations
Age
I
Sex M M F F M M M F F M M M M F M
37 26
54 18 38 51 23 52 25 25 22 36 23 40 39
Mean SEM
1
(umol/g protein) 1st
1
2.9 3.3 2.1 2.3 2.1 2.3 2.3 2.5 2.9 2.5 3.4 2.1 2.3 2.8 2.0
1.7 2.6 2.2 1.9 1.5 1.7 2.1 1.9 2.2 1.4 1.o 2.7 1.9 1.7 1.3
2.5 0.11
1.9 0.12
they are. The individual values vary greatly but in regard to serotonin uptake the order of populations is almost constant among all subjects. A variance analysis perfomied on individual results shows no difference between subjects but a very significant difference between the four populations (p < 0.001). DISCUSSION
Blood platelets are separated by a sucrose
1
2nd
.
3rd
1
4th
2.9 3.7 3.9 4.1 4.9 4.1 3.1 1.9 3.2 2.1 1.5 2.5 1.7 2.0 1.1
4.8 6.7 3.7 4.5 3.9 3.0 5.0 2.6 4.4 3.8 2.9 3.7 3.2 4.2 3.1
2.8 0.29
4.0 0.27
gradient which is hyperosmolar and it may be possible that the platelets were obviously harmed by the isolation procedure to different extents along the gradient. That hypothesis may account for the variations observed in platelet recovery and serotonin uptake between the 4 populations. But we have to point out that the 4th population (the lightest) is isolated in sucrose 50 % (the smaller concentration) and even despite that the loss during incubation is particularly heavy for this population.
SEROTONIN UPTAKE BY PLATELETS
Blood platelets take up serotonin by an active energy-dependent mechanism (Hardisty & Stacey 1955), which is inhibited by metabolic inhibitors such as cyanide (Born & Gillson 1959) and fluoride (Weissbach et a1 1960). The serotonin taken up by platelets from the surrounding medium is localized at least partly in dense osmiophilic organelles (Tranzer et a1 1968) where it forms a complex with ATP (Da Prada et a1 1972). According to Born et a1 (1958) there is a direct relationship between ATP and serotonin concentrations. ATP concentrations in platelet populations have been investigated by Karpatkin (1969). His separation method enabled the separation of only two populations: a large heavy population with a density greater than 1.055 and a light small platelet population with a density below 1.046. The quantity of ATP contained in loll platelets came to 5.90 pmol for the heavy platelets and 2.08 pmol for the light platelets. The difference was less if the concentrations were expressed by ml of platelet volume (5.0 and 4.1 respectively), and cancelled out altogether if the ATP concentrations were related to the protein concentrations (25.6 and 23.3 pmoVg protein respectively, which scarcely differs from concentrations observed in whole blood platelets: 25.7 pmol/g protein). In our investigation the serotonin uptake was expressed against protein. It is conceivable therefore that the observed variations are independent of ATP concentrations. But since the protein content per platelet was distinctly less in the light populations [lightheavy ratio 0.39 for Karpatkin (1969)], conceivably if the serotonin uptake had been expressed in pmol per platelet it would have eliminated the differences observed between populations.
269
However, this does not explain the discordance observed between our results and those of Rendu & Caen (1973). They stated that serotonin uptake (expressed as pmoVg protein) is greater for the heavy platelets than for the light ones. The technique used by Rendu & Caen (1973) differs considerably from ours, because they separated the platelets into 4 populations only after serotonin uptake and without elimination of sucrose. Moreover, the platelets were incubated in plasma together with citric acid and without EDTA. Despite the serotonin concentration (5.4 1 c 6 M) used by these workers uptake for 30 min incubation was about 5 times less than that observed by us. Finally the authors do not account for the interference due to sucrose in determining protein content by the method of Lowry et a1 (1951). Our study is in agreement with the results of Hardeman (1974): platelet conservation at 37O C for 4 days with dialysis renewal of the plasma conservation medium does not modify serotonin uptake (expressed as pM/109 platelets). Since the life span of platelets is 7 days the absence of any overall variation in the serotonin uptake expressed by platelet would seem to be in favour of better serotonin uptake by the older (lighter) platelets than by the younger (heavier) ones when this uptake had been expressed against protein. ACKNOWLEDGEMENT These investigations were supported by a Grant from INSERM no. 874-24. REFERENCES Booyse F M, Hoveke T & Rafelson M E Jr (1968) Studies on human platelets. 11. Protein synthetic activity of various platelet populations. Biochim Biophys Acta 157, 660-63.
270
P. KAMOUN, G. LAFOURCADE & H. JEROME
Booyse F M & Rafelson M E J r (1969) Protein synthesis and platelet function. In S Johnson & M M Guest (eds) Dynamics o f thrombus formation and dissolution, pp 149-71. J B Lippincot, Philadelphia. Born G V R & Gillson R E (1959) Studies on the uptake of 5-hydroxytryptamine by blood platelets. J Physiol 146, 472-91. Born G V R, Ingram G I C & Stacey P S (1958) The relationship between 5-hydroxytryptamine and adenosine triphosphate in blood platelets. Brit J Pharmacol 13, 62-64. D a Prada M, Tranzer J P & Pletscher A (1972) Storage of 5-hydroxytryptamine in human blood platelets. Experientia 28, 1328-29. Dillard G H L, Brecher G & Cronkite E P (1951) Separation, concentration and transfusion of platelets. Proc Soc Exp Biol Med 78, 796-99. Hardeman M R (1974) The effect of continuous dialysis on platelet preservation. In C F Hogman, H W Krijnen & C R Valeri (eds) Symposium on platelet preservation and transfusion. Uppsala Offset Center, Uppsala, Sweden. Hardisty R M & Stacey R S (1955) 5-hydroxytryptamine in normal human platelets. 1 Physiol 130, 711-20. Hardisty R M & Stacey R S (1957) Platelet 5hydroxytryptamine (HT) in disorders of the blood. Br J Haematol 3, 292-98. Jerome H & Kamoun P (1970) Platelet binding of serotonin. Ann N Y Acad Sci 171, 543-50. Kamoun P & Jerome H (1976) Blood serotonin in
mental disorders. In D J Boullin (ed) Serotonin in mental disorders. John Wiley, London (in press). Karnoun P, Kleinknecht D, Ducrot H & Jerome H (1970) Platelet serotonin in uraemia. Lancet 1, 782. Karpatkin S (1969) Heterogeneity of human platelets. I. Metabolic and kinetic evidence suggestive of young and old platelets. J CIin Invest 48, 1073-82. Lowry 0 H, Rosebrough N J, Fan- A L & Randall R J (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-75. Polli E E, Bianchi P A & Crosti P F (1958) La serotonina piastrinica nelle leucemie. Haematologica Latina 1, 2 5 M 4 . Rendu F & Caen J P (1973) Effect of reserpine on 14C serotonin uptake by human platelet populations. Haemostasis 1, 161-68. Tranzer J P, Da Prada M & Pletscher A (1968) Electron microscopic study of the storage site of 5-hydroxytryptamine in blood platelets. Adv Pharmacol Chemother 6 A , 125-28. Vainer H, Besson P & Caen J (1971) La glycoghe synthktase dans les plaquettes et les populations plaquettaires anormales de syndromes myelo prolifkratifs. Nouv Rev Fr Hematol 11, 769-80. Weissbach H, Redfield B G & Titus E (1960) Effect of cardiac glycosides and inorganic ions on binding of serotonin by platelets. Nature 185, 99-100.
