- Email: [email protected]
A simple and efficient method for platelet isolation from their plasma
THROMBOSIS RESEARCH 17 ; 581-588 Printed in the United States © Pergamon Press Ltd . 1980 . o049-3848/8o/o215-0581 $02 .00/0
B R I E F
C 0 M M U N I C A .T 3: O N
A SIMPLE AND EFFICIENT METHOD FOR PLATELET ISOLATION FROM THEIR PLASMA
M . LAGARDEU, P .A . BRYON, M . GUICAABDANT and M . DECHAVANNE Inserm U 63, Institut Pasteur, Laboratoire d'hfimobiologie, Facult€ Alexis Carrel, 69372 Lyon Cedex 2, France (Received 23 .5 .1979 ; in revised form 29 .10 .1979 Accepted by Editor M . Samama)
INTRODUCTION Several methods have been used for platelet isolation from plasma . Such methods utilize albumin (1-4) or EDTA (5,6) in suspension medium, except for centrifugation on sodium motrizoate (7) . Most of these isolation techniques alter platelet ultrastructure with the exception of Mustard's isolation technique (8) . Isolated platelets by the various methods have shown a decreased radioactivity to inducers (9) . Moreover, albumin is not convenient since it binds a large quantity of fatty acids and particularly fatty acids used as substrates of platelet prostaglandin synthetase such as arachidonic acid (10, 11) . We describe here a quick, simple and convenient technique for separating human platelets from their plasma . METHODS Reagents : Sodium arachidonate was prepared by equimolar treatment of arachidonic acid (Sigma, St Louis) with sodium hydroxyde and diluted in distilled water . Bovine thrombin provided from Hoffman-La Roche, Basel, collagen from Horm, Munchen, ADP from Boehringer, Mannheim, human fibrinogen from Kabi, Stockholm, He as, apyrase and hirudin from Sigma, St Louis . [ 125 -I]-human albumin and Nap 1 Cr0 4 were purchased by CIS-Sorin ; [ 125 1]-human fibrinogen and [2- 14 C] serotonin from the Radiochemical Centre of Amersham . Other reagents were furnished by Prolabo, Paris . 9-methano analogue of prostaglandin H2 was a generous gift from Dr . J .E . Pike (Upjohn Co, Kalamazoo) .
Key Words : Platelet suspension, platelet isolation
•
to whom correspondence should be addressed .
581
582
HUMAN PLATELET ISOLATION
Vol .17,No .3/4
Bloodcollection Venous punction of blood was done on ACD as anticoagulant (citric acid 0 .8 %, trisodium citrate 2 .2 %, dextrose 2 .45 %, pH 4 .5) with I volume for 9 volumes of blood . Platelet preparation : All operations were done at room temperature . Blood was centrifuged at 1OOg during 15 min . After removal, platelet rich plasma (PRP) was acidified at pH 6 .4 with citric acid 0 .15 M and fractions of 6 ml were centrifuged at 900g during 10 min . i n siliconized conical tubes . For each tube, supernatant (platelet poor plasma) was completely removed and pellet was suspended in 6 ml of modified Tyrode-Hepes buffer pH 7 .35 containing NaCl 0 .137 M, KCI : 2 .68 mM, NaHC0 3 : 11 .9 mM, NaH2P0" : 0 .416 mM, MgC1 2 : 1 mM, dextrose : 0 .555 mM, Hepes : 5 mM . Platelet suspensions were maintained at room temperature all day . Their concentration was adjusted to 3 .10 8 platelet/ ml before testing . Contamination by plasma proteins : To test contamination of the platelet suspension by plasma components, [" 5 I]-human albumin or fibrinogen were incubated with plasma before platelet centrifugation . Radioactivity was determined in platelet poor plasma supernatant and in platelet suspension . Electron microscopy : For electron microscopy studies, samples (I ml) of platelet suspensions were prewarmed 10 min . a t 37 ° C before fixation, then they were fixed initially by addition of 0 .5 ml of 0 .15 % glutaraldehyde in Tyrode's solution at 20 ° C for 10 min ., the supernatant was removed, and the pellets were fixed for 30 min . i n Tyrode's solution containing 1 .5 Z glutaraldehyde, and post-fixed in I % osmic acid at 4 ° C for 60 min . The specimens were embedded in Epon 812 . Thin section were stained with uranyl acetate and lead citrate and examined in a JEOL . JEMT 7 microscope at 60 kV . An ultrastructural morphometric investigation was performed using a method previously described (12) to evaluate the possible influence of the preparative method on the size and shapes of platelet surface-connected system (S .C .S .) and granules . Platelet aggregation studies : Aggregations were performed according to the method of Born (13) and inducer addition was preceeded by addition of 2 mM CaCl 2 (10 yl of a 80 mM solution in 400 Ill of platelet suspension) . Only ADPinduced aggregation was performed immediately after platelet isolation . Release of platelet[ 1 YC serotonin : Platelet rich plasma was incubated with ['"C]-serotonin (1 pCi/ml) during 30 m in . a t room temperature before acidification . Thereafter, platelets were separated from plasma as described above . Release was measured at 37 ° C as previously described (14) . Platelet lysis : To measure the lytic effect of thrombin, platelet rich plasma was incubated with Na 2 51 Cr" (specific radioactivity : 2 .10 5 Ci/mole during 30 min . a t room temperature) before acidification . The radioactivity of supernatants was expressed as a percentage of total radioactivity . RESULTS Contamination of platelet suspension by plasma components, tested by retained radioactive albumin and fibrinogen was respectively 0 .7 % and 0 .6 % of initial content in plasma (measured twice) . The ultrastructural appearance of platelets isolated by the method is shown figure 1 . They retained their disc shape, with uniform distribution of cytoplasmic structures . Microtubules were evident, and the surface-connected canalicular system (S .C .S .) did not appear to be dilated . The structure of these platelets was similar to the resting platelets from PRP .
Vol .17,No .3/ 4
HUMAN PLATELET ISOLATION
FIG . 7 PHOTOMICROGRAPHS OF PLATELETS ISOLATED USING THE TYRODE-HEPES BUFFER MEDIUM (X 29 .500)
583
HUMAN PLATELET ISOLATION
584
Vol .17,No .3/4
The relative volume of specific granules (including a-granules and dense granules) was 0 .1091 ± 0 .0053 (n - 3) for the control platelets from PRP and 0 .1122 ± 0 .0047 (n - 3) for the isolated platelets . The relative volume of S .C .S . was 0 .1016 ± 0 .0082 (n - 3) for the P .R .P . platelets and 0 .0964 ± 0 .0056 (n - 3) for the isolated platelets . Direct measurements of the long and short axes showed the same results : no significant differences could be demonstrated for any of the measured parameters . Aggregability of platelet suspensions, plotted in figure 2, shows a responsiveness at low doses of different aggregating agents . ADP-induced aggregation was obtained in the presence of human fibrinogen 2 g/l . Sodium arachidonate PG112 Fibrinogen Thrombin Collagen 0 .02 U/ml 1 .25 jig/ml 2 .5 uM 9 m€thanoADP 2 {1M analogous 0 .1 u8/ml l j
I
1 min . FIG .2 Aggregation of platelet suspended in Tyrode-Hepes buffer stimulated by different inducers . Platelet aggregation induced by low concentration of collagen was not inhibited by hirudin (figure 3) .
Tyrodel ' Thrombin 0 .1 U/ml
Hirudi~_I U/ml Thrombi n 0 .1 U/ml
Collagen 1 .25 Mg/ml
Collagen 1 .25 ug/ml
FIG .3 . Effect of hirudin on platelet aggregation induced by thrombin or collagen .
Vol .17,No .3/ 4
HUMAN PLATELET
ISOLATION
585
Results of 04C ]-serotonin release, shown in table I, demonstrate a good reactivity of platelets although the release control is low . TABLE I
2 .3 . 0 .7 Z
Control Collagen
2 .5 11g/ml
46 .1 ± 1 .0 Z
0 .04 U/ml
48 .4 i 1 .6 Z
0 .1 U/ml
67 .7 ± 0 .7 Z
5 .10-6 M
25 .0 ± 2 .4 Z
10-5 M
49 .9 t 2 .4 Z
2 uH
1 .0 t 0 .1 Z
Thrombin
Arachidonate ADP
[ 1 °C]-serotonin release from platelets in Tyrode-Rapes . Results are expressed as percentage of total radioactivity in platelets . Each value represents the mean ± a standard deviation of 10 determinations in two different suspensions . Platelets lysis, induced by thrombin is very low in the presence of high doses of the agent (table II) . TABLE II
Concentrations of thrombin Percentage of released radioactivity
0 .1 U/ml
0 .3 t 0 .3
1 U/ml
0 .7 ± 0 .2
5 U/ml
1 .8 t 1 .2
[s 1 Cr] from platelets after incubation with thrombin at 37 ° C for 4 min . Results are expressed as percentage of total radioactivity in platelets . Each value represents the mean ± a standard deviation of 4 determinations . The method was transformed in a washing technique with slight modifications . For this, the first medium of platelet resuspersion contained serumalbumin 3 .5 g/l . Thereafter, the suspension was acidified as described above, centrifuged at 700g for 10 min . and platelets resuspended in the buffer without albumin . The responsiveness of these platelets was about the same that described above (results not shown) . DISCUSSION The method described in this paper makes use of a simple platelet isolation technique with a weak contamination of platelet suspension by present plasma proteins . However it was necessary to suspend platelets in a calcium free medium to avoid fibrin formation : calcium 2 .10 y M was added just before testing .
586
HUMAN PLATELET ISOLATION
Vol .17,No .3/4
Platelets separated from blood by the present method had no significant ultrastructural change compared to platelets in PEP . Previous- studies had town that the separation of human platelets from blood may produce ultrastructural alterations of varying degrees ; irregularity of cytoplasmic outlines, formation of pseudopods, dilatation of surface-connected canalicular system, central position of granules, formation of empty cytoplasmic sacs . In considering these ultrastructural features, the isolation method of MUSTARD (I) showed the least changes (8) . The platelets separated by the method described in this paper have a similar ultrastructural appearance, retaining their disc shape, with uniform distribution of cytoplasmic structures and without any dilatation of the surface connected canalicular system . A pre-warming at 37 ° C was necessary to obtain this excellent ultrastructural morphology . Aggregation of these platelets was obtained with low doses of inducers and particularly with sodium arachidonate . Absence of albumin in the medium explains the very good responsiveness to arachidonate . A dose as low as 2 .5 .10-6 M of arachidonate was sufficient to provoke aggregation while platelets washed with Mustard's method (albumin 3 .5 g/1) required at least 50 .10 -6M for the similar results . ADP-induced aggregation was done as soon as platelets were resuspended in Tyrode-Repes buffer because, in contrast to other inducers, aggtr gability to this diminished with time . However it was possible to maintain aggregatility to ADP by pre-incubation of PRP with apyrase 100 mg/1 at room temperature during 30 min . Platelet aggregation induced by low doses of an agent such as collagen was not inhibited by hirudin, a specific antithrombin component . This fact showed that no traces of thrombin were generated during activation . An important percentage of release was obtained with various inducers although the release of controls were very low . These results indicate a high stability of unstismlated platelets and a good responsiveness . Platelet lysis, investigated with s 1 Cr as tool, is very low and shows a good stability of these platelets . Preparation of these platelet suspensions is a convenient method to study prostaglandin biosynthesis from either exogenous or endogenous arachidonic acid, since this technique does not use albumin which strongly binds the acid . Because of the above mentioned reasons, labelling of platelets with traces of arachidonic acid was very efficient (results not shown) . Lastly, this method of platelet preparation is suitable to assay the release reaction induced by sera from patients with idiopathic thrombopenic purpura (15-16) . In conclusions, the reported method is very convenient to work with a small volume of PRP (6 ml) yielding platelets with a high stability and a good ultrastructural state . ACKNOWLEDGEMENT This work was supported by grants INSERM, ASR N ° 5 and U .E .R . Lyon-Nord . REFERENCES I . MUSTARD, J .F ., PERRY, D .W ., ARDLIE, M .G . and PAG1M}I, M .A . Preparation of
Vo1 .17,No . 3 /4
HUMAN PLATELET ISOLATION
Suspensions of washed platelets from humans . &t it . J . 1972 .
587
Haemat . 22,
193,
2 . NICHOLLS, D .G . and HAMPTON, J .R . Density gradient separation of human platelet from-plasma and the role of plasma in adenosine diphosphate induced platelet electrophoretic mobility changes . Thnombo6 . V.Lathes . Haemonnh . 27, 425, 1972 . 3.
TANGEN, 0 ., ANDRAE, M .L . and NILSSON, B .E . Nucleotide leakage from platelets in artificial media : prevention by albumin and other macromolecules and relation to ADP-induced platelet aggregation . Scand . J . Haemat . 11, 241, 1973 .
4 . LACES, B ., SCRUTTON, M .C . and HOLMSEN, H . Studies on gel-filtered human platelets : isolation and characterization in a medium containing no added Ca 2+ , Mgt+ or K . J . Lab . C.Un . Med . 85, 811, 1975 . 5 . TANGEN, 0 ., BERMAN, H .L . and MERFEY, P . Gel filtration . A new technique for separation of blood platelets from plasma . Thumb . D.iatheb . Haemarth . 25, 268, 1971 . 6 . CRONBERG, S . and CAEN, J .P . Platelet aggregation in washed suspensions . Scand . J . Haemat . 8, 161, 1971 . 7 . GANGULY, P . and SONNICHSEN, W .J . A simple method for the isolation of blood platelets . J . Ctin . Pathot . 26, 635, 1973 . 8 . 2UCKER, W .H ., SHERMER, R .W . and MASON, R .G . Ultrastructural comparison of human platelets separated from blood by various means . Am . J . Patho .t . 77, 255, 1974 . 9 . MASON, R .G ., READ, S . and SHERMER, R .W . Comparison of certain functions of human platelets separated from blood by variousreans . Am . J . Patho .t . 76, 323, 1974 . 10 . WHITE, H .L . and GLASSMAN, A .T . Biochemical properties of the prostaglandin/thromboxane synthetase of human blood platelets and comparison with the synthetase of bovine seminal vesicles . Pho4tag.tandLyb 12, 811, 1976 . 11 . YOSHIDA, N . and AOKI, N . Release of arachidonic acid from human platelets . A key role for the potentiation of platelet aggregability in normal subjects as well as those nephrotic syndrome . Stood 52, 969, 1978 . 12 . BRYON, P .A ., LAGARDE, M . et DECHAVANNE M . Etude ultrastructurale quantitative de la degranulation et de la concentration des plaquettes sous 1'induction de la thrombine "in vitro" . Nouv . Rev . Haemat . 20, 173, 1978 . 13 . BORN, G .V .R . Aggregation of blood platelets by adenosine diphosphate and its reversal . Na.un.e 194, 927, 1962 . 14 . LAGARDE, M ., BRYON, P .A ., VARGAFTIG, B . and DECHAVANNE, M . Impairment of platelet thromboxane A 2 generation and of the platelet release reaction in two patients with congenital deficiency of platelet cyclo-oxygenase . BxLt . J . Haemat . 38, 251, 1978 . 15 . HIRSCHMAN, R .J . and SHULNAN, N .R . The use of platelet serotonin release
588
HUMAN PLATELET ISOLATION
Vol .17,No .3/4
as a sensitive method for detecting anti-platelet antibodies and a plasma anti-platelet factor in patients with idiopathic thrombocytopenic purpura . &tit . J . Haemat . 24, 793, 1973 . 16 . DECHAVANNE, M ., LAGARDE, M ., ARNAUD, P ., THOUVEREZ, J .P . CREYSSEL, R . at VIALA, J .J . Miss en Evidence "in vitro" d'anticorps antiplaquettaires au cours du purpura thrombop€pique idiopathique par Is meanre de 1'excr€tion plaquettaire de [ 3° C]-serotonine . Path . Fa.oL . 22, 17, 1*74 .
B R I E F
C 0 M M U N I C A .T 3: O N
A SIMPLE AND EFFICIENT METHOD FOR PLATELET ISOLATION FROM THEIR PLASMA
M . LAGARDEU, P .A . BRYON, M . GUICAABDANT and M . DECHAVANNE Inserm U 63, Institut Pasteur, Laboratoire d'hfimobiologie, Facult€ Alexis Carrel, 69372 Lyon Cedex 2, France (Received 23 .5 .1979 ; in revised form 29 .10 .1979 Accepted by Editor M . Samama)
INTRODUCTION Several methods have been used for platelet isolation from plasma . Such methods utilize albumin (1-4) or EDTA (5,6) in suspension medium, except for centrifugation on sodium motrizoate (7) . Most of these isolation techniques alter platelet ultrastructure with the exception of Mustard's isolation technique (8) . Isolated platelets by the various methods have shown a decreased radioactivity to inducers (9) . Moreover, albumin is not convenient since it binds a large quantity of fatty acids and particularly fatty acids used as substrates of platelet prostaglandin synthetase such as arachidonic acid (10, 11) . We describe here a quick, simple and convenient technique for separating human platelets from their plasma . METHODS Reagents : Sodium arachidonate was prepared by equimolar treatment of arachidonic acid (Sigma, St Louis) with sodium hydroxyde and diluted in distilled water . Bovine thrombin provided from Hoffman-La Roche, Basel, collagen from Horm, Munchen, ADP from Boehringer, Mannheim, human fibrinogen from Kabi, Stockholm, He as, apyrase and hirudin from Sigma, St Louis . [ 125 -I]-human albumin and Nap 1 Cr0 4 were purchased by CIS-Sorin ; [ 125 1]-human fibrinogen and [2- 14 C] serotonin from the Radiochemical Centre of Amersham . Other reagents were furnished by Prolabo, Paris . 9-methano analogue of prostaglandin H2 was a generous gift from Dr . J .E . Pike (Upjohn Co, Kalamazoo) .
Key Words : Platelet suspension, platelet isolation
•
to whom correspondence should be addressed .
581
582
HUMAN PLATELET ISOLATION
Vol .17,No .3/4
Bloodcollection Venous punction of blood was done on ACD as anticoagulant (citric acid 0 .8 %, trisodium citrate 2 .2 %, dextrose 2 .45 %, pH 4 .5) with I volume for 9 volumes of blood . Platelet preparation : All operations were done at room temperature . Blood was centrifuged at 1OOg during 15 min . After removal, platelet rich plasma (PRP) was acidified at pH 6 .4 with citric acid 0 .15 M and fractions of 6 ml were centrifuged at 900g during 10 min . i n siliconized conical tubes . For each tube, supernatant (platelet poor plasma) was completely removed and pellet was suspended in 6 ml of modified Tyrode-Hepes buffer pH 7 .35 containing NaCl 0 .137 M, KCI : 2 .68 mM, NaHC0 3 : 11 .9 mM, NaH2P0" : 0 .416 mM, MgC1 2 : 1 mM, dextrose : 0 .555 mM, Hepes : 5 mM . Platelet suspensions were maintained at room temperature all day . Their concentration was adjusted to 3 .10 8 platelet/ ml before testing . Contamination by plasma proteins : To test contamination of the platelet suspension by plasma components, [" 5 I]-human albumin or fibrinogen were incubated with plasma before platelet centrifugation . Radioactivity was determined in platelet poor plasma supernatant and in platelet suspension . Electron microscopy : For electron microscopy studies, samples (I ml) of platelet suspensions were prewarmed 10 min . a t 37 ° C before fixation, then they were fixed initially by addition of 0 .5 ml of 0 .15 % glutaraldehyde in Tyrode's solution at 20 ° C for 10 min ., the supernatant was removed, and the pellets were fixed for 30 min . i n Tyrode's solution containing 1 .5 Z glutaraldehyde, and post-fixed in I % osmic acid at 4 ° C for 60 min . The specimens were embedded in Epon 812 . Thin section were stained with uranyl acetate and lead citrate and examined in a JEOL . JEMT 7 microscope at 60 kV . An ultrastructural morphometric investigation was performed using a method previously described (12) to evaluate the possible influence of the preparative method on the size and shapes of platelet surface-connected system (S .C .S .) and granules . Platelet aggregation studies : Aggregations were performed according to the method of Born (13) and inducer addition was preceeded by addition of 2 mM CaCl 2 (10 yl of a 80 mM solution in 400 Ill of platelet suspension) . Only ADPinduced aggregation was performed immediately after platelet isolation . Release of platelet[ 1 YC serotonin : Platelet rich plasma was incubated with ['"C]-serotonin (1 pCi/ml) during 30 m in . a t room temperature before acidification . Thereafter, platelets were separated from plasma as described above . Release was measured at 37 ° C as previously described (14) . Platelet lysis : To measure the lytic effect of thrombin, platelet rich plasma was incubated with Na 2 51 Cr" (specific radioactivity : 2 .10 5 Ci/mole during 30 min . a t room temperature) before acidification . The radioactivity of supernatants was expressed as a percentage of total radioactivity . RESULTS Contamination of platelet suspension by plasma components, tested by retained radioactive albumin and fibrinogen was respectively 0 .7 % and 0 .6 % of initial content in plasma (measured twice) . The ultrastructural appearance of platelets isolated by the method is shown figure 1 . They retained their disc shape, with uniform distribution of cytoplasmic structures . Microtubules were evident, and the surface-connected canalicular system (S .C .S .) did not appear to be dilated . The structure of these platelets was similar to the resting platelets from PRP .
Vol .17,No .3/ 4
HUMAN PLATELET ISOLATION
FIG . 7 PHOTOMICROGRAPHS OF PLATELETS ISOLATED USING THE TYRODE-HEPES BUFFER MEDIUM (X 29 .500)
583
HUMAN PLATELET ISOLATION
584
Vol .17,No .3/4
The relative volume of specific granules (including a-granules and dense granules) was 0 .1091 ± 0 .0053 (n - 3) for the control platelets from PRP and 0 .1122 ± 0 .0047 (n - 3) for the isolated platelets . The relative volume of S .C .S . was 0 .1016 ± 0 .0082 (n - 3) for the P .R .P . platelets and 0 .0964 ± 0 .0056 (n - 3) for the isolated platelets . Direct measurements of the long and short axes showed the same results : no significant differences could be demonstrated for any of the measured parameters . Aggregability of platelet suspensions, plotted in figure 2, shows a responsiveness at low doses of different aggregating agents . ADP-induced aggregation was obtained in the presence of human fibrinogen 2 g/l . Sodium arachidonate PG112 Fibrinogen Thrombin Collagen 0 .02 U/ml 1 .25 jig/ml 2 .5 uM 9 m€thanoADP 2 {1M analogous 0 .1 u8/ml l j
I
1 min . FIG .2 Aggregation of platelet suspended in Tyrode-Hepes buffer stimulated by different inducers . Platelet aggregation induced by low concentration of collagen was not inhibited by hirudin (figure 3) .
Tyrodel ' Thrombin 0 .1 U/ml
Hirudi~_I U/ml Thrombi n 0 .1 U/ml
Collagen 1 .25 Mg/ml
Collagen 1 .25 ug/ml
FIG .3 . Effect of hirudin on platelet aggregation induced by thrombin or collagen .
Vol .17,No .3/ 4
HUMAN PLATELET
ISOLATION
585
Results of 04C ]-serotonin release, shown in table I, demonstrate a good reactivity of platelets although the release control is low . TABLE I
2 .3 . 0 .7 Z
Control Collagen
2 .5 11g/ml
46 .1 ± 1 .0 Z
0 .04 U/ml
48 .4 i 1 .6 Z
0 .1 U/ml
67 .7 ± 0 .7 Z
5 .10-6 M
25 .0 ± 2 .4 Z
10-5 M
49 .9 t 2 .4 Z
2 uH
1 .0 t 0 .1 Z
Thrombin
Arachidonate ADP
[ 1 °C]-serotonin release from platelets in Tyrode-Rapes . Results are expressed as percentage of total radioactivity in platelets . Each value represents the mean ± a standard deviation of 10 determinations in two different suspensions . Platelets lysis, induced by thrombin is very low in the presence of high doses of the agent (table II) . TABLE II
Concentrations of thrombin Percentage of released radioactivity
0 .1 U/ml
0 .3 t 0 .3
1 U/ml
0 .7 ± 0 .2
5 U/ml
1 .8 t 1 .2
[s 1 Cr] from platelets after incubation with thrombin at 37 ° C for 4 min . Results are expressed as percentage of total radioactivity in platelets . Each value represents the mean ± a standard deviation of 4 determinations . The method was transformed in a washing technique with slight modifications . For this, the first medium of platelet resuspersion contained serumalbumin 3 .5 g/l . Thereafter, the suspension was acidified as described above, centrifuged at 700g for 10 min . and platelets resuspended in the buffer without albumin . The responsiveness of these platelets was about the same that described above (results not shown) . DISCUSSION The method described in this paper makes use of a simple platelet isolation technique with a weak contamination of platelet suspension by present plasma proteins . However it was necessary to suspend platelets in a calcium free medium to avoid fibrin formation : calcium 2 .10 y M was added just before testing .
586
HUMAN PLATELET ISOLATION
Vol .17,No .3/4
Platelets separated from blood by the present method had no significant ultrastructural change compared to platelets in PEP . Previous- studies had town that the separation of human platelets from blood may produce ultrastructural alterations of varying degrees ; irregularity of cytoplasmic outlines, formation of pseudopods, dilatation of surface-connected canalicular system, central position of granules, formation of empty cytoplasmic sacs . In considering these ultrastructural features, the isolation method of MUSTARD (I) showed the least changes (8) . The platelets separated by the method described in this paper have a similar ultrastructural appearance, retaining their disc shape, with uniform distribution of cytoplasmic structures and without any dilatation of the surface connected canalicular system . A pre-warming at 37 ° C was necessary to obtain this excellent ultrastructural morphology . Aggregation of these platelets was obtained with low doses of inducers and particularly with sodium arachidonate . Absence of albumin in the medium explains the very good responsiveness to arachidonate . A dose as low as 2 .5 .10-6 M of arachidonate was sufficient to provoke aggregation while platelets washed with Mustard's method (albumin 3 .5 g/1) required at least 50 .10 -6M for the similar results . ADP-induced aggregation was done as soon as platelets were resuspended in Tyrode-Repes buffer because, in contrast to other inducers, aggtr gability to this diminished with time . However it was possible to maintain aggregatility to ADP by pre-incubation of PRP with apyrase 100 mg/1 at room temperature during 30 min . Platelet aggregation induced by low doses of an agent such as collagen was not inhibited by hirudin, a specific antithrombin component . This fact showed that no traces of thrombin were generated during activation . An important percentage of release was obtained with various inducers although the release of controls were very low . These results indicate a high stability of unstismlated platelets and a good responsiveness . Platelet lysis, investigated with s 1 Cr as tool, is very low and shows a good stability of these platelets . Preparation of these platelet suspensions is a convenient method to study prostaglandin biosynthesis from either exogenous or endogenous arachidonic acid, since this technique does not use albumin which strongly binds the acid . Because of the above mentioned reasons, labelling of platelets with traces of arachidonic acid was very efficient (results not shown) . Lastly, this method of platelet preparation is suitable to assay the release reaction induced by sera from patients with idiopathic thrombopenic purpura (15-16) . In conclusions, the reported method is very convenient to work with a small volume of PRP (6 ml) yielding platelets with a high stability and a good ultrastructural state . ACKNOWLEDGEMENT This work was supported by grants INSERM, ASR N ° 5 and U .E .R . Lyon-Nord . REFERENCES I . MUSTARD, J .F ., PERRY, D .W ., ARDLIE, M .G . and PAG1M}I, M .A . Preparation of
Vo1 .17,No . 3 /4
HUMAN PLATELET ISOLATION
Suspensions of washed platelets from humans . &t it . J . 1972 .
587
Haemat . 22,
193,
2 . NICHOLLS, D .G . and HAMPTON, J .R . Density gradient separation of human platelet from-plasma and the role of plasma in adenosine diphosphate induced platelet electrophoretic mobility changes . Thnombo6 . V.Lathes . Haemonnh . 27, 425, 1972 . 3.
TANGEN, 0 ., ANDRAE, M .L . and NILSSON, B .E . Nucleotide leakage from platelets in artificial media : prevention by albumin and other macromolecules and relation to ADP-induced platelet aggregation . Scand . J . Haemat . 11, 241, 1973 .
4 . LACES, B ., SCRUTTON, M .C . and HOLMSEN, H . Studies on gel-filtered human platelets : isolation and characterization in a medium containing no added Ca 2+ , Mgt+ or K . J . Lab . C.Un . Med . 85, 811, 1975 . 5 . TANGEN, 0 ., BERMAN, H .L . and MERFEY, P . Gel filtration . A new technique for separation of blood platelets from plasma . Thumb . D.iatheb . Haemarth . 25, 268, 1971 . 6 . CRONBERG, S . and CAEN, J .P . Platelet aggregation in washed suspensions . Scand . J . Haemat . 8, 161, 1971 . 7 . GANGULY, P . and SONNICHSEN, W .J . A simple method for the isolation of blood platelets . J . Ctin . Pathot . 26, 635, 1973 . 8 . 2UCKER, W .H ., SHERMER, R .W . and MASON, R .G . Ultrastructural comparison of human platelets separated from blood by various means . Am . J . Patho .t . 77, 255, 1974 . 9 . MASON, R .G ., READ, S . and SHERMER, R .W . Comparison of certain functions of human platelets separated from blood by variousreans . Am . J . Patho .t . 76, 323, 1974 . 10 . WHITE, H .L . and GLASSMAN, A .T . Biochemical properties of the prostaglandin/thromboxane synthetase of human blood platelets and comparison with the synthetase of bovine seminal vesicles . Pho4tag.tandLyb 12, 811, 1976 . 11 . YOSHIDA, N . and AOKI, N . Release of arachidonic acid from human platelets . A key role for the potentiation of platelet aggregability in normal subjects as well as those nephrotic syndrome . Stood 52, 969, 1978 . 12 . BRYON, P .A ., LAGARDE, M . et DECHAVANNE M . Etude ultrastructurale quantitative de la degranulation et de la concentration des plaquettes sous 1'induction de la thrombine "in vitro" . Nouv . Rev . Haemat . 20, 173, 1978 . 13 . BORN, G .V .R . Aggregation of blood platelets by adenosine diphosphate and its reversal . Na.un.e 194, 927, 1962 . 14 . LAGARDE, M ., BRYON, P .A ., VARGAFTIG, B . and DECHAVANNE, M . Impairment of platelet thromboxane A 2 generation and of the platelet release reaction in two patients with congenital deficiency of platelet cyclo-oxygenase . BxLt . J . Haemat . 38, 251, 1978 . 15 . HIRSCHMAN, R .J . and SHULNAN, N .R . The use of platelet serotonin release
588
HUMAN PLATELET ISOLATION
Vol .17,No .3/4
as a sensitive method for detecting anti-platelet antibodies and a plasma anti-platelet factor in patients with idiopathic thrombocytopenic purpura . &tit . J . Haemat . 24, 793, 1973 . 16 . DECHAVANNE, M ., LAGARDE, M ., ARNAUD, P ., THOUVEREZ, J .P . CREYSSEL, R . at VIALA, J .J . Miss en Evidence "in vitro" d'anticorps antiplaquettaires au cours du purpura thrombop€pique idiopathique par Is meanre de 1'excr€tion plaquettaire de [ 3° C]-serotonine . Path . Fa.oL . 22, 17, 1*74 .