Kurze Mitteilung Blut, Band 34, Seite 329-332 (1977) Centre of Blood Transfusion, Red Cross and Department of Physiology, School of Medicine, Namur
Cycl ic 3",5"-Adenosi ne M onophosphate Level a nd Adenylate Cyclase Activity in Human Blood Platelets during Storage in ACD Solution Evelyne Lammerant-Guillemare, Fran~oise Corcelle-Cerf and Jacques Lammerant Summary The level of cyclic 3',5'-adenosine monophosphate (cAMP) in human blood platelets and the activity of platelet adenylate cyctase in response to prostaglandin E1 stimulation do not change during two days storage at room temperature in ACD solution. However, the level of cyclic AMP is lower in platelets stored in ACD solution than in platelets from blood anticoagulated by ethylenediamine tetra-acetic acid. Zusammenfassung Die Konzentration yon zyklischem AMP und die Aktivit~t der Adenylat-Zyklase nach Stimulation mit Prostaglandin E1 in menschlichen Thrombozyten ~indert sich nicht, wenn diese zwei Tage bei Zimmertemperatur in ACD-L6sung gelagert werden. Die Konzentration yon zyklischem AMP in derart gelagerten Blutplitttchen ist niedriger als in Thrombozyten, die aus EDTA ungerinnbar gemachtem Blut gewonnen werden. Key words: Platelet storage, cAMP, prostaglandin El, ACD, EDTA.
Despite the current interest in the biochemistry and pharmacology of platelets [1 ], the level of cyclic 3',5'-adenosine monophosphate (cAMP) in human blood platelets during their storage for transfusion remains unsettled. Accordingly, we have examined the natural course of cyclic AMP in platelets stored during two days ~t room temperature in ACD solution and the activity of platelet adenylate cyclase in response to prostaglandin E1 stimulation during the storage. A Fenwall blood pack, containing 450 ml whole blood mixed with 67.5 ml ACD solution (0.8% citric acid, 2.2% sodium citrate and 2.24% dextrose) and three Fenwall transfer packs, the first containing 25 ml ACE) solution, were used under sterile conditions. The blood pack was centrifuged at 1,700 g for 5 min at 20 ~ C. The resultant platelet rich plasma was further mixed with the ACD solution in the first Eingegangen am 11.8. 1976
2~. Lammerant-Guillemare, F. Corcelle-Cerf and J. Lammerant
330
transfer pack and evenly distributed between the three transfer packs. Thus, 270 ml platelet rich plasma, approximately, were mixed with 92.5 ml ACD solution, in accordance with the usual procedure for platelet storage in Fenwall packs. The three transfer packs were stored at room temperature under constant agitation and used for cyclic AMP assay in the platelets on days 0 ( • 2 h storage), 1 and 2, respectively. The level of cyclic AMP in the platelets was measured by the method of Tovey, Oldham and Whelan [3], using the assay kit marketed by The Radiochemical Centre, Amersham, England. The platelet rich plasma stored in ACD solution was centrifuged at 4 ~ C, first at 120 g for 15 min to eliminate residual red cells, and again at 27,700 g for 60 min to separate a pellet of platelets from platelet poor plasma. The number of platelets in the pellet was taken as the difference between the count in platelet rich and platelet poor plasma, as determined by the phase microscopy technique. The pellet was re-suspended in 1 ml saline solution, deproteinized with ethanol and extracted as described for plasma samples in the leaflet furnished with the assay kit. Three extracts were prepared from each transfer pack and stored at 4 ~ C until the assay. The results were corrected for recoveries and expressed in terms of picomoles of cyclic AMP per 109 platelets.
Day of storage
Platelet concentration (109/1) n = 12
Cylic AMP in platelets (pmol/10 ~ platelets) n = 36
0 ( ! 2h) 1 2
253 _+ 14 246 _+ 14 241 _+ 11 p > 0.10
6.7 i 0.6 6.5 • 0.6 5.9 i 0.4 p > 0.10
Tab. 1 : Level of cyclic AMP in human blood platelets during storage of platelet rich plasma in ACD solution. Results are from 12 blood donors. Three platelet extracts were assayed for each donor. Data expressed as mean i s.e., p by variance analysis.
Platelets obtained from 12 unselected blood donors were stored and assayed. There was a slight but significant increase in the p H of the platelet rich plasma in the transfer packs, from 6.51 • 0.03 (s.e.) on day 0 to 6.74 + 0.03 (s.e.) on day 2 (p < 0.001 by variance analysis). As shown in Tab. 1, the platelet count in platelet rich plasma mixed with the ACD solution and the level of cyclic AMP in the platelets did not change during two days storage (p > 0.10 by variance analysis). However, the level of cyclic AMP in the platelets on day 0, or 6.7 • 0.6 (s.e.) pmol per 109 platelets, was significantly lower (p < 0.01) than that measured in platelets separated from blood anticoagulated by ethylene-diamine tetra-acetic acid, i.e. 9.6 • 0.6 (s.e.) pmol per 109 platelets (49 donors). Platelets from 10 additional unselected blood donors were examined to evaluate the effect of storage in ACD solution on their responsiveness to prostaglandin E1 in terms of adenylate cyclase stimulation. The effect of prostaglandin E1 was tested using the procedure described by Robison, Arnold, and I-Iartmann [2]. As shown in
Cyclic 3',5"-adenosine monophosphate level and adenyla/e cydase activity
331
Fig. 1, the response to prostaglandin E1 was essentially the same on day 0 and on day 2. The highest concentration of prostaglandin E~, 1000 ng per ml, elicited a 7-fold increase in the level of cyclic A M P in the platelets, as compared to the 10-fold increase observed by Robison, Arnold and Hartmann in platelets separated from blood collected into 3.2% sodium citrate (10% v/v).
4O
o
3oi
0 E
0 O 20 -~. a. ,r _J 10 >CO
C)
L
//
,
0 1 CONCENTRATION
,
,
10 100 OF PROSTAGLANDIN
,
1000 E 1 (nglml)
Fig. 1 : Effect of prostaglandin E 1 on the level of cyclic AMP in human blood platelets after 2 h (o) and 2 days (e) storage of platelet rich plasma inACD solution. The procedure used is that described by Robison, Arnold and Hartmann [1]. Results are from 10 blood donors. Values are means + s.e. of 30 measurements.
We thus found that the level of cyclic A M P in human blood platelets and the activity of platelet adenylate cyclase in response to prostaglandin E 1 stimulation do not change during two days storage at r o o m temperature in A C D solution. However, the level of cyclic A M P is lower in platelets stored in A C D solution than in platelets from blood anticoagulated by ethylene-diamine tetra-acetic acid.
Acknowledgments We thank Mrs. Christine 2Veveu and Mrs. Dani&le Ruelle for technical assistance. We also thank Dr. Jean de ViNe de Goyet, Director of the Centre de Transfusion Sanguine de la Croix Rouge in Namur, for his help and advice and Professor Ernest tVeytmans, Department of Quantitative Biology, Faculty of Sciences, Namur, for statistical treatment of our data. Prostaglandin E 1 was a gift of the Upjohn Company.
332
E. Lammerant-Guillemare, F. Corcelle-Cerf and.[. Lammerant
References 1. Biochemistry and Pharmacology of Platelets, Ciba Foundation Symposium 35, new series (Elsevier, Excerpta Medica, NorthHolland, Amsterdam, 1975). 2. Robison G. A., Arnold A. & Hartmann R. C. : Divergent effects of epinephrine and prostaglandin E1 on the level of cyclic
AMP in human blood platelets. Pharmacol. Res. Commun. 1, 325 (1969). 3. Tovey K. C., Oldham K. G. & Whelan J. A. M. : A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique. Clin. chim. Acta 56, 221 (1974).
Author's address : Prof. J. Lammerant, School of Medicine, Department of Physiology, rue de Brnxelles 61, B-5000 Namur, Belgium.
Cycl ic 3",5"-Adenosi ne M onophosphate Level a nd Adenylate Cyclase Activity in Human Blood Platelets during Storage in ACD Solution Evelyne Lammerant-Guillemare, Fran~oise Corcelle-Cerf and Jacques Lammerant Summary The level of cyclic 3',5'-adenosine monophosphate (cAMP) in human blood platelets and the activity of platelet adenylate cyctase in response to prostaglandin E1 stimulation do not change during two days storage at room temperature in ACD solution. However, the level of cyclic AMP is lower in platelets stored in ACD solution than in platelets from blood anticoagulated by ethylenediamine tetra-acetic acid. Zusammenfassung Die Konzentration yon zyklischem AMP und die Aktivit~t der Adenylat-Zyklase nach Stimulation mit Prostaglandin E1 in menschlichen Thrombozyten ~indert sich nicht, wenn diese zwei Tage bei Zimmertemperatur in ACD-L6sung gelagert werden. Die Konzentration yon zyklischem AMP in derart gelagerten Blutplitttchen ist niedriger als in Thrombozyten, die aus EDTA ungerinnbar gemachtem Blut gewonnen werden. Key words: Platelet storage, cAMP, prostaglandin El, ACD, EDTA.
Despite the current interest in the biochemistry and pharmacology of platelets [1 ], the level of cyclic 3',5'-adenosine monophosphate (cAMP) in human blood platelets during their storage for transfusion remains unsettled. Accordingly, we have examined the natural course of cyclic AMP in platelets stored during two days ~t room temperature in ACD solution and the activity of platelet adenylate cyclase in response to prostaglandin E1 stimulation during the storage. A Fenwall blood pack, containing 450 ml whole blood mixed with 67.5 ml ACD solution (0.8% citric acid, 2.2% sodium citrate and 2.24% dextrose) and three Fenwall transfer packs, the first containing 25 ml ACE) solution, were used under sterile conditions. The blood pack was centrifuged at 1,700 g for 5 min at 20 ~ C. The resultant platelet rich plasma was further mixed with the ACD solution in the first Eingegangen am 11.8. 1976
2~. Lammerant-Guillemare, F. Corcelle-Cerf and J. Lammerant
330
transfer pack and evenly distributed between the three transfer packs. Thus, 270 ml platelet rich plasma, approximately, were mixed with 92.5 ml ACD solution, in accordance with the usual procedure for platelet storage in Fenwall packs. The three transfer packs were stored at room temperature under constant agitation and used for cyclic AMP assay in the platelets on days 0 ( • 2 h storage), 1 and 2, respectively. The level of cyclic AMP in the platelets was measured by the method of Tovey, Oldham and Whelan [3], using the assay kit marketed by The Radiochemical Centre, Amersham, England. The platelet rich plasma stored in ACD solution was centrifuged at 4 ~ C, first at 120 g for 15 min to eliminate residual red cells, and again at 27,700 g for 60 min to separate a pellet of platelets from platelet poor plasma. The number of platelets in the pellet was taken as the difference between the count in platelet rich and platelet poor plasma, as determined by the phase microscopy technique. The pellet was re-suspended in 1 ml saline solution, deproteinized with ethanol and extracted as described for plasma samples in the leaflet furnished with the assay kit. Three extracts were prepared from each transfer pack and stored at 4 ~ C until the assay. The results were corrected for recoveries and expressed in terms of picomoles of cyclic AMP per 109 platelets.
Day of storage
Platelet concentration (109/1) n = 12
Cylic AMP in platelets (pmol/10 ~ platelets) n = 36
0 ( ! 2h) 1 2
253 _+ 14 246 _+ 14 241 _+ 11 p > 0.10
6.7 i 0.6 6.5 • 0.6 5.9 i 0.4 p > 0.10
Tab. 1 : Level of cyclic AMP in human blood platelets during storage of platelet rich plasma in ACD solution. Results are from 12 blood donors. Three platelet extracts were assayed for each donor. Data expressed as mean i s.e., p by variance analysis.
Platelets obtained from 12 unselected blood donors were stored and assayed. There was a slight but significant increase in the p H of the platelet rich plasma in the transfer packs, from 6.51 • 0.03 (s.e.) on day 0 to 6.74 + 0.03 (s.e.) on day 2 (p < 0.001 by variance analysis). As shown in Tab. 1, the platelet count in platelet rich plasma mixed with the ACD solution and the level of cyclic AMP in the platelets did not change during two days storage (p > 0.10 by variance analysis). However, the level of cyclic AMP in the platelets on day 0, or 6.7 • 0.6 (s.e.) pmol per 109 platelets, was significantly lower (p < 0.01) than that measured in platelets separated from blood anticoagulated by ethylene-diamine tetra-acetic acid, i.e. 9.6 • 0.6 (s.e.) pmol per 109 platelets (49 donors). Platelets from 10 additional unselected blood donors were examined to evaluate the effect of storage in ACD solution on their responsiveness to prostaglandin E1 in terms of adenylate cyclase stimulation. The effect of prostaglandin E1 was tested using the procedure described by Robison, Arnold, and I-Iartmann [2]. As shown in
Cyclic 3',5"-adenosine monophosphate level and adenyla/e cydase activity
331
Fig. 1, the response to prostaglandin E1 was essentially the same on day 0 and on day 2. The highest concentration of prostaglandin E~, 1000 ng per ml, elicited a 7-fold increase in the level of cyclic A M P in the platelets, as compared to the 10-fold increase observed by Robison, Arnold and Hartmann in platelets separated from blood collected into 3.2% sodium citrate (10% v/v).
4O
o
3oi
0 E
0 O 20 -~. a. ,r _J 10 >CO
C)
L
//
,
0 1 CONCENTRATION
,
,
10 100 OF PROSTAGLANDIN
,
1000 E 1 (nglml)
Fig. 1 : Effect of prostaglandin E 1 on the level of cyclic AMP in human blood platelets after 2 h (o) and 2 days (e) storage of platelet rich plasma inACD solution. The procedure used is that described by Robison, Arnold and Hartmann [1]. Results are from 10 blood donors. Values are means + s.e. of 30 measurements.
We thus found that the level of cyclic A M P in human blood platelets and the activity of platelet adenylate cyclase in response to prostaglandin E 1 stimulation do not change during two days storage at r o o m temperature in A C D solution. However, the level of cyclic A M P is lower in platelets stored in A C D solution than in platelets from blood anticoagulated by ethylene-diamine tetra-acetic acid.
Acknowledgments We thank Mrs. Christine 2Veveu and Mrs. Dani&le Ruelle for technical assistance. We also thank Dr. Jean de ViNe de Goyet, Director of the Centre de Transfusion Sanguine de la Croix Rouge in Namur, for his help and advice and Professor Ernest tVeytmans, Department of Quantitative Biology, Faculty of Sciences, Namur, for statistical treatment of our data. Prostaglandin E 1 was a gift of the Upjohn Company.
332
E. Lammerant-Guillemare, F. Corcelle-Cerf and.[. Lammerant
References 1. Biochemistry and Pharmacology of Platelets, Ciba Foundation Symposium 35, new series (Elsevier, Excerpta Medica, NorthHolland, Amsterdam, 1975). 2. Robison G. A., Arnold A. & Hartmann R. C. : Divergent effects of epinephrine and prostaglandin E1 on the level of cyclic
AMP in human blood platelets. Pharmacol. Res. Commun. 1, 325 (1969). 3. Tovey K. C., Oldham K. G. & Whelan J. A. M. : A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique. Clin. chim. Acta 56, 221 (1974).
Author's address : Prof. J. Lammerant, School of Medicine, Department of Physiology, rue de Brnxelles 61, B-5000 Namur, Belgium.
