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Platelet aggregation on whole blood after administration of ultra low dosage acetylsalicylic acid in healthy volunteers
THROMBOSIS RESEARCH 47; 373-377, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
BRIEF
COMMUNICATION
PLATELET AGGREGATION ON WHOLE BLOOD AFTER ADMINISTRATION OF ULTRA LOW DOSAGE ACETYLSALICYLIC ACID IN HEALTHY VOLUNTEERS.
C. DOUTREMEPUICH, 0. de SEZE, M.C. ANNE, E. HARIVEAU, R. QUILICHINI. Laboratoire d'Hematologie, Faculte de Pharmacie Place de la Victoire 33076 Bordeaux, France. (Received 15.5.1987; Accepted in original form 20.5.1987 by Editor L.J. Berliner)
INTRODUCTION Usually, acetylsalicylic acid (ASA) is administered for preventing arterial thrombosis. The dosages used, which are between 50 and 600 mg per 24 hr, involve the inhibition of platelet aggregation, and an extension of bleeding time (1). In a previous study, ultra low dosages of ASA were used in healthy adult volunteers (n=lO):for a single dose of ASA much lower than lmg, two hours after the drug administration, bleeding time was shorter (~(0.05) than for a dose of placebo solution (2). In order to examine this effect, the present study in healthy subjects (n=17) explored platelet aggregation in whole blood after ingestion of the same ultra low dosage of ASA.
MATERIALS AND METHODS Materials: Healthy volunteers: the subjects were healthy male (n=9) or female (n=8) adults (mean age 2 SD= 34.52 + 5.38 years) who had not ingested any antiinflammatory, antiaggregant or salicylate drugs for at least 10 days before the initiation of the study. Drugs: France):1 geometric to 334 x used, the Placebo France).
acetylsalicylic acid was prepared by Laboratory BOIRON (Lyon, mg of ASA was diluted in 1 ml of ethyl alcohol 70%. Then several dilutions (ratio l/100) were performed, and the last corresponded 109molecules per ml of solvant. On account of the small quantities concentration was calculated in molecules per ml. solution was ethyl alcohol supplied by Laboratory BOIRON (Lyon,
Keys words: Platelet Aggregation, Acetylsalicylic Acid 373
374
PLATELET AGGREGATION, ASPIRIN
Vol. 47, No. 3
Methods: Protocol: the administration of the tested drugs was randomized. After fasting or a light meal, the subjects kept 2 ml of ASA solution (A) or placebo solution (B), under their tongues for 30 seconds. At least 8 days separated the ingestion of the two products. No antiplatelet drug could be taken between these two study days. The order of drug ingestion was determined by a random table and was noted: A then B or B then A. The administration of the product corresponded to time T = 0. At T = 120 minutes, blood samples (IO ml) were collected from an antecubital vein, on 9% tri-sodium citrate (Laboratory STAGO), nineteen parts blood to one part anticoagulant. Biology: Platelet count was performed by optic microscopy. Platelet aggregation on whole blood was performed in the Chrono Log Whole Blood Aggregometer 500VS (Coultronics, Margency, France): 490 ~1 of blood was diluted half and half with isotonic saline (NaCl 0.9%) and was warmed for 2-3 minutes at 37OC in a non-siliconised glass cuvette (10 x 44 mm) with a teflon coated stir bar. The impedance electrode was inserted in the cuvette, the whole apparatus was put on steady state and then initial adjustement was made. The impedance platelet aggregation was calibrated on 2 units for 5 ohms. Then, 20 1-11of aggregating reagent were added, corresponding to the final concentration in whole blood: -ADP 10 PM (Laboratory STAGO) -Collagen 50 pg/ml (Laboratory STAGO) -Arachidonic acid 30 fig/ml(Laboratory SIGMA) 15 minutes after the addition of aggregating agents (for ensuring a plateau in the aggregation curve), the change of impedance (ohms) was measured by a program on a HP 85 computer (Hewlett-Packard). Velocity (% aggregation/minutes) and response time (seconds) were also calculated by the program. Statistical analysis: The mean and the standard deviation were calculated. Both the Student's t-test and the Hills and Armitage test were performed for each measurement between each series (ASA and placebo).
RESULTS
Platelet count: Platelet count was similar in the ASA and the placebo groups: no statistical difference was observed between A and B. Aggregating reagents: The study of platelet aggregation in whole blood, has, in recent years, become a subject of discussion (3). This technique of estimating platelet function is more physiological and provides better results than the classical optic method (5,6). Several parameters may thus be studied: - Platelet impedance aggregation: platelet aggregation is detected by passing a very small current between two electrodes immersed in a sample of blood. The technique was developed by the Wellcome Research Laboratories, Beckenham (3).
375
PLATELET AGGREGATION, ASPIRIN
Vol. 47, No. 3
- The response time is the time separating the addition of the aggregating reagent and the beginning of aggregation. The velocity is the speed of aggregation expressed in $ aggregation per minute.
Table no
1
Results of platelet aggregation (mean + SD and the Student's t-test between A and B.
A
ADP Impedance (ohms)
B
Student'st-test
9.59
+
3.97
11.68
+
4.61
P< 0.009
Responsetime(sec) 27.75
+
9.57
30.90
+
11.26
N.S.
Velocity ($/min) 24.75
5
9.64
23.43
z
10.27
N.S.
i COLLAGEN Impedance (ohms)
12.01
z
4.71
10.79
+
5.21
N.S.
Responsetime(sec) 80.88
+
20.50
78.80
+
17.43
N.S.
Velocity (%/min)
16.38
+
9.77
14.15
+
6.84
N.S.
A.A. Impedance (ohms)
12.50 +
3.94
12.22 +
4.06
N.S.
36.23 + 18.36
N.S.
Responsetime(sec) 34.66 + 10.13
Velocity (%/min) 30.40 2 13.64 29.20 2 JO.51
N.S.
N.S.:not significant
Table no1 shows a statistical difference (~(0.05) for platelet impedance aggregation induced by ADP: in the presence of ASA, impedance iS lower than in the presence of placebo. To study the impedance difference more precisely, the Hills and Armitage test was conducted: this test was used to show a possible order effect of product ingestion and/or an eventual interaction between the two drugs. For the ADP impedance item, no order effect or interaction resulted from the test. Response time and velocity were similar in the two groups: these results may be explained by the effect of ASA on the inhibition of secondary but not primary platelet aggregation.
376
PLATELET AGGREGATION, ASPIRIN
Vol. 47, No. 3
Impedance, response time and velocity values of collagen-induced aggregation are not statistically different between the two groups (Table noI): the collagen concentration used (50 pg/ml) may be too great to detect the effect of ASA on platelet aggregation; however, we did not want collagen concentration to be a limiting factor of aggregation. The last aggregating reagent, Arachidonic Acid (A.A.), gives results similar to collagen: no value (impedance,velocity or response time) presents a statistical difference between the two groups (Table noI). Sex influence on aggregation: The different results were studied in males and females to investigate any sex influence on aggregation values. Statistical analysis showed no difference between males and females for aggregation tests,apart from velocity induced by A.A. in the ASA group (~(0.05): the platelets of females reacted more quickly than the platelets of males to A.A.-induced aggregation.
DISCUSSION
A first study of ASA used at ultra low dosage showed a reduced bleeding time (2). It was therefore of interest to observe platelet aggregation in healthy subjects having such low dosages. This work did not include usual dosage of ASA (50 - 500 mg) because the actionsof ASA on platelets functions are known since a long time (1). The aim was to research whether reduced bleeding time observed previously was confirmed by a response time shorter or a change of impedance induced "in vitro" by aggregating reagent.However, our results do not show any difference between the ASA group and the placebo group apart from ADP impedance. A recent study, using low doses of ASA (10 mg daily during 3 weeks),showed that bleeding time could be normal when Thromboxane B2 (TX B2) synthesis was inhibited (4). However 10 mg of ASA have no influence on the urinary excretion of 6-keto-PGFlti.According the doses, ASA have different effects and it would be interesting to study TX B2 production and 6-keto-PGFlv excretion after administration of ASA at ultra low dosages. This study could not explain the action of ASA at ultra low dosage but point to another mode of action.
ACKNOWLEDGEMENTS
The authors are indebited to Mr A. MASSE, Mr H. PELLEVRAULT, Mr D. FAUDOT for their excellent assistance.
Vol. 47, No. 3
PLATELET AGGREGATION, ASPIRIN
377
REFERENCES
1. BUCHANAN M.R., HIRSH J. Effect of Aspirin on Hemostasis and Thrombosis. New Engl. Reg. Allergy ProcA, 26 - 32, 1986. 2. DOUTREMEPUICH C., PAILLEY D., DE SEZE O., ANNE M.C., PACCALIN J., QUILICHINI R. Variation du temps de saignement apres administration, a differentes posologies, d'acide acetylsalicylique chez le volontaire sain. In press. 3. CARDINAL D.C., FLOWER R.J. The electronic aggregometer: a novel device for assessing platelet behavior in blood. J. Pharm. Meth.,3,135 - 158, 1980. 4. KALLMANN R., NIEUWENHUIS H.K., DE GROOT P.G., VAN GIJN J., SIXMA J.J. Effects of low doses of Aspirin, 10 mg and 30 mg daily, on bleeding time,thromboxane production and 6-keto-PGFl excretion in healthy subjects Thromb. Res., 45, 355 - 361, 1987. 5. MACKIE I.J., JONES R., MACHIN S.J. Platelet impedance aggregation in whole blood and its inhibition by antiplatelet drugs. J. Clin. Pathol, 37, 874 - 878, 1984. 6. ZWIERZINA W.D., KUNZ F. A method of testing platelet aggregation in native whole blood. Thromb. Res., 38, 91 - 100, 1985.
BRIEF
COMMUNICATION
PLATELET AGGREGATION ON WHOLE BLOOD AFTER ADMINISTRATION OF ULTRA LOW DOSAGE ACETYLSALICYLIC ACID IN HEALTHY VOLUNTEERS.
C. DOUTREMEPUICH, 0. de SEZE, M.C. ANNE, E. HARIVEAU, R. QUILICHINI. Laboratoire d'Hematologie, Faculte de Pharmacie Place de la Victoire 33076 Bordeaux, France. (Received 15.5.1987; Accepted in original form 20.5.1987 by Editor L.J. Berliner)
INTRODUCTION Usually, acetylsalicylic acid (ASA) is administered for preventing arterial thrombosis. The dosages used, which are between 50 and 600 mg per 24 hr, involve the inhibition of platelet aggregation, and an extension of bleeding time (1). In a previous study, ultra low dosages of ASA were used in healthy adult volunteers (n=lO):for a single dose of ASA much lower than lmg, two hours after the drug administration, bleeding time was shorter (~(0.05) than for a dose of placebo solution (2). In order to examine this effect, the present study in healthy subjects (n=17) explored platelet aggregation in whole blood after ingestion of the same ultra low dosage of ASA.
MATERIALS AND METHODS Materials: Healthy volunteers: the subjects were healthy male (n=9) or female (n=8) adults (mean age 2 SD= 34.52 + 5.38 years) who had not ingested any antiinflammatory, antiaggregant or salicylate drugs for at least 10 days before the initiation of the study. Drugs: France):1 geometric to 334 x used, the Placebo France).
acetylsalicylic acid was prepared by Laboratory BOIRON (Lyon, mg of ASA was diluted in 1 ml of ethyl alcohol 70%. Then several dilutions (ratio l/100) were performed, and the last corresponded 109molecules per ml of solvant. On account of the small quantities concentration was calculated in molecules per ml. solution was ethyl alcohol supplied by Laboratory BOIRON (Lyon,
Keys words: Platelet Aggregation, Acetylsalicylic Acid 373
374
PLATELET AGGREGATION, ASPIRIN
Vol. 47, No. 3
Methods: Protocol: the administration of the tested drugs was randomized. After fasting or a light meal, the subjects kept 2 ml of ASA solution (A) or placebo solution (B), under their tongues for 30 seconds. At least 8 days separated the ingestion of the two products. No antiplatelet drug could be taken between these two study days. The order of drug ingestion was determined by a random table and was noted: A then B or B then A. The administration of the product corresponded to time T = 0. At T = 120 minutes, blood samples (IO ml) were collected from an antecubital vein, on 9% tri-sodium citrate (Laboratory STAGO), nineteen parts blood to one part anticoagulant. Biology: Platelet count was performed by optic microscopy. Platelet aggregation on whole blood was performed in the Chrono Log Whole Blood Aggregometer 500VS (Coultronics, Margency, France): 490 ~1 of blood was diluted half and half with isotonic saline (NaCl 0.9%) and was warmed for 2-3 minutes at 37OC in a non-siliconised glass cuvette (10 x 44 mm) with a teflon coated stir bar. The impedance electrode was inserted in the cuvette, the whole apparatus was put on steady state and then initial adjustement was made. The impedance platelet aggregation was calibrated on 2 units for 5 ohms. Then, 20 1-11of aggregating reagent were added, corresponding to the final concentration in whole blood: -ADP 10 PM (Laboratory STAGO) -Collagen 50 pg/ml (Laboratory STAGO) -Arachidonic acid 30 fig/ml(Laboratory SIGMA) 15 minutes after the addition of aggregating agents (for ensuring a plateau in the aggregation curve), the change of impedance (ohms) was measured by a program on a HP 85 computer (Hewlett-Packard). Velocity (% aggregation/minutes) and response time (seconds) were also calculated by the program. Statistical analysis: The mean and the standard deviation were calculated. Both the Student's t-test and the Hills and Armitage test were performed for each measurement between each series (ASA and placebo).
RESULTS
Platelet count: Platelet count was similar in the ASA and the placebo groups: no statistical difference was observed between A and B. Aggregating reagents: The study of platelet aggregation in whole blood, has, in recent years, become a subject of discussion (3). This technique of estimating platelet function is more physiological and provides better results than the classical optic method (5,6). Several parameters may thus be studied: - Platelet impedance aggregation: platelet aggregation is detected by passing a very small current between two electrodes immersed in a sample of blood. The technique was developed by the Wellcome Research Laboratories, Beckenham (3).
375
PLATELET AGGREGATION, ASPIRIN
Vol. 47, No. 3
- The response time is the time separating the addition of the aggregating reagent and the beginning of aggregation. The velocity is the speed of aggregation expressed in $ aggregation per minute.
Table no
1
Results of platelet aggregation (mean + SD and the Student's t-test between A and B.
A
ADP Impedance (ohms)
B
Student'st-test
9.59
+
3.97
11.68
+
4.61
P< 0.009
Responsetime(sec) 27.75
+
9.57
30.90
+
11.26
N.S.
Velocity ($/min) 24.75
5
9.64
23.43
z
10.27
N.S.
i COLLAGEN Impedance (ohms)
12.01
z
4.71
10.79
+
5.21
N.S.
Responsetime(sec) 80.88
+
20.50
78.80
+
17.43
N.S.
Velocity (%/min)
16.38
+
9.77
14.15
+
6.84
N.S.
A.A. Impedance (ohms)
12.50 +
3.94
12.22 +
4.06
N.S.
36.23 + 18.36
N.S.
Responsetime(sec) 34.66 + 10.13
Velocity (%/min) 30.40 2 13.64 29.20 2 JO.51
N.S.
N.S.:not significant
Table no1 shows a statistical difference (~(0.05) for platelet impedance aggregation induced by ADP: in the presence of ASA, impedance iS lower than in the presence of placebo. To study the impedance difference more precisely, the Hills and Armitage test was conducted: this test was used to show a possible order effect of product ingestion and/or an eventual interaction between the two drugs. For the ADP impedance item, no order effect or interaction resulted from the test. Response time and velocity were similar in the two groups: these results may be explained by the effect of ASA on the inhibition of secondary but not primary platelet aggregation.
376
PLATELET AGGREGATION, ASPIRIN
Vol. 47, No. 3
Impedance, response time and velocity values of collagen-induced aggregation are not statistically different between the two groups (Table noI): the collagen concentration used (50 pg/ml) may be too great to detect the effect of ASA on platelet aggregation; however, we did not want collagen concentration to be a limiting factor of aggregation. The last aggregating reagent, Arachidonic Acid (A.A.), gives results similar to collagen: no value (impedance,velocity or response time) presents a statistical difference between the two groups (Table noI). Sex influence on aggregation: The different results were studied in males and females to investigate any sex influence on aggregation values. Statistical analysis showed no difference between males and females for aggregation tests,apart from velocity induced by A.A. in the ASA group (~(0.05): the platelets of females reacted more quickly than the platelets of males to A.A.-induced aggregation.
DISCUSSION
A first study of ASA used at ultra low dosage showed a reduced bleeding time (2). It was therefore of interest to observe platelet aggregation in healthy subjects having such low dosages. This work did not include usual dosage of ASA (50 - 500 mg) because the actionsof ASA on platelets functions are known since a long time (1). The aim was to research whether reduced bleeding time observed previously was confirmed by a response time shorter or a change of impedance induced "in vitro" by aggregating reagent.However, our results do not show any difference between the ASA group and the placebo group apart from ADP impedance. A recent study, using low doses of ASA (10 mg daily during 3 weeks),showed that bleeding time could be normal when Thromboxane B2 (TX B2) synthesis was inhibited (4). However 10 mg of ASA have no influence on the urinary excretion of 6-keto-PGFlti.According the doses, ASA have different effects and it would be interesting to study TX B2 production and 6-keto-PGFlv excretion after administration of ASA at ultra low dosages. This study could not explain the action of ASA at ultra low dosage but point to another mode of action.
ACKNOWLEDGEMENTS
The authors are indebited to Mr A. MASSE, Mr H. PELLEVRAULT, Mr D. FAUDOT for their excellent assistance.
Vol. 47, No. 3
PLATELET AGGREGATION, ASPIRIN
377
REFERENCES
1. BUCHANAN M.R., HIRSH J. Effect of Aspirin on Hemostasis and Thrombosis. New Engl. Reg. Allergy ProcA, 26 - 32, 1986. 2. DOUTREMEPUICH C., PAILLEY D., DE SEZE O., ANNE M.C., PACCALIN J., QUILICHINI R. Variation du temps de saignement apres administration, a differentes posologies, d'acide acetylsalicylique chez le volontaire sain. In press. 3. CARDINAL D.C., FLOWER R.J. The electronic aggregometer: a novel device for assessing platelet behavior in blood. J. Pharm. Meth.,3,135 - 158, 1980. 4. KALLMANN R., NIEUWENHUIS H.K., DE GROOT P.G., VAN GIJN J., SIXMA J.J. Effects of low doses of Aspirin, 10 mg and 30 mg daily, on bleeding time,thromboxane production and 6-keto-PGFl excretion in healthy subjects Thromb. Res., 45, 355 - 361, 1987. 5. MACKIE I.J., JONES R., MACHIN S.J. Platelet impedance aggregation in whole blood and its inhibition by antiplatelet drugs. J. Clin. Pathol, 37, 874 - 878, 1984. 6. ZWIERZINA W.D., KUNZ F. A method of testing platelet aggregation in native whole blood. Thromb. Res., 38, 91 - 100, 1985.