THROMBOSIS RESEARCH 65; l-11,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press plc. All rights reserved.
- CONTRIBUTION
DOSE-RESPONSE AGGmMETRY TO TBE PRECISE pLAT&LET FUNCTION EVALUATION
Viera Jancinova, Rado Nosal and Margita Petrikova Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska 2, 842 16 Bratislava, Czecho-Slovakia
(Received 28.7.1991; accepted in revised form 16.9.1991 by Editor J. Dyr)
ABSTRACT A method for the determination of blood platelet aggregability as the function of ADP concentration is described. Individual platelet aggregability was characterised by means of parameters of the siqmoidal relationship between the dose of the aggregating reagent and the aggregation curve amplitude. The method was applied to establish ADP-induced aggregability of various mammalian species. The aggregation curves of rabbit and rat platelets reached significantly lower maximum amplitudes than those of human and dog platelets. The mean concentration of ADP was significantly higher in dog platelets and the slope of the dose-response curve was significantly steeper in rat platelets compared to the other species studied. Some of the causes of individual intra-species ADP-induced aggregation variability revealed by means of doseresponse aggregometry are discussed.
INTRODUCTION The ADP-stimulated aggregation test is one of the common measurements of blood platelet functions. It is used both for quantification of altered platelet activity, e.g. in patients with ischaemic heart disease (1,2,3) and in testing the antiaggregatory activity of drugs in man (4,5,6,7) as well as in experimental animals (8,9,10,11). The platelet aggregability is Key words: Platelet aggregation, dose-response ADP, species variation 1
aggregometry,
2
DOSE-RESPONSE AGGREGOMETRY...
Vol. 65, No. 1
influenced by various factors, e.g. the biological species, sex, health and drug therapy of the platelet donor age, (12,13), as well as by conditions under which aggregation tests are performed (bleeding technique, preparation of platelet suspension, aggregometry itself). The concentrations of ADP, used for stimulation of human (14,15,16,17),rat (13,18,19,20,21), dog (22) and rabbit platelets (23) ranged from 0.1 to 20 pmol/l. Moreover, a single dose of aggregation reagent was used in almost 50% of the studies published. In view of the nonlinear relationship between the extent of platelet aggregation and stimulus concentration, only a broader range of doses can be expected to yield reliable results. The aim of the present study was: 1/ to determine a standardized method for full, objective and precise characterisation of the platelet aggregability of each individual studied; 2/ to compare the ADP-stimulated aggregability of platelets in various mammalian species; 3/ to characterize the variability of ADP-stimulated aggregation among individuals of the same biological species and variations of aggregation in each individual over a longterm observation period; 4/ to determine the optimal ADP concentration range for aggregation studies of platelets in various mammalian species. METHODS Platelet
preparation
Blood samples were obtained by venepuncture (human,dog) or by cathetrisation of the common carotid artery (rat, rabbit under light ether anaesthesia), using Na -citrate as anticoagulant (final concentration Mostly single blood 0.0129 mol/lf. samplings were performed, while in dogs blood was taken,6 times within a period of 4 months. Blood was centrifuged at 140xg (dog), 240xg (rabbit) or 300xg (human, rat) for 15 min at 22OC to obtain platelet rich plasma (PHP) and then again at 2000xg for 30 min at 22OC to obtain platelet poor plasma (PPP). The number of platelets in PHP was determined in the Thromand adjusted to LTD.,DE) bocoun er C (Coulter Electronics 2.5x10 5 /l ~1 by autologous PPP. Platelet
aggregation
Platelet aggregation was measured by the turbidimetric method of Born (24) in a dual channel aggregometer (Chrono-log. aggrometer, USA), using 450 ~1 samples. After preincubation of was initiated by adding platelets (2 min at 37OC) aggregation 0.1, 0.2, 0.4, 1, 2, 4, 10, 20 pl of ADP (final concentrations 20, 40 or 100 pmol/l) and was recorded for 3 min by means of aggregation curves (Fig.1). Fresh working dilutions of ADP were prepared each day from the stored in small aliquots at stock solution (2.35 mmol/l) -2OOC. Aggregometry was done between 60 and 180 min after blood sampling.
Vol. 65, No.
1
DOSE-RESPONSE AGGREGOMETRY...
2 0.1,0.2,0.4 I
0
1
.
3
2 TIME
Cminl
Figure 1 Typical aggregation curves of rat platelets stimulated by various concentrations (~ol/l) of ADP. The increase of the amplitude by 1 cm represents the increase of light transmission by 11% or the decrease of voltage by 0.88 mV. Calculation of dose-response curves A method of nonlinear regression (25) was used to fit the dose-response curve of each individual, i.e. the relationship between the amplitude of the aggregation curve and the dose of the stimulus. The dose-response curve was characterised by the eciuation
where A is the dose of the stimulus, PA the amplitude of the aggregation curve read in the first minute, the estimated maximum amplitude, A5o the estimated dose of%D P yielding 50% and q characterlses the slope of the sigmoidal curve. of &, These three parameters were used to characterise individual platelet aggregability. The coefficient characterizing the correlation between actual and fitted sigmoidal dose-response curves was mostly above 0.99.
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Materials Donors of platelets: male albino Wistar rats (350-400 g) and chinchilla rabbits (3.0-3.5 kg); beagle dogs (7.0-12.0 kg) and healthy human volunteers of both sexes. ADP (adenosine-5'-diphosphate): Serva,BRD. All other chemicals were of analytical grade from commercial sources. RESULTS Fig.1 illustrates the effect of increasing concentrations of ADP on the amplitudes of the aggregation curves. ADP in the concentration range from 1 to 40 pmol/l induced a dose-depen-dent aggregation of rat platelets. The aggregation was reversible in the ADP concentration of 1,2 and 4 pmol/l and irreversible in the concentration of 10, 20, 40 and 100 pmol/l. The lower ADP concentrations tested (0.1, 0.2 and 0.4 lupol/l)did not stimulate aggregation. The amplitudes of the aggregation curves read in the 60th second of aggregation, given as the function of ADP concentration, are demonstrated in Fig.2. This dose-response aggregation
Figure2 Dose-response aggregation curve of rat platelets stimulated by ADP, with parameters s = maximum amplitude, A50 = concentration of ADP yielding 50% of EW, and q characterising the slope of the sigmoida1 curve: amplitude = amplitude of aggregation curve read in the first minute of aggregation. curve of rat platelets was characterised by the maximum amplitude (BW) 50.22 + 1.47 mm and by the mean concentration of ADP yielding 50% of WW 0.97 + 0.12 wol/l. Parameter q is proportionate to the slope of the tangent to the sigmoid at A = A50. It reached the value of 2.73 f 0.29.
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1
5
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The variation observed in the aggregability of platelets Of various mammalian species is presented in Fig.3; the parameters of individual dose-response curves are shown in Table 1. The maximum amplitudes of rat and rabbit platelet aggregation curves (50.22 and 47.27 mm,respectively) were significantly lower than the amplitudes of dog and human platelet aggregation curves (60.37 and 57.90 mm, respectively). The mean ADP concentration needed for the stimulation of dog platelets (A5 ) was significantly higher than that required to stimulate Euman, rat and rabbit platelets (4.45 pmol/l in comparison to 1.39, 0.97 and 1.04 crmol/l),and parameter q decreased in the rank order: rat(2.73) - dog(2.20) - human (2.08) - rabbit platelets (1.81). I)-
HUMAN RAT RABBIT
6
4. 2*
Figure3 Species variation in ADP-stimulated platelet aggregation dose-response curves. Each curve is characterised by mean parameters from 10 individuals. TAB=1 Parameters of ADP-stimulated aggregation dose-response curves in human, rat. rabbit and dog platelets. & is the maximum amplitude,'A of ADP yielding 50% of s, and q characterises5&let%op?z dose-response curve: n = 10, mean + SEM Species Human Rat Rabbit
A& (mm) 57.90 50.22 47.27 60.37
_+2.87 + 1.47 + 1.31 -+1.83
A50 (~rmol/l) 1.39 0.97 1.04 4.45
+ f + +
0.16 0.12 0.10 0.58
q 2.08 2.73 1.81 2.20
+ + f k
0.09 0.29 0.18 0.17
Individual variations in ADP-induced aggregability among members of the same animal species are shown on the example of four rabbits in Fig.4. The aggregation curves of three of these animals reached the maximum amplitudes of 47.18, 48.39 and
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48.60 mm; the mean concentration of ADP ranged from 1.16 .to 1.3 pmol/l, and the values of parameter q were 1.39, 1.73 and 2.36. The platelets of the fourth rabbit showed increased ag-
/I , 0.1
1’
10
I
loo
ADO l@nol/lJ Figure 4 ADP-stimulated platelet aggregation dose-response curves of four control rabbits.
Figure5 Individual variation in aggregability of platelets during long-term observation. A = dog with high, B = with low variability in platelet aggregation: 0,2,4,8,12,16. = weeks in which aggregation was tested.
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gregability, characterised mainly by a lower parameter A50 (0.61 )Lmol/l)and a higher parameter q (4.05). Fig.5 illustrates the individual variation in platelet aggregability.The dose-response curves of dog ,cAn(left part of the figure), obtained at week 0, 2, 4, 8, 12 and 16 of platelet testing, reached maximum amplitudes ranging from 40.7 mm to 84.6 mm, the mean concentrations of ADP were within 3.73 pmol/l to 16.98 pmol/l; the minimum and maximum values of parameter q were 1.07 and 2.91. The individual ADP-aggregation variability of dog IIB)I (right part of the figure) was, with exception of week 0, very low and the mean values of parameters and q were 78.7 + 1.17 mm, 6.83 + 0.56 clmol/land 1.35 EM, A + 0.02: respectively. DISCUSSION A method for testing platelet aggregation as the function of ADP concentration, supplemented by mathematical evaluation of the data, was applied to characterize species and individual variations in aggregability. The method proved reliable for precise statistical evaluation of individual variability in the sensitivity of platelets to ADP, as well as for characterization of species differences. Although the advantages of dose-response aggregometry have been demonstrated (26,27) a single-dose method is generally used for the evaluation of platelet aggregability. Our results confirmed that a single dose of ADP may not be sufficient to determine individual and species variability in platelet aggregability and, moreover, it may lead to incorrect conclusions (Fig.3 and 4). For the evaluation of platelet dose-response aggregation curves different approaches were used, such as graphical description (28), graphical determination of the mean concentration of the stimulus (29) or determination of "threshold concentration" of ADP (4,5,7,15). Different approaches to the evaluation of platelet aggregability described by different authors may lead to conflicting conclusions, as demonstrated for the antiaggregatory activity of propranolol (6,30,31). The evaluation of aggregability by means of the maximum amplitude (EW), the mean concentration of ADP (A50), and parameter q used in our experiments was found to yield exact and complex information about platelet responsiveness to aggregation stimuli. Dose-response aggregometry was applied to establish platelet aggregability in four mammalian species. It results from Fig. 3 and Table 1 that the tested parameters of aggregability of rat, rabbit and human platelets were very similar, especially the values of A The mean concentration of ADP for stimulation of human plate?O' ets, 1.39 pmol/l, correlated well with 1.0 pmol/l described by Thompson and Vickers (26). We found that aggregation curves of human platelets reached higher maximum amplitudes than of rabbit and rat platelets. The higher amplitudes, indicating increased transmission through platelet suspension, might be associated with the larger size of human platelets - 8.5 fl - in comparison to 4.9 fl of rat and rabbit platelets (32), and consequently with their increased sedimentation. Dog platelets responded in a different way to ADP than did
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human, rat and rabbit platelets. The dose-response sigmoidal curves of dog platelets were characterized by the ,highest maximum amplitudes (E = 60.37 f 1.83 mm) as well as by the highest mean concentraF# ions of ADP (A50 = 4.45 + 0.58 ~mol/l), which correlated with the results of Macmillan and Sim (22). The species variations in aggregability may be due to differences in the platelet membrane structure resulting from variations in membrane phospholipid content, stimulated arachidonit acid displacement, or availability in membrane glycoproteins for signal transduction (33). Moreover, different participation of some amplification mechanisms in aggregation (PAF, arachidonic acid or dense granule feedback pathways) probably induces different sensitivity to ADP in platelets of various mammalian species. For instance, the amount of secretable ADP in dog platelets is much lower than in rabbit and human platelets (34) and therefore higher concentrations of external ADP are needed to stimulate platelet aggregation. The mean species variability in parameter A$0' g iven as SEM, was 12% (Table 1). The minimum and maximum es imated mean concentration of ADP was in rabbit platelets 0.56 pmol/l and 1.58 pmol/l, and in dog platelets 1.60 pmol/l and 7.24 ~mol/l. Moreover, individual variation in aggregability within repeated platelet tests was observed (see left part of Fig.5). Several factors may account for this phenomenon. To obtain correct results it is necessary to observe strictly the standardized techniques for blood sampling, platelet preparation and aggregometry itself (8,35). Yet several factors, e.g. stress or partial mechanical platelet activation during bleeding procedure, cannot be fully excluded. Stress (36,37,38), but also pathological events such as inflammatory and degenerative processes (14,39) and hormonal alterations (29) may significantly alter sensitivity of platelets to stimuli and contribute to individual variability in platelet aggregation. While single-dose aggregometry failed, the dose-response method proved to be suitable for detecting the intricate pattern of species-specific and individual variability in platelet aggregation. Moreover, the latter is considered to yield more reliable results in evaluating the effect of antiaggregatory drugs. Acknowledgements The authors wish to thank Ing. Maria Duri&ova,CSc. for her help with mathematical evaluation of the experiments. REFERENCES 1. ZAHAVI, M., ZAHAVI, J., SCHAFER, R., FIRSTETER.,E., LANIADO, S. Abnormal typical pattern of platelet function and thromboxa62, ne generation in unstable angina. TJzrplnb.Haemustas. 840-845, 1989 2. ZANNAD, F., HANSEN, C., VOISIN, P., HHALIFE, K;, STOLTZ, J.-F., GILGENKRANTZ, J.-M. Hemorheology and platelet function in unstable angina. Clin. Hemorbeol. 9, 219-235, 1989
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3. RENAUD, s. Linoleic acid, platelet aggregation al infarction. Atherosclerosis 80, 255-256, 1990
9
and myocardi-
4. WINTHER, K., KNUDSEN, J.B., GORMSEN,J., JENSEN, J. Effect of metoprolol and propranolol on platelet aggregation and CAMP level in hypertensive patients. Eur. J. Clin. I Pharmacol. 29, 561-564, 1986 5. WIN!CHER,'K., KNUDSEN, J.B.,'JORGENSEN, E.O.,ELDRUP, E. Differential effects of timolol and metoprolol on platelet function at rest and during exercise. Eur. J. Clin. Pharmacol, 33, 587-592, 1988 6. MEACCI, E., LA VILLA, G., LAB, G., COMINELLI, F., DI DONATO, M ., BIZZI, P., ALBANI, F., GENTILINI, P. Systemic haemodynamics, renal and platelet function during chronic propranolol administration in patients with compensated cirrhosis. Liver 7, 110-115, 1987 7. GLFZRUP, G., WINTHER, K. Differential effects of non-specific beta-blockade and calcium antagonism on blood-clotting mechanisms. Am. J. Med. 4A), 127-129, 1989 86 (Suppl. 8. DWYER, S.D., MEYERS, K.M. Anesthetics and anticoagulants used in the preparation of rat platelet rich plasma alter rat platelet aggregation. Thromb. Res. 42, 139-151, 1986 9. JANCINOVA, V., NOSAL, R., BENES, L. Interaction of local anaesthetics with blood platelet aggregation. Drugs Exptl. Clin. Res. 12, 857-860, 1986
10. NOSAL, R., JAN&NOVA, V., ONDRIASOVA, E., PI&&, H. Effect of beta-adrenergic receptor blockers on aggregation and secretion response of blood platelets. Bratisl. lek. Listy 87, 129-138, 1987 11. TERRES, W., BECKER, B.F., SCHRODL, W., GERLACH, E. Effects of chronic treatment with adrenaline or propranolol on platelet function and CAMP levels in the rat. Cardiovasc. Res. 23, 112-116, 1989 12. HWANG, D.H. Species variation in platelet aggregation. In: The Platelets: Physiology and Pharmacology, G.L.Longenecker (Ed) Orlando, Academic Press, 1985, pp. 289-305 13. DWYER, S.D., MEYERS, K.M. Rat platelet aggregation: and stock variations. Thromb. Res. 42, 49-53, 1986
Strain
14. NO&L, R., NO&L, M., ‘JAN&NOVA, V. The alteration in platelet aggregability and plasma coagulation factors in patients with chronic liver disease. In: New Trends in Pathological Php siology, J.Vasku, M.Dostal (Eds) Brno, Acta Facult. Med. Univ. Brunensis 92, 1985, pp.289-294
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15. WINTHER, K., TRAP-JENSEN, J. Effects of three beta-blockers with different pharmacodynamic properties on platelet aggregaand platelet and plasma cyclic AMP. Eur. J.Clin. Pharmation col. 35, 17-20, 1988
16. HEINTZ, B., FEMERS, C., MAURIN, N., KIERDORF, II., BRILON, C ., WIENERT, V. Beziehung zwischen postthrombotischen syndrom, ADP-induzierter thrombozytenaggregation und intrathrombozytarem calcium-gehalt. Klin. Wochenschr. 67, 1194-1198, 1989 17. MACHLEIDT, C., METTANG, T., STARZ, E., WEBER, J., RISLER, T KUHLMANN, u. Multifactorial genesis of enhanced platelet a&egability in patients with nephrotic syndrome. Kidney Int. 36, 1119-1124, 1989 18. NOSAL, R., JANCINOVA, V., PETRfKOVA, M. On the relationship between aggregation and cyclic nucleotides in platelets treated with betaadrenoceptor blocking drugs. Gen. Physiol. Biophys. 2, 353-362, 1983 19. NOSti, R., JAN&NOVA, V., ONDRIAS, K., JAKUBOVSKY, J., BALP. The interaction of beta-adrenoceptor blocking drugs GAVY, with platelet aggregation, calcium displacement and fluidization of the membrane. Biochim. Biophys., Acta 821, 217-228, 1985 platelet aggregation in 20. my,, M., O'DELL, B.L. Biphasic and the effect of calcium flux modulators. Thromb. rat plasma Res. 46, 617-623, 1987 21. SALADINO, C.F., FOX, R.L., YEH, Q., KARPOWICZ, F. FEFFER, S.E., JONAS, E.A. Platelet aggregability in rat with early atherosclerotic changes induced by parenterally-administered lipid emulsions& Atherosclerosis 66, 19-28, 1987 22. MACMILLAN, D.C., SIM, A.K. A comparative study of platelet animals. Thromb. Diathes. in man and laboratory aggregation Haemorrh. 24, 385-394, 1970 23. PACKHAM, M.A., BRYANT, N.L., GUCCIONE, MA., KINLOUGH-ypBONE, R.L. MUSTARD, J.F. Effect of the concentration of Ca in the suspending medium on the responses of human and rabbit platelets to aggregating agents. Thromb. Haemostas.62,968-976,1989 24. BORN, G.V.R. Aggregation of blood platelets by ADP and its reversal. Nature 194, 927-929, 1962 K., HEBAK, P., KUBACEK, L. Tvorba empirickych 25. KVETON, modelu metodami regresni analyzy. CSVTS - FEL -&AJT, Praha 1979 26. THOMPSON, S.G., VICKERS, M.V. Methods in dose-response telet aggregometry. Thromb. Haemostas. 54, 216-218, 1985
pla-
27. VI-, M.V., THOMPSON, S.G. Sources of variability in Thromb. Haemostas. 54, dose-response platelet aggregometry. 219-220, 1985
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28. THOMPSON, N.T., SCRUTTON, M.C., WALLIS, R.B. Synergistic responses in human platelets2+Comparison between aggregation, concentration. Eur. J. Biochem. and cytosolic Ca secretion 161, 399-408, 1986 29. THEODOROU, A.E., WISTRY, H., DAVIES, S-L., YAMAGUCHI, Y., HORTON, R.W. Platelet alpha -adrenoceptor binding and function during the menstrual cycle. 3 . pychiat. Res. 21, 163-169, 1987 30. GREEN, D., ROSSI, E.C., HARING, attack trial: Studies of platelets Res. 28, 261-267, 1982
0. The beta-blocker heart and factor VIII. Thromb.
31. RING, M.E., CORRIGAN, J.J.Jr., FENSTER, P.E. Antiplatelet effects of oral diltiazem, propranolol, and their combination. Br. J. clin. Pharmac. 24, 615-620, 1987 32. EASON, C.T., PATTISON, A., BONNER, F.W. Platelet population cies variation and drug-induced 437-441, 1986
HOWELLS, D.D., MITCHESON, J., profiles: Significance of spechanges. J. Appl. Toxicol. 6,
33. SIESS, W. Multiple signal-transduction pathways synergize in platelet activation. News Physiol. Sci 6, 51-56, 1991 34. MEYERS, K.M. Pathobiology of animal platelets. Advances in Veterinary Science and Comparative Medicine 30, 131-165, 1985 35. NOSAL, R. Essential platelet functions and their practical implications. In: R.NosBl, V.Jancinovb: Blood Platelets in Biology and Medicine, F.Kornalik (Ed.), Bratislava, Veda, 1990, pp.38-81 36. ANDREN, L., WADENVIK, H., KUTTI, J., HANSSON, L. Stress and platelet activation. Acta haemat. 70, 302-306, 1983 37. LEVINE, S.P., TOWELL, B.L., SUAREZ, A.M., KNIERIEM, L.K., HARRIS, M.M., GEORGE, J.N. Platelet activation and secretion associated with emotional stress. Circulation 71, 1129-1134, 1985 38. LANZA, F. CAZENAVE, J.-P. Studies of alpha -adrenergic receptors 0 intact and functional w shed human p ?atelets by binpng of 5H-dihydroergocryptine and 9H-yohimbine. Correlation of H-yohimbine binding with the potentiation by adrenaline of ADP-induced aggregation. Thromb. Haemostas. 54, 402-408, 1985 39. CORDOVA, C., MUSCA, A., VIOLI, F., ALESSANDRI, C., PERRONE, A BALSANO, F. Platelet hyperfunction in patients with chronic a&iays obstruction. Eur. J. Respir. Dis.. 66, 9-12, 1985
11
- CONTRIBUTION
DOSE-RESPONSE AGGmMETRY TO TBE PRECISE pLAT&LET FUNCTION EVALUATION
Viera Jancinova, Rado Nosal and Margita Petrikova Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska 2, 842 16 Bratislava, Czecho-Slovakia
(Received 28.7.1991; accepted in revised form 16.9.1991 by Editor J. Dyr)
ABSTRACT A method for the determination of blood platelet aggregability as the function of ADP concentration is described. Individual platelet aggregability was characterised by means of parameters of the siqmoidal relationship between the dose of the aggregating reagent and the aggregation curve amplitude. The method was applied to establish ADP-induced aggregability of various mammalian species. The aggregation curves of rabbit and rat platelets reached significantly lower maximum amplitudes than those of human and dog platelets. The mean concentration of ADP was significantly higher in dog platelets and the slope of the dose-response curve was significantly steeper in rat platelets compared to the other species studied. Some of the causes of individual intra-species ADP-induced aggregation variability revealed by means of doseresponse aggregometry are discussed.
INTRODUCTION The ADP-stimulated aggregation test is one of the common measurements of blood platelet functions. It is used both for quantification of altered platelet activity, e.g. in patients with ischaemic heart disease (1,2,3) and in testing the antiaggregatory activity of drugs in man (4,5,6,7) as well as in experimental animals (8,9,10,11). The platelet aggregability is Key words: Platelet aggregation, dose-response ADP, species variation 1
aggregometry,
2
DOSE-RESPONSE AGGREGOMETRY...
Vol. 65, No. 1
influenced by various factors, e.g. the biological species, sex, health and drug therapy of the platelet donor age, (12,13), as well as by conditions under which aggregation tests are performed (bleeding technique, preparation of platelet suspension, aggregometry itself). The concentrations of ADP, used for stimulation of human (14,15,16,17),rat (13,18,19,20,21), dog (22) and rabbit platelets (23) ranged from 0.1 to 20 pmol/l. Moreover, a single dose of aggregation reagent was used in almost 50% of the studies published. In view of the nonlinear relationship between the extent of platelet aggregation and stimulus concentration, only a broader range of doses can be expected to yield reliable results. The aim of the present study was: 1/ to determine a standardized method for full, objective and precise characterisation of the platelet aggregability of each individual studied; 2/ to compare the ADP-stimulated aggregability of platelets in various mammalian species; 3/ to characterize the variability of ADP-stimulated aggregation among individuals of the same biological species and variations of aggregation in each individual over a longterm observation period; 4/ to determine the optimal ADP concentration range for aggregation studies of platelets in various mammalian species. METHODS Platelet
preparation
Blood samples were obtained by venepuncture (human,dog) or by cathetrisation of the common carotid artery (rat, rabbit under light ether anaesthesia), using Na -citrate as anticoagulant (final concentration Mostly single blood 0.0129 mol/lf. samplings were performed, while in dogs blood was taken,6 times within a period of 4 months. Blood was centrifuged at 140xg (dog), 240xg (rabbit) or 300xg (human, rat) for 15 min at 22OC to obtain platelet rich plasma (PHP) and then again at 2000xg for 30 min at 22OC to obtain platelet poor plasma (PPP). The number of platelets in PHP was determined in the Thromand adjusted to LTD.,DE) bocoun er C (Coulter Electronics 2.5x10 5 /l ~1 by autologous PPP. Platelet
aggregation
Platelet aggregation was measured by the turbidimetric method of Born (24) in a dual channel aggregometer (Chrono-log. aggrometer, USA), using 450 ~1 samples. After preincubation of was initiated by adding platelets (2 min at 37OC) aggregation 0.1, 0.2, 0.4, 1, 2, 4, 10, 20 pl of ADP (final concentrations 20, 40 or 100 pmol/l) and was recorded for 3 min by means of aggregation curves (Fig.1). Fresh working dilutions of ADP were prepared each day from the stored in small aliquots at stock solution (2.35 mmol/l) -2OOC. Aggregometry was done between 60 and 180 min after blood sampling.
Vol. 65, No.
1
DOSE-RESPONSE AGGREGOMETRY...
2 0.1,0.2,0.4 I
0
1
.
3
2 TIME
Cminl
Figure 1 Typical aggregation curves of rat platelets stimulated by various concentrations (~ol/l) of ADP. The increase of the amplitude by 1 cm represents the increase of light transmission by 11% or the decrease of voltage by 0.88 mV. Calculation of dose-response curves A method of nonlinear regression (25) was used to fit the dose-response curve of each individual, i.e. the relationship between the amplitude of the aggregation curve and the dose of the stimulus. The dose-response curve was characterised by the eciuation
where A is the dose of the stimulus, PA the amplitude of the aggregation curve read in the first minute, the estimated maximum amplitude, A5o the estimated dose of%D P yielding 50% and q characterlses the slope of the sigmoidal curve. of &, These three parameters were used to characterise individual platelet aggregability. The coefficient characterizing the correlation between actual and fitted sigmoidal dose-response curves was mostly above 0.99.
DOSE-RESPONSE AGGREGOMETRY...
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Materials Donors of platelets: male albino Wistar rats (350-400 g) and chinchilla rabbits (3.0-3.5 kg); beagle dogs (7.0-12.0 kg) and healthy human volunteers of both sexes. ADP (adenosine-5'-diphosphate): Serva,BRD. All other chemicals were of analytical grade from commercial sources. RESULTS Fig.1 illustrates the effect of increasing concentrations of ADP on the amplitudes of the aggregation curves. ADP in the concentration range from 1 to 40 pmol/l induced a dose-depen-dent aggregation of rat platelets. The aggregation was reversible in the ADP concentration of 1,2 and 4 pmol/l and irreversible in the concentration of 10, 20, 40 and 100 pmol/l. The lower ADP concentrations tested (0.1, 0.2 and 0.4 lupol/l)did not stimulate aggregation. The amplitudes of the aggregation curves read in the 60th second of aggregation, given as the function of ADP concentration, are demonstrated in Fig.2. This dose-response aggregation
Figure2 Dose-response aggregation curve of rat platelets stimulated by ADP, with parameters s = maximum amplitude, A50 = concentration of ADP yielding 50% of EW, and q characterising the slope of the sigmoida1 curve: amplitude = amplitude of aggregation curve read in the first minute of aggregation. curve of rat platelets was characterised by the maximum amplitude (BW) 50.22 + 1.47 mm and by the mean concentration of ADP yielding 50% of WW 0.97 + 0.12 wol/l. Parameter q is proportionate to the slope of the tangent to the sigmoid at A = A50. It reached the value of 2.73 f 0.29.
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1
5
DOSE-RESPONSE AGGREGOMETRY...
The variation observed in the aggregability of platelets Of various mammalian species is presented in Fig.3; the parameters of individual dose-response curves are shown in Table 1. The maximum amplitudes of rat and rabbit platelet aggregation curves (50.22 and 47.27 mm,respectively) were significantly lower than the amplitudes of dog and human platelet aggregation curves (60.37 and 57.90 mm, respectively). The mean ADP concentration needed for the stimulation of dog platelets (A5 ) was significantly higher than that required to stimulate Euman, rat and rabbit platelets (4.45 pmol/l in comparison to 1.39, 0.97 and 1.04 crmol/l),and parameter q decreased in the rank order: rat(2.73) - dog(2.20) - human (2.08) - rabbit platelets (1.81). I)-
HUMAN RAT RABBIT
6
4. 2*
Figure3 Species variation in ADP-stimulated platelet aggregation dose-response curves. Each curve is characterised by mean parameters from 10 individuals. TAB=1 Parameters of ADP-stimulated aggregation dose-response curves in human, rat. rabbit and dog platelets. & is the maximum amplitude,'A of ADP yielding 50% of s, and q characterises5&let%op?z dose-response curve: n = 10, mean + SEM Species Human Rat Rabbit
A& (mm) 57.90 50.22 47.27 60.37
_+2.87 + 1.47 + 1.31 -+1.83
A50 (~rmol/l) 1.39 0.97 1.04 4.45
+ f + +
0.16 0.12 0.10 0.58
q 2.08 2.73 1.81 2.20
+ + f k
0.09 0.29 0.18 0.17
Individual variations in ADP-induced aggregability among members of the same animal species are shown on the example of four rabbits in Fig.4. The aggregation curves of three of these animals reached the maximum amplitudes of 47.18, 48.39 and
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48.60 mm; the mean concentration of ADP ranged from 1.16 .to 1.3 pmol/l, and the values of parameter q were 1.39, 1.73 and 2.36. The platelets of the fourth rabbit showed increased ag-
/I , 0.1
1’
10
I
loo
ADO l@nol/lJ Figure 4 ADP-stimulated platelet aggregation dose-response curves of four control rabbits.
Figure5 Individual variation in aggregability of platelets during long-term observation. A = dog with high, B = with low variability in platelet aggregation: 0,2,4,8,12,16. = weeks in which aggregation was tested.
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gregability, characterised mainly by a lower parameter A50 (0.61 )Lmol/l)and a higher parameter q (4.05). Fig.5 illustrates the individual variation in platelet aggregability.The dose-response curves of dog ,cAn(left part of the figure), obtained at week 0, 2, 4, 8, 12 and 16 of platelet testing, reached maximum amplitudes ranging from 40.7 mm to 84.6 mm, the mean concentrations of ADP were within 3.73 pmol/l to 16.98 pmol/l; the minimum and maximum values of parameter q were 1.07 and 2.91. The individual ADP-aggregation variability of dog IIB)I (right part of the figure) was, with exception of week 0, very low and the mean values of parameters and q were 78.7 + 1.17 mm, 6.83 + 0.56 clmol/land 1.35 EM, A + 0.02: respectively. DISCUSSION A method for testing platelet aggregation as the function of ADP concentration, supplemented by mathematical evaluation of the data, was applied to characterize species and individual variations in aggregability. The method proved reliable for precise statistical evaluation of individual variability in the sensitivity of platelets to ADP, as well as for characterization of species differences. Although the advantages of dose-response aggregometry have been demonstrated (26,27) a single-dose method is generally used for the evaluation of platelet aggregability. Our results confirmed that a single dose of ADP may not be sufficient to determine individual and species variability in platelet aggregability and, moreover, it may lead to incorrect conclusions (Fig.3 and 4). For the evaluation of platelet dose-response aggregation curves different approaches were used, such as graphical description (28), graphical determination of the mean concentration of the stimulus (29) or determination of "threshold concentration" of ADP (4,5,7,15). Different approaches to the evaluation of platelet aggregability described by different authors may lead to conflicting conclusions, as demonstrated for the antiaggregatory activity of propranolol (6,30,31). The evaluation of aggregability by means of the maximum amplitude (EW), the mean concentration of ADP (A50), and parameter q used in our experiments was found to yield exact and complex information about platelet responsiveness to aggregation stimuli. Dose-response aggregometry was applied to establish platelet aggregability in four mammalian species. It results from Fig. 3 and Table 1 that the tested parameters of aggregability of rat, rabbit and human platelets were very similar, especially the values of A The mean concentration of ADP for stimulation of human plate?O' ets, 1.39 pmol/l, correlated well with 1.0 pmol/l described by Thompson and Vickers (26). We found that aggregation curves of human platelets reached higher maximum amplitudes than of rabbit and rat platelets. The higher amplitudes, indicating increased transmission through platelet suspension, might be associated with the larger size of human platelets - 8.5 fl - in comparison to 4.9 fl of rat and rabbit platelets (32), and consequently with their increased sedimentation. Dog platelets responded in a different way to ADP than did
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human, rat and rabbit platelets. The dose-response sigmoidal curves of dog platelets were characterized by the ,highest maximum amplitudes (E = 60.37 f 1.83 mm) as well as by the highest mean concentraF# ions of ADP (A50 = 4.45 + 0.58 ~mol/l), which correlated with the results of Macmillan and Sim (22). The species variations in aggregability may be due to differences in the platelet membrane structure resulting from variations in membrane phospholipid content, stimulated arachidonit acid displacement, or availability in membrane glycoproteins for signal transduction (33). Moreover, different participation of some amplification mechanisms in aggregation (PAF, arachidonic acid or dense granule feedback pathways) probably induces different sensitivity to ADP in platelets of various mammalian species. For instance, the amount of secretable ADP in dog platelets is much lower than in rabbit and human platelets (34) and therefore higher concentrations of external ADP are needed to stimulate platelet aggregation. The mean species variability in parameter A$0' g iven as SEM, was 12% (Table 1). The minimum and maximum es imated mean concentration of ADP was in rabbit platelets 0.56 pmol/l and 1.58 pmol/l, and in dog platelets 1.60 pmol/l and 7.24 ~mol/l. Moreover, individual variation in aggregability within repeated platelet tests was observed (see left part of Fig.5). Several factors may account for this phenomenon. To obtain correct results it is necessary to observe strictly the standardized techniques for blood sampling, platelet preparation and aggregometry itself (8,35). Yet several factors, e.g. stress or partial mechanical platelet activation during bleeding procedure, cannot be fully excluded. Stress (36,37,38), but also pathological events such as inflammatory and degenerative processes (14,39) and hormonal alterations (29) may significantly alter sensitivity of platelets to stimuli and contribute to individual variability in platelet aggregation. While single-dose aggregometry failed, the dose-response method proved to be suitable for detecting the intricate pattern of species-specific and individual variability in platelet aggregation. Moreover, the latter is considered to yield more reliable results in evaluating the effect of antiaggregatory drugs. Acknowledgements The authors wish to thank Ing. Maria Duri&ova,CSc. for her help with mathematical evaluation of the experiments. REFERENCES 1. ZAHAVI, M., ZAHAVI, J., SCHAFER, R., FIRSTETER.,E., LANIADO, S. Abnormal typical pattern of platelet function and thromboxa62, ne generation in unstable angina. TJzrplnb.Haemustas. 840-845, 1989 2. ZANNAD, F., HANSEN, C., VOISIN, P., HHALIFE, K;, STOLTZ, J.-F., GILGENKRANTZ, J.-M. Hemorheology and platelet function in unstable angina. Clin. Hemorbeol. 9, 219-235, 1989
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28. THOMPSON, N.T., SCRUTTON, M.C., WALLIS, R.B. Synergistic responses in human platelets2+Comparison between aggregation, concentration. Eur. J. Biochem. and cytosolic Ca secretion 161, 399-408, 1986 29. THEODOROU, A.E., WISTRY, H., DAVIES, S-L., YAMAGUCHI, Y., HORTON, R.W. Platelet alpha -adrenoceptor binding and function during the menstrual cycle. 3 . pychiat. Res. 21, 163-169, 1987 30. GREEN, D., ROSSI, E.C., HARING, attack trial: Studies of platelets Res. 28, 261-267, 1982
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