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In vitro platelet hypoaggregability to ADP after body hypothermia in anaesthesized dogs is due to phenobarbital
THROMBOSIS RESEARCH 66; 609-612,1992 0049-8848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
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IN VITRO PLATELET HYPOAGGREGABILITY TO ADP AFTER BODY HYPOTHERMIA IN ANAESTHESIZED DOGS IS DUE TO PHENOBARBITAL
F. Sansonetty Goncalves, A. Almeida-Dias, P. Dinis Oliveira, J.M. Pina-Cabral, Centro de Fisiologia da Hemostase (INIC), Service de Fisiologia da Faculdade de Medicina do Porto, 4200 Potto, Portugal
(Received 20.8.1991; accepted in revised form 14.2.1992 by Editor M.B. Donati)
DUCTION During body cooling there is, mainly at hepatic level, a platelet sequestratioln, which is reversible after rewarming (l-l 1). Sequestered platelets undergo shape change and microplatelet clumps may be observed inside liver microvessels. In order to evaluate if platelet function is altered by body hypothermia, platelet aggregation induced by ADP was studied before cooling and immediately after rewarming in anaesthesized dogs. Since anaesthesia alone may inhibit platelet aggregation(12) the influence of anaesthesia on the observed results was also evaluated.
Twelve mongrel dogs weighing between ten and fifteen kilograms were anaesthesized
Key words: Platelets, aggregation, hypothermia, phenobarbital. 609
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PLATELETSAND ANAESTHESIA
Vol. 66, No. 5
with intravenous phenobarbital (30 mg/kg ) and divided in two groups. In group I thirty minutes after barbital injection the animals were submitted to deep hypothermia by immersion in ice water until oesophageal temperature of 20’ C was attained, and rewarmed to 36°C with water at 40°C. Blood samples were collected before anaesthesia, thirty minutes after injection of phenobarbital and immediately after rewarming. In group II, the control group, the animals received only an injection of phenobarbital. Blood samples were drawn before, 30 and 120 minutes after the induction of anaesthesia. All samples were colllected from the femural vein in one tenth volume of 3.8% trisodium citrate. Platelet rich plasma was obtained according to Austin and Rhymes (13). After adjusting platelet concentration to 30000041L, aggregation was studied in a Payton Dual Channel aggregometer using ten different ADP concentrations: .78, 1.56, 3.13, 6.20, 12.5, 25, 50, 100,200,500 PM. Platelet rich plasma obtained from the blood samples drawn before anaesthesia was treated in vitro with phenobarbital in order to attain a concentration similar to that obtained in vivo through anaesthesia induction. Aggregation of plasma thus obtained was studied as above. Aggregability was evaluated according to an original procedure which allowed a good correlation between dose and aggregation induced by ADP. Aggregometric tracing amplitude was measured for each individual dose at 30, 60, 120, and 180 seconds after addition of ADP. The product of the amplitudes observed at these four moments was considered arbitrarily the “aggregability” for each dose. Given the different platelet sensitivities among dogs, and that maximal answer was always obtained with 500 PM of ADP, the aggregability obtained with this dose was considered as 100% and all the other “aggregabilities” were transformed in percent of that value. Differences between groups were analysed by the Mann Whitney U test and a “p” value under 0.05 was considered statiscally significant.
In figure I comparison of aggregability values obtained in group I before and after anaesthesia, 30 minutes after anaesthesia and immediately after rewarming is done.A decrease in platelet answer to ADP is observed after rewarming. However the same level of reduced aggregability was already present 30 minutes after the induction of phenobarbital anaesthesia, before starting hypothermia. In order to evaluate the responsibility of the anaesthesia in the observed results we repeated the experiences in Group II, studying platelet activity before, 30 and 120 minutes after the injection of pentobarbital, withholding hypothermia. As we can see in figure II a decrease in platelet aggregability was also observed, and the results of this group were not significantly different from those obtained in dogs submitted to anaesthesia plus body cooling and rewarming. To further substantiate our findings we carried out studies of platelet aggregation using platelets drawn from dogs neither submitted to anaesthesia nor hypothermia and treated in vitro with phenobarbital in order to attain a concentration similar to that obtained in vivo after anaesthesia and also a reduction in platelet aggregability was found (data not shown).
Vol. 66, No. 5
Fig I (platelet aggregability with anaesthesia plus body hypothermia)
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611
AND ANAESTHESIA
Fig II (platelet aggregability after phenobarbital injection)
We conclude that in our experimental conditions the decrease of platelet aggregation during body hypothermia is mainly due to phenobarbital injection. The study of the effect of deep body cooling on platelet function implies an experimental model taking into account the proved interference of anaesthesia with that function.
1 Helmsworth, G. A., Stiles, W. J., and Elstun, W. Changes in blood cellular elements in dogs during hypothermia. m 38, 843-846, 1955. 2 Wilson, J.T. Miller, W. R., and Elliot, T. S. Blood studies in the hypothermic dog;43,979-989, 1958 3 Villalobos, T. J., Adelson, E Riley, P A, and Jr. Crosby, W H A cause of the thrombocitopenia and leukopenia that occur in dogs during deep hypothermia. J. -37, 1-7, 1958 4 Wensel, R. H. and Bigelow, W.G. The use of heparin to minimize thrombocitopenia and bleeding tendency during hypothermia. U@%y 45, 223-228, 1959.
5 Biorck, G., Johanssen, B. W. and Nilsson, I. M Blood coagulation studies in hedgehogs, in an hibernating and a non hibernating state, and in dogs hypothermic and normothermic. Physiol. 56, 334-348, 1962.
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PLATELETSAND ANAESTHESIA
Vol. 66, No. 5
6 Johanssen. B. W., Nilsson, I. M. The effect of heparin and I- aminocaproic acid On the coagulation in hypothermic dogs. Acta Phym 60, 267-277, 1964 7 Malmejac, R., Dosne, A. and Rieu, M. Modifications thromboelastographiques au tours de I’hypothermie profonde provoquee chez le chien. Pathol. 14, 429-432, 1966. 8 Pina-Cabral, J.M. A coagulacao sanguinea no cZio submetido a hipotermia profunda. In . . . . . do Se@+!@de Fm da Faculdade do Ports - Homenagem ao Prof. Dr. J. Afonso Guimara’is, pp 93-102 ;1970. 9 Pina-Cabral, J M, lzolett Amaral, Macedo-Pinto, M., Guerra e Paz, LH. Hepatic and Splenic platelet sequestration during deep hypothermia in the dog Hemostasis. 74, 2: 235-244, 1973. 10 Paul, J., Cornillon, B., Baguet, J., Dureau, G.and Belleville, J., In vivo release of an heparin like factor in dogs during profound hypothermia. J. Thor-. Surg 82, 4548,198l 11 Pina Cabral, J.M., Ribeiro-da-Silva, A. and Almeida-Dias, A. Platelet sequestration during hypothermia in dogs treated with sulphinpirazone and ticlopidine. Reversibility accelerated after intra-abdominal rewarming. Thromb. l-&xa~& 54(4), 838-841, 1985. 12 Dwyer, S.D. and Meyers, K. M. Anaesthetics and anticoagulants used in preparation of rat PRP altered rat platelet aggregation. Thromb.42 (2) 139-151, 1986 13 Austen, D. E. G. and Rhymes, I.L. Al Blackwell Scientific publications, Oxford 1975; p 101
of Rlood
BRIEF
COMMUNICATIOI’(
IN VITRO PLATELET HYPOAGGREGABILITY TO ADP AFTER BODY HYPOTHERMIA IN ANAESTHESIZED DOGS IS DUE TO PHENOBARBITAL
F. Sansonetty Goncalves, A. Almeida-Dias, P. Dinis Oliveira, J.M. Pina-Cabral, Centro de Fisiologia da Hemostase (INIC), Service de Fisiologia da Faculdade de Medicina do Porto, 4200 Potto, Portugal
(Received 20.8.1991; accepted in revised form 14.2.1992 by Editor M.B. Donati)
DUCTION During body cooling there is, mainly at hepatic level, a platelet sequestratioln, which is reversible after rewarming (l-l 1). Sequestered platelets undergo shape change and microplatelet clumps may be observed inside liver microvessels. In order to evaluate if platelet function is altered by body hypothermia, platelet aggregation induced by ADP was studied before cooling and immediately after rewarming in anaesthesized dogs. Since anaesthesia alone may inhibit platelet aggregation(12) the influence of anaesthesia on the observed results was also evaluated.
Twelve mongrel dogs weighing between ten and fifteen kilograms were anaesthesized
Key words: Platelets, aggregation, hypothermia, phenobarbital. 609
610
PLATELETSAND ANAESTHESIA
Vol. 66, No. 5
with intravenous phenobarbital (30 mg/kg ) and divided in two groups. In group I thirty minutes after barbital injection the animals were submitted to deep hypothermia by immersion in ice water until oesophageal temperature of 20’ C was attained, and rewarmed to 36°C with water at 40°C. Blood samples were collected before anaesthesia, thirty minutes after injection of phenobarbital and immediately after rewarming. In group II, the control group, the animals received only an injection of phenobarbital. Blood samples were drawn before, 30 and 120 minutes after the induction of anaesthesia. All samples were colllected from the femural vein in one tenth volume of 3.8% trisodium citrate. Platelet rich plasma was obtained according to Austin and Rhymes (13). After adjusting platelet concentration to 30000041L, aggregation was studied in a Payton Dual Channel aggregometer using ten different ADP concentrations: .78, 1.56, 3.13, 6.20, 12.5, 25, 50, 100,200,500 PM. Platelet rich plasma obtained from the blood samples drawn before anaesthesia was treated in vitro with phenobarbital in order to attain a concentration similar to that obtained in vivo through anaesthesia induction. Aggregation of plasma thus obtained was studied as above. Aggregability was evaluated according to an original procedure which allowed a good correlation between dose and aggregation induced by ADP. Aggregometric tracing amplitude was measured for each individual dose at 30, 60, 120, and 180 seconds after addition of ADP. The product of the amplitudes observed at these four moments was considered arbitrarily the “aggregability” for each dose. Given the different platelet sensitivities among dogs, and that maximal answer was always obtained with 500 PM of ADP, the aggregability obtained with this dose was considered as 100% and all the other “aggregabilities” were transformed in percent of that value. Differences between groups were analysed by the Mann Whitney U test and a “p” value under 0.05 was considered statiscally significant.
In figure I comparison of aggregability values obtained in group I before and after anaesthesia, 30 minutes after anaesthesia and immediately after rewarming is done.A decrease in platelet answer to ADP is observed after rewarming. However the same level of reduced aggregability was already present 30 minutes after the induction of phenobarbital anaesthesia, before starting hypothermia. In order to evaluate the responsibility of the anaesthesia in the observed results we repeated the experiences in Group II, studying platelet activity before, 30 and 120 minutes after the injection of pentobarbital, withholding hypothermia. As we can see in figure II a decrease in platelet aggregability was also observed, and the results of this group were not significantly different from those obtained in dogs submitted to anaesthesia plus body cooling and rewarming. To further substantiate our findings we carried out studies of platelet aggregation using platelets drawn from dogs neither submitted to anaesthesia nor hypothermia and treated in vitro with phenobarbital in order to attain a concentration similar to that obtained in vivo after anaesthesia and also a reduction in platelet aggregability was found (data not shown).
Vol. 66, No. 5
Fig I (platelet aggregability with anaesthesia plus body hypothermia)
PLATELETS
611
AND ANAESTHESIA
Fig II (platelet aggregability after phenobarbital injection)
We conclude that in our experimental conditions the decrease of platelet aggregation during body hypothermia is mainly due to phenobarbital injection. The study of the effect of deep body cooling on platelet function implies an experimental model taking into account the proved interference of anaesthesia with that function.
1 Helmsworth, G. A., Stiles, W. J., and Elstun, W. Changes in blood cellular elements in dogs during hypothermia. m 38, 843-846, 1955. 2 Wilson, J.T. Miller, W. R., and Elliot, T. S. Blood studies in the hypothermic dog;43,979-989, 1958 3 Villalobos, T. J., Adelson, E Riley, P A, and Jr. Crosby, W H A cause of the thrombocitopenia and leukopenia that occur in dogs during deep hypothermia. J. -37, 1-7, 1958 4 Wensel, R. H. and Bigelow, W.G. The use of heparin to minimize thrombocitopenia and bleeding tendency during hypothermia. U@%y 45, 223-228, 1959.
5 Biorck, G., Johanssen, B. W. and Nilsson, I. M Blood coagulation studies in hedgehogs, in an hibernating and a non hibernating state, and in dogs hypothermic and normothermic. Physiol. 56, 334-348, 1962.
612
PLATELETSAND ANAESTHESIA
Vol. 66, No. 5
6 Johanssen. B. W., Nilsson, I. M. The effect of heparin and I- aminocaproic acid On the coagulation in hypothermic dogs. Acta Phym 60, 267-277, 1964 7 Malmejac, R., Dosne, A. and Rieu, M. Modifications thromboelastographiques au tours de I’hypothermie profonde provoquee chez le chien. Pathol. 14, 429-432, 1966. 8 Pina-Cabral, J.M. A coagulacao sanguinea no cZio submetido a hipotermia profunda. In . . . . . do Se@+!@de Fm da Faculdade do Ports - Homenagem ao Prof. Dr. J. Afonso Guimara’is, pp 93-102 ;1970. 9 Pina-Cabral, J M, lzolett Amaral, Macedo-Pinto, M., Guerra e Paz, LH. Hepatic and Splenic platelet sequestration during deep hypothermia in the dog Hemostasis. 74, 2: 235-244, 1973. 10 Paul, J., Cornillon, B., Baguet, J., Dureau, G.and Belleville, J., In vivo release of an heparin like factor in dogs during profound hypothermia. J. Thor-. Surg 82, 4548,198l 11 Pina Cabral, J.M., Ribeiro-da-Silva, A. and Almeida-Dias, A. Platelet sequestration during hypothermia in dogs treated with sulphinpirazone and ticlopidine. Reversibility accelerated after intra-abdominal rewarming. Thromb. l-&xa~& 54(4), 838-841, 1985. 12 Dwyer, S.D. and Meyers, K. M. Anaesthetics and anticoagulants used in preparation of rat PRP altered rat platelet aggregation. Thromb.42 (2) 139-151, 1986 13 Austen, D. E. G. and Rhymes, I.L. Al Blackwell Scientific publications, Oxford 1975; p 101
of Rlood