- Email: [email protected]
The effect of moderate doses of chlorpromazine on the haemostasis in dogs defibrinogenated with defibrase
THROMBOSIS RESEARCH Printed in the United
vol. States
4, pp. 229-236, Pergamon Press,
1974 Inc.
THE EFFECT OF MODERATE DOSES OF CHLORPROMAZINE ON THE HAEMOSTASIS IN DOGS DEFIBRINOGENATED WITH DEFIBRASE
H. Johnsson and P.M. Niklasson Department of Blood Coagulation Research, The Thoracic Surgery Research Laboratory, Karolinska Sjukhuset, and Roslagstulls Sjukhus, Stockholm, Sweden.
(Received
24.7.1973; in revised Accepted by Editor H.C.
form 1.12.1973. Godal)
ABSTRACT Three dogs were kept defibrinogenated by daily injections of the thrombin-like enzyme Defibrase. The bleeding from skin flap wounds was measured on the third day when the concentration of fibrinogen degradation products, secondary to the defibrinogenation, was fairly constant. Administration of chlorpromazine did not increase the bleeding. Acetylsalicylic acid, previously reported to increase bleeding significantly in this model by inhibiting the platelet function, was used as control. It is concluded that moderate doses of chlorpromazine, which affects platelet function in vitro, does not significantly influence haemostasis in defibrinogenated dogs.
INTRODUCTION Administration of drugs known to impair haemostasis should be restricted in patients with coagulation disorders. We have recently reported unusually severe bleeding complications in patients with meningococcal septicemia and shock (1). The bleeding tendency in these patients is at least partly due to increased consumption of coagulation factors and platelets, but drugs could not be excluded as contributory causes of bleeding. One of the drugs with possible influence on haemostasis was chlorpromazine, which has been reported to inhibit platelet function ,$ vitro (2, 3).
Chlorpromazine is widely used in small doses
as an alfa-receptor-blocking agent to improve microcirculation in shock. This study was undertaken to investigate the effect of chlorpromazine on haemostasis in vivo, using a previously described experimental model with dogs defibrinogenated with the thrombin-like enzyme Defibrase (4). Previously
229
DEFIBFUNOGENATED
230
DOGS
Vol.4,No.2
we have shown that acetylsalicylicacid (ASA) increasesbleeding simificantly in this model due to an inhibitionof platelet function (4). ASA was therefore used as a control in this investigation.
MATERIALS ANDMETHODS Doas_cMongrel dogs, weight 12 - 16 kg. Acetylsalicylicacid (ASA): One gram dissolved in 20 ml 0.9 $ NaCl containing 0.7 g Trishydroxyaminomethan. Chlorpromazine:(AB Leo, Stockholm,Sweden) 10 mg/ml, dissolved in 0.9 $ NaCl to a final concentrationof 1 mg/ml. Enzymes: Defibrase enzyme per ml in --_-__-_- (Pentapharm,Basle, Switzerland).Fifty ug active,\ 0.9 $ NaCl and 0.3 $ phenol, correspondingto about 3-4 NIHI' units of thrombin. Thrombin, ________ 225 NIH units/mg, prepared according to BlombBck and Yamashina (5). Dog fibrinogen, ----_------- fraction I-2F (12), dissolved in 0.15 M NaCl to a final concentration of 2 $ (6). Enzyme inhibitors: Epsilon-aminocaproic acid (Epsikapron,AB Kabi, Sweden) 0.4 g per ml. _^_______________________ Antiserum -------_-against Bothrops venom (BehringwerkeAG, Marburg/Lahn,West Germany) No. S 1211, polyvalent serum from horse. One volume of antiserum inhibits the thrombin-likeactivity of the same volume of Defibrase measured as inhibition of Defibrase clotting time. Plasma samples were obtained by drawing 9 ml of blood from an arterial catheter into a test tube containing1 ml of trisodlum citrate (0.13 M) and 0.1 ml of venom-antiserum.The latter was added to prevent in vitro digestion of fibrinogen by Defibrase.The blood was centrifugedat 2000 g and the plasma was kept deep-frozenuntil fibrinogendeterminationswere made. Serum samples were obtained after storing arterial blood in glass tubes for four hours at room temperature,The samples were drawn Into tubes containing epsilon-aminocaproicacid in a final concentrationof 8 mg per ml in order to prevent in vitro fibrinolysis. Platelet countingwas performed according to Kristensson (7) using phase contrast microscope. Fibrinogen concentrationIn plasma was determinedaccording to Bergstrijmet al. (a).
1) National Institutesof Health
Vo1.4,No.2
DEFIBRINOGENATED
DOGS
231
Fibrinogendegradationproducts (FDP) were determinedaccording to Merskey -et al. (9). Determinationof acetylsalicylicacid in serum was kindly performed by Dr. A. Kallner using the method described by Saltzmann (10). Normotest:Normotest-reagentfrom Nyegaard and Co AS, Oslo, Norway, was used. Partial thromboplastintime (PIT): ThrombofaxReagent from Ortho Diagnostics, New Jersey, USA, was used. To defibrinogenatedplasma (0.9 ml) dog fibrinogen (0.1 ml) was added prior to determinationof PIT. Thrombin time: To 0.2 ml titrated plasma (37’ C) 0.1 ml thrombin, 3 NIH units/ml was added and clottingtime was determined.To defibrinogenatedplasma (0.9 ml) dog fibrinogen (0.1 ml) was added prior to thrombin. Injection:All injectionswere given intravenously. EXPERIMENTALPROCEDURE Three mongrel dogs were defibrinogenatedwith Defibrase. Defibrasewas administeredintravenouslyonce daily for three days in a dose of 0.15 ml per kg. The experimentswere performed on the third day about two hours after the last Defibrase injection. The concentrationsof fibrinogen,fibrinogendegradationproducts (FDP) and plateletswere measured repeatedly.Blood samples were taken before Defibrase administrationthe first two days and during the experimenton the third day. During the experimentsthe animals were anaesthetizedby intravenousinjection of thiopentalsodium. Artificial respirationwas maintainedwith air utilizing an EngstrijmRespirator (Mivab,Stockholm,Sweden). Blood pressure was monitored via a catheter in a femoral artery with a strain gauge manometer.The bleeding was provoked and measured in the followingway: One full thickness skin flap, 5 by 5 cm square, was raised. Small bleeding arteries in the edges of the skin were cauterized,The inside of the skin flap and the bottom of the wound were roughed with sandpaperto provoke, what was believed, capillarybleeding.Each skin flap thus created a wound surface of 2 x 25 cm2. The amount of bleeding from the wound was measured by weighing a sponge placed on the wound and exchanged every five minutes. After a control period of twenty minutes i.e. four five-minutesperiods with stationarybleeding p
mg of chlorpromazinewas given to the dogs during
fifteen minutes and the bleeding was measured during the following fourty minutes. After that a new skin flap was raised and after another control period of twenty minutes one gram of ASA was administeredand the bleeding was measured in the two skin flaps during another fourty-minutesperiod. Blood samples for determina-
232
DEFIBRINOGENATED
DOGS
Vo1.4,~0.2
tion of salicylates in serum were drawn fourtyfive minutes after the injection of ASA. Normotest, partial thromboplastin-time, thrombin-time and platelet counts were assayed before and twentyfive ,minutes after chlorpromazine administration, and fourtyfive minutes after injection of ASA.
RESULTS On the third day of Defibrase-treatment when the bleeding was provoked the dogs were depleted of measurable fibrinogen in plasma. The fibrinogen degradation products had increased to about ten times compared with the initial concentration measured before Defibrase-treatment. There was no change in platelet concentration during this period of Defibrase-treatment (Table I). There were no changes in normotest, partial thromboplastin-time, thrombin-time, or platelet counts after administration of chlorpromazine. The bleeding in the skin flaps did not increase after chlorpromazine compared with that during the control period i.e. before chlorpromazine was'administered (Fig. 1 and Fig. 2).
TABLE I Fibrinogen, FDP, and platelets in three dogs during Defibrinogenation with Defibrase. The blood samples were drawn before Defibrase injection on day one and day two, and during the experiment on the third day.
Dog No.
I
Day
Fibrinogen mg/ml
FDP ug/ml plasma
Platelets 103/mm3
15
390
0
400
320
0
100
330
3.4
10
225
0
800
250
0
200
270
3.2
16
260
0.6
400
280
0
200
250
1
3.2
2 3
On the other hand, when the acetylsalicylic acid ASA was given to the same dogs about 45 minutes after chlorpromazine the bleeding in all skin
Vo1.4,No.Z
DEFIBRINOGENATED
Y5mln
Chkw p~lln
DOGS
233
A.5.S.A
s-
J-4
e-
7-
B-
5-
4-
3-
2-
,-Ld
-
7
0.7 M
O., 0.7 a8 M
0
20
i 2. c
a5 OS 05 OA 05 a4 an OA 0.4
40
50
FIG. 1 Average bleeding from skin flap wounds in three dogs during the control period, after administration of chlorpromazine, and after administration of ASA. Bleeding gi5min
,ftr
A.S.A.
FIG. 2 Mean bleeding from skin flap wounds in three dogs during five-minutes periods before and after administration of chlorpromazine and ASA.
234
DEFIBRINOGENATED
DOGS
vol.4,~0.2
flaps increased excessivelywithout any noticed change in normotest, partial thromboplastin-time,thrombin-timeor platelet counts (Fig. 1 and Fig. 2). The concentrationsof salicylatesin serum 45 minutes after injection were 13.7, 14.2, 13.0 mg/lOO ml, respectivelyin three dogs. All dogs were clinicallyhealthy during the Defibrase-treatment.The blood pressure did not vary more than 25 mm Hg during the experimentalperiod. NaCl, 0.9 k, was infused when there was a tendency of falling in blood pressure. No dog received more than 500 ml of NaCl.
DISCUSSION Earlier experiments in dogs have shown that depletion of measurable amounts of fibrinogeninduced by the thrombin-likeenzyme Defibrase results in a peak concentrationof FDP, about one hundred times the normal serum level, four to six hours after the first injectionof Defibrase.The dogs can than be kept defibrinogenatedby repeated injectionsof Defibrase every twentyfourhours and the FDP concentrationcan be maintainedat a fairly constant level at about ten times the normal concentration(4;6;1.0;11). The experimentswere performed on the third day to avoid the hi& and uneven concentrationsof FDP, which are known to increase bleeding tendency (4). Platelet counts did not decrease during defibrfnogenation,which is in agreement with earlier experiments (6). The coagulationtime was not measured in this study but has earlier been shown to be significantlyprolonged in defibrinogenatedanimals (6). In spite of this and no detectable fibrinogenthe bleeding tendency in the skin wounds was very moderate, and therefore the haemostatic function is supposed to depend mainly on intact platelet function (4). There is a large body of evidence that platelet function is influenced by ASA in vitro as well as in vivo throuefian inhibitionof the platelet release reaction (13;14;15;16).We have noted no changes induced by ASA in normotest, partial thromboplastin-timeor thrombin-timein this study, and preliminaryresults (17) indicate that ASA does not interferewith the coagulationfactors, at least not as early as fourtyfiveminutes after injection.Administrationof ASA to the defibrinogenateddogs caused dramaticallyincreasedbleeding in these and in previous experiments.The sensitivityof the experimentalmodel as a measure of platelet haemostatic function is illustratedby the fact that no increased bleeding was noticed in identical skin flap wounds in non-defibrinogenated dogs (4). Whether or not the clearly demonstratedASA induced bleedings are caused solely by an inhibitionof platelet function or also by some unknown effects on blood coagulation,it can be concludedthat the use of ASA should be
vo1.4,No.2
DEFIBRINOGENATED
DOGS
235
avoided in patients with coagulation disorders. Chlorpromazine has been reported to inhibit platelet function in vitro (2,
3). We have, however, not been able to demonstrate any chlorpromazine indu-
ced changes in the haemostatic function in defibrinogenated dogs. Nor were there any changes in normotest, partial thromboplastin-time or thrombin-time. The dosage of chlorpromazine was about 2 mg/kg body weight, which is well above commonly used dosage in humans to prevent shock. This does not exclude impairment of haemostasis in man, but it is unlikely that the inhibitory effect on platelet function in vitro would be of any clinical importance.
ACKNOWLEDGEMENTS The authors wish to express their appreciation for informative discussions to Dr. Margareta Blomback. This work was supported by grants from the Swedish Medical Research Council (19X - 520).
1.
NIKLASSON, P.M., LUNDBERG, P., STRANDELL, T. and BLOMBACK, M. Trombocytopenia and bleeding complications in severe cases of meningococcal infections, treated with heparin, dextran 70 and chlorpromazine. Stand. J. Inf. dis. 2, 17, 1971.
2.
O'BRIEN, J.R. The adhesiveness of native platelets and its prevention. J. Clin. Pathol. g, 140, 1961.
3.
MILLS, D.C.B. and ROBERTS, G.C.K. Membrane active drugs and the aggregation of human blood platelets. Nature (London) 219, 35, 1967.
4.
OLSSON, P.I. and JOHNSSON, H. Interference of acetyl salicylic acid, heparin and fibrinogen degradation products in haemostasis of Reptilase defibrinogenated dogs. Thrombosis Research l_, 135, 1972.
5.
BLOMBACK, B. and YAMASHINA, I. On the N-terminal amino acids in fibrinogen and fibrin. Arkiv Kemi I& 299, 1958.
6.
EGBERG, N. and JOHNSSON, H. Platelet aggreetion induced by ADP and thrombin in Reptilase defibrinogenated dogs. Thrombosis Research 1, 95, 1972.
7.
KRISTENSSON, A.A. A new method for the direct counting of the so-called blood platelets in man. Acta Med. Stand. 57, 301, 1922-1923.
a.
BERCSTRiiM,K., BLOMBACK, B. and KLEEN, G. Studies on the plasma fibrinolytic activity in a case of liver cirrhosis. Acta Med. Stand. 168, 291, 1960.
9.
MERSKEY, C., KLEINER, G.J. and JOHNSON, A. Quantitative estimation of split products of fibrinogen in human serum, relation to diagnosis and treatment. Blood 28, 1, 1966.
236
DEFIBRINOGENATED
DOGS
Vo1.4,No.2
10.
Fluorophotometricmethod for estimation of salicylate SALTZMAN,A. in blood. J. Biol. Chem. 174, 399, 1948.
11.
ECBERG, N. and NORDSTROM,S. Effects of Reptilase induced intravascular coagulationin dogs. Acta Physiol. Stand. D, 493, 1970.
12.
ECBERG, N., BLOMBACK, M., JOHNSSON,H., ABILDCAARD,;., BMMBACK, B., DIENER, L., McDONAGH Jr., R.P., McDONAGH, J,, EKESTROM, S., G&ANSSON, L ., JOHANSSON, S.A., NILSSON, S.E., NORDSTROM,S., OLSSON, P. and WIMAN, B. Clinical and experimentalstudies on Reptilase. Thromb. Diathes. haemorrhag.,suppl. 3, 379, 1971.
13.
O'BRIEN, J.R. Effects of salicylateson human platelets. Lancet.l, 779, 1968.
14.
WEISS, H.J., ALEDORT, L.M. and KOCHWA, S. The effect of salicylates on the haemostaticproperties of platelets in man. J. Clin. Invest. 5, 2169, 1968.
15.
ZUCKER, M.D. and PETERSON,J. Inhibitionof adenosindisphosphateinduced secondary aggregationand other platelet functionsby acetylsalicylic acid ingestion. Proc. Sot. Exp. Biol. Med. 127, 547, 1968.
16.
QUICK, A.J. Salicylatesand bleeding. The aspirin tolerance test. Am. J. Med. Sci. 252, 265, 1966.
17.
JOHNSSON, H., BOOK, "" K. and OLSSON, P. To be published.
vol. States
4, pp. 229-236, Pergamon Press,
1974 Inc.
THE EFFECT OF MODERATE DOSES OF CHLORPROMAZINE ON THE HAEMOSTASIS IN DOGS DEFIBRINOGENATED WITH DEFIBRASE
H. Johnsson and P.M. Niklasson Department of Blood Coagulation Research, The Thoracic Surgery Research Laboratory, Karolinska Sjukhuset, and Roslagstulls Sjukhus, Stockholm, Sweden.
(Received
24.7.1973; in revised Accepted by Editor H.C.
form 1.12.1973. Godal)
ABSTRACT Three dogs were kept defibrinogenated by daily injections of the thrombin-like enzyme Defibrase. The bleeding from skin flap wounds was measured on the third day when the concentration of fibrinogen degradation products, secondary to the defibrinogenation, was fairly constant. Administration of chlorpromazine did not increase the bleeding. Acetylsalicylic acid, previously reported to increase bleeding significantly in this model by inhibiting the platelet function, was used as control. It is concluded that moderate doses of chlorpromazine, which affects platelet function in vitro, does not significantly influence haemostasis in defibrinogenated dogs.
INTRODUCTION Administration of drugs known to impair haemostasis should be restricted in patients with coagulation disorders. We have recently reported unusually severe bleeding complications in patients with meningococcal septicemia and shock (1). The bleeding tendency in these patients is at least partly due to increased consumption of coagulation factors and platelets, but drugs could not be excluded as contributory causes of bleeding. One of the drugs with possible influence on haemostasis was chlorpromazine, which has been reported to inhibit platelet function ,$ vitro (2, 3).
Chlorpromazine is widely used in small doses
as an alfa-receptor-blocking agent to improve microcirculation in shock. This study was undertaken to investigate the effect of chlorpromazine on haemostasis in vivo, using a previously described experimental model with dogs defibrinogenated with the thrombin-like enzyme Defibrase (4). Previously
229
DEFIBFUNOGENATED
230
DOGS
Vol.4,No.2
we have shown that acetylsalicylicacid (ASA) increasesbleeding simificantly in this model due to an inhibitionof platelet function (4). ASA was therefore used as a control in this investigation.
MATERIALS ANDMETHODS Doas_cMongrel dogs, weight 12 - 16 kg. Acetylsalicylicacid (ASA): One gram dissolved in 20 ml 0.9 $ NaCl containing 0.7 g Trishydroxyaminomethan. Chlorpromazine:(AB Leo, Stockholm,Sweden) 10 mg/ml, dissolved in 0.9 $ NaCl to a final concentrationof 1 mg/ml. Enzymes: Defibrase enzyme per ml in --_-__-_- (Pentapharm,Basle, Switzerland).Fifty ug active,\ 0.9 $ NaCl and 0.3 $ phenol, correspondingto about 3-4 NIHI' units of thrombin. Thrombin, ________ 225 NIH units/mg, prepared according to BlombBck and Yamashina (5). Dog fibrinogen, ----_------- fraction I-2F (12), dissolved in 0.15 M NaCl to a final concentration of 2 $ (6). Enzyme inhibitors: Epsilon-aminocaproic acid (Epsikapron,AB Kabi, Sweden) 0.4 g per ml. _^_______________________ Antiserum -------_-against Bothrops venom (BehringwerkeAG, Marburg/Lahn,West Germany) No. S 1211, polyvalent serum from horse. One volume of antiserum inhibits the thrombin-likeactivity of the same volume of Defibrase measured as inhibition of Defibrase clotting time. Plasma samples were obtained by drawing 9 ml of blood from an arterial catheter into a test tube containing1 ml of trisodlum citrate (0.13 M) and 0.1 ml of venom-antiserum.The latter was added to prevent in vitro digestion of fibrinogen by Defibrase.The blood was centrifugedat 2000 g and the plasma was kept deep-frozenuntil fibrinogendeterminationswere made. Serum samples were obtained after storing arterial blood in glass tubes for four hours at room temperature,The samples were drawn Into tubes containing epsilon-aminocaproicacid in a final concentrationof 8 mg per ml in order to prevent in vitro fibrinolysis. Platelet countingwas performed according to Kristensson (7) using phase contrast microscope. Fibrinogen concentrationIn plasma was determinedaccording to Bergstrijmet al. (a).
1) National Institutesof Health
Vo1.4,No.2
DEFIBRINOGENATED
DOGS
231
Fibrinogendegradationproducts (FDP) were determinedaccording to Merskey -et al. (9). Determinationof acetylsalicylicacid in serum was kindly performed by Dr. A. Kallner using the method described by Saltzmann (10). Normotest:Normotest-reagentfrom Nyegaard and Co AS, Oslo, Norway, was used. Partial thromboplastintime (PIT): ThrombofaxReagent from Ortho Diagnostics, New Jersey, USA, was used. To defibrinogenatedplasma (0.9 ml) dog fibrinogen (0.1 ml) was added prior to determinationof PIT. Thrombin time: To 0.2 ml titrated plasma (37’ C) 0.1 ml thrombin, 3 NIH units/ml was added and clottingtime was determined.To defibrinogenatedplasma (0.9 ml) dog fibrinogen (0.1 ml) was added prior to thrombin. Injection:All injectionswere given intravenously. EXPERIMENTALPROCEDURE Three mongrel dogs were defibrinogenatedwith Defibrase. Defibrasewas administeredintravenouslyonce daily for three days in a dose of 0.15 ml per kg. The experimentswere performed on the third day about two hours after the last Defibrase injection. The concentrationsof fibrinogen,fibrinogendegradationproducts (FDP) and plateletswere measured repeatedly.Blood samples were taken before Defibrase administrationthe first two days and during the experimenton the third day. During the experimentsthe animals were anaesthetizedby intravenousinjection of thiopentalsodium. Artificial respirationwas maintainedwith air utilizing an EngstrijmRespirator (Mivab,Stockholm,Sweden). Blood pressure was monitored via a catheter in a femoral artery with a strain gauge manometer.The bleeding was provoked and measured in the followingway: One full thickness skin flap, 5 by 5 cm square, was raised. Small bleeding arteries in the edges of the skin were cauterized,The inside of the skin flap and the bottom of the wound were roughed with sandpaperto provoke, what was believed, capillarybleeding.Each skin flap thus created a wound surface of 2 x 25 cm2. The amount of bleeding from the wound was measured by weighing a sponge placed on the wound and exchanged every five minutes. After a control period of twenty minutes i.e. four five-minutesperiods with stationarybleeding p
mg of chlorpromazinewas given to the dogs during
fifteen minutes and the bleeding was measured during the following fourty minutes. After that a new skin flap was raised and after another control period of twenty minutes one gram of ASA was administeredand the bleeding was measured in the two skin flaps during another fourty-minutesperiod. Blood samples for determina-
232
DEFIBRINOGENATED
DOGS
Vo1.4,~0.2
tion of salicylates in serum were drawn fourtyfive minutes after the injection of ASA. Normotest, partial thromboplastin-time, thrombin-time and platelet counts were assayed before and twentyfive ,minutes after chlorpromazine administration, and fourtyfive minutes after injection of ASA.
RESULTS On the third day of Defibrase-treatment when the bleeding was provoked the dogs were depleted of measurable fibrinogen in plasma. The fibrinogen degradation products had increased to about ten times compared with the initial concentration measured before Defibrase-treatment. There was no change in platelet concentration during this period of Defibrase-treatment (Table I). There were no changes in normotest, partial thromboplastin-time, thrombin-time, or platelet counts after administration of chlorpromazine. The bleeding in the skin flaps did not increase after chlorpromazine compared with that during the control period i.e. before chlorpromazine was'administered (Fig. 1 and Fig. 2).
TABLE I Fibrinogen, FDP, and platelets in three dogs during Defibrinogenation with Defibrase. The blood samples were drawn before Defibrase injection on day one and day two, and during the experiment on the third day.
Dog No.
I
Day
Fibrinogen mg/ml
FDP ug/ml plasma
Platelets 103/mm3
15
390
0
400
320
0
100
330
3.4
10
225
0
800
250
0
200
270
3.2
16
260
0.6
400
280
0
200
250
1
3.2
2 3
On the other hand, when the acetylsalicylic acid ASA was given to the same dogs about 45 minutes after chlorpromazine the bleeding in all skin
Vo1.4,No.Z
DEFIBRINOGENATED
Y5mln
Chkw p~lln
DOGS
233
A.5.S.A
s-
J-4
e-
7-
B-
5-
4-
3-
2-
,-Ld
-
7
0.7 M
O., 0.7 a8 M
0
20
i 2. c
a5 OS 05 OA 05 a4 an OA 0.4
40
50
FIG. 1 Average bleeding from skin flap wounds in three dogs during the control period, after administration of chlorpromazine, and after administration of ASA. Bleeding gi5min
,ftr
A.S.A.
FIG. 2 Mean bleeding from skin flap wounds in three dogs during five-minutes periods before and after administration of chlorpromazine and ASA.
234
DEFIBRINOGENATED
DOGS
vol.4,~0.2
flaps increased excessivelywithout any noticed change in normotest, partial thromboplastin-time,thrombin-timeor platelet counts (Fig. 1 and Fig. 2). The concentrationsof salicylatesin serum 45 minutes after injection were 13.7, 14.2, 13.0 mg/lOO ml, respectivelyin three dogs. All dogs were clinicallyhealthy during the Defibrase-treatment.The blood pressure did not vary more than 25 mm Hg during the experimentalperiod. NaCl, 0.9 k, was infused when there was a tendency of falling in blood pressure. No dog received more than 500 ml of NaCl.
DISCUSSION Earlier experiments in dogs have shown that depletion of measurable amounts of fibrinogeninduced by the thrombin-likeenzyme Defibrase results in a peak concentrationof FDP, about one hundred times the normal serum level, four to six hours after the first injectionof Defibrase.The dogs can than be kept defibrinogenatedby repeated injectionsof Defibrase every twentyfourhours and the FDP concentrationcan be maintainedat a fairly constant level at about ten times the normal concentration(4;6;1.0;11). The experimentswere performed on the third day to avoid the hi& and uneven concentrationsof FDP, which are known to increase bleeding tendency (4). Platelet counts did not decrease during defibrfnogenation,which is in agreement with earlier experiments (6). The coagulationtime was not measured in this study but has earlier been shown to be significantlyprolonged in defibrinogenatedanimals (6). In spite of this and no detectable fibrinogenthe bleeding tendency in the skin wounds was very moderate, and therefore the haemostatic function is supposed to depend mainly on intact platelet function (4). There is a large body of evidence that platelet function is influenced by ASA in vitro as well as in vivo throuefian inhibitionof the platelet release reaction (13;14;15;16).We have noted no changes induced by ASA in normotest, partial thromboplastin-timeor thrombin-timein this study, and preliminaryresults (17) indicate that ASA does not interferewith the coagulationfactors, at least not as early as fourtyfiveminutes after injection.Administrationof ASA to the defibrinogenateddogs caused dramaticallyincreasedbleeding in these and in previous experiments.The sensitivityof the experimentalmodel as a measure of platelet haemostatic function is illustratedby the fact that no increased bleeding was noticed in identical skin flap wounds in non-defibrinogenated dogs (4). Whether or not the clearly demonstratedASA induced bleedings are caused solely by an inhibitionof platelet function or also by some unknown effects on blood coagulation,it can be concludedthat the use of ASA should be
vo1.4,No.2
DEFIBRINOGENATED
DOGS
235
avoided in patients with coagulation disorders. Chlorpromazine has been reported to inhibit platelet function in vitro (2,
3). We have, however, not been able to demonstrate any chlorpromazine indu-
ced changes in the haemostatic function in defibrinogenated dogs. Nor were there any changes in normotest, partial thromboplastin-time or thrombin-time. The dosage of chlorpromazine was about 2 mg/kg body weight, which is well above commonly used dosage in humans to prevent shock. This does not exclude impairment of haemostasis in man, but it is unlikely that the inhibitory effect on platelet function in vitro would be of any clinical importance.
ACKNOWLEDGEMENTS The authors wish to express their appreciation for informative discussions to Dr. Margareta Blomback. This work was supported by grants from the Swedish Medical Research Council (19X - 520).
1.
NIKLASSON, P.M., LUNDBERG, P., STRANDELL, T. and BLOMBACK, M. Trombocytopenia and bleeding complications in severe cases of meningococcal infections, treated with heparin, dextran 70 and chlorpromazine. Stand. J. Inf. dis. 2, 17, 1971.
2.
O'BRIEN, J.R. The adhesiveness of native platelets and its prevention. J. Clin. Pathol. g, 140, 1961.
3.
MILLS, D.C.B. and ROBERTS, G.C.K. Membrane active drugs and the aggregation of human blood platelets. Nature (London) 219, 35, 1967.
4.
OLSSON, P.I. and JOHNSSON, H. Interference of acetyl salicylic acid, heparin and fibrinogen degradation products in haemostasis of Reptilase defibrinogenated dogs. Thrombosis Research l_, 135, 1972.
5.
BLOMBACK, B. and YAMASHINA, I. On the N-terminal amino acids in fibrinogen and fibrin. Arkiv Kemi I& 299, 1958.
6.
EGBERG, N. and JOHNSSON, H. Platelet aggreetion induced by ADP and thrombin in Reptilase defibrinogenated dogs. Thrombosis Research 1, 95, 1972.
7.
KRISTENSSON, A.A. A new method for the direct counting of the so-called blood platelets in man. Acta Med. Stand. 57, 301, 1922-1923.
a.
BERCSTRiiM,K., BLOMBACK, B. and KLEEN, G. Studies on the plasma fibrinolytic activity in a case of liver cirrhosis. Acta Med. Stand. 168, 291, 1960.
9.
MERSKEY, C., KLEINER, G.J. and JOHNSON, A. Quantitative estimation of split products of fibrinogen in human serum, relation to diagnosis and treatment. Blood 28, 1, 1966.
236
DEFIBRINOGENATED
DOGS
Vo1.4,No.2
10.
Fluorophotometricmethod for estimation of salicylate SALTZMAN,A. in blood. J. Biol. Chem. 174, 399, 1948.
11.
ECBERG, N. and NORDSTROM,S. Effects of Reptilase induced intravascular coagulationin dogs. Acta Physiol. Stand. D, 493, 1970.
12.
ECBERG, N., BLOMBACK, M., JOHNSSON,H., ABILDCAARD,;., BMMBACK, B., DIENER, L., McDONAGH Jr., R.P., McDONAGH, J,, EKESTROM, S., G&ANSSON, L ., JOHANSSON, S.A., NILSSON, S.E., NORDSTROM,S., OLSSON, P. and WIMAN, B. Clinical and experimentalstudies on Reptilase. Thromb. Diathes. haemorrhag.,suppl. 3, 379, 1971.
13.
O'BRIEN, J.R. Effects of salicylateson human platelets. Lancet.l, 779, 1968.
14.
WEISS, H.J., ALEDORT, L.M. and KOCHWA, S. The effect of salicylates on the haemostaticproperties of platelets in man. J. Clin. Invest. 5, 2169, 1968.
15.
ZUCKER, M.D. and PETERSON,J. Inhibitionof adenosindisphosphateinduced secondary aggregationand other platelet functionsby acetylsalicylic acid ingestion. Proc. Sot. Exp. Biol. Med. 127, 547, 1968.
16.
QUICK, A.J. Salicylatesand bleeding. The aspirin tolerance test. Am. J. Med. Sci. 252, 265, 1966.
17.
JOHNSSON, H., BOOK, "" K. and OLSSON, P. To be published.