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Effects of verapamil on some parameters of haemostasis in rats with experimental chronic renal failure
THROMBOSIS RESEARCH 60; 99-l 03,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
BRIEF
COMMUNICATION
EFFECTS OF VERAPAMIL ON SOME PARAMETERS OF HAEMOSTASIS IN RATS WITH EXPERIMENTAL CHRONIC RENAL FAILURE
A. Azzadin, Department of Biaaystok,
M.H. Pietraszek Pharmacodynamics, 2c Mickiewicz
and W. Euczko Medical Academy Str., Poland
(Received 1.3.1990; accepted in revised form 3.8.1990 by Editor M.B. Donati)
INTRODUCTION
Chronic renal failure is often associated with haemorrhagic tendency that is evidenced by a prolonged bleeding time (1,2,3). In such cases a defective platelet aggregation (3) and adhesion to vessel wall (4) have been found. On the lother hand it is well documented that in patients as well as laboratory animals with renal insufficiency one of the most frequent complications is an arterial hypertension (5). Some data have been shown that in lowering of blood pressure in patients with chronic renal failure the calcium antagonists were very effective (6). One of which is verapamil. This drug has been shown to influence the function It inhibits the aggregation of platelets of blood platelets. induced by various factors (71, and decreases the synthesis and release of tromboxane from human platelets (8). Our recent study indicates that verapamil does not prolong the bleeding time in healthy
rats
(9).
The purpose of this study verapamil on the some parameters experimental uremia. MATERIAL
was to evaluate of haemostasis AND
the in
effect rats
of with
METHODS
Male Wistar rats weighing 200-250 g were used. Studies were performed in four groups of animals; (A) sham-operated rats; (i3> sham-operated rats treated with verapamil; (C) partially nephrectomized rats; (D) group C rats chronically treated with Induction verapamil. of chronic renal insufficiency was performed according to surgical method described by Rutkowski et al. Key words:
Verapamil,
haemostasis,
uraemia, 99
rats
URAEMIAAND VERAPAMIL
Vol. 60, No. 1
(10). The rats were anaesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg body weight.Then 2-3 cm long parallel incisions were made on both sides of animal dorsum, about 1 cm from the spine. The right kidney was removed totally. Also half of the left kidney was removed and the remaining part was covered with spongostan. The wound was closed using catgut suteres for muscles and stilon suteres forskin. After 2 weeks the rats were used for experiments. They had elevated concentration of urea and creatinine in the blood. Additionally after each experiment histopatological changes were investigated. Only those rats in which the morphological and histoenzymatic changes indicated the development of chronic renal injury, were taken under consideration. Sham-operated animals with incisions of skin and muscles were used as controls. The “transection” techniques of tail bleeding time was performed according to Dejana et al. (11). Initial blood loss was performed as described previously (9). In other experiments blood was collected from ether-anaesthetized rats by intracardiac puncture and decalcified with 3.13% sodium citrate, 1:9. For platelet rich plasma (PRP) the blood was centrifuged at 490 x g for two minutes. Platelet poor plasma (PPP) was prepared by centrifuging the remaining blood at 490 x g for 20 minutes. Platelet counts, done by the phase microscopy method varied between 300-400 G/l. Platelet aggregation induced by ADP wa,s produced in an Elvi 840 aggregometer at 37” by the method of Born (12). The prothrombin time was performed according to Quick (13) and the partial activated thromboplastin time was performed by the method described by Rodman et al. (14). Platelet cyclic AMP levels were determined by a protein-binding assay as described by Towey et a1.(15). Verapamil (Isoptin, Lek) was injected intraperitoneally for 4 weeks (once a day) in a dose 5 mg/kg body weight (starting 2 weeks after the operation). Student s t-test was used for statistical analysis. RESULTS Table 1 summarizes the effect of verapamil on the tail bleeding time and the initial blood loss. Verapamil had no significant effect on both parameters in sham-operated rats. A significant prolongation of bleeding time in uraemic rats was observed (from 220 + 60 to 372 + 40 sec.). Verapamil further intensified this effect and also increases the initial blood loss in uraemic rats. Blood platelets obtained from sham-operated rats pretreated with verapamil showed a decrease in aggregability of about 25% (from 75.2 + 3.9 to 55.0 + 2.6% LT) while in uraemic rats verapamil produces inhibition of platelet aggregation induced by ADP of about 40% (from 79.0 + 5.2 to 47.4+ 4.5% LT) (Table 2). As shown in Table 3 a significantly higherplatelet CAMP level was found after treatment with verapamil in sham-operated as well as in uraemic animals as compared to untreated group. Verapamil does not affect the prothrombin time and activated partial thromboplastin time in all experimental groups. The control values in sham-operated rats were 17.2 + 0.6 set and 26.5 + 1.4 set, respectively. In the uraemic animals we have observed an insignificant reduction of hematocrit in comparison to the shame-operated
Vol. 60, No. 1
rats
(data
101
URAEMIAAND VERAPAMIL
not
shown). TABLE 1
Effects of Verapamil on the Bleeding Time and Initial Blood Loss in Studied Four Groups of Animals. Means + s.d. of 10 rats for each group. ___---____---__-------------~--____-____----__-----~----_~~~--_ Blood loss (u1/30 set> Bleeding time (set> Group _____-_____--____----___-----_-__________---__------------__--20.0 + 7.1 A sham -operated 220 + 60 sham-ooerated B + verapamil
255
+ 42
29.0
+ 4.2
C uraemic
372
+ 40
22.0
+ 5.0
840
+ 80
94.0
+ 8.8
0
uraemic + veraoamil
PA-B
NS
PA-8
N5
PA-C
PA-C
PB-0
PB-D <
NS Oeol
PC_* (0.01 -------------___-----____----_____--_____---__------__-----___-
PC-G < 0.01
TABLE 2 Inhibition of 4 uM ADP-induced Platelet Aggregation by Verapamil. The values represents means + s.d. of 10 experiments. LT - light transmission with 4 uM ADP. -------------_--__--------------------------------------------Group Platelets aggregation (%LT) -_-__~-----------_----------___-----__-----~_----------_---_--A sham-operated 75.2 + 3.9 PA-B < 0.001 B sham-operated 55.0 + 2.6 PA-C NS + verapamil C uraemic
19.0
+ 5.2
PB_,, <
Oeool
uraemic D 47.4 + 4.5 + verapamil _____________--_____-_-_--~-___---___-_--___~~~~-~-~:~~!--__--TABLE 3 Platelet CAMP Level in Studied Groups of Rats. presents the mean + s.d. of 8 determinations. ~~~~~~----___---------------_____~~~---__~~~--___~~~---_------Group pmol CAMP / lo9 platelets -----_____---_______-__________-----------------------_-.---____
Each
level
A sham-operated 3.2 + 0.6 PA-B < 0.05 B sham-operated + verapamil 4.6 + 0.8 PA-C NS C uraemic 3.5 f 0.9 pB_G < “01 uraemic _~_~_~~~~~~~~~_______5_19__~_l_:l________________PC,~_~_~~~!~_______
re-
102
URAEMIA
AND VERAPAMIL
Vol. 60, No.
1
OISCUSSION It is well known that patients with chronic renal failure have increased bleeding tendency (1,2).. This effect is accompanied with defective platelets aggregation and adhesion to the vessel wall (3). The interaction of platelets with blood vessel subendothelium seems to be of great importance in haemostasis, as measured by the bleeding time and initial blood loss (2,9). In the present experiments performed on rats with experimental chronic renal failure verapamil significantly augments the bleeding time and the initial blood loss. However, our previous findings have shown that verapamil does not prolong the bleeding time in healthy rats (9). Verapamil as an antihypertensive drugs has antivasoconstrictive properties, but no correlation between the hypotensive effect of this drug and the prolongation of the bleeding time or augmentation of blood loss were observed (9). These data suggest involvement of studied drug in the hemostatic process. Since verapamil did not affect both PT and APTT in healthy rats (9) as well as in rats with chronic renal failure we can exclude the anticoagulant properties of this drug. Verapamil potently inhibits human and animal platelet aggregation induced by various factors (7). We have obtained similar effects using blood platelets of rats with experimental chronic renal failure. However, our experiments have shown that verapamil decreases with high potency platelet aggregation in rats with uraemia. Moore et al. (16) have reported that’verapamil in healthy rats inhibits the rabbit platelet phosphodiesterase activity leading to the increase in platelet CAMP. It is well known that a small increase in platelet CAMP level is required to achieve a significat inhibitory effect on platelet aggregation (16). The observed inhibition of aggregation in uraemic rats seems to be dependent on the rise of intraplatelet CAMP levels after verapamil administration (Tab. 3). This suggestion is supported by our experiments where we have shown that platelets obtained from rats with chronic renal insufficiency and pretreated with verapamil contained higher concentration of CAMP than platelets.from healthy rats. However, lack of correlation between bleeding time reduction of platelet aggregation and augmentation of CAMP in uraemic rats seems to be associated with a more complex defect. Thus, at present, the exact mechanism of these changes is unknown. In conclusion, our observation provides evidence that a increased bl eetherapeutic dose of verapamil in uraemia may evoke ding as compared with healthy subjects. These results show that verapamil may cause haemorrhagic disturbances in patients with chronic renal insufficiency. REFERENCES 1.
ANDRASSY,K. and RITZ,E. J.Nephrol. 5,313-316,1985.
2.
REMUZZI ,G.
Bleeding
Uremia
in renal
failure.
as
a cause Lancet
of
Bleeding.
1,1205-1211,1988.
Am,-
Vol.60, No. 1
URAEMIAANDVERAPAMIL
103
3.
JUBELIRER,S.J. Hemostatic abnormalities Am.J.Kidney Dis. 5,219-223,1985.
4.
CASTILLO,R., LORAND,T., ESCOLAR,G., REVERT,L., LOPEZ,J. and ORDINAS,A. Defective platelet adhesion on vessel subendothelium in uremic patients. Blood 63,337-340,1986.
5.
ACOSTA,J.H. Hypertension Int. 22,702-712,1982.
6.
BALDWIN,D.S. and NEUGARTEN,J. Treatment of hypertension renal disease. Am.J.Kidney gis. 5,57-61,1985.
7.
ADDONIZIO,V.P., FISHER,C.A. verapamil and nifedipine on tion 62,326-330,1986.
8.
DAHL,M.L. and V$llILA,P. Verapamil decreases the formation from exogenous C arachidonic acid in human platelets in vitro. ProstaglandinsLeuk.Med. 17,191-202,1985.
9.
PIETRASZEK,M.H., CHABIELSKA,E. and BUCZKO W. Effects of verapamil, diltiazem and nifedipine on some parameters of hemostasis in rat. Thromb,Res. 50,325-328,1988.
10.
RUTKOWSKI,B., glucose and with chronic
11.
DEJANA,E., VILLA,S. and DE GAETANO,G. A comparison of different experimental Haemost 48,108-111,1982.
12.
BORN,B.V.R. diphosphate
13.
PUICK,A.J. The prothrombin tive jaudice. J.Biol.Chem.
14.
RODMAN,N.F., BARROW,E.M. and GRAHAM,J.B. Diagnosis and control of the hemophilioid states with the partial thromboplastin time (PTT). Am.J.Clin.Path. 29,525-538,1958.
15.
TOVEY,K.C., OLDHAM,K.G. and WHELAN,J.A.M. A simple assay for cyclic AMP in plasma and other biological ples using an improved competitive protein binding nique. Clin.Chim.Acta 56,221-234,1974.
16.
MOORE,J.B., FULLER,B.L., FALOTICO,R. and bition of rabbit platelet phosphodiesterase aggregation by calcium channel blockers. 401-411,1985.
in
chronic
in
renal
renal
disease.
disease.
Kidney
in
and EDMUNDS,L.H. Effects of platelet activation. Circula-
HOPPE,A., MANITIUS,A. and ANGIELSKI,S. Blood insulin levels following glucose load in rats renal failure. Acta Med.Pol. 3,22-30,1981. Bleeding time rats. conditions. ,Thromb.
Aggregation of blood platelets by adenosin and its reversal. Nature 194,937-941,1962. in haemophilia 109,73-77,1935.
and
in
obstruc-
TOLMAN,E.L. activity Thromb.Res.
direct samtech-
Inhiand 40,
BRIEF
COMMUNICATION
EFFECTS OF VERAPAMIL ON SOME PARAMETERS OF HAEMOSTASIS IN RATS WITH EXPERIMENTAL CHRONIC RENAL FAILURE
A. Azzadin, Department of Biaaystok,
M.H. Pietraszek Pharmacodynamics, 2c Mickiewicz
and W. Euczko Medical Academy Str., Poland
(Received 1.3.1990; accepted in revised form 3.8.1990 by Editor M.B. Donati)
INTRODUCTION
Chronic renal failure is often associated with haemorrhagic tendency that is evidenced by a prolonged bleeding time (1,2,3). In such cases a defective platelet aggregation (3) and adhesion to vessel wall (4) have been found. On the lother hand it is well documented that in patients as well as laboratory animals with renal insufficiency one of the most frequent complications is an arterial hypertension (5). Some data have been shown that in lowering of blood pressure in patients with chronic renal failure the calcium antagonists were very effective (6). One of which is verapamil. This drug has been shown to influence the function It inhibits the aggregation of platelets of blood platelets. induced by various factors (71, and decreases the synthesis and release of tromboxane from human platelets (8). Our recent study indicates that verapamil does not prolong the bleeding time in healthy
rats
(9).
The purpose of this study verapamil on the some parameters experimental uremia. MATERIAL
was to evaluate of haemostasis AND
the in
effect rats
of with
METHODS
Male Wistar rats weighing 200-250 g were used. Studies were performed in four groups of animals; (A) sham-operated rats; (i3> sham-operated rats treated with verapamil; (C) partially nephrectomized rats; (D) group C rats chronically treated with Induction verapamil. of chronic renal insufficiency was performed according to surgical method described by Rutkowski et al. Key words:
Verapamil,
haemostasis,
uraemia, 99
rats
URAEMIAAND VERAPAMIL
Vol. 60, No. 1
(10). The rats were anaesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg body weight.Then 2-3 cm long parallel incisions were made on both sides of animal dorsum, about 1 cm from the spine. The right kidney was removed totally. Also half of the left kidney was removed and the remaining part was covered with spongostan. The wound was closed using catgut suteres for muscles and stilon suteres forskin. After 2 weeks the rats were used for experiments. They had elevated concentration of urea and creatinine in the blood. Additionally after each experiment histopatological changes were investigated. Only those rats in which the morphological and histoenzymatic changes indicated the development of chronic renal injury, were taken under consideration. Sham-operated animals with incisions of skin and muscles were used as controls. The “transection” techniques of tail bleeding time was performed according to Dejana et al. (11). Initial blood loss was performed as described previously (9). In other experiments blood was collected from ether-anaesthetized rats by intracardiac puncture and decalcified with 3.13% sodium citrate, 1:9. For platelet rich plasma (PRP) the blood was centrifuged at 490 x g for two minutes. Platelet poor plasma (PPP) was prepared by centrifuging the remaining blood at 490 x g for 20 minutes. Platelet counts, done by the phase microscopy method varied between 300-400 G/l. Platelet aggregation induced by ADP wa,s produced in an Elvi 840 aggregometer at 37” by the method of Born (12). The prothrombin time was performed according to Quick (13) and the partial activated thromboplastin time was performed by the method described by Rodman et al. (14). Platelet cyclic AMP levels were determined by a protein-binding assay as described by Towey et a1.(15). Verapamil (Isoptin, Lek) was injected intraperitoneally for 4 weeks (once a day) in a dose 5 mg/kg body weight (starting 2 weeks after the operation). Student s t-test was used for statistical analysis. RESULTS Table 1 summarizes the effect of verapamil on the tail bleeding time and the initial blood loss. Verapamil had no significant effect on both parameters in sham-operated rats. A significant prolongation of bleeding time in uraemic rats was observed (from 220 + 60 to 372 + 40 sec.). Verapamil further intensified this effect and also increases the initial blood loss in uraemic rats. Blood platelets obtained from sham-operated rats pretreated with verapamil showed a decrease in aggregability of about 25% (from 75.2 + 3.9 to 55.0 + 2.6% LT) while in uraemic rats verapamil produces inhibition of platelet aggregation induced by ADP of about 40% (from 79.0 + 5.2 to 47.4+ 4.5% LT) (Table 2). As shown in Table 3 a significantly higherplatelet CAMP level was found after treatment with verapamil in sham-operated as well as in uraemic animals as compared to untreated group. Verapamil does not affect the prothrombin time and activated partial thromboplastin time in all experimental groups. The control values in sham-operated rats were 17.2 + 0.6 set and 26.5 + 1.4 set, respectively. In the uraemic animals we have observed an insignificant reduction of hematocrit in comparison to the shame-operated
Vol. 60, No. 1
rats
(data
101
URAEMIAAND VERAPAMIL
not
shown). TABLE 1
Effects of Verapamil on the Bleeding Time and Initial Blood Loss in Studied Four Groups of Animals. Means + s.d. of 10 rats for each group. ___---____---__-------------~--____-____----__-----~----_~~~--_ Blood loss (u1/30 set> Bleeding time (set> Group _____-_____--____----___-----_-__________---__------------__--20.0 + 7.1 A sham -operated 220 + 60 sham-ooerated B + verapamil
255
+ 42
29.0
+ 4.2
C uraemic
372
+ 40
22.0
+ 5.0
840
+ 80
94.0
+ 8.8
0
uraemic + veraoamil
PA-B
NS
PA-8
N5
PA-C
PA-C
PB-0
PB-D <
NS Oeol
PC_* (0.01 -------------___-----____----_____--_____---__------__-----___-
PC-G < 0.01
TABLE 2 Inhibition of 4 uM ADP-induced Platelet Aggregation by Verapamil. The values represents means + s.d. of 10 experiments. LT - light transmission with 4 uM ADP. -------------_--__--------------------------------------------Group Platelets aggregation (%LT) -_-__~-----------_----------___-----__-----~_----------_---_--A sham-operated 75.2 + 3.9 PA-B < 0.001 B sham-operated 55.0 + 2.6 PA-C NS + verapamil C uraemic
19.0
+ 5.2
PB_,, <
Oeool
uraemic D 47.4 + 4.5 + verapamil _____________--_____-_-_--~-___---___-_--___~~~~-~-~:~~!--__--TABLE 3 Platelet CAMP Level in Studied Groups of Rats. presents the mean + s.d. of 8 determinations. ~~~~~~----___---------------_____~~~---__~~~--___~~~---_------Group pmol CAMP / lo9 platelets -----_____---_______-__________-----------------------_-.---____
Each
level
A sham-operated 3.2 + 0.6 PA-B < 0.05 B sham-operated + verapamil 4.6 + 0.8 PA-C NS C uraemic 3.5 f 0.9 pB_G < “01 uraemic _~_~_~~~~~~~~~_______5_19__~_l_:l________________PC,~_~_~~~!~_______
re-
102
URAEMIA
AND VERAPAMIL
Vol. 60, No.
1
OISCUSSION It is well known that patients with chronic renal failure have increased bleeding tendency (1,2).. This effect is accompanied with defective platelets aggregation and adhesion to the vessel wall (3). The interaction of platelets with blood vessel subendothelium seems to be of great importance in haemostasis, as measured by the bleeding time and initial blood loss (2,9). In the present experiments performed on rats with experimental chronic renal failure verapamil significantly augments the bleeding time and the initial blood loss. However, our previous findings have shown that verapamil does not prolong the bleeding time in healthy rats (9). Verapamil as an antihypertensive drugs has antivasoconstrictive properties, but no correlation between the hypotensive effect of this drug and the prolongation of the bleeding time or augmentation of blood loss were observed (9). These data suggest involvement of studied drug in the hemostatic process. Since verapamil did not affect both PT and APTT in healthy rats (9) as well as in rats with chronic renal failure we can exclude the anticoagulant properties of this drug. Verapamil potently inhibits human and animal platelet aggregation induced by various factors (7). We have obtained similar effects using blood platelets of rats with experimental chronic renal failure. However, our experiments have shown that verapamil decreases with high potency platelet aggregation in rats with uraemia. Moore et al. (16) have reported that’verapamil in healthy rats inhibits the rabbit platelet phosphodiesterase activity leading to the increase in platelet CAMP. It is well known that a small increase in platelet CAMP level is required to achieve a significat inhibitory effect on platelet aggregation (16). The observed inhibition of aggregation in uraemic rats seems to be dependent on the rise of intraplatelet CAMP levels after verapamil administration (Tab. 3). This suggestion is supported by our experiments where we have shown that platelets obtained from rats with chronic renal insufficiency and pretreated with verapamil contained higher concentration of CAMP than platelets.from healthy rats. However, lack of correlation between bleeding time reduction of platelet aggregation and augmentation of CAMP in uraemic rats seems to be associated with a more complex defect. Thus, at present, the exact mechanism of these changes is unknown. In conclusion, our observation provides evidence that a increased bl eetherapeutic dose of verapamil in uraemia may evoke ding as compared with healthy subjects. These results show that verapamil may cause haemorrhagic disturbances in patients with chronic renal insufficiency. REFERENCES 1.
ANDRASSY,K. and RITZ,E. J.Nephrol. 5,313-316,1985.
2.
REMUZZI ,G.
Bleeding
Uremia
in renal
failure.
as
a cause Lancet
of
Bleeding.
1,1205-1211,1988.
Am,-
Vol.60, No. 1
URAEMIAANDVERAPAMIL
103
3.
JUBELIRER,S.J. Hemostatic abnormalities Am.J.Kidney Dis. 5,219-223,1985.
4.
CASTILLO,R., LORAND,T., ESCOLAR,G., REVERT,L., LOPEZ,J. and ORDINAS,A. Defective platelet adhesion on vessel subendothelium in uremic patients. Blood 63,337-340,1986.
5.
ACOSTA,J.H. Hypertension Int. 22,702-712,1982.
6.
BALDWIN,D.S. and NEUGARTEN,J. Treatment of hypertension renal disease. Am.J.Kidney gis. 5,57-61,1985.
7.
ADDONIZIO,V.P., FISHER,C.A. verapamil and nifedipine on tion 62,326-330,1986.
8.
DAHL,M.L. and V$llILA,P. Verapamil decreases the formation from exogenous C arachidonic acid in human platelets in vitro. ProstaglandinsLeuk.Med. 17,191-202,1985.
9.
PIETRASZEK,M.H., CHABIELSKA,E. and BUCZKO W. Effects of verapamil, diltiazem and nifedipine on some parameters of hemostasis in rat. Thromb,Res. 50,325-328,1988.
10.
RUTKOWSKI,B., glucose and with chronic
11.
DEJANA,E., VILLA,S. and DE GAETANO,G. A comparison of different experimental Haemost 48,108-111,1982.
12.
BORN,B.V.R. diphosphate
13.
PUICK,A.J. The prothrombin tive jaudice. J.Biol.Chem.
14.
RODMAN,N.F., BARROW,E.M. and GRAHAM,J.B. Diagnosis and control of the hemophilioid states with the partial thromboplastin time (PTT). Am.J.Clin.Path. 29,525-538,1958.
15.
TOVEY,K.C., OLDHAM,K.G. and WHELAN,J.A.M. A simple assay for cyclic AMP in plasma and other biological ples using an improved competitive protein binding nique. Clin.Chim.Acta 56,221-234,1974.
16.
MOORE,J.B., FULLER,B.L., FALOTICO,R. and bition of rabbit platelet phosphodiesterase aggregation by calcium channel blockers. 401-411,1985.
in
chronic
in
renal
renal
disease.
disease.
Kidney
in
and EDMUNDS,L.H. Effects of platelet activation. Circula-
HOPPE,A., MANITIUS,A. and ANGIELSKI,S. Blood insulin levels following glucose load in rats renal failure. Acta Med.Pol. 3,22-30,1981. Bleeding time rats. conditions. ,Thromb.
Aggregation of blood platelets by adenosin and its reversal. Nature 194,937-941,1962. in haemophilia 109,73-77,1935.
and
in
obstruc-
TOLMAN,E.L. activity Thromb.Res.
direct samtech-
Inhiand 40,