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Prolonged bleeding time in experimental cirrhosis: role of nitric oxide
Journal of Hepatology 1999; 30:456-460 Printed in Denmark . All rights reserved Munksgaard" Copenhagen
Copyright © European Association for the Stuct~ of the Liver 1999
Journal of Hepatology ISSN 0168-8278
Prolonged bleeding time in experimental cirrhosis: role of nitric oxide Liliana Albornoz ~, Juan Carlos Bandi 1, Juan Carlos Otaso 2, Oscar Laudanno ~ and Ricardo Mastai 1 1Secci6n de Higado, Servicio de Clinica Mkdica and 2Secci6n de Hemostasia, Laboratorio Central. Hospital Italiano, Buenos Aires, Argentina
Background~Aims: Nitric oxide is a powerful in vitro inhibitor of platelet adhesion and aggregation. Our aim was to investigate whether the in vivo inhibition of nitric oxide release shortens bleeding time, in rats with cirrhosis induced by chronic bile duct ligation. Methods: Mean arterial pressure and bleeding time were measured under basal conditions and 5, 15 and 30 min after administration of vehicle (0.9% saline) or an inhibitor of nitric oxide synthesis, Nw-nitro-Larginine (5 mg/kg, iv). Mean arterial pressure was measured with an intra-arterial catheter and bleeding time with a standardized Simplate device. Results: Cirrhotic rats showed a lower mean arterial pressure (116-+4 mmHg) and a prolonged bleeding time (177-+40 s) compared to control animals (133-+6 mmHg and 95-+12 S, respectively, p<0.01). In cirrhotic rats, Nw-nitro-L-arginine significantly in-
creased mean arterial pressure (from 116_+5 to 141_+ 11 mmHg, p<0.05) and completely normalized bleeding time (from 170-+39 to 103-+21 S, p<0.05) 15 min after administration. Pretreatment with L-arginine (300 mg/kg, iv) prevented the hemodynamic and hemostatic changes induced by Nw-nitro-L-arginine. A trend to normalize platelet adhesion was observed in cirrhotic rats after the inhibition of nitric oxide production. In control animals, Nw-nitro-L-arginine increased mean arterial pressure, while no effect on bleeding time was observed. Conclusions: These findings support the concept that nitric oxide may be a mediator in the bleeding time abnormalities associated with experimental cirrhosis.
HRONIC LIVER diseases are often associated with impaired primary hemostasis (1). Prolonged bleeding time, which is a reliable indicator of platelet function in vivo (2), is a well-known complication of cirrhosis (3,4). The mechanisms underlying this abnormal platelet function have not been elucidated. It has recently been demonstrated that nitric oxide, a powerful endogenous vasodilator produced by vascular endothelial cells among others in response to different stimuli (5), plays a regulatory role in platelet function (6). Experimental studies have shown that nitric oxide causes inhibition of platelet adhesion and aggregation through activation of the soluble guanylate cyclase and elevation of intracellular cyclic G M P (6). On the other hand, an overproduction of nitric oxide has been involved in the hyperdynamic state and the vascular hy-
poreactivity to vasoconstrictors observed in portal hypertension (7-10). The aim of the present study was to investigate whether an increased formation of nitric oxide might contribute to platelet function disorders observed in cirrhosis. We investigated the effect of Nwnitro-L-arginine (L-NNA), a specific inhibitor of nitric oxide synthesis, on primary hemostasis in rats with cirrhosis induced by common bile duct ligation.
C
Received 3 June; revised 25 August; accepted 28 August 1998
Correspondence." Ricardo Mastai, Secci6n de Higado, Servicio de Clinica Medica, Hospital Italiano, Potosi 4215, Buenos Aires (1199), Argentina. Tel: 541 959 0346. Fax: 541 981 4041. E-mail: [email protected] 456
Key words: Bleeding time; Cirrhosis; Nitric oxide.
Materials and Methods Animals The study was performed in male Wistar rats. The animals were housed in a controlled environment and allowed free access to food and water until the time of the study. Animal treatment during experiments was approved by the local committee for animal experimentation. The animals were divided into two groups. In one group a secondary biliary cirrhosis with portal hypertension was induced by bile duct ligation, according to a previously reported method (11). Under ether anesthesia, a midline abdominal incision was made, and the c o m m o n bile duct exposed, doubly ligated with 4.0 silk and sectioned between the two ligatures. A second group consisted of control rats. In this latter group, rats underwent a sham operation, during which the c o m m o n bile duct was exposed but not ligated. The hemodynamic and hemostatic studies were performed 4 weeks after surgical procedures. The diagnosis of cirrhosis was confirmed by macroand microscopic examination of the liver.
Nitric oxide and bleeding time in cirrhosis Protocol To evaluate the effect of L-NNA on bleeding time and mean arterial pressure, cirrhotic animals were divided into three groups: a control group that received an intravenous bolus injection of saline (n=8), a second group that received an intravenous bolus of L-NNA at a dose of 5 mg/kg (n=8), and a third group in which 300 mg/kg of L-arginine was administered 5 min before L-NNA (5 mg/kg, n=8). In shamoperated animals, one group received saline (n=6), while in a further group L-NNA (5 mg/kg, iv; n=6) was administered. In all groups, mean arterial pressure and bleeding time were measured under basal conditions and 5, 15 and 30 min after vehicle or drug administration. Twenty-one animals (sham-operated rats, n=7; cirrhotics, n=14) were used to evaluate whether shortening of bleeding time induced by L-NNA was associated with an effect on in vivo platelet adhesion. This effect was studied 15 min after vehicle and L-NNA injection. This time was chosen because it is the maximum shortening of bleeding time observed in rats with biliary cirrhosis after L-NNA treatment. In this group of animals the effect of L-NNA on ex vivo platelet aggregation was also evaluated. Another group of cirrhotic (n=12) and control rats (n=12) was included to evaluate the effect of L-NNA or its vehicle over portal pressure, hematocrit and platelet count. Measurements were made 15 rain after vehicle or drug administration, i.e., at the time of maximum shortening of bleeding time. Hemodynamic and hemostatic studies The animals were anesthetized with ether. The right carotid artery was cannulated with a PE-50 catheter (Porter Ltd., Kent, UK) that was connected to a transducer (P-23-Gould Staham), and permanent recordings of arterial pressure were made on a Dyne MCD recorder. The external zero reference limit was placed at the midportion of the animal. Another PE-50 catheter was placed in the right jugular vein and used for drug administration. The animals were kept in a plastic cylinder with several openings, from one of which the rat's tail emerged; they were allowed to waken and stabilize for a period of 30 min so as to avoid any effect of anesthesia on blood-vessel interplay and to prevent possible interactions between anesthetics and other drugs administered (12). The tails were left outside the cylinder and the animals were maintained at room temperature. For bleeding time measurements, a standardized Simplate 1 device (Organon Teknika, USA) was applied longitudinally on the dorsal part of the tail between 6 and 9 cm from the tip, taking care to avoid large veins. Bleeding time, expressed in seconds, was measured from the moment of incision until bleeding stopped (no rebleeding within 30 s). In vivo platelet adhesion was measured by the method of Borchgrevink (13). Briefly, platelets were counted in blood collected from the abdominal aorta and in the drops from a standardized incision in the tail performed with Simplate I device. The difference in the two counts represents the platelets which are retained at the wound surface; i.e. the adhesive platelets. Aggregation tests were performed according to
Born & Cross (14). Blood was collected from the abdominal aorta in 3.1% trisodium citrate (1:10). Platelet-rich plasma (PRP) was obtained by centrifugation at 200×g for 15 min at room temperature. Platelet-poor plasma (PPP) was obtained by centrifugation blood at 2000xg for 10 min. Platelet count in PRP was adjusted to 2.5x 105/ ~1 with autologous PPP. Platelet aggregation was induced by 20/zM ADP and 25 pg/ml collagen (both from Meditech, CA, USA). Aggregation was monitored in an aggregometer (Chrono-Log, WholeBlood Aggro-Meter) and expressed as a percentage of the maximally possible increase in light transmission (100%) as defined by the difference in light transmission between the PPP and the non-stimulated PRP. Portal pressure was measured in anesthetized rats by inserting a 16 G "butterfly" needle into the portal vein. Hematocrit and platelet count were counted in a hematology autoanalyzer (Coulter 790, Coulter Electronics, Ltd., USA). L-NNA and L-arginine were obtained from Sigma Chemical Co (St. Louis, MO, USA), dissolved in saline immediately before use, and administered in 0.3 ml. Data analysis Results are expressed as mean_+SD. Student's t-test for paired and non-paired data and one-way ANOVA with the Tukey test were used in the statistical analysis of the results. Significance was taken at p<0.05.
Results Hemodynamic data In cirrhotic rats, L-NNA treatment significantly increased mean arterial pressure (Table 1). These changes were evident at 5 min after administration and reached their maximal value after 15 min (Table 1). L-NNA treatment also resulted in an increase in mean arterial pressure in control rats (Table 1). The percentages of variation produced by L-NNA administration significantly differed between control and cirrhotic rats (13% vs 25%, p<0.05). Pretreatment with L-arginine prevented the effect of L-NNA on mean arterial pressure in cirrhotic animals (Table 1). After 15 min of vehicle or L-NNA administration, similar values of portal pressure in cirrhotic (L-NNA: 14.9_+2.2 and vehicle: 15.1___2.5 mmHg) and control rats (data not shown) were observed.
TABLE 1 Mean arterial pressure and bleeding time under basal conditions and after intravenous administration of L-NNA or vehicle in cirrhotic and control rats. The effects of pretreatment with L-arginine in cirrhotic animals are also shown Parameters
Cirrhotic rats Basal
Control rats 5 min
15 min
30 min
Basal
5 min
15 min
30 min
Mean arterial pressure (mmHg) Vehicle 116_+4 L-NNA 116-+5 L-arginine + L-NNA 115-+4
115_+4 125-+12 116±3
116---3 141-+11" 115-+3
117-+5 143-+6" 117-+4
133-+6 126---9
137___5 126-+8
134+-5 143-+9"*
137-+4 146+--10**
Bleeding time (s) Vehicle L-NNA L-arginine + L-NNA
180_+38 139±32 179±41
173___33 103±21" 176±28
179±57 173±38 171-+36
95_+12 100±15
90-+19 90_+12
177±40 170±39 177___35
95±22 95-+20
95_+12 110±15
Data expressed as mean±SD, * p<0.05 vs basal and L-arginine + L-NNA. ** p<0.05 vs basal.
457
L. Albornoz et al. TABLE 2
Platelet function
The intrasubject reproducibility of the method was ensured by one observer (L.A) performing bleeding time measurements over a 4-h period in the same animal. For each measurement performed, the coefficient of variation was less than 8%, suggesting a high degree of reproducibility. Bleeding time was significantly prolonged in cirrhotic rats compared to control animals (177_+40 vs 95_+ 12 s, p<0.01). In cirrhotic rats, intravenous administration of L-NNA caused a significant shortening in bleeding time. This effect was detected at 5 min following administration and reached its maximal value at 15 min (Fig. 1). Bleeding time at 15 min in cirrhotic rats treated with L-NNA did not significantly differ from that observed in the control group under basal conditions (cirrhotic: 103_+21; control: 95_+12 s). At 30 min after L-NNA administration, bleeding time returned to near basal values (Fig. 1). Pretreatment of cirrhotic rats with L-arginine 5 min before L-NNA prevented its effect on bleeding time (Table 1). Vehicle administration had no effect on bleeding time. In contrast to the findings in cirrhotic animals, L-NNA treatment in control rats did not modify bleeding time (Table 1). No correlation was observed between mean arterial pressure and bleeding time changes in cirrhotic rats. As shown in Table 2, cirrhotic rats showed a significantly lower platelet adhesion and aggregation than control animals. A trend to normalize platelet adhesion after L-NNA treatment was observed in cirrhotic rats. However, this difference did not reach statistical significance. No changes were observed in platelet aggregation after nitric oxide inhibition in cirrhotic animals.
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200 e I.u
150
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_= ci
100
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[• •
1
Vehicle L-NNA
]
0
0
5
15
30
T I M E (mln)
Fig. 1. Effects of vehicle and L - N N A administration on bleeding time in cirrhotic rats. Results are mean+SD. * Significantly different from basal values @<0.05). 458
Effect of L - N N A on platelet adhesion and aggregation in cirrhotic and control rats Parameters
Controls +vehicle
Cirrhotics +vehicle
Cirrhotics +L-NNA
Platelet adhesion (%)
55.2_+15.2
45.6+_9.0 *
51.5_+4.8
Platelet aggregation (%) A D P (20/~M) Collagen (25 Fg/ml)
51.7_+16.3 53.8_+ 17.5
26.7+_15.5" 37.5--_18.0"
27.9_+14.3" 30.5+- 11.5"
Data expressed as mean+-SD. * p<0.05 vs control rats.
After 15 min of vehicle or L-NNA administration in cirrhotic rats, similar values of hematocrit (L-NNA: 4125 and vehicle: 4426%) and platelet count (LNNA: 8529 and vehicle: 88_+11×104//A) were observed. No changes in hematocrit and platelet count were found in control animals after treatments (data not shown).
Discussion The present study shows that inhibition of nitric oxide formation by administration of L-NNA normalizes the prolonged bleeding time in an experimental model of cirrhosis in the rat, induced by bile duct ligation, supporting a role for nitric oxide in the pathogenesis of primary hemostatic disorders associated with chronic liver diseases. Bleeding tendency is a well-known feature in patients with cirrhosis (1). This hemostatic disorder has been related to a complex coagulation defect and impaired platelet function (1). Several studies have reported thrombocytopenia (15,16) and abnormalities in platelet function, such as reduced aggregation (17,18) and prolonged bleeding time (3,4) in patients with cirrhosis. Moreover, a direct correlation has been found between hepatocellular function, evaluated by ChildPugh class, and bleeding time (4), suggesting that in cirrhosis, worsening of platelet function is closely related to the degree of liver failure. However, the mechanisms underlying these hemostatic disorders are not well defined. Nitric oxide is a powerful endothelium-derived vasedilator that, in addition to the relaxation effect on vascular smooth muscle, is a potent inhibitor of platelet function (5,6). It has been demonstrated that nitric oxide synthesized by the constitutive nitric oxide synthase in both platelets and vascular endothelium, inhibits platelet aggregation and adhesion, exerting its action through activation of the soluble guanylate cyclase and elevation of cyclic GMP (6). Under pathological conditions, platelet function may also be modified as a re-
Nitr& oxide and bleeding time in cirrhos&
suit of the expression of the inducible nitric oxide synthase (6). Recent studies suggest that increased nitric oxide formation plays a role in the pathogenesis of the hemodynamic abnormalities associated with portal hypertension. Several authors have presented evidence that nitric oxide inhibition normalizes the systemic and splanchnic vasodilation and restores the vascular hyporesponsiveness to endogenous and exogenous vasoconstrictors in portal hypertensive rats (7-10). Therefore, and in view of the influence of nitric oxide on vascular tone and platelet function (5,6), we hypothesized that the increased nitric oxide formation observed in portal hypertension states could contribute to the abnormal primary hemostatic disorders observed in cirrhosis. In this study, we investigated whether treatment with LNNA, which competitively inhibits the production of nitric oxide (19), could improve platelet function, assessed by bleeding time measurement, in cirrhotic rats. Bleeding time is an easily performed in vivo test of primary hemostasis and a reliable parameter of platelet function as shown by previous pharmacological data (2,20). It has also been proposed as a useful test for studying the in vivo effects of compounds interfering with the platelet adhesion-aggregation reaction (21). In our study, bleeding time was significantly prolonged in cirrhotic animals as compared with controls, demonstrating that experimental cirrhosis is associated with an acquired defect of primary hemostasis, in accordance with results in humans (3,4). Moreover, the inhibition of nitric oxide production by L - N N A administration normalized the prolonged bleeding time. The determination of bleeding time is influenced by platelet-related (i.e., adhesion and aggregation) and vascular (i.e., vasoconstriction) factors. In our study, cirrhotic rats showed reduced platelet adhesion and aggregation. L-NNA treatment partially corrected platelet adhesion at 15 min after administration, i.e. at the time of maximum shortening of bleeding time, but had no effect on platelet aggregation. The discrepancy between these findings with regard to the effects of LN N A on platelet adhesion and aggregation is not clearly understood. However, it could be related to a methodologic difference between the two assessments, as platelet aggregation is an ex vivo, while adhesion is an in vivo assay which seems more physiological because platelet adhesiveness is tested against a damaged vascular wall. On the other hand, as was expected, LN N A treatment increased mean arterial pressure in conscious cirrhotic rats. This hemodynamic change did not correlate with shortening of bleeding time, suggesting that the hemostatic effect of nitric oxide inhibition could not be mediated by the vasoconstriction
observed after L-NNA treatment. However, new experiments with a greater number of animals are needed to confirm these findings. Finally, in the current study the effects of L-NNA were prevented by previous administration of L-arginine, suggesting that the hemodynamic and hemostatic effects of L-NNA result from inhibition of nitric oxide biosynthesis. Similar results have been recently reported (22). In conclusion, our study demonstrates that the model of bile duct ligation in the rat is suitable for evaluating primary hemostasis in cirrhosis. As in humans, bleeding time is prolonged in this experimental model and is normalized by inhibition of nitric oxide synthesis, suggesting a role of this vasodilator in regulating the platelet hemostatic response in cirrhosis. Moreover, this is the first report of in vivo involvement of nitric oxide in the pathogenesis of this primary hemostatic disorder.
References 1. Kelly DA, Summerfield JA. Hemostasis in liver disease. Semin Liver Dis 1987; 7: 182-91. 2. Rodgers RPC. A critical reappraisal of bleeding time. Semin Thromb Haemost 1990; 3: 233-9. 3. Blake JC, Sprengers D, Grech P, McCormick PA, Mclntyre N, Burroughs AK. Bleeding time in patients with hepatic cirrhosis. Br Med J 1990; 301: 12-5. 4. Violi E Leo R, Vezza E, Basili S, Cordova C, Balsano E C.A.L.C. Group. Bleeding time in patients with cirrhosis: relation to degree of liver failure and clotting abnormalities. J Hepatol 1994; 20: 531-6. 5. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991; 43: 10942. 6. Radomski MW, Moncada S. Regulation of platelet function by nitric oxide. Thromb Haemorrh Disorders 1993; 7: 1-7. 7. Pizcueta ME Pique JM, Bosch J, Whittle BJR, Moncada S. Effects of inhibiting nitric oxide biosynthesis on the systemic and splanchnic circulation of rats with portal hypertension. Br J Pharmacol 1992; 105: 184-90. 8. Pizcueta ME Pique JM, Fernandez M, Bosch J, Rodes J, Whittle BJR, et al. Modulation of the hyperdynamic circulation of cirrhotic rats by nitric oxide inhibition. Gastroenterology 1992; 103: 1909-15. 9. Lee E Albillos A, Colombato LA, Groszmann RJ. The role of nitric oxide in the vascular hyporesponsiveness to methoxamine in portal hypertensive rats. Hepatology 1992; 16: 1043-8. 10. Claria J, Jim6nez W, Ros J, Rigol M, Angeli P, Arroyo V, et al. Increased nitric oxide-dependent vasorelaxation in aortic rings of cirrhotic rats with ascites. Hepatology 1994; 20: 1615-21. 11. Lee SS, Girod C, Braillon A, Hadengue A, Lebrec D. Hemodynamic characterization of chronic bile-duct ligated rats: effects of pentobarbital sodium. Am J Physiol 1986; 251: G176-GI80. 12. Dejana E, Callioni A, Quintana A, de Gaetano G. Bleeding time in laboratory animals. II. A comparison of different assay conditions in rats. Thromb Res 1979; 15: 191-7. 13. Borchgrevink CE A method for measuring platelet adhesiveness in vivo. Acta Med Scand 1960; 168: 157-65. 14. Born GVL, Cross MJ. The aggregation of blood platelets. J Physiol 1963; 168: 178-95. 15. Jorgensen B, Fischer E, Ingeberg G, Hollaender N, Ring-Larsen H, Henriksen JH. Decreased blood platelet volume and count in patients with liver disease. Scand J Gastroenterol 1984; 19: 4926.
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16. Boks AL, Brommer EJP, Schalm SW, Van Vliet HHDM. Hemostasis and fibrinolysis in severe liver failure and their relation to hemorrhage. Hepatology 1986; 6: 79-86. 17. Ballard HS, Marcus AJ. Platelet aggregation in portal cirrhosis. Arch Intern Med 1976; 136: 316-9. 18. Ingeberg S, Jacobsen P, Fisher E, Bentsen KD. Platelet aggregation and release of ATP in patients with hepatic cirrhosis. Scand ,i Gastroenterol 1985; 20: 285-8. 19. Ishii K, Chang B, Kerwin ,IF, Huang ZJ, Murad E N-nitro-Larginine: a potent inhibitor of EDRF formation. Eur ,i Pharmacol 1990; 176: 21944.
460
20. Dejana E, Villa S, de Gaetano G. Bleeding time in rats: a comparison of different experimental conditions. Thromb Haemost 1982; 48: 108-1h 21. Praga C, Cortellaro M, Pogliani E. Standardized bleeding time in the study of drugs interfering with platelet function. Adv Exp Med Biol 1972; 34: 149-58. 22. Lopez Talavera JC, Groszmann R,I. La inhibicion de la sintesis de oxido nitrico mejora el sindrome hiperdinamico y el tiempo de sangria en ratas con hipertension portal [abstract]. Gastroenterol Hepatol 1995; 18: 219.
Copyright © European Association for the Stuct~ of the Liver 1999
Journal of Hepatology ISSN 0168-8278
Prolonged bleeding time in experimental cirrhosis: role of nitric oxide Liliana Albornoz ~, Juan Carlos Bandi 1, Juan Carlos Otaso 2, Oscar Laudanno ~ and Ricardo Mastai 1 1Secci6n de Higado, Servicio de Clinica Mkdica and 2Secci6n de Hemostasia, Laboratorio Central. Hospital Italiano, Buenos Aires, Argentina
Background~Aims: Nitric oxide is a powerful in vitro inhibitor of platelet adhesion and aggregation. Our aim was to investigate whether the in vivo inhibition of nitric oxide release shortens bleeding time, in rats with cirrhosis induced by chronic bile duct ligation. Methods: Mean arterial pressure and bleeding time were measured under basal conditions and 5, 15 and 30 min after administration of vehicle (0.9% saline) or an inhibitor of nitric oxide synthesis, Nw-nitro-Larginine (5 mg/kg, iv). Mean arterial pressure was measured with an intra-arterial catheter and bleeding time with a standardized Simplate device. Results: Cirrhotic rats showed a lower mean arterial pressure (116-+4 mmHg) and a prolonged bleeding time (177-+40 s) compared to control animals (133-+6 mmHg and 95-+12 S, respectively, p<0.01). In cirrhotic rats, Nw-nitro-L-arginine significantly in-
creased mean arterial pressure (from 116_+5 to 141_+ 11 mmHg, p<0.05) and completely normalized bleeding time (from 170-+39 to 103-+21 S, p<0.05) 15 min after administration. Pretreatment with L-arginine (300 mg/kg, iv) prevented the hemodynamic and hemostatic changes induced by Nw-nitro-L-arginine. A trend to normalize platelet adhesion was observed in cirrhotic rats after the inhibition of nitric oxide production. In control animals, Nw-nitro-L-arginine increased mean arterial pressure, while no effect on bleeding time was observed. Conclusions: These findings support the concept that nitric oxide may be a mediator in the bleeding time abnormalities associated with experimental cirrhosis.
HRONIC LIVER diseases are often associated with impaired primary hemostasis (1). Prolonged bleeding time, which is a reliable indicator of platelet function in vivo (2), is a well-known complication of cirrhosis (3,4). The mechanisms underlying this abnormal platelet function have not been elucidated. It has recently been demonstrated that nitric oxide, a powerful endogenous vasodilator produced by vascular endothelial cells among others in response to different stimuli (5), plays a regulatory role in platelet function (6). Experimental studies have shown that nitric oxide causes inhibition of platelet adhesion and aggregation through activation of the soluble guanylate cyclase and elevation of intracellular cyclic G M P (6). On the other hand, an overproduction of nitric oxide has been involved in the hyperdynamic state and the vascular hy-
poreactivity to vasoconstrictors observed in portal hypertension (7-10). The aim of the present study was to investigate whether an increased formation of nitric oxide might contribute to platelet function disorders observed in cirrhosis. We investigated the effect of Nwnitro-L-arginine (L-NNA), a specific inhibitor of nitric oxide synthesis, on primary hemostasis in rats with cirrhosis induced by common bile duct ligation.
C
Received 3 June; revised 25 August; accepted 28 August 1998
Correspondence." Ricardo Mastai, Secci6n de Higado, Servicio de Clinica Medica, Hospital Italiano, Potosi 4215, Buenos Aires (1199), Argentina. Tel: 541 959 0346. Fax: 541 981 4041. E-mail: [email protected] 456
Key words: Bleeding time; Cirrhosis; Nitric oxide.
Materials and Methods Animals The study was performed in male Wistar rats. The animals were housed in a controlled environment and allowed free access to food and water until the time of the study. Animal treatment during experiments was approved by the local committee for animal experimentation. The animals were divided into two groups. In one group a secondary biliary cirrhosis with portal hypertension was induced by bile duct ligation, according to a previously reported method (11). Under ether anesthesia, a midline abdominal incision was made, and the c o m m o n bile duct exposed, doubly ligated with 4.0 silk and sectioned between the two ligatures. A second group consisted of control rats. In this latter group, rats underwent a sham operation, during which the c o m m o n bile duct was exposed but not ligated. The hemodynamic and hemostatic studies were performed 4 weeks after surgical procedures. The diagnosis of cirrhosis was confirmed by macroand microscopic examination of the liver.
Nitric oxide and bleeding time in cirrhosis Protocol To evaluate the effect of L-NNA on bleeding time and mean arterial pressure, cirrhotic animals were divided into three groups: a control group that received an intravenous bolus injection of saline (n=8), a second group that received an intravenous bolus of L-NNA at a dose of 5 mg/kg (n=8), and a third group in which 300 mg/kg of L-arginine was administered 5 min before L-NNA (5 mg/kg, n=8). In shamoperated animals, one group received saline (n=6), while in a further group L-NNA (5 mg/kg, iv; n=6) was administered. In all groups, mean arterial pressure and bleeding time were measured under basal conditions and 5, 15 and 30 min after vehicle or drug administration. Twenty-one animals (sham-operated rats, n=7; cirrhotics, n=14) were used to evaluate whether shortening of bleeding time induced by L-NNA was associated with an effect on in vivo platelet adhesion. This effect was studied 15 min after vehicle and L-NNA injection. This time was chosen because it is the maximum shortening of bleeding time observed in rats with biliary cirrhosis after L-NNA treatment. In this group of animals the effect of L-NNA on ex vivo platelet aggregation was also evaluated. Another group of cirrhotic (n=12) and control rats (n=12) was included to evaluate the effect of L-NNA or its vehicle over portal pressure, hematocrit and platelet count. Measurements were made 15 rain after vehicle or drug administration, i.e., at the time of maximum shortening of bleeding time. Hemodynamic and hemostatic studies The animals were anesthetized with ether. The right carotid artery was cannulated with a PE-50 catheter (Porter Ltd., Kent, UK) that was connected to a transducer (P-23-Gould Staham), and permanent recordings of arterial pressure were made on a Dyne MCD recorder. The external zero reference limit was placed at the midportion of the animal. Another PE-50 catheter was placed in the right jugular vein and used for drug administration. The animals were kept in a plastic cylinder with several openings, from one of which the rat's tail emerged; they were allowed to waken and stabilize for a period of 30 min so as to avoid any effect of anesthesia on blood-vessel interplay and to prevent possible interactions between anesthetics and other drugs administered (12). The tails were left outside the cylinder and the animals were maintained at room temperature. For bleeding time measurements, a standardized Simplate 1 device (Organon Teknika, USA) was applied longitudinally on the dorsal part of the tail between 6 and 9 cm from the tip, taking care to avoid large veins. Bleeding time, expressed in seconds, was measured from the moment of incision until bleeding stopped (no rebleeding within 30 s). In vivo platelet adhesion was measured by the method of Borchgrevink (13). Briefly, platelets were counted in blood collected from the abdominal aorta and in the drops from a standardized incision in the tail performed with Simplate I device. The difference in the two counts represents the platelets which are retained at the wound surface; i.e. the adhesive platelets. Aggregation tests were performed according to
Born & Cross (14). Blood was collected from the abdominal aorta in 3.1% trisodium citrate (1:10). Platelet-rich plasma (PRP) was obtained by centrifugation at 200×g for 15 min at room temperature. Platelet-poor plasma (PPP) was obtained by centrifugation blood at 2000xg for 10 min. Platelet count in PRP was adjusted to 2.5x 105/ ~1 with autologous PPP. Platelet aggregation was induced by 20/zM ADP and 25 pg/ml collagen (both from Meditech, CA, USA). Aggregation was monitored in an aggregometer (Chrono-Log, WholeBlood Aggro-Meter) and expressed as a percentage of the maximally possible increase in light transmission (100%) as defined by the difference in light transmission between the PPP and the non-stimulated PRP. Portal pressure was measured in anesthetized rats by inserting a 16 G "butterfly" needle into the portal vein. Hematocrit and platelet count were counted in a hematology autoanalyzer (Coulter 790, Coulter Electronics, Ltd., USA). L-NNA and L-arginine were obtained from Sigma Chemical Co (St. Louis, MO, USA), dissolved in saline immediately before use, and administered in 0.3 ml. Data analysis Results are expressed as mean_+SD. Student's t-test for paired and non-paired data and one-way ANOVA with the Tukey test were used in the statistical analysis of the results. Significance was taken at p<0.05.
Results Hemodynamic data In cirrhotic rats, L-NNA treatment significantly increased mean arterial pressure (Table 1). These changes were evident at 5 min after administration and reached their maximal value after 15 min (Table 1). L-NNA treatment also resulted in an increase in mean arterial pressure in control rats (Table 1). The percentages of variation produced by L-NNA administration significantly differed between control and cirrhotic rats (13% vs 25%, p<0.05). Pretreatment with L-arginine prevented the effect of L-NNA on mean arterial pressure in cirrhotic animals (Table 1). After 15 min of vehicle or L-NNA administration, similar values of portal pressure in cirrhotic (L-NNA: 14.9_+2.2 and vehicle: 15.1___2.5 mmHg) and control rats (data not shown) were observed.
TABLE 1 Mean arterial pressure and bleeding time under basal conditions and after intravenous administration of L-NNA or vehicle in cirrhotic and control rats. The effects of pretreatment with L-arginine in cirrhotic animals are also shown Parameters
Cirrhotic rats Basal
Control rats 5 min
15 min
30 min
Basal
5 min
15 min
30 min
Mean arterial pressure (mmHg) Vehicle 116_+4 L-NNA 116-+5 L-arginine + L-NNA 115-+4
115_+4 125-+12 116±3
116---3 141-+11" 115-+3
117-+5 143-+6" 117-+4
133-+6 126---9
137___5 126-+8
134+-5 143-+9"*
137-+4 146+--10**
Bleeding time (s) Vehicle L-NNA L-arginine + L-NNA
180_+38 139±32 179±41
173___33 103±21" 176±28
179±57 173±38 171-+36
95_+12 100±15
90-+19 90_+12
177±40 170±39 177___35
95±22 95-+20
95_+12 110±15
Data expressed as mean±SD, * p<0.05 vs basal and L-arginine + L-NNA. ** p<0.05 vs basal.
457
L. Albornoz et al. TABLE 2
Platelet function
The intrasubject reproducibility of the method was ensured by one observer (L.A) performing bleeding time measurements over a 4-h period in the same animal. For each measurement performed, the coefficient of variation was less than 8%, suggesting a high degree of reproducibility. Bleeding time was significantly prolonged in cirrhotic rats compared to control animals (177_+40 vs 95_+ 12 s, p<0.01). In cirrhotic rats, intravenous administration of L-NNA caused a significant shortening in bleeding time. This effect was detected at 5 min following administration and reached its maximal value at 15 min (Fig. 1). Bleeding time at 15 min in cirrhotic rats treated with L-NNA did not significantly differ from that observed in the control group under basal conditions (cirrhotic: 103_+21; control: 95_+12 s). At 30 min after L-NNA administration, bleeding time returned to near basal values (Fig. 1). Pretreatment of cirrhotic rats with L-arginine 5 min before L-NNA prevented its effect on bleeding time (Table 1). Vehicle administration had no effect on bleeding time. In contrast to the findings in cirrhotic animals, L-NNA treatment in control rats did not modify bleeding time (Table 1). No correlation was observed between mean arterial pressure and bleeding time changes in cirrhotic rats. As shown in Table 2, cirrhotic rats showed a significantly lower platelet adhesion and aggregation than control animals. A trend to normalize platelet adhesion after L-NNA treatment was observed in cirrhotic rats. However, this difference did not reach statistical significance. No changes were observed in platelet aggregation after nitric oxide inhibition in cirrhotic animals.
250
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200 e I.u
150
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_= ci
100
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[• •
1
Vehicle L-NNA
]
0
0
5
15
30
T I M E (mln)
Fig. 1. Effects of vehicle and L - N N A administration on bleeding time in cirrhotic rats. Results are mean+SD. * Significantly different from basal values @<0.05). 458
Effect of L - N N A on platelet adhesion and aggregation in cirrhotic and control rats Parameters
Controls +vehicle
Cirrhotics +vehicle
Cirrhotics +L-NNA
Platelet adhesion (%)
55.2_+15.2
45.6+_9.0 *
51.5_+4.8
Platelet aggregation (%) A D P (20/~M) Collagen (25 Fg/ml)
51.7_+16.3 53.8_+ 17.5
26.7+_15.5" 37.5--_18.0"
27.9_+14.3" 30.5+- 11.5"
Data expressed as mean+-SD. * p<0.05 vs control rats.
After 15 min of vehicle or L-NNA administration in cirrhotic rats, similar values of hematocrit (L-NNA: 4125 and vehicle: 4426%) and platelet count (LNNA: 8529 and vehicle: 88_+11×104//A) were observed. No changes in hematocrit and platelet count were found in control animals after treatments (data not shown).
Discussion The present study shows that inhibition of nitric oxide formation by administration of L-NNA normalizes the prolonged bleeding time in an experimental model of cirrhosis in the rat, induced by bile duct ligation, supporting a role for nitric oxide in the pathogenesis of primary hemostatic disorders associated with chronic liver diseases. Bleeding tendency is a well-known feature in patients with cirrhosis (1). This hemostatic disorder has been related to a complex coagulation defect and impaired platelet function (1). Several studies have reported thrombocytopenia (15,16) and abnormalities in platelet function, such as reduced aggregation (17,18) and prolonged bleeding time (3,4) in patients with cirrhosis. Moreover, a direct correlation has been found between hepatocellular function, evaluated by ChildPugh class, and bleeding time (4), suggesting that in cirrhosis, worsening of platelet function is closely related to the degree of liver failure. However, the mechanisms underlying these hemostatic disorders are not well defined. Nitric oxide is a powerful endothelium-derived vasedilator that, in addition to the relaxation effect on vascular smooth muscle, is a potent inhibitor of platelet function (5,6). It has been demonstrated that nitric oxide synthesized by the constitutive nitric oxide synthase in both platelets and vascular endothelium, inhibits platelet aggregation and adhesion, exerting its action through activation of the soluble guanylate cyclase and elevation of cyclic GMP (6). Under pathological conditions, platelet function may also be modified as a re-
Nitr& oxide and bleeding time in cirrhos&
suit of the expression of the inducible nitric oxide synthase (6). Recent studies suggest that increased nitric oxide formation plays a role in the pathogenesis of the hemodynamic abnormalities associated with portal hypertension. Several authors have presented evidence that nitric oxide inhibition normalizes the systemic and splanchnic vasodilation and restores the vascular hyporesponsiveness to endogenous and exogenous vasoconstrictors in portal hypertensive rats (7-10). Therefore, and in view of the influence of nitric oxide on vascular tone and platelet function (5,6), we hypothesized that the increased nitric oxide formation observed in portal hypertension states could contribute to the abnormal primary hemostatic disorders observed in cirrhosis. In this study, we investigated whether treatment with LNNA, which competitively inhibits the production of nitric oxide (19), could improve platelet function, assessed by bleeding time measurement, in cirrhotic rats. Bleeding time is an easily performed in vivo test of primary hemostasis and a reliable parameter of platelet function as shown by previous pharmacological data (2,20). It has also been proposed as a useful test for studying the in vivo effects of compounds interfering with the platelet adhesion-aggregation reaction (21). In our study, bleeding time was significantly prolonged in cirrhotic animals as compared with controls, demonstrating that experimental cirrhosis is associated with an acquired defect of primary hemostasis, in accordance with results in humans (3,4). Moreover, the inhibition of nitric oxide production by L - N N A administration normalized the prolonged bleeding time. The determination of bleeding time is influenced by platelet-related (i.e., adhesion and aggregation) and vascular (i.e., vasoconstriction) factors. In our study, cirrhotic rats showed reduced platelet adhesion and aggregation. L-NNA treatment partially corrected platelet adhesion at 15 min after administration, i.e. at the time of maximum shortening of bleeding time, but had no effect on platelet aggregation. The discrepancy between these findings with regard to the effects of LN N A on platelet adhesion and aggregation is not clearly understood. However, it could be related to a methodologic difference between the two assessments, as platelet aggregation is an ex vivo, while adhesion is an in vivo assay which seems more physiological because platelet adhesiveness is tested against a damaged vascular wall. On the other hand, as was expected, LN N A treatment increased mean arterial pressure in conscious cirrhotic rats. This hemodynamic change did not correlate with shortening of bleeding time, suggesting that the hemostatic effect of nitric oxide inhibition could not be mediated by the vasoconstriction
observed after L-NNA treatment. However, new experiments with a greater number of animals are needed to confirm these findings. Finally, in the current study the effects of L-NNA were prevented by previous administration of L-arginine, suggesting that the hemodynamic and hemostatic effects of L-NNA result from inhibition of nitric oxide biosynthesis. Similar results have been recently reported (22). In conclusion, our study demonstrates that the model of bile duct ligation in the rat is suitable for evaluating primary hemostasis in cirrhosis. As in humans, bleeding time is prolonged in this experimental model and is normalized by inhibition of nitric oxide synthesis, suggesting a role of this vasodilator in regulating the platelet hemostatic response in cirrhosis. Moreover, this is the first report of in vivo involvement of nitric oxide in the pathogenesis of this primary hemostatic disorder.
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