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8 Antithrombin prevents endotoxin-induced hypotension in rats by inhibiting excess production of nitric oxide
ORAL PRESENTATIONS: Experimental animal studies I
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7 Melagatran, a novel thrombin inhibitor,
reduces renal and haemodynamic effects of endotoxin in the pig *ERIKSSON M, **LARSSON A, ***SALDEEN T. and ****MATTSSON C Departments of *Anaesthesiology and lntensive Care, **Clinical Chemistry and ***Forensic Medicine, University Hospital, Uppsala, and ****Astra Hassle Preclinical Research & Development, M6lndal, Sweden Thrombin is of crucial importance in the development of septic shock. This is a most serious condition, which can be replicated in the endotoxaemic pig. Melagatran, a novel low molecular weight competitive inhibitor of thrombin, was evaluated in this model. Methods. Fifteen anaesthetised pigs were given a continuous infusion ofE. coli endotoxin (10mg × kg t × h l ) for 6 hours. Six of those pigs were given melagatran as a bolus dose (0.3 mg × kg -l) followed by a melagatran infusion (0.1 mg × kg L × h -l) during the first 3 hours o f e n dotoxaemia. The other nine pigs were given the corresponding volume of saline for 3 hours. Blood samples were taken for analysis of creatinine, APTT, melagatran, and fibrinogen. Haemodynamics and oxygen extraction were monitored during the experimental period.
Results. One o f the 6 pigs in the melagatran group and 4 out of 9 pigs in the control group died during the experimental period. Plasma creatinine (p < 0.01) as well as left ventricular stroke work index, systemic vascular resistance index, oxygen extraction, and urinary output were all less affected (p < 0.05) in melagatran-treated pigs as compared to endotoxaemic controls. The plasma concentration of melagatran declined as soon as this infusion was stopped. APTT increased during melagatran infusion and reached a maximum two hours after discontinuation of this infusion (= 6-fold prolongation of the pre-treatment value). In endotoxaemic control pigs APTT was prolonged 1.3 times (p < 0.001). Defibrinogenation did not cause this difference. Conclusions. Our findings indicate a beneficial effect of melagatran in porcine endotoxaemia. This may be of interest in human septic shock. It is an important observation that APTT in plasma of endotoxaemic pigs "spiked" with a decreasing concentration of melagatran.
m
8 Antithrombin prevents endotoxin-induced
hypotension in rats by inhibiting excess production of nitric oxide ISOBE H, OKAJIMA K, UCHIBA M, MURAKAM1 K, and HARADA N Department of Laboratory Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860, Japan
tivity was determined in lung samples by measuring the conversion rate of L-arginine to L-citrulline. Plasma tumor necrosis factor-a (TNF-a) levels were determined by ELISA.
Introduction. Antithrombin (AT) is an important inhibitor of the coagulation system. AT inhibits cytokine production by promoting the endothelial release of PGI2 through interacting with the cell surface heparin-like substance. Since cytokines promote the production of nitric oxide (NO) which plays an important role in septic shock. In the present study, we examined whether AT may prevent endotoxininduced hypotension in rats by inhibiting excess production o f nitric oxide.
Results. AT significantly prevented decrease in MABP after 1.5 hr of ET administration, while DEGR-Xa, a selective inhibitor of thrombin generation, did not. AT inhibited increases in plasma levels of TNF-~, nitrite and nitrate, and lung inducible NOS activity after ET administration, but DEGR-Xa did not. Leukocyte depletion and Iloprost, a stable derivative of PGI2, produced similar effects as those induced by AT in animals given ET.
Methods. Mean arterial blood pressure (MABP) was continuously measured in anesthetized rats after endotoxin administration (ET, 5 mg/kg, iv). Test drugs such as AT (250 U/kg) were administered intravenously 30 rain before ET administration. Plasma nitrite and nitrate levels were measured by Griess method. Lung NO syntase (NOS) ac-
Conclusion. AT prevents ET-induced hypotension in rats by inhibiting production o f TNF-ot which excessively stimulates NO synthesis. Such preventive effects produced by AT might be mediated by PGI2 released from endothelial cells.
m
9 Effect of unfractionated heparin and a heparin
pentasaccharide on the thrombolytic process in an experimental model in rabbits *KAISER B, **WALENGA JM, and **FAREED J *FSU dena, Centerfor Vascular Biology and Medicine, Erfurt, Germany and **Department of Pathology, Loyola University Medical Center, Maywood, IL, USA In the jugular vein of rabbits, thrombus formation was induced by a vessel wall damage using a balloon catheter and following reduction of blood flow by 8090% for 60 min. The thrombus formed was treated with rt-PA (Actilyse®) administered as i.v. bolus injection of 600 lag/kg followed by i.v. infusion of 600 lag/kg over 60 rain. The blood flow in the vein was measured continuously and the time until lysis with rt-PA as well as the incidence of rethrombosis after lysis were determined. At the end of the experiment (3 h after stopping the rt-PA administration), the wet weight of the thrombus formed in the vessel was measured. For the determination of haemostaseological parameters, blood was drawn from the cannulated femoral artery. To study the effectivenessof heparin and a heparin pentasaccharide (Sanofi Thrombose, France) on rethrombosis after lysis, the substances were injected i.v. at the start of rt-PA administration. In saline-treated control animals, a reocclusion of the vessel due to a rethrombosis after lysis was seen in 80% of the animals. Heparin at a dose of 160 U/kg (=1
mg/kg) prevented the complete thrombotic reocclusion in 2 of 12 animals, 4 animals developed a complete thrombosis (mean thrombus wet weight=29.5 mg), whereas in 6 animals a partial reocclusion with a mean thrombus wet weight of 4.2 mg was found. The heparin administration led to strong anticoagulant effects; clotting times in the TT assay were not measurable up to 3 h after injection and were strongly prolonged in the aPTT assay. The synthetic pentasaccharide given at a dose of 250 U/kg (=0.35 mg/kg) completely prevented the thrombotic reocclusion after lysis in 80% of the animals treated (n=10). Furthermore, the time to lysis with rt-PA was markedly shortened, i.e. mean lysis time after pentasaccharide was 25.6 min compared to 44.8 min in controls. At the dose used, pentasaccharide caused only modest anticoagulant effects. It led to a prolongation of clotting times in the Heptest®assay up to 4 h after injection, whereas TT, aPTT and PT were not significantly influenced. The results show that in a rabbit model of thrombotic reocclusion after lysis, heparin pentasaccharide is a potent antithrombotic agent which effectively inhibits the continuation of thrombotic processes over an extended period of time.
S
i
7 Melagatran, a novel thrombin inhibitor,
reduces renal and haemodynamic effects of endotoxin in the pig *ERIKSSON M, **LARSSON A, ***SALDEEN T. and ****MATTSSON C Departments of *Anaesthesiology and lntensive Care, **Clinical Chemistry and ***Forensic Medicine, University Hospital, Uppsala, and ****Astra Hassle Preclinical Research & Development, M6lndal, Sweden Thrombin is of crucial importance in the development of septic shock. This is a most serious condition, which can be replicated in the endotoxaemic pig. Melagatran, a novel low molecular weight competitive inhibitor of thrombin, was evaluated in this model. Methods. Fifteen anaesthetised pigs were given a continuous infusion ofE. coli endotoxin (10mg × kg t × h l ) for 6 hours. Six of those pigs were given melagatran as a bolus dose (0.3 mg × kg -l) followed by a melagatran infusion (0.1 mg × kg L × h -l) during the first 3 hours o f e n dotoxaemia. The other nine pigs were given the corresponding volume of saline for 3 hours. Blood samples were taken for analysis of creatinine, APTT, melagatran, and fibrinogen. Haemodynamics and oxygen extraction were monitored during the experimental period.
Results. One o f the 6 pigs in the melagatran group and 4 out of 9 pigs in the control group died during the experimental period. Plasma creatinine (p < 0.01) as well as left ventricular stroke work index, systemic vascular resistance index, oxygen extraction, and urinary output were all less affected (p < 0.05) in melagatran-treated pigs as compared to endotoxaemic controls. The plasma concentration of melagatran declined as soon as this infusion was stopped. APTT increased during melagatran infusion and reached a maximum two hours after discontinuation of this infusion (= 6-fold prolongation of the pre-treatment value). In endotoxaemic control pigs APTT was prolonged 1.3 times (p < 0.001). Defibrinogenation did not cause this difference. Conclusions. Our findings indicate a beneficial effect of melagatran in porcine endotoxaemia. This may be of interest in human septic shock. It is an important observation that APTT in plasma of endotoxaemic pigs "spiked" with a decreasing concentration of melagatran.
m
8 Antithrombin prevents endotoxin-induced
hypotension in rats by inhibiting excess production of nitric oxide ISOBE H, OKAJIMA K, UCHIBA M, MURAKAM1 K, and HARADA N Department of Laboratory Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860, Japan
tivity was determined in lung samples by measuring the conversion rate of L-arginine to L-citrulline. Plasma tumor necrosis factor-a (TNF-a) levels were determined by ELISA.
Introduction. Antithrombin (AT) is an important inhibitor of the coagulation system. AT inhibits cytokine production by promoting the endothelial release of PGI2 through interacting with the cell surface heparin-like substance. Since cytokines promote the production of nitric oxide (NO) which plays an important role in septic shock. In the present study, we examined whether AT may prevent endotoxininduced hypotension in rats by inhibiting excess production o f nitric oxide.
Results. AT significantly prevented decrease in MABP after 1.5 hr of ET administration, while DEGR-Xa, a selective inhibitor of thrombin generation, did not. AT inhibited increases in plasma levels of TNF-~, nitrite and nitrate, and lung inducible NOS activity after ET administration, but DEGR-Xa did not. Leukocyte depletion and Iloprost, a stable derivative of PGI2, produced similar effects as those induced by AT in animals given ET.
Methods. Mean arterial blood pressure (MABP) was continuously measured in anesthetized rats after endotoxin administration (ET, 5 mg/kg, iv). Test drugs such as AT (250 U/kg) were administered intravenously 30 rain before ET administration. Plasma nitrite and nitrate levels were measured by Griess method. Lung NO syntase (NOS) ac-
Conclusion. AT prevents ET-induced hypotension in rats by inhibiting production o f TNF-ot which excessively stimulates NO synthesis. Such preventive effects produced by AT might be mediated by PGI2 released from endothelial cells.
m
9 Effect of unfractionated heparin and a heparin
pentasaccharide on the thrombolytic process in an experimental model in rabbits *KAISER B, **WALENGA JM, and **FAREED J *FSU dena, Centerfor Vascular Biology and Medicine, Erfurt, Germany and **Department of Pathology, Loyola University Medical Center, Maywood, IL, USA In the jugular vein of rabbits, thrombus formation was induced by a vessel wall damage using a balloon catheter and following reduction of blood flow by 8090% for 60 min. The thrombus formed was treated with rt-PA (Actilyse®) administered as i.v. bolus injection of 600 lag/kg followed by i.v. infusion of 600 lag/kg over 60 rain. The blood flow in the vein was measured continuously and the time until lysis with rt-PA as well as the incidence of rethrombosis after lysis were determined. At the end of the experiment (3 h after stopping the rt-PA administration), the wet weight of the thrombus formed in the vessel was measured. For the determination of haemostaseological parameters, blood was drawn from the cannulated femoral artery. To study the effectivenessof heparin and a heparin pentasaccharide (Sanofi Thrombose, France) on rethrombosis after lysis, the substances were injected i.v. at the start of rt-PA administration. In saline-treated control animals, a reocclusion of the vessel due to a rethrombosis after lysis was seen in 80% of the animals. Heparin at a dose of 160 U/kg (=1
mg/kg) prevented the complete thrombotic reocclusion in 2 of 12 animals, 4 animals developed a complete thrombosis (mean thrombus wet weight=29.5 mg), whereas in 6 animals a partial reocclusion with a mean thrombus wet weight of 4.2 mg was found. The heparin administration led to strong anticoagulant effects; clotting times in the TT assay were not measurable up to 3 h after injection and were strongly prolonged in the aPTT assay. The synthetic pentasaccharide given at a dose of 250 U/kg (=0.35 mg/kg) completely prevented the thrombotic reocclusion after lysis in 80% of the animals treated (n=10). Furthermore, the time to lysis with rt-PA was markedly shortened, i.e. mean lysis time after pentasaccharide was 25.6 min compared to 44.8 min in controls. At the dose used, pentasaccharide caused only modest anticoagulant effects. It led to a prolongation of clotting times in the Heptest®assay up to 4 h after injection, whereas TT, aPTT and PT were not significantly influenced. The results show that in a rabbit model of thrombotic reocclusion after lysis, heparin pentasaccharide is a potent antithrombotic agent which effectively inhibits the continuation of thrombotic processes over an extended period of time.