© 1991 S. Karger AG. Basel 0301 -0147/91 /0217-0142S2.75/0
Haemostasis 1991 ;21(suppl 1): 142-148
Hirudin in Disseminated Intravascular Coagulation G. Nowak, F. Markwardt Institute of Pharmacology and Toxicology, Medical Academy Erfurt, FRG
Key Words. Disseminated intravascular coagulation • Recombinant hirudin • Thrombokinase • Endotoxin • Adrenaline • Thrombin
Introduction Many clinical and experimental investi gations revealed that several diseases and injuries may result in microcirculatory dis turbances due to intravascular coagulation. This microthrombosis following clotting ac tivation in streaming blood may be induced directly or indirectly by multiple pathomechanisms in which the close relation between hemostatic processes and organ functions plays a decisive role [1,2]. Acute or chronic disseminated micro thrombosis have multiple etiologies, such as hypocirculation, intravascular hemolysis, blocking of the reticuloendothelial system or endothelial cell injury [3].
These events may lead to severe or even fatal complications of the underlying disease unless there is pharmacological control of the intravascular coagulation processes. Controlled clinical studies in comparable groups of patients are complicated by the etiological diversity combined with diagnos tic difficulties. Hence, further findings have predominantly been gained in animal exper iments. There is a wide variety of experi mental models that facilitate studies on the pathogenesis of disseminated intravascular coagulation (DIC) [4], Normally, clotting en zymes and bacterial endotoxins were used as triggers, but in special investigations, bio genic amines were also employed. Rats, rab bits, dogs and pigs served as experimental
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Abstract. Various reactions of disseminated intravascular coagulation (DIC) were experi mentally induced by infusion of thrombokinase in rats, by administration of endotoxin in rabbits and pigs and by infusion of adrenaline and thrombin in dogs. Low plasma concen trations of recombinant hirudin (r-hirudin; 20-200 ng/ml) were sufficient for the inhibi tion of the different triggering mechanisms. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy of this specific tight-binding thrombin inhib itor.
Hirudin in DIC
Materials and Methods Materials Thrombin: Topostasin (3,000 NIH-U) (Hoffmann-La Roche, Basel, Switzerland); thrombokinase: brain thrombokinase, prepared and purified accord ing to Deutsch et al. [10]; adrenaline: epinephrinehydrochloride (Suprarenin®) (Hoechst, Frankfurt am Main, FRG); endotoxin (a): lipid A fraction (VM 030877) from Escherichia coli (strain VO 149 K) pre pared and purified by Dr. H. Klie, Institut fur Impfstoffe, Dessau; (b): lipopolysaccaride W E. coli 026:B6, Difco Laboratories Detroit, Mich. (USA); m In-oxin: 37 MBq/ml, Amersham-Buchler, Braun schweig (FRG); ,25I-fbrinogen: homologous fibrino gen, purified and labeled according to the iodogen method [11]; r-hirudin: CGP 39393 (expressed in and secreted from yeast) from Ciba-Geigy, Biotechnology Department, Basel (Switzerland) and Plantorgan KG, Bad Zwischenahn (FRG). Animals Shoe-Wistar strain rats of either sex, body weight 220-250 g; Beagle dogs from our Beagle farm, both sexes, body weight 11-18 kg; New Zealand White rab bits, both sexes, body weight 3-4 kg; young pigs (Deutsches Landschwein), both sexes, body weight 811 kg. All animals were kept under conventional breeding conditions, pellet feeding and water ad libi tum. Methods Microthrombosis in Rat Lungs. Microthrombosis in the rat lung was continuously measured in vivo after application of l25I-fibrinogen and/or 11'In-la beled platelets [labeled according to Lotter et al., 12], The radioactivity was continuously recorded by means of a gamma measurement system [for details, see Koth et al, 13]. Localized Shwartzman Reaction in Rabbits. The localized Shwartzman reaction was induced in rabbits
by a preparatory intradermal injection of endotoxin (50 pg) followed by a provocative application of in travenous endotoxin (200 pg) on the next day. The size of the affected area was measured and the haem orrhage was graded by a score from 1 to 4. Radiola beled fibrinogen was used for quantifying the fibrin accumulation at the affected skin area. Six hours af ter the provocative endotoxin dose, the animals were sacrificed and skinned. The affected areas were cut out (16 mm diameter) and the l25I-radioactivity measured. Generalized Shwartzman Reaction in Young Pigs. Experimental endotoxinemia was induced in pigs by infusion of the endotoxin fraction lipid A [for details of test design and measurement, see Nowak and Markwardt, 14], Adrenaline-Thrombin-Induced Platelet Retention in Dogs. The combined administration of thrombin (75 NIH-U/kg X h) and adrenaline (lOpg/kg X h) evokes an in vivo potentiation of platelet aggregation and retention of these circulating aggregates in var ious organs [15]. This reaction was continuously re corded by measuring the distribution o f 111In-labeled platelets. Recording, data compilation as well as eval uation were performed by means of a Picker gamma camera computer system (Picker International Ltd.) [for details, see Nowak and Glusa, 15].
Results and Discussion We investigated thrombokinase-induced microthrombosis in rat lungs. In parallel, we recorded 125I-ftbrin accumulation as well as 11'In-platelet retention in the lungs of the animals. Immediately after starting the thrombokinase infusion, the radioactivity over the lungs increased and lasted until the end of the infusion. The increase in 125Iradioactivity became demonstrable about 10 min after starting the infusion (fig. la). Administration of 1.5 pg r-hirudin/kg/min inhibited the thrombokinase-induced fibrin accumulation (fig. lb), whereas a dose of 6.5 pg r-hirudin/kg/min was necessary to in hibit platelet retention (fig. lc). This means
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
animals [5-8], For pharmacological studies on the influence of recombinant hirudin (r-hirudin) on DIC we used animal models which allow to continuously follow altera tions in the clotting variables and the forma tion of microthrombi [9].
143
Nowak/Markwardt
144
Fig. 1. Influence of r-hirudin on the thrombokinase-induced 125I-fibrin accumulation and 11'In platelet retention in the lungs of rats, a Control: infu sion of thrombokinase. b Additional infusion of 1.5 pg/kg X min hirudin, c Additional infusion of 6.5 pg/kg X min hirudin.
that a higher plasma level of hirudin is re quired to antagonize this specific plateletthrombin interaction. These results eluci date the specific affinity of thrombin to tight-binding platelet receptors. Previous investigations have shown that the activation of the clotting system and the
Affected skin area
Control n=16
Hirudin n-16
Diameter, cm Hemorrhage (score 1-4) Fibrin accumulation, l25I-radioactivity, cpm X 103
2.4±0.5 3.8 ±0.2
1.6 ± 0.7 1.5 ±0.3
4.2±0.3
1.2±0.4
development of fibrin deposits in the micro vasculature are important starting points for the pathomechanism of the endotoxin-in duced localized or generalized Shwartzman reaction. At first, we investigated the influence of r-hirudin on a model of localized DIC. After initial ischemia, inside the preparatory intradermal injection point, petechial bleed ings were observed and after 4-5 h a more or less pronounced haemorrhagic necrosis oc curred (table 1). The rabbits received r-hirudin hourly at a dose of 100 pg/kg. The results are summarized in table 1. Both the size of the affected skin area and the hemorrhage were minimized. The results revealed that hirudin was able to prevent microthrombo sis in the superficial microcirculation of the skin. Furthermore, we investigated DIC exper imentally induced in pigs by infusion of pu rified endotoxin (lipid A) from pathogenic coli strains. We attempted to clarify wether the activation of the clotting system is im portant for the pathomechanism of endotoxinemia in young pigs. Moreover, we studied the influence of r-hirudin of the changes in the clotting system, on microthrombosis and the circulation, r-hirudin was continuously
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
T ablet. Influence of r-hirudin (5 X 0.1 mg/kg i.v.) on endotoxin-induced localized Shwartzman reaction
Hirudin in DIC
145
infused at a dose of 200 pg/kg/h. Endotoxin caused typical consumption changes in the clotting system: 60 min after starting the in fusion the fibrinogen level began to decrease continuously until the end of the experiment (fig. 2c). After 80 min, fibrin monomers could be observed. The number of circulat ing platelets decreased. Three hours after starting the endotoxin infusion, only 30% of platelets were found in blood (fig. 2a). Ad ministration of 200 pg r-hirudin/kg/h re duced the endotoxin-induced changes in the clotting system while monomers appeared only after 160 min. Microthrombosis in lungs, liver, spleen and kidneys was esti mated by measuring the radioactivity in the
tissue samples after exchanging blood against dextran solution. After endotoxin in fusion, a 3- to 4-fold increase in radioactivity was found in the organs investigated (fig. 2d). Upon simultaneous infusion of r-hirudin this increase was significantly re duced. The investigation of cardiovascular functions elicited the following results: dur ing endotoxin infusion, the right ventricular pressure increased by about 180% within the first 30 min (fig. 3a), whereas the left ven tricular pressure decreased by 30% (fig. 3b). The contractility parameter, dp/dtmax, of the right ventricle increased at the beginning of the endotoxin administration, reaching a maximum of about 45 kPa/s, after 30 min
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Fig. 2. Influence of recombinant hirudin on the lipid-A-induced changes in platelet count (a), fibrinogen level (b), l25I-fibrinogen uptake in the plasma (c) and on microthrombosis in various organs (d). o, □ - Control without endotoxin (lipid A); •, H = endotoxin (lipid A) infusion; A, B = endotoxin (lipid A) and hirudin infusion.
Nowak/Markwardt
146
jS
(fig. 3c). However, the contractility of the left heart was not significantly changed. Both heart and respiratory rate were measured simultaneously and showed a continuous in crease during the endotoxin application (fig. 3d). Administration of r-hirudin re duced the increase in both the right ventric ular pressure and the contractility, r-hirudin was also able to normalize the disturbed respiratory and heart rate. Animals who re ceived an endotoxin infusion alone died whereas those who received an additional r-hirudin infusion survived. Of particular pathophysiological impor tance is the thrombin-induced platelet reac tion, potentiated by adrenaline, since - via this pathway low thrombin levels cause ag gregation of platelets, thus mediating the possible development of thrombi. In Beagle
dogs, the simultaneous infusion of adrena line and thrombin at nonaggregating concen trations caused the development of platelet thrombi as well as the activation of the microcirculatory clotting system. Microcirculatory disturbances induced by platelet aggre gates were found in the lungs, the heart and the liver of the dogs, whereas the spleen was platelet depleted (fig. 4a). Perfusion scintig raphy of the lungs using 99TC microspheres revealed perfusion disturbances, particularly with regard to the basal lung lobes. Infusion of r-hirudin at a dose of 200 pg/kg/h pre vented the accumulation of 11‘In-labeled platelets in the lungs and in the heart (fig. 4b). Perfusion disturbances, as men tioned above, did not occur. Summing up, it may be said that in our studies in experimental animals a blood level
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Fig. 3. Influence of recombinant hirudin on the lipid-A-induced changes in right ventricular pressure (a), left ventricular pressure (b), right ventricular dp/dtmax (c) and heart rate (d). o = Control without endotoxin (lipid A); • = endotoxin (lipid A)-infusion; A = endotoxin (lipid A)-and hirudin-infusion.
147
Hirudin in DIC
of 20-200 ng r-hirudin/ml proved to be suf ficient to block DIC reactions. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy and specificity of the tight-binding thrombin-in hibitor.
4
5
References 1 McKay DG: Disseminated Intravascular Coagula tion. An Intermediary Mechanism of Disease. New York, Harper & Row, 1964. 2 Hardaway RM III: Syndromes of Disseminated Intravascular Coagulation. With Special Refer ence to Shock and Hemorrhage. Springfield, Thomas, 1966. 3 Muller-Berghaus G: Pathophysiologic and bio chemical events in disseminated intravascular co
6
7
agulation: Dyregulation of procoagulant and anti coagulant pathways. Semin Thromb Hemost 1989;15:58-87. Müller-Berghaus G, Lasch HG: Microcirculatory disturbances induced by generalized intravascular coagulation; in Schmier J, Eichler O (eds): Hand book of Experimental Pharmacology. Berlin, Springer, 1975, vol 16/3, pp 429-514. Markwardt F, Nowak G, Meerbach W, Rüdiger KD: Studies in experimental animals on dissemi nated intravascular coagulation (DIC). Thromb Diath Haemorrh 1975;34:513-521. Markwardt F, Nowak G, Meerbach W, Rüdiger KD: The influence of drugs on disseminated intravscular coagulation (DIC). I. Effects of antifi brinolytics and fibrinolytics on thrombin-induced DIC in rats. Thromb Res 1976;9:143-152. Markwardt F, Nowak G, Hoffmann J: The in fluence of drugs on disseminated intravascular coagulation (DIC). II. Effects of naturally occur
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Fig. 4. Distribution of 11'In-labeled platelets in the lungs, heart, liver and spleen in dogs during infusion of adrena line (10 pg/kg X h) and thrombin (75 NIH-U/kg X h). a Control, b Infusion of r-hirudin (0.2 mg/kg X h).
8
9
10
11
12
ring and synthetic thrombin inhibitors. Thromb Res 1977;11:275-283. Markwardt F, Nowak G, Hoffmann J: Compara tive studies on thrombin inhibitors in experimen tal microthrombosis. Thromb Haemost 1983;49: 235-237. Nowak G, Markwardt F: Influence of hirudin on endotoxin-induced disseminated intravascular co agulation (DIC) in weaned pigs. Exp Pathol 1980; 18:438-443. Deutsch E, Irsigler K, Lomoschitz H: Studien über Gewebsthromboplastin. 1. Reinigung, chemische Charakterisierung und Trennung in einen Eiweiss- und Lipoidanteil. Thromb Diath Haemorrh 1964;12:12-34. Richter M, Paintz M, Nowak G, Schmidt HE; Herzmann H: Untersuchungen zur Gerinnbar keit von 125I-Rinderfibrinogen. Folia Haematol (Leipz) 1984;111:862-869. Lotter MG, Badenhorst PN, Heyns AduP, van Reenen OR, Pieters H, Minnaar PC: Kinetics, dis tribution and sites of destruction of canine blood platelets with In-111 oxine. J Nucl Med 1980;21: 36-40.
Nowak/Markwardt
13 Koth W, Nowak G, Markwardt F: Monitoring of microthrombosis in experimental animals by con tinuous recording of 12SI-fibrin deposits and slCrlabelled platelets in the lungs. Acta Biol Med Ger 1980;39:157-162. 14 Nowak G, Markwardt F: Influence of cyprohepta dine on endotoxin-induced dissseminated intra vascular coagulation (DIC) in weaned pigs. Thromb Haemost 1985;53:252-254. 15 Nowak G, Glusa E: Pharmakologische Untersu chungen zur Adrenalin-Thrombin-induzierten Plättchenaggregation bei Hunden. Folia Haema tol (Leipz) 1989;116:831-839.
Doz. Dr. Götz Nowak Medizinische Akademie Erfurt Institut für Pharmakologie und Toxikologie Nordhäuser Strasse 74 D-O-5010 Erfurt (FRG)
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
148
Haemostasis 1991 ;21(suppl 1): 142-148
Hirudin in Disseminated Intravascular Coagulation G. Nowak, F. Markwardt Institute of Pharmacology and Toxicology, Medical Academy Erfurt, FRG
Key Words. Disseminated intravascular coagulation • Recombinant hirudin • Thrombokinase • Endotoxin • Adrenaline • Thrombin
Introduction Many clinical and experimental investi gations revealed that several diseases and injuries may result in microcirculatory dis turbances due to intravascular coagulation. This microthrombosis following clotting ac tivation in streaming blood may be induced directly or indirectly by multiple pathomechanisms in which the close relation between hemostatic processes and organ functions plays a decisive role [1,2]. Acute or chronic disseminated micro thrombosis have multiple etiologies, such as hypocirculation, intravascular hemolysis, blocking of the reticuloendothelial system or endothelial cell injury [3].
These events may lead to severe or even fatal complications of the underlying disease unless there is pharmacological control of the intravascular coagulation processes. Controlled clinical studies in comparable groups of patients are complicated by the etiological diversity combined with diagnos tic difficulties. Hence, further findings have predominantly been gained in animal exper iments. There is a wide variety of experi mental models that facilitate studies on the pathogenesis of disseminated intravascular coagulation (DIC) [4], Normally, clotting en zymes and bacterial endotoxins were used as triggers, but in special investigations, bio genic amines were also employed. Rats, rab bits, dogs and pigs served as experimental
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Abstract. Various reactions of disseminated intravascular coagulation (DIC) were experi mentally induced by infusion of thrombokinase in rats, by administration of endotoxin in rabbits and pigs and by infusion of adrenaline and thrombin in dogs. Low plasma concen trations of recombinant hirudin (r-hirudin; 20-200 ng/ml) were sufficient for the inhibi tion of the different triggering mechanisms. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy of this specific tight-binding thrombin inhib itor.
Hirudin in DIC
Materials and Methods Materials Thrombin: Topostasin (3,000 NIH-U) (Hoffmann-La Roche, Basel, Switzerland); thrombokinase: brain thrombokinase, prepared and purified accord ing to Deutsch et al. [10]; adrenaline: epinephrinehydrochloride (Suprarenin®) (Hoechst, Frankfurt am Main, FRG); endotoxin (a): lipid A fraction (VM 030877) from Escherichia coli (strain VO 149 K) pre pared and purified by Dr. H. Klie, Institut fur Impfstoffe, Dessau; (b): lipopolysaccaride W E. coli 026:B6, Difco Laboratories Detroit, Mich. (USA); m In-oxin: 37 MBq/ml, Amersham-Buchler, Braun schweig (FRG); ,25I-fbrinogen: homologous fibrino gen, purified and labeled according to the iodogen method [11]; r-hirudin: CGP 39393 (expressed in and secreted from yeast) from Ciba-Geigy, Biotechnology Department, Basel (Switzerland) and Plantorgan KG, Bad Zwischenahn (FRG). Animals Shoe-Wistar strain rats of either sex, body weight 220-250 g; Beagle dogs from our Beagle farm, both sexes, body weight 11-18 kg; New Zealand White rab bits, both sexes, body weight 3-4 kg; young pigs (Deutsches Landschwein), both sexes, body weight 811 kg. All animals were kept under conventional breeding conditions, pellet feeding and water ad libi tum. Methods Microthrombosis in Rat Lungs. Microthrombosis in the rat lung was continuously measured in vivo after application of l25I-fibrinogen and/or 11'In-la beled platelets [labeled according to Lotter et al., 12], The radioactivity was continuously recorded by means of a gamma measurement system [for details, see Koth et al, 13]. Localized Shwartzman Reaction in Rabbits. The localized Shwartzman reaction was induced in rabbits
by a preparatory intradermal injection of endotoxin (50 pg) followed by a provocative application of in travenous endotoxin (200 pg) on the next day. The size of the affected area was measured and the haem orrhage was graded by a score from 1 to 4. Radiola beled fibrinogen was used for quantifying the fibrin accumulation at the affected skin area. Six hours af ter the provocative endotoxin dose, the animals were sacrificed and skinned. The affected areas were cut out (16 mm diameter) and the l25I-radioactivity measured. Generalized Shwartzman Reaction in Young Pigs. Experimental endotoxinemia was induced in pigs by infusion of the endotoxin fraction lipid A [for details of test design and measurement, see Nowak and Markwardt, 14], Adrenaline-Thrombin-Induced Platelet Retention in Dogs. The combined administration of thrombin (75 NIH-U/kg X h) and adrenaline (lOpg/kg X h) evokes an in vivo potentiation of platelet aggregation and retention of these circulating aggregates in var ious organs [15]. This reaction was continuously re corded by measuring the distribution o f 111In-labeled platelets. Recording, data compilation as well as eval uation were performed by means of a Picker gamma camera computer system (Picker International Ltd.) [for details, see Nowak and Glusa, 15].
Results and Discussion We investigated thrombokinase-induced microthrombosis in rat lungs. In parallel, we recorded 125I-ftbrin accumulation as well as 11'In-platelet retention in the lungs of the animals. Immediately after starting the thrombokinase infusion, the radioactivity over the lungs increased and lasted until the end of the infusion. The increase in 125Iradioactivity became demonstrable about 10 min after starting the infusion (fig. la). Administration of 1.5 pg r-hirudin/kg/min inhibited the thrombokinase-induced fibrin accumulation (fig. lb), whereas a dose of 6.5 pg r-hirudin/kg/min was necessary to in hibit platelet retention (fig. lc). This means
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
animals [5-8], For pharmacological studies on the influence of recombinant hirudin (r-hirudin) on DIC we used animal models which allow to continuously follow altera tions in the clotting variables and the forma tion of microthrombi [9].
143
Nowak/Markwardt
144
Fig. 1. Influence of r-hirudin on the thrombokinase-induced 125I-fibrin accumulation and 11'In platelet retention in the lungs of rats, a Control: infu sion of thrombokinase. b Additional infusion of 1.5 pg/kg X min hirudin, c Additional infusion of 6.5 pg/kg X min hirudin.
that a higher plasma level of hirudin is re quired to antagonize this specific plateletthrombin interaction. These results eluci date the specific affinity of thrombin to tight-binding platelet receptors. Previous investigations have shown that the activation of the clotting system and the
Affected skin area
Control n=16
Hirudin n-16
Diameter, cm Hemorrhage (score 1-4) Fibrin accumulation, l25I-radioactivity, cpm X 103
2.4±0.5 3.8 ±0.2
1.6 ± 0.7 1.5 ±0.3
4.2±0.3
1.2±0.4
development of fibrin deposits in the micro vasculature are important starting points for the pathomechanism of the endotoxin-in duced localized or generalized Shwartzman reaction. At first, we investigated the influence of r-hirudin on a model of localized DIC. After initial ischemia, inside the preparatory intradermal injection point, petechial bleed ings were observed and after 4-5 h a more or less pronounced haemorrhagic necrosis oc curred (table 1). The rabbits received r-hirudin hourly at a dose of 100 pg/kg. The results are summarized in table 1. Both the size of the affected skin area and the hemorrhage were minimized. The results revealed that hirudin was able to prevent microthrombo sis in the superficial microcirculation of the skin. Furthermore, we investigated DIC exper imentally induced in pigs by infusion of pu rified endotoxin (lipid A) from pathogenic coli strains. We attempted to clarify wether the activation of the clotting system is im portant for the pathomechanism of endotoxinemia in young pigs. Moreover, we studied the influence of r-hirudin of the changes in the clotting system, on microthrombosis and the circulation, r-hirudin was continuously
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
T ablet. Influence of r-hirudin (5 X 0.1 mg/kg i.v.) on endotoxin-induced localized Shwartzman reaction
Hirudin in DIC
145
infused at a dose of 200 pg/kg/h. Endotoxin caused typical consumption changes in the clotting system: 60 min after starting the in fusion the fibrinogen level began to decrease continuously until the end of the experiment (fig. 2c). After 80 min, fibrin monomers could be observed. The number of circulat ing platelets decreased. Three hours after starting the endotoxin infusion, only 30% of platelets were found in blood (fig. 2a). Ad ministration of 200 pg r-hirudin/kg/h re duced the endotoxin-induced changes in the clotting system while monomers appeared only after 160 min. Microthrombosis in lungs, liver, spleen and kidneys was esti mated by measuring the radioactivity in the
tissue samples after exchanging blood against dextran solution. After endotoxin in fusion, a 3- to 4-fold increase in radioactivity was found in the organs investigated (fig. 2d). Upon simultaneous infusion of r-hirudin this increase was significantly re duced. The investigation of cardiovascular functions elicited the following results: dur ing endotoxin infusion, the right ventricular pressure increased by about 180% within the first 30 min (fig. 3a), whereas the left ven tricular pressure decreased by 30% (fig. 3b). The contractility parameter, dp/dtmax, of the right ventricle increased at the beginning of the endotoxin administration, reaching a maximum of about 45 kPa/s, after 30 min
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Fig. 2. Influence of recombinant hirudin on the lipid-A-induced changes in platelet count (a), fibrinogen level (b), l25I-fibrinogen uptake in the plasma (c) and on microthrombosis in various organs (d). o, □ - Control without endotoxin (lipid A); •, H = endotoxin (lipid A) infusion; A, B = endotoxin (lipid A) and hirudin infusion.
Nowak/Markwardt
146
jS
(fig. 3c). However, the contractility of the left heart was not significantly changed. Both heart and respiratory rate were measured simultaneously and showed a continuous in crease during the endotoxin application (fig. 3d). Administration of r-hirudin re duced the increase in both the right ventric ular pressure and the contractility, r-hirudin was also able to normalize the disturbed respiratory and heart rate. Animals who re ceived an endotoxin infusion alone died whereas those who received an additional r-hirudin infusion survived. Of particular pathophysiological impor tance is the thrombin-induced platelet reac tion, potentiated by adrenaline, since - via this pathway low thrombin levels cause ag gregation of platelets, thus mediating the possible development of thrombi. In Beagle
dogs, the simultaneous infusion of adrena line and thrombin at nonaggregating concen trations caused the development of platelet thrombi as well as the activation of the microcirculatory clotting system. Microcirculatory disturbances induced by platelet aggre gates were found in the lungs, the heart and the liver of the dogs, whereas the spleen was platelet depleted (fig. 4a). Perfusion scintig raphy of the lungs using 99TC microspheres revealed perfusion disturbances, particularly with regard to the basal lung lobes. Infusion of r-hirudin at a dose of 200 pg/kg/h pre vented the accumulation of 11‘In-labeled platelets in the lungs and in the heart (fig. 4b). Perfusion disturbances, as men tioned above, did not occur. Summing up, it may be said that in our studies in experimental animals a blood level
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Fig. 3. Influence of recombinant hirudin on the lipid-A-induced changes in right ventricular pressure (a), left ventricular pressure (b), right ventricular dp/dtmax (c) and heart rate (d). o = Control without endotoxin (lipid A); • = endotoxin (lipid A)-infusion; A = endotoxin (lipid A)-and hirudin-infusion.
147
Hirudin in DIC
of 20-200 ng r-hirudin/ml proved to be suf ficient to block DIC reactions. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy and specificity of the tight-binding thrombin-in hibitor.
4
5
References 1 McKay DG: Disseminated Intravascular Coagula tion. An Intermediary Mechanism of Disease. New York, Harper & Row, 1964. 2 Hardaway RM III: Syndromes of Disseminated Intravascular Coagulation. With Special Refer ence to Shock and Hemorrhage. Springfield, Thomas, 1966. 3 Muller-Berghaus G: Pathophysiologic and bio chemical events in disseminated intravascular co
6
7
agulation: Dyregulation of procoagulant and anti coagulant pathways. Semin Thromb Hemost 1989;15:58-87. Müller-Berghaus G, Lasch HG: Microcirculatory disturbances induced by generalized intravascular coagulation; in Schmier J, Eichler O (eds): Hand book of Experimental Pharmacology. Berlin, Springer, 1975, vol 16/3, pp 429-514. Markwardt F, Nowak G, Meerbach W, Rüdiger KD: Studies in experimental animals on dissemi nated intravascular coagulation (DIC). Thromb Diath Haemorrh 1975;34:513-521. Markwardt F, Nowak G, Meerbach W, Rüdiger KD: The influence of drugs on disseminated intravscular coagulation (DIC). I. Effects of antifi brinolytics and fibrinolytics on thrombin-induced DIC in rats. Thromb Res 1976;9:143-152. Markwardt F, Nowak G, Hoffmann J: The in fluence of drugs on disseminated intravascular coagulation (DIC). II. Effects of naturally occur
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/16/2018 10:27:26 AM
Fig. 4. Distribution of 11'In-labeled platelets in the lungs, heart, liver and spleen in dogs during infusion of adrena line (10 pg/kg X h) and thrombin (75 NIH-U/kg X h). a Control, b Infusion of r-hirudin (0.2 mg/kg X h).
8
9
10
11
12
ring and synthetic thrombin inhibitors. Thromb Res 1977;11:275-283. Markwardt F, Nowak G, Hoffmann J: Compara tive studies on thrombin inhibitors in experimen tal microthrombosis. Thromb Haemost 1983;49: 235-237. Nowak G, Markwardt F: Influence of hirudin on endotoxin-induced disseminated intravascular co agulation (DIC) in weaned pigs. Exp Pathol 1980; 18:438-443. Deutsch E, Irsigler K, Lomoschitz H: Studien über Gewebsthromboplastin. 1. Reinigung, chemische Charakterisierung und Trennung in einen Eiweiss- und Lipoidanteil. Thromb Diath Haemorrh 1964;12:12-34. Richter M, Paintz M, Nowak G, Schmidt HE; Herzmann H: Untersuchungen zur Gerinnbar keit von 125I-Rinderfibrinogen. Folia Haematol (Leipz) 1984;111:862-869. Lotter MG, Badenhorst PN, Heyns AduP, van Reenen OR, Pieters H, Minnaar PC: Kinetics, dis tribution and sites of destruction of canine blood platelets with In-111 oxine. J Nucl Med 1980;21: 36-40.
Nowak/Markwardt
13 Koth W, Nowak G, Markwardt F: Monitoring of microthrombosis in experimental animals by con tinuous recording of 12SI-fibrin deposits and slCrlabelled platelets in the lungs. Acta Biol Med Ger 1980;39:157-162. 14 Nowak G, Markwardt F: Influence of cyprohepta dine on endotoxin-induced dissseminated intra vascular coagulation (DIC) in weaned pigs. Thromb Haemost 1985;53:252-254. 15 Nowak G, Glusa E: Pharmakologische Untersu chungen zur Adrenalin-Thrombin-induzierten Plättchenaggregation bei Hunden. Folia Haema tol (Leipz) 1989;116:831-839.
Doz. Dr. Götz Nowak Medizinische Akademie Erfurt Institut für Pharmakologie und Toxikologie Nordhäuser Strasse 74 D-O-5010 Erfurt (FRG)
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