Endotoxin-induced Disseminated Intravascular Coagulation Research Articles

Abstract 039: A Small Fragment Derived from von Willebrand Factor Improves Survival in a Mouse Model of Endotoxemia-Induced Disseminated Intravascular Coagulation

Introduction: Disseminated intravascular coagulation (DIC) is a life-threatening condition that results in wide-spread clot formation and organ failure. Sepsis-induced DIC is associated with a high mortality rate, prompting the need for an effective therapeutic intervention. Our lab has identified the A2 domain of the von Willebrand factor (VWF) protein as a potent treatment for sepsis-induced DIC. Historically, VWF has only been described as a contributor of clot formation; however, our lab has demonstrated that purified recombinant full length A2 domain of the VWF protein can dampen fibrin-rich microthrombi formation in mice with endotoxin. The mode of action appears to be via the interaction with the cell-surface expressed vimentin on endothelium, inhibiting VWF strings formation and thus, platelet adhesion. Goal: Since the VWF protein is naturally cleaved in circulation by the enzyme ADAMTS-13 via the A2 domain, one can argue whether the beneficial effect observed with full len gth A2 domain is terminated by the cleavage of ADAMTS-13. We produced recombinant proteins encompassing the sequences of the two proteolytic products of the A2 domain; the A2Nt and A2Ct proteins. I will identify which fragment retains the mode of action of the full length A2 domain in a mouse model of endotoxin-induced DIC. Methods: The recombinant A2 and vimentin proteins were produced using the bacterial expression system. Protein: protein interactions were determined by optical interferometry. The endotoxin-induced DIC model was achieved by injecting C57B/6 mice with lipopolysaccharide (LPS). Treatment with A2 or its fragments was started approximately 2 hours after LPS insult. Results: The A2N t protein has a higher affinity for vimentin than the A2Ct. Treatment of A2Nt in a LPS-induced DIC mouse model was effective in maintaining survival in males, a comparable finding that mimics treatment of mice with full-length recombinant A2. Interestingly, A2Nt showed less survival than full length A2 in females. These findings highlight the potential differences in therapy responses between genders, an occurrence that some studies have described to also occur in humans.

Crocetin administration ameliorates endotoxin-induced disseminated intravascular coagulation in rabbits

Disseminated intravascular coagulation (DIC), a life-threatening secondary complication in several diseases, is characterized by large amounts of thrombin that lead to fibrin deposition and microthrombus formation throughout the microcirculation. Recent in-vitro studies suggest that crocin, crocetin or safranal, carotenoid constituents of the spice Crocus sativus L. (saffron), have antithrombotic properties, especially anti-Xa activity. The present study was designed to evaluate the effect of crocetin on thrombosis procedure using a rabbit model of bacterial endotoxin-induced DIC. Crocetin administration (3 mg/kg), 30 min before the beginning of endotoxin infusion, improved DIC-related haemostatic indices such as platelet blood counts (P≤ 0.05), blood plasma fibrinogen and protein C concentration (P≤ 0.05). In addition, it ameliorated DIC-associated disease and fibrin deposition in the glomeruli (P≤ 0.05). These results indicate that crocetin reveals a preventive antithrombotic role in vivo and prescribe further investigation on the possibility of developing crocetin-based DIC treatment modalities.

Rivaroxaban Has Protective Effects in a Model of Disseminated Intravascular Coagulation (DIC) in Rats.

Introduction: DIC is a complication that occurs during serious infection with Gram-negative bacteria. Endotoxin is a component of the bacterial cell wall that elicits a cytokine-mediated cascade of tissue factor-dependent hypercoagulable reactions. The resulting hypercoagulable state may be inhibited by potent anticoagulation. Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. The aim of this study was to examine the effects of rivaroxaban in a rat model of endotoxin-induced DIC.Methods: Rivaroxaban (0.1–10 mg/kg p.o.) or vehicle control (PEG400/H2O 60/40%, 5 mL/kg p.o.) were administered 30 minutes before endotoxin injection (lipopolysaccharide O55 B5 [LPS], 250 μg/kg i.v.) to conscious rats. Blood samples were withdrawn from anesthetized animals by heart puncture 4 hours after LPS injection and fibrinogen, platelet count, thrombin-antithrombin (TAT) complex levels and IL-6 were measured.Results: The induction of DIC was indicated in the placebo + LPS group vs vehicle control by decreased fibrinogen (2.12±0.08 vs 2.69±0.10 g/L) and platelet count (571±28 vs 904±30×109/L) an increase in TAT (75±9 vs 2±1 μg/L) and IL-6 (8.6±1.23 μg/L vs 0.028±0.020 μg/L). Pretreatment with rivaroxaban (0.1–10 mg/kg p.o.) dose-dependently ameliorated the effects of LPS on fibrinogen, platelets and TAT. Rivaroxaban 10 mg/kg p.o. normalized fibrinogen (2.58±0.07 g/L) and TAT (5.6±1.2 μg/L) and increased platelet count (703±19×109/L) (Table). Rivaroxaban also slightly reduced the plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) (4.24±0.67 μg/L).Conclusions: These results show that direct, selective inhibition of FXa by rivaroxaban effectively normalized the hypercoagulable reactions of endotoxemia with a slight modulating effect on the generation of pro-inflammatory active cytokines, such as IL-6, in the rat DIC model. Further research into the use of rivaroxaban in the management of DIC is therefore warranted.Effects of rivaroxaban in an endotoxin-induced DIC model in rats 4 hours after LPS injection. Results show mean ± SEM.Vehicle controlPlacebo + LPSRivaroxaban 1 mg/kg + LPSRivaroxaban 10 mg/kg + LPSFibrinogen (g/L)2.69±0.10 n=52.12±0.08## n=142.41±0.09 n=142.58± 0.07** n=7Platelets (×109/l)904±30 n=5571±28### n=8655±23 n=6703±19* n=7TAT (μg/L)1.8±0.8 n=575.2±9.4### n=1465.3±8.0 n=145.6±1.2*** n=7IL-6 (μg/ml)0.028±0.020 n=58.60±1.23### n=65.01±0.47 n=64.24±0.67 n=7DIC, disseminated intravascular coagulation; IL-6, interleukin-6; LPS, lipopolysaccharide O55 B5; SEM, standard error of the mean; TAT, thrombin-antithrombin ## p<0.01 vs sham, ### p<0.001 vs sham, *p<0.05 vs LPS/placebo, ** p<0.01 vs LPS/placebo, *** p<0.001 vs LPS/placebo

Gene Expression Profiling Identifies C/EBPδ as a Candidate Regulator of Endotoxin-induced Disseminated Intravascular Coagulation

A runaway inflammatory response to systemic infection or severe trauma is characterized by the activation of a diversity of pathways, ultimately resulting in the development of disseminated intravascular coagulation (DIC) and multiorgan failure. Despite increased fundamental knowledge of the pathogenesis of DIC, the exact molecular mechanisms remain elusive. We aimed therefore to improve our understanding of the molecular pathways underlying endotoxin-induced DIC. We performed large-scale gene expression profiling in the liver of mice during the onset of endotoxin-induced DIC. The relevance of an identified candidate gene involved in endotoxin-induced DIC was subsequently assessed in the generalized Shwartzman reaction. Approximately 5% of over 20,000 genes were differentially regulated. In addition to well-established sepsis-associated genes, such as macrophage inflammatory protein 1, plasminogen activator inhibitor 1, CD14, and A20, we identified several novel candidates for inflammatory disease of which the transcription factor C/EBPdelta (CAAT/enhancer binding protein delta) was studied further. Induction of DIC in C/EBPdelta-deficient mice decreased endotoxin-induced systemic inflammation as compared with wild-type mice, as evident from decreased plasma levels of tumor necrosis factor-alpha and IL-6. In addition, C/EBPdelta deficiency partly protected against DIC-induced mortality. Interestingly, C/EBPdelta deficiency seemed mainly protective by improving renal function. This latter notion was confirmed in an experimental model of renal ischemia/reperfusion injury in which C/EBPdelta deficiency reduced ischemia/reperfusion-induced creatinine and urea levels. Our results endorse the usefulness of gene expression profiling in identifying novel mediators of DIC by showing that C/EBPdelta regulates specific pathologic features of this endotoxin-induced syndrome.

Effect of pentoxifylline on endotoxin-induced haemostatic disturbances in rabbits

Pentoxifylline (PTX, a methylxanthine derivative) has been found to interrupt early gene activation for tumour necrosis factor, interleukin-1, interleukin-6 and tissue factor production and to improve survival from experimental sepsis. During endotoxaemia, lipopolysaccharide (LPS, endotoxin) and proinflammatory cytokines trigger the development of disseminated intravascular coagulation (DIC) via the tissue factor-dependent pathway of coagulation. The present study was undertaken to determine whether pentoxifylline could prevent coagulation disturbances in LPS-treated rabbits. Endotoxaemia was induced with E. coli lipopolysaccharide in New Zealand White rabbits. Forty rabbits were used and divided into four equal groups. Group 1 served as a control group; Group 2: lipopolysaccharide was injected intravenously, Group 3: pentoxifylline was injected intraperitoneally, Group 4: lipopolysaccharide and pentoxifylline were injected simultaneously. Blood samples were collected 6 h after the treatments. In rabbits with endotoxin-induced DIC, platelet count, leukocyte count, percentage of differential leukocyte values, fibrinogen level, antithrombin III (AT-III) and protein C (PC) activity were decreased. Moreover, activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged when compared to the control group. In conclusion, haemostatic disturbances associated with endotoxin-induced DIC were moderately suppressed by the administration of PTX.

Prevention of Renal Fibrin Deposition in Endotoxin-induced DIC through Inhibition of PAI-1

Plasminogen activator inhibitor-1 (PAI-1) increases in endotoxemia thus possibly cooperating in altering the hemostatic balance in a prothrombotic direction. The effect of the inhibition of PAI-1 with the monoclonal antibody MA-33B8 was studied systemically and in kidneys in a lapine model of endotoxin-induced disseminated intravascular coagulation (DIC). The increase in plasmatic PAI activity in the control group (n = 9) was inhibited in the MA-33B8 treated rabbits (n = 5). Control rabbits showed renal fibrin deposits, whereas only one of the MA-33B8 rabbits did so. These results were confirmed immunohistochemically in kidneys as PAI-1 immunostaining was seen inside the glomeruli and larger vessels in the control group, whereas MA-33B8 rabbits showed a remarkable decrease, demonstrating that MA-33B8 successfully inhibited PAI-1 in the kidneys as well. Therefore evidence for the important role of PAI-1 in fibrin generation in endotoxin-induced DIC is presented, suggesting that strategies aiming at its reduction can be useful in this pathology.

Prednisolone inhibits endotoxin-induced disseminated intravascular coagulation and improves mortality in rats: importance of inflammatory cytokine suppression.

In order to determine whether prednisolone has a protective effect against the development of disseminated intravascular coagulation (DIC), we measured the effect of prednisolone on changes in hemostatic parameters and plasma levels of inflammatory cytokines in endotoxin-treated rats. Decreases in platelet count and fibrinogen levels, prolongation of prothrombin time, and increases in the plasma fibrin degradation products and levels of thrombin-antithrombin III (TAT) complex following the administration of endotoxin, all of which are associated with DIC, were significantly suppressed by the administration of prednisolone. Heparin administration significantly suppressed changes in all these parameters except for the decrease in platelet count. The combination of prednisolone and heparin was more effective than either treatment alone. In order to determine whether these effects of prednisolone are correlated with the suppression of inflammatory cytokine production, we examined the relationship between changes in plasma levels of cytokine, the hemostatic parameters listed above, and mortality using a number of intervention regimens designed to alter events of the experimentally induced DIC. Changes in hemostatic parameters associated with DIC following 30 mg/kg per 4 h of endotoxin infusion were significantly suppressed by treatment with 1 mg/kg prednisolone 30 min before beginning endotoxin infusion, followed by administration of 250 U/kg heparin 2 h after the start of endotoxin infusion (prednisolone-endotoxin-heparin regimen). The heparin and prednisolone were administrated subcutaneously. The administration of prednisolone and heparin in the reverse order (i.e. heparin first and prednisolone second: heparin-endotoxin-prednisolone regimen) also suppressed changes in hemostatic parameters, albeit to a smaller degree. Cytokine production was also significantly suppressed by the first treatment, but was not affected by the regimen in which heparin was administered first. Administration of prednisolone alone or heparin alone 30 min before endotoxin significantly reduced the number of renal glomeruli with fibrin thrombi. Plasma levels of creatinine and alanine transferase were reduced only by prednisolone. Increased plasma levels of interleukin-1beta, tissue necrosis factor-alpha and interleukin-6 were suppressed by prednisolone but not by heparin, and there were significant correlations between plasma levels of TAT and cytokines. Prednisolone was more effective than heparin in reducing mortality at 24 h after 100 mg/kg over 4 h of endotoxin infusion (four of 20 versus 15 of 20 deaths for prednisolone and heparin, respectively). These findings suggest that prednisolone inhibits the development of endotoxin-induced DIC and reduces mortality by a different mechanism than heparin, possibly through suppressing the production of inflammatory cytokines. Prednisolone may be efficacious in preventing DIC and multiple organ dysfunction caused by endotoxin.

Dithiocarbamates ameliorate the effects of endotoxin in a rabbit model of disseminated intravascular coagulation.

Induction of tissue factor (TF) activity by endotoxin and cytokines is an important mechanism for initiation of disseminated intravascular coagulation (DIC) seen in patients with gram-negative sepsis. Based on data from an in vitro study in which dithiocarbamates abrogated endothelial cell TF activity by inhibition of the NF-kappaB pathway, we investigated whether dithiocarbamates had in vivo activity in an animal model of DIC. Dithiocarbamates ameliorated the adverse clinical and histological effects of endotoxin-induced DIC, including morbidity, hypofibrinogenemia, and target organ damage, especially in the liver and kidney, even when given up to 1 hour after administration of endotoxin. This pilot study confirms the key role of the nuclear factor-kappa beta (NF-kappaB) pathway in induction of TF activity in initiating sepsis-associated DIC and suggests that dithiocarbamates may be useful in treatment of DIC associated with excessive TF expression because of gram-negative sepsis. Additional studies of dithiocarbamates in DIC models are warranted.

Effect of Reduqing"Equation missing" on plasma lnterleukin-8 and nitric oxide in rabbits with endotoxin induced disseminated intravascular coagulation

Objective: To investigate the effects of Reduqing (RDQ, Open image in new window ) on plasma interleukin-8 (IL-8), NO2 - /NO3 -, complement 5a (C5a) and polymorphonuclear neutrophilic leukocyte (PMN) chemotaxis Index (CI) in rabbits with endotoxin-induced disseminated intravascular coagulation (DIC).Methods: Endotoxin-in-duced DIC model made by injection of Lipopolysacchrides (LPS) was used in the experiment. Above-mentioned indexes were determined before and after RDQ treatment and compared with blank and dexamethasone treated group.Results: Plasma IL-8, NO2 - /NO3 -, C5a and CI level of PMN increased markedly in the model group, which were confirmed pathologically with obvious damage of tissues or organs. In the RDQ group, the abovem entiond parameters and damage of tissues or organs were reduced significantly (P < 0.01).Conclusion: IL-8 and NO might be involved in pathogenesis of endotoxin-induced DIC, and RDQ could be used in preventing or treating DIC through the mechanism of regulation of cytokines network.

Treatment of disseminated intravascular coagulation (DIC) with all-trans retinoic acid in an endotoxin-induced rat model.

Anticoagulant drugs such as heparin are often administered to patients with disseminated intravascular coagulation (DIC) who are also being treated for their underlying disease. The pathophysiology of DIC is so varied that treatment with medications other than anticoagulants may be useful. All-trans retinoic acid (ATRA), which is used for the treatment of acute promyelocytic leukemia (APL), improves DIC in APL. In vitro studies have reported that ATRA caused downregulation of tissue factor and upregulation of thrombomodulin (TM) on endothelial cells as well as APL cells. We examined the effect of ATRA in an endotoxin-induced rat DIC model. DIC was induced in male Wistar rats with a 4-h sustained infusion of endotoxin at a dose of 30 mg/kg. ATRA (20 mg/day) was given every day for 1 week before the injection of endotoxin. ATRA improved the increase in thrombin-antithrombin III (TAT) complex and D-dimer in this model. Fibrin deposition in renal glomeruli was inhibited by ATRA administration, with an increase in the intensity of immunohistochemical TM staining. These findings suggest that ATRA has beneficial effects in the endotoxin-induced rat DIC model. The mechanism may be an upregulation of TM expression on endothelial cells.

Increased anticoagulant activity of recombinant thrombomodulin modified with glycosaminoglycan.

Thrombomodulin (TM) is a thrombin receptor on the endothelial cell surface, effective as an anticoagulant by changing procoagulant thrombin to an anticoagulant one. As rabbit TM with glycosaminoglycan (GAG) has a more potent anticoagulant activity than that without GAG, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C-127 cells. The effect of an additional GAG chain on anticoagulant activity was investigated in comparison with unmodified recombinant UTM (r-UTM). In vitro, the activity of cleavage of fibrinogen by thrombin or prothrombinase activity was more potently depressed by GAG-UTM than by r-UTM, and the generation of activated protein C by TM-thrombin complex was accelerated by GAG modification. The acceleration of antithrombin III-dependent anticoagulant activity was shown only by GAG-UTM. Parameters like thrombin time, prothrombin time and activated partial thromboplastin time in human plasma were prolonged by GAG-UTM more than by r-UTM. In vivo, the effect of GAG-UTM and r-UTM in endotoxin-induced disseminated intravascular coagulation (DIC) rats was investigated using hematological parameters. GAG-UTM and r-UTM significantly reduced the decrease in fibrinogen and platelet number induced by endotoxin at the dosage of 0.1 and 1.0 mg/kg/h, respectively, suggesting that the antithrombotic effect of GAG-UTM in endotoxin-induced DIC rats was 10-fold as potent as that of r-UTM. GAG-UTM reduced the prolongation of the bleeding time induced by endotoxin, while r-UTM accelerated it. These results suggest that the addition of a GAG chain may increase availability as an anticoagulant.

Effect of Reduqing on TNF-α, IL-1, IL-6, IL-8 and PAF levels in endotoxin-lnduced DIC model of rabbits) on TNF-α, IL-1, IL-6, IL-8 and PAF levels in endotoxin-lnduced DIC model of rabbits

Objective: To study the mechanism of anti-endotoxemic effect of Reduqing Injection (RDQ) and to explore the essence of traditional Chinese “heat-clearing and detoxifying therapy”.Methods: A disseminated intravascular coagulation (DIC) model was made in rabbits by intravenous injection with E. coli endotoxin. Increased plasma levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and platelet activating factor (PAF), as well as the ex-vivo LPS-induced interleukin-1 (IL-1) production in peripheral blood monocytes were observed in model (M) group.Results: Same parameters were significantly lower in RDQ and dexamethasone (DXM) groups than those in the M group (P<0.01,P<0.05). Histopathological examination showed severe damage of the liver, lungs and kidneys in the rabbits of M group, in contrast, only mild affects were seen in the RDQ and DXM groups.Conclusions: RDQ exhibits protective effect on rabbits against endotoxin-induced DIC. The suppression of cytokines and inflammatory mediator PAF by RDQ may play a central role in the inhibition of endotoxin-induced DIC cascade.

The preventive-therapeutic effects of reduqing (热毒清) on endotoxin-induced DIC in rabbits (Continued) protection of hepatocytic microsomes, maintenance of calcium homeostasis and reduction of oxygen free radicals

Disseminated intravascular coagulation (DIC) model of rabbits was made by the injection of endotoxin. Cytochrome P-450 content and aniline hydroxylase activity in hepatocytic microsomes in DIC rabbits were significantly reduced. The microviscosity of mitochondrial membrane was elevated, while the fluidity of the membrane, and the activities of calcium-magnesium-adenosine triphosphatase and calcium-adenosine triphosphatase were reduced. Lipid peroxide and xanthine oxidase activities were increased, while superoxide dismutase and glutathione peroxidase activities were significantly decreased (P<0.05 or <0.01). The changes in the Reduqing test group were not prominent, and the parameters of the test group were close to those of the control group. This study suggests that in endotoxin induced DIC rabbits the hepatocytic microsomes are injured, calcium homeostasis is disturbed and oxygen free radicals are notably accumulated. Whereas Reduqing may have a protective effect on hepatocytic microsomes from the injury, maintain calcium homeostasis and reduce oxygen free radicals in DIC rabbits.

Effects of DX-9065a, an Orally Active, Newly Synthesized and Specific Inhibitor of Factor Xa, against Experimental Disseminated Intravascular Coagulation in Rats

We investigated the protective effects of DX-9065a, an orally active, newly synthesized and specific inhibitor of factor Xa, against two kinds of experimental disseminated intravascular coagulation (DIC) in rats. Endotoxin-induced experimental DIC was induced by a 4-h sustained infusion of endotoxin at a dose of 100 mg/kg. Thromboplastin-induced experimental DIC was induced by a bolus injection of thromboplastin at a dose of 150 mg/kg. The rats were orally administered DX-9065a at 10, 30 or 100 mg/kg 30 min before endotoxin or thromboplastin injection. In both DIC models, DX-9065a showed a protective effect against DIC, at all doses and in all parameters, including fibrin/fibrinogen degradation products (FDP), fibrinogen level, prothrombin time, activated partial thromboplastin time, platelet count and the number of renal glomeruli with fibrin thrombi. When DX-9065a was orally administrated at 100 mg/kg without endotoxin or thromboplastin, no significant changes were seen in hemostatic parameters except PT and APTT, and no fibrin thrombi or abnormal bleeding were seen in renal specimens. These findings suggest that the new oral anti-Xa drug, DX-9065a, has an effect in reducing the severity of DIC. However, further dose-finding and safety studies of this drug have still to be assessed.

The effect of two-domain tissue factor pathway inhibitor on endotoxin-induced disseminated intravascular coagulation in rabbits

Disseminated intravascular coagulation (DIC) is a common complication in sepsis, and may result from endotoxin-induced exposure of tissue factor on the surface of monocytes and endothelial cells. Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent feedback inhibitor of the tissue factor-factor VIIa complex. In the present study the effect on DIC of a two-domain TFPI analogue (2D-TFPI), consisting of the first two Kunitz domains of TFPI but lacking the third domain, was tested. DIC was induced in rabbits by two intravenous bolus injections of endotoxin from Escherichia coli (10 and 50 μg/kg) 24 h apart. Simultaneously with the last endotoxin injection an infusion of 2D-TFPI (0, 0.3, 1.0 or 3.0 mg/kg/h) was given. Blood samples were obtained at 0 h, 24 h and 31 h. At 31 h the animals were sacrificed and the kidneys were submitted to histological examination. The degree of fibrin deposition in glomeruli was scored blindly using an arbitrary scale from 0 to 3. Between 24 and 31 h the group receiving endotoxin alone showed a significant decrease in platelet count (65%), plasma fibrinogen (41%), antithrombin III (25%), and factor VIII (63%), and a significant prolongation of the aPTT (14%). Furthermore, massive fibrin deposition was detected in the renal glomeruli at 31 h. Infusions of 2D-TFPI inhibited all the endotoxininduced changes in a dose-dependent manner. In conclusion, the data demonstrate that inhibition of the TF/FVIIa complex by infusion of 2D-TFPI significantly counteracts endotoxin-induced coagulopathy in rabbits, and might thus be an attractive drug for treatment of endotoxin-induced DIC in humans.

The effect of two-domain tissue factor pathway inhibitor on endotoxin-induced disseminated intravascular coagulation in rabbits

Disseminated intravascular coagulation (DIC) is a common complication in sepsis, and may result from endotoxin-induced exposure of tissue factor on the surface of monocytes and endothelial cells. Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent feedback inhibitor of the tissue factor-factor VIIa complex. In the present study the effect on DIC of a two-domain TFPI analogue (2D-TFPI), consisting of the first two Kunitz domains of TFPI but lacking the third domain, was tested. DIC was induced in rabbits by two intravenous bolus injections of endotoxin from Escherichia coli (10 and 50 micrograms/kg) 24 h apart. Simultaneously with the last endotoxin injection an infusion of 2D-TFPI (0, 0.3, 1.0 or 3.0 mg/kg/h) was given. Blood samples were obtained at 0 h, 24 h and 31 h. At 31 h the animals were sacrificed and the kidneys were submitted to histological examination. The degree of fibrin deposition in glomeruli was scored blindly using an arbitrary scale from 0 to 3. Between 24 and 31 h the group receiving endotoxin alone showed a significant decrease in platelet count (65%), plasma fibrinogen (41%), antithrombin III (25%), and factor VIII (63%), and a significant prolongation of the aPTT (14%). Furthermore, massive fibrin deposition was detected in the renal glomeruli at 31 h. Infusions of 2D-TFPI inhibited all the endotoxin-induced changes in a dose-dependent manner. In conclusion, the data demonstrate that inhibition of the TF/FVIIa complex by infusion of 2D-TFPI significantly counteracts endotoxin-induced coagulopathy in rabbits, and might thus be an attractive drug for treatment of endotoxin-induced DIC in humans.

Inhibitory effect of TCV-309, a novel platelet activating factor (PAF) antagonist, on endotoxin-induced disseminated intravascular coagulation in rats: Possible role of PAF in tissue factor generation

The possible involvement of platelet activating factor (PAF) in the pathogenesis of endotoxin-induced disseminated intravascular coagulation (DIC) was investigated in rats using a novel potent PAF antagonist, TCV-309. TCV-309 (> 1 mg/kg, i.v.) showed beneficial effects in rats with experimental DIC induced by a 4-hour sustained infusion of endotoxin (1 mg/kg) in a dose-dependent manner. TCV-309 (1 mg/kg) significantly ameliorated the decrease in platelet count and plasma fibrinogen, the prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) and the increase in fibrin and fibrinogen degradation products (FDP) and inhibited glomerular fibrin deposition. Furthermore, plasma tissue factor (TF) activity was greatly increased in the DIC rats, and this was also significantly decreased by TCV-309 (1 mg/kg). TCV-309 (1 mg/kg) did not affect these parameters in normal rats. A 4-hour sustained infusion of PAF (60 μg/kg) caused mild but significant changes in some DIC parameters such as PT, fibrinogen and FDP concentration and increased the plasma TF activity. TCV-309 (1 mg/kg) inhibited all these PAF-induced changes. TCV-309 (0.1 mM) itself had no direct in vitro effects on the blood coagulation system including TF activity. These results strongly suggest that PAF plays a role in the pathogenesis of endotoxin-induced DIC via the generation of TF. Prophylactic use of PAF antagonists may therefore be useful for the treatment of DIC with sepsis.

Anticoagulant Profile of Low Molecular Weight Heparinoids (KB-101) and its in Vivo Effects on Experimental DIC

Anticoagulant profile of low molecular weight heparinoids (KB-101) and its in vivo effects on the experimental DIC (disseminated intravascular coagulation) were investigated in comparison with unfractionated heparin (UFH). KB-101 displayed considerably less effects than UFH on the conventional laboratory tests as activated partial thromboplastin, prothrombin and thrombin time. Both of these agents catalyzed the inactivation of factor Xa by antithrombin III (AT III) and the inactivation of thrombin by AT III and heparin cofactor II (HC II). The potencies of KB-101 on the inactivation of these activated coagulation factors were far lesser than those of UFH. On the other hand, the anti-Xa to anti-thrombin activity ratio of KB-101 was about 28 times higher than that of UFH. Either KB-101 and UFH enhanced the antiplasmin activity in plasma, which was less prominent in KB-101 than UFH.The in vivo effects of these heparin on the endotoxin-induced experimental DIC using rats, were assessed by coagulation and fibrinolytic parameters and the degree of fibrin deposition in the renal glomeruli. The inhibitory effects of KB-101 on DIC were proved to be as equivalent to those of UFH. From these results it was concluded that KB-101 showed a higher anti-Xa to anti-thrombin activity ratio than UFH and revealed a potent inhibitory effects on experimental DIC, which suggested its therapeutic efficacy for DIC.

Effects of nicardipine in rats subjected to endotoxic shock

1. 1. The calcium channel blocker, nicardipine, produced a dose-dependent reduction in the mortality caused by endotoxin in rats. 2. 2. The drug also reduced most of the hematological and gross pathological manifestations of disseminated intravascular coagulation (DIC) caused by endotoxin. 3. 3. The endotoxin-induced monocytopenia but not the granulocytopenia, lymphocytopenia or thrombocytopenia was inhibited by the drug. 4. 4. The results suggest that the protective action of nicardipine is causally related to prevention of the endotoxin-induced DIC and that an effect of the drug on monocytes may be of importance.

A purified antithrombin III-heparin complex as a potent inhibitor of thrombin in porcine endotoxin shock

Inhibition of activated clotting factors is an important therapeutic approach in disseminated intravascular coagulation (DIC). We examined the possible protective effect of a purified complex of human antithrombin III (AT III) and heparin in endotoxin-induced DIC in pigs. Two groups of endotoxemic pigs were studied. AT III-heparin group pigs (n=8) were pretreated with a bolus injection of 500 units AT III-heparin complex, followed by a continuous infusion of 1000 units of the complex for 6 hours given simultaneously with the infusion of 10 μg/kgh of S. abortus equi endotoxin. Controls (n=9) were given saline in addition to the continuous infusion of endotoxin. AT III activity, prothrombin and soluble fibrin in plasma were determined by chromogenic substrate methods. Fibrinogen was measured turbidimetrically. Human AT III antigen in the treated group was 64 ± 3% at 2 hours and increased to 84 ± 4% until the end of the experiment. AT III activity in the AT III - heparin group was elevated throughout the whole observation period (> 100%), whereas it was significantly lower in the controls. Prothrombin decreased similarly in both groups by approximately 35% until the end of the experiment. AT III-heparin treatment significantly attenuated the endotoxin-induced consumption of fibrinogen and completely prevented the increase in soluble fibrin in plasma. However, no significant effect of AT III-heparin was observed on endotoxin-induced mortality and dysfunction in pulmonary gas exchange. Therefore we conclude that the purified AT III - heparin complex inhibits thrombin effects and prevents development of DIC, but fails to significantly influence clinical outcome in endotoxin shock of the pig.