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1. Disseminated intravascular coagulation frequently accompanies Gram-negative sepsis and may contribute to widespread deposition of microthrombi. Besides the endotoxin-induced activation of coagulation, an important role for the fibrinolytic system has been postulated. The precise mechanisms underlying these fibrinolytic changes during endotoxaemia are not known but have been suggested to be mediated directly by cytokines or secondary to thrombin generation. 2. In the present study we have delineated in detail the fibrinolytic response to a bolus injection of endotoxin in non-human primates and analysed the contribution of cytokines and thrombin generation to the endotoxin-induced release of tissue-type plasminogen activator and plasminogen activator inhibitor 1. Chimpanzees received a bolus injection of endotoxin alone or in combination with blocking monoclonal antibodies directed against tumour necrosis factor or interleukin 6 or in combination with pentoxifylline. Furthermore, to assess the effect of coagulation activation on the activation of fibrinolysis, another group of chimpanzees received endotoxin in combination with either anti-tissue factor antibodies or recombinant hirudin. 3. Infusion of endotoxin induced a rapid increase in plasminogen activator activity and tissue-type plasminogen activator antigen levels and subsequent plasmin generation, reaching peak levels 2h after endotoxin administration. Plasminogen activator inhibitor 1 levels remained constant for the first 2 h, after which time a steep increase was observed. Plasminogen activator activity and plasmin generation decreased simultaneously with the rise in plasminogen activator inhibitor 1 levels. Fibrinolytic activity remained suppressed during the remainder of the study owing to sustained increased levels of plasminogen activator inhibitor 1. The administration of pentoxifylline strongly attenuated the release of tissue-type plasminogen activator and plasminogen activator inhibitor 1, whereas the antitumour necrosis factor antibodies blocked the fibrinolytic response entirely. In contrast, interleukin 6-neutralizing antibodies did not affect the fibrinolytic response. Although endotoxin-induced generation of thrombin was completely prevented by the administration of tissue factor-neutralizing antibodies or by hirudin, no effect on the fibrinolytic response was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

TNF is considered to be an intermediate factor in endotoxin-induced release of other cytokines and endotoxin-induced neutrophil degranulation. Little is known about the effect of postponed treatment with anti-TNF in primate endotoxin models. To assess the effect of delayed treatment with anti-TNF in endotoxaemia, six healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In three of these animals the administration of endotoxin was followed after 30 min by a bolus i.v. injection of the anti-TNF F(ab')2 fragment MAK 195F (0.1 mg/kg). Post-treatment with MAK 195F completely prevented the appearance of TNF activity in serum elicited by endotoxin, and markedly reduced the rises in the serum concentrations of IL-6 and IL-8. In addition, the endotoxin-induced increases in the type I and type II soluble TNF receptors were also profoundly inhibited by MAK 195F, suggesting that TNF is involved in the release of its own soluble receptors in endotoxaemia. Neutrophilic leucocytosis was not affected by MAK 195F. In contrast, MAK 195F did significantly abrogate neutrophil degranulation, as measured by the plasma concentrations of lactoferrin. These results indicate that treatment with anti-TNF 30 min after the administration of endotoxin is still effective in attenuating the induction of the cytokine network and of neutrophil degranulation.

Gram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin-induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees. Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 micrograms/kg (n = 2) or 100 micrograms/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1 + 2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor (TNF) is considered to be a pivotal mediator of endotoxin-induced lethality. To assess the intermediate role of TNF in specific systemic inflammatory responses known to contribute to tissue injury in endotoxemia, eight healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In four of these animals the administration of endotoxin was followed immediately by a bolus intravenous injection of an anti-TNF monoclonal antibody (15 mg/kg). Treatment with anti-TNF completely prevented the endotoxin-induced increase in serum TNF activity, and profoundly reduced the appearance of interleukin-6 and -8 (both P < .05). Neutrophilia and lymphopenia were not affected by anti-TNF, whereas neutrophil degranulation, as measured by the plasma concentrations of elastase-alpha 1-antitrypsin complexes, was only slightly reduced (peak levels after endotoxin alone 31.0 +/- 3.4 ng/mL, versus 25.5 +/- 3.4 ng/mL after endotoxin with anti-TNF; P < .05). Anti-TNF did not influence endotoxin-induced activation of the coagulation system, as reflected by unchanged increases in the plasma concentrations of the prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes. In contrast, anti-TNF strongly attenuated the activation of the fibrinolytic system, ie, peak plasma levels of plasmin-alpha 2-antiplasmin were 33.8 +/- 11.1 nmol/L after endotoxin alone and 17.0 +/- 2.9 nmol/L after endotoxin with anti-TNF (P < .05). These results suggest that TNF is not the common mediator of systemic inflammatory changes in low-grade endotoxemia. Moreover, the finding that in this mild model anti-TNF specifically inhibited fibrinolysis suggests that treatment with anti-TNF potentially may enhance the tendency towards microvascular thrombosis in sepsis.