Prevention of endotoxin-induced mortality by antitissue factor immunization.

Abstract

Microvascular thrombosis with intravascular fibrin deposition is a characteristic pathologic alteration during endotoxic shock. This effect is predominantly mediated by expression of the cellular procoagulant tissue factor by endothelial cells and cells of monocyte or macrophage lineage, resulting in acceleration of the coagulation cascade and fibrin deposition. To determine whether modulation of this response by treatment with an antitissue factor antibody might have beneficial effects. A polyclonal antibody to murine tissue factor was prepared by injecting rabbits with a synthesized peptide sequence of murine tissue factor. To determine the activity of the antibody, elicited murine peritoneal macrophages were treated for 4 hours with 10-micrograms/mL lipopolysaccharide (LPS), and procoagulant activity was determined via a clotting assay (milliunits of activity per 10(6) macrophages). The tissue factor antibody abrogated LPS-induced macrophage procoagulant activity, confirming activity of the antibody (macrophages, 236 +/- 28 mU/10(6) macrophages; macrophages/LPS, 3801 +/- 190* mU/10(6) macrophages; macrophages/LPS/alpha-tissue factor, 753 +/- 92* mU/10(6) macrophages; n = 3; the asterisk indicates P < .05 by an analysis of variance). Additionally, antibody-protein affinity was confirmed by Western blot analysis. Having determined the activity of the antibody in vitro, we tested its efficacy in vivo in a lethal endotoxemia model. Mice were immunized with 200 microL of antiserum intraperitoneally 2 hours before injection with 250 micrograms of LPS intraperitoneally and 24 hours later. Control animals received 200 microL of saline solution. All animals initially exhibited lethargy and piloerection, characteristic of the predicted response to LPS. However, immunized animals had a significantly (P < .05) reduced mortality compared with control animals. Fibrinogen levels were significantly (P < .05) higher in the immunized mice, suggesting decreased consumption of coagulation factors, a finding consistent with an antitissue factor effect. Further, plasma tumor necrosis factor levels 90 minutes after LPS injection were similar in both groups, suggesting normal induction of the cytokine cascade. Modulation of microvascular fibrin deposition by abrogating tissue factor-mediated coagulation significantly (P < .05) improved survival in this model without attenuating the initiation of the cytokine cascade. These findings suggest a pathogenic role for coagulation in the induction of acute organ injury during sepsis.