compstatin and Sepsis

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation.

Topics: Antithrombins; Blood Coagulation; C-Reactive Protein; Complement C3; Escherichia coli; Hirudins; Humans; Lipopolysaccharide Receptors; Lipopolysaccharides; Peptide Fragments; Peptides, Cyclic; Prothrombin; Recombinant Proteins; RNA, Messenger; Sepsis; Serum Amyloid P-Component; Thromboplastin; Up-Regulation

Combined inhibition of CD14 and complement, two main inducers of the inflammatory response, have proved particularly effective in attenuating Gram-negative bacteria-induced inflammation. Approaching possible clinical relevance, we investigated the effect of such inhibition in a post-challenge setting. Human whole blood was anti-coagulated with lepirudin. Anti-CD14, compstatin (C3 inhibitor) and the combination thereof were added 5 min prior to or 5, 15 or 30 min after adding Escherichia coli. Total incubation time with Escherichia coli was 120 min. Cytokines, myeloperoxidase (MPO) and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. Delayed combined inhibition significantly attenuated the inflammatory response. IL-1β, IL-8 and TNF-α were significantly inhibited in the range of 20-40%, even when adding the inhibitors with up to 30 min delay. IL-6 was significantly inhibited with 15 min delay, and MIP-1α and MPO with 5 min delay. Complement activation (TCC) was blocked completely at each time point compstatin was added, whereas the cytokines and MPO increased steadily between the time points. The combined regimen was significantly more effective than single inhibition in the pre-challenge setting. The attenuation of Escherichia coli-induced inflammation in a post-challenge setting suggests a potential therapeutic window for this treatment in sepsis.

Topics: Antibodies, Blocking; Blood; Complement Activation; Complement C3; Complement Membrane Attack Complex; Cytokines; Drug Combinations; Drug Synergism; Escherichia coli; Hot Temperature; Humans; Immunity, Innate; Immunization; Immunotherapy; Inflammation Mediators; Lipopolysaccharide Receptors; Peptides, Cyclic; Peroxidase; Sepsis

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Topics: Animals; Biomarkers; Blood Coagulation; Blood Pressure; Complement Activation; Complement Inactivator Proteins; Cytokines; Disease Models, Animal; Escherichia coli Infections; Multiple Organ Failure; Papio; Peptides, Cyclic; Sepsis