Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli-induced coagulation accompanied by Toll-like receptor 4.

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.

Staphylococcus aureus-induced complement activation promotes tissue factor-mediated coagulation.

Essentials Complement, Toll-like receptors and coagulation cross-talk in the process of thromboinflammation. This is explored in a unique human whole-blood model of S. aureus bacteremia. Coagulation is here shown as a downstream event of C5a-induced tissue factor (TF) production. Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation. SUMMARY: Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

Systemic inflammation in acute intermittent porphyria: a case-control study.

This study aimed to examine whether acute intermittent porphyria (AIP) is associated with systemic inflammation and whether the inflammation correlates with disease activity. A case-control study with 50 AIP cases and age-, sex- and place of residence-matched controls was performed. Plasma cytokines, insulin and C-peptide were analysed after an overnight fast using multiplex assay. Long pentraxin-3 (PTX3) and complement activation products (C3bc and TCC) were analysed using enzyme-linked immunosorbent assay (ELISA). Urine porphobilinogen ratio (U-PBG, µmol/mmol creatinine), haematological and biochemical tests were performed using routine methods. Questionnaires were used to register AIP symptoms, medication and other diseases. All 27 cytokines, chemokines and growth factors investigated were increased significantly in symptomatic AIP cases compared with controls (P < 0·0004). Hierarchical cluster analyses revealed a cluster with high visfatin levels and several highly expressed cytokines including interleukin (IL)-17, suggesting a T helper type 17 (Th17) inflammatory response in a group of AIP cases. C3bc (P = 0·002) and serum immunoglobulin (Ig)G levels (P = 0·03) were increased significantly in cases with AIP. The U-PBG ratio correlated positively with PTX3 (r = 0·38, P = 0·006), and with terminal complement complex (TCC) levels (r = 0·33, P = 0·02). PTX3 was a significant predictor of the biochemical disease activity marker U-PBG in AIP cases after adjustment for potential confounders in multiple linear regression analyses (P = 0·032). Prealbumin, C-peptide, insulin and kidney function were all decreased in the symptomatic AIP cases, but not in the asymptomatic cases. These results indicate that AIP is associated with systemic inflammation. Decreased C-peptide levels in symptomatic AIP cases indicate that reduced insulin release is associated with enhanced disease activity and reduced kidney function.

C1-inhibitor efficiently delays clot development in normal human whole blood and inhibits Escherichia coli-induced coagulation measured by thromboelastometry.

INTRODUCTION: C1-inhibitor (C1-INH), a serine protease inhibitor in plasma plays a central role in the cross-talk among the complement, coagulation, fibrinolytic and kallikrein-kinin systems. However, previous reports indicate thrombotic risks in children following supraphysiological dosing with C1-INH. OBJECTIVE: To investigate the role of supraphysiological C1-INH concentrations in clot development with and without addition of Escherichia coli (E. coli) in fresh human whole blood using thromboelastometry. MATERIALS AND METHODS: Blood was collected in citrate tubes, and C1-INH (3.0 to 47.6µM) or human serum albumin (HSA) was added as a control. Activated partial thromboplastin time (aPTT) was analysed in the plasma. The analyses non-activated thromboelastometry (NATEM), extrinsic (EXTEM) or intrinsic thromboelastometry (INTEM) were performed using rotational thromboelastometry. RESULTS: C1-INH increased aPTT 1.8-fold (p< 0.05), whereas HSA had no effect. C1-INH increased NATEM clotting time (CT) from 789s to 2025 s (p< 0.05) in a dose-dependent manner. C1-INH reduced the NATEM alpha angle from 47 to 28° (p<0.05) and increased the NATEM clot formation time from 261s to 595s (p< 0.05). E. coli significantly reduced the NATEM CT after 120min of incubation. C1-INH prevented E. coli-induced activation (p< 0.05). C1-INH significantly increased the INTEM CT (p< 0.05), but had no effect on EXTEM CT. C1-INH (47.6µM) significantly reduced fibrinolysis measured as NATEM and EXTEM lysis indices LI60. CONCLUSIONS: Supraphysiological C1-INH concentrations have dose-dependent anticoagulant effects in human whole blood in vitro. At very high levels C1-INH also inhibits fibrinolysis.

The key roles of complement and tissue factor in Escherichia coli-induced coagulation in human whole blood.

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.

Do soccer and Zumba exercise improve fitness and indicators of health among female hospital employees? A 12-week RCT.

This randomized controlled study investigated the effectiveness of soccer and Zumba on fitness and health indicators in female participants recruited from a workplace. One hundred seven hospital employees were cluster-randomized to either a soccer group (SG), Zumba group (ZG), or control group (CG). Intervention effects for the two training groups were compared with CG. The training was conducted outside working hours as 2-3 1-h sessions per week for 12 weeks. Peak oxygen uptake (VO2peak ), fat percentage, fat mass, bone mineral content, and plasma osteocalcin were measured before and after the intervention period. Based on intention-to-treat-analyses, SG significantly improved the VO2peak relative to body mass (5%; P = 0.02) and decreased heart rate during 100-W cycle exercise (-7 bpm; P = 0.01), total body fat percentage (-1.1%; P = 0.002), and total body fat mass (-1.0 kg; P = 0.001) compared with CG. ZG significantly improved the VO2peak relative to body mass (5%; P = 0.03) and decreased total fat mass (-0.6 kg; P < 0.05) compared with CG. Plasma osteocalcin increased in SG (21%; P < 0.001) and ZG (10%; P = 0.01) compared with CG. The present study indicates that workplace initiated short-term soccer training as well as Zumba outside working hours may result in fitness and modest health benefits among female hospital employees.

C1-inhibitor efficiently inhibits Escherichia coli-induced tissue factor mRNA up-regulation, monocyte tissue factor expression and coagulation activation in human whole blood.

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses.

The effects of selective complement and CD14 inhibition on the E. coli-induced tissue factor mRNA upregulation, monocyte tissue factor expression, and tissue factor functional activity in human whole blood.

BACKGROUND: The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood. METHODS: Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')2 monoclonal antibody. RESULTS: The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1beta, IL-6, IL-8, and platelet-derived growth factor bb. CONCLUSION: Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

Differential effect of inhibiting MD-2 and CD14 on LPS- versus whole E. coli bacteria-induced cytokine responses in human blood.

BACKGROUND: Sepsis is a major world-wide medical problem with high morbidity and mortality. Gram-negative bacteria are among the most important pathogens of sepsis and their LPS content is regarded to be important for the systemic inflammatory reaction. The CD14/myeloid differentiation factor 2 (MD-2)/TLR4 complex plays a major role in the immune response to LPS . The aim of this study was to compare the effects of inhibiting MD-2 and CD14 on ultra-pure LPS - versus whole E. coli bacteria-induced responses. METHODS: Fresh human whole blood was incubated with upLPS or whole E. coli bacteria in the presence of MD-2 or CD14 neutralizing monoclonal antibodies, or their respective controls, and/or the specific complement-inhibitor compstatin. Cytokines were measured by a multiplex (n = 27) assay. NFκB activity was examined in cells transfected with CD14, MD-2 and/or Toll-like receptors. RESULTS: LPS-induced cytokine response was efficiently and equally abolished by MD-2 and CD14 neutralization. In contrast, the response induced by whole E. coli bacteria was only modestly reduced by MD-2 neutralization, whereas CD14 neutralization was more efficient. Combination with compstatin enhanced the effect of MD-2 neutralization slightly. When compstatin was combined with CD14 neutralization, however, the response was virtually abolished for all cytokines, including IL-17, which was only inhibited by this combination. The MD-2-independent effect observed for CD14 could not be explained by TLR2 signaling. CONCLUSION: Inhibition of CD14 is more efficient than inhibition of MD-2 on whole E. coli-induced cytokine response, suggesting CD14 to be a better target for intervention in Gram-negative sepsis, in particular when combined with complement inhibition.