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.

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.

The artificial surface-induced whole blood inflammatory reaction revealed by increases in a series of chemokines and growth factors is largely complement dependent.

Exposing blood to an artificial surface results in a systemic inflammatory response, including cytokine release and complement activation. We studied the artificial surface-induced inflammation in human whole blood using an extensive panel of inflammatory mediators including proinflammatory cytokines, chemokines and growth-factors and investigated the role of the complement system in the induction of this response. Using multiplex technology, 27 different inflammatory mediators were measured after circulating blood for 4 hours in polyvinyl chloride tubing. The C3 inhibitor compstatin was used to block complement activation. A significant (p < 0.05) increase in 14 of the 27 mediators was induced by the surface, of which 7 were chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, RANTES, eotaxin and IP-10) and 5 were growth-factors (G-CSF, GM-CSF, VEGF, PDGF and FGF). The traditional proinflammatory cytokines like IL-1beta, TNFalpha and IL-6 were not induced, although IL-6, as well as IL-15 and IL-17 increased if the surface was coated with highly bioincompatible laminaran. Inhibition of complement activation with compstatin significantly (p < 0.05) reduced the formation of 12 of the 14 mediators. For 10 of the 12 mediators, the inhibition was by 2/3 or more, for the remaining two the inhibition was more moderate. A highly biocompatible heparin-coated PVC surface was used as negative control and completely abolished the whole inflammatory response. The artificial surface PVC markedly induced a broad spectrum of chemokines and growth-factors, which was largely dependent on activation of complement.

A novel enzyme immunoassay for plasma thrombospondin. Comparison with beta-thromboglobulin as platelet activation marker in vitro and in vivo.

A novel enzyme immunoassay for plasma thrombospondin (TSP) based on commercially available monoclonal antibodies was established. The following conditions for correct collection and preservation of blood samples were required: venipuncture directly into a vacutainer containing citrate, theophylline, adenosine and dipyridamole, storage on ice, and separation of plasma within 30 minutes. Thereafter, the plasma TSP concentration remained constant at room temperature and after five times of freezing and thawing. Both inter- and intraassay variation coefficients were 5%. The lower detection limit was 20 microg/L. Median TSP concentration among 40 healthy blood donors was 43 microg/L, slightly lower than previously published. The assay is valid, reliable, and has certain advantages compared with previously published methods. TSP and beta-thromboglobulin (BTG) were then compared as platelet activation and biocompatibility markers in vivo: 23 patients undergoing cardiopulmonary bypass (CPB); and in vitro: effect of coating polyvinyl chloride with heparin. The kinetic patterns of TSP and BTG were markedly different in vivo but virtually identical in vitro, explained by different in vivo clearance mechanisms during CPB. We conclude that BTG is superior to TSP for evaluation of platelet activation during in vivo CPB, whereas TSP and BTG are virtually identical as markers in vitro.

Platelet compatibility of an artificial surface modified with functionally active heparin.

Platelet compatibility after coating an artificial material with functionally active heparin was investigated. Blood was circulated in uncoated or heparin coated PVC tubing. In one hour platelet counts decreased from 155 (113-184)x10(9)/l to 124 (100-148)x10(9)/l with uncoated compared to 164 (132-192)x10(9)/l with heparin coated tubing (intergroup p = 0.02). Beta-thromboglobulin increased from 116 (80-148) microg/l to 1039 (757-1298) microg/l with uncoated and to 352 (229-638) microg/l with heparin coated tubing (intergroup p = 0.005). Platelet counts and beta-thromboglobulin correlated closely with complement activation. Solid-phase enzyme immunoassay demonstrated substantial deposition of CD42a/GPIbIX and CD61/GPIIIa on uncoated, but not on heparin coated tubing (intergroup p<0.0005). Scanning electron microscopy demonstrated activated platelets and aggregates on uncoated in contrast to heparin coated tubing, where scattered, unactivated platelets were found. Changes in P-selectin and microparticles were minor. In conclusion, this heparin surface substantially improved platelet compatibility. Markers of choice for in vitro evaluation were platelet counts, beta-thromboglobulin and platelet deposition.

Effect of time, temperature and anticoagulants on in vitro complement activation: consequences for collection and preservation of samples to be examined for complement activation.

The effects of time, temperature, ethylene-diamine-tetra-acetic acid (EDTA), citrate and heparin on in vitro complement activation were examined in enzyme immuno assays (EIA) for detection of C3 activation products and the terminal complement complex (TCC). In vitro complement activation occurred during coagulation since baseline concentrations of activation products were considerably higher in serum than in plasma. EDTA was more efficient than citrate and heparin in inhibiting in vitro activation. Minimal activation was observed in all preparations when samples were kept at 4 degrees C for up to ten days, whereas a very rapid increase in activation products occurred even in EDTA plasma when the temperature was elevated. Based on the data obtained, guidelines for the collection and preservation of samples to be examined for complement activation are given.