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

Abstract

SummaryThe 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 anticoagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 3 10 7 /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab 0 ) 2 antibody and a control F(ab 0 ) 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 112 (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 < 0Á05 for all). The combined inhibition was significantly (P < 0Á05) 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 < 0Á05) with combined C3 and CD14 inhibition. LPS-and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0Á05) 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.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

“…However, the demonstrated activation of complement on platelets raises the question whether complement may contribute to activation of coagulation in the setting of thrombosis. Although the complement product C5a can induce tissue factor (TF) expression in leukocytes, 16,17 in vitro studies indicated that complement activation is particularly required for the rapid posttranslational conversion of monocyte-expressed TF to a fully procoagulant molecule. 18 In this study, antibody-mediated complement activation was shown to cause the conversion of TF to a procoagulant The online version of this article contains a data supplement.…”

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

“…We have previously reported that the E. coli-induced coagulation is predominantly TF-dependent [4]. We have previously reported that the E. coli-induced coagulation is predominantly TF-dependent [4].…”

“…We have previously demonstrated that the Escherichia coli-induced coagulation in human whole blood measured by prothrombin fragment 1+2 (PTF1·2) is TF-dependent, using a blocking antibody against TF [4]. We have previously demonstrated that the Escherichia coli-induced coagulation in human whole blood measured by prothrombin fragment 1+2 (PTF1·2) is TF-dependent, using a blocking antibody against TF [4].…”

Alginate microbeads are coagulation compatible, while alginate microcapsules activate coagulation secondary to complement or directly through FXII

“…TF is expressed by monocytes following stimulation by complement, LPS [12, 23] and the interleukins IL-6, IL-8, MCP-1 or PDGF-BB [24, 25], and could therefore potentially be induced by the various alginate microspheres. The PMCG, APA and AP microcapsules significantly increased monocyte TF surface expression compared to the saline control (Fig.…”

“…For instance, the coagulation proteases (FXIa, FXa, FIXa, FIIa) are capable of directly cleave C3 and C5 [8], a finding that might be of physiological relevance [9]. Complement can also be a gateway into coagulation activation as C5a is shown to induce endothelial TF [10], monocyte TF [11, 12], TF on microparticles [13] and in certain cases on neutrophils [14]. In addition, platelets contain complement receptors for C3a and C5a [15], and C5b-9 triggers platelet prothrombinase activity [16].…”