Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

Abstract

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Quantitative Tissue Factor Gene Expression Analysis in Whole Blood: Development and Evaluation of a Real-Time PCR Platform

“…Whiskers indicate the highest and lowest values, excluding outliers (E) and extreme outliers ‫)ء(‬ increasing amounts of TF mRNA were quickly generated, and peak concentrations were reached between 1 and 2 h after exposure to LPS, followed by a progressive decrease thereafter. This pattern of TF mRNA expression agrees well with previously published data (11)(12)(13). The LPS receptor CD14 has been proposed to be a suitable marker for the number of monocytic cells in blood (14 ).…”

“…Induction of monocytic TF expression by lipopolysaccharide (LPS) plays an important role in the pathogenesis of sepsis-associated thrombotic complications and has been investigated extensively in several in vitro studies (8,(11)(12)(13). We therefore used the LPS model to evaluate the newly established PCR methods.…”

Lipopolysaccharide Induction of THP-1 Cells Activates Binding of c-Jun, Ets, and Egr-1 to the Tissue Factor Promoter

“…Serine phosphorylation by a Ha-Ras-induced c-Jun nuclear kinase causes an increase in DNA binding (35), whereas phosphorylation by mitogen activated protein kinase enhances transcriptional activation (36). Expression of TF in THP-1 cells is rapidly activated within 60 min after LPS addition and is mediated by protein kinase C (12,37). The TF promoter contains two AP-1 sites that are required for optimal induction (12).…”

Induction of interleukin‐1 and subsequent tissue factor expression by anti‐proteinase 3 antibodies in human umbilical vein endothelial cells

“…The amount of factor Xa measured using the chromogenic substrate S2765 is proportional to the TF concentration. HUVEC in 96-well plates (4 X lo4 cells per well) were washed and incubated for various times (0.5, 6, 12, 24, and 48 hours) with culture medium and 2 pdml LPS (a potent inducer of TF [32]), or patients' sera, or Ig from normal sera, or irrelevant IgG, human Fc fragments, or affinity-purified anti-PR3 antibodies. We checked that endotoxin at a concentration of 0.05 ndml did not induce TF expression (unpublished data).…”