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Expression of tissue factor and tissue factor pathway inhibitor in monocytes in response to bacterial
C P van der Logt1, R J Dirven, P H Reitsma
1Haemostasis and Thrombosis Research Centre, University Hospital, Leiden, The Netherlands.
Abstract:
The monocyte is the only circulating cell type capable of initiating blood coagulation by the expression of tissue factor (TF). The mechanism and kinetics of TF mRNA and TF activity induction in human peripheral blood monocytes (HPBM) in response to bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) were investigated. Northern blot analysis showed that both LPS and PMA induce a transient accumulation of TF mRNA in HPBM, that reaches maximum levels after 3-6 h and rapidly declines thereafter. Nuclear run-on experiments demonstrated that the accumulation of TF mRNA requires de novo transcription of the TF gene. Since cycloheximide alone also caused an increase of TF mRNA levels and gene transcription it is concluded that the transcriptional activation of the TF gene does not require protein synthesis. Using specific protein kinase inhibitors, it was further demonstrated that activation of the protein kinase C pathway is involved in the induction of TF mRNA in HPBM. The accumulation of TF mRNA in LPS-stimulated HPBM is followed by an increase of TF activity on the cell surface. The kinetics of TF mRNA induction were found to be very similar in HPBM stimulated with LPS or PMA. However, in the latter case TF activity appeared considerably later on the cell membrane than in the LPS-stimulated cells. Non-stimulated HPBM contain very low levels of mRNA of the tissue factor pathway inhibitor (TFPI). No induction of TFPI (mRNA, activity or antigen) in HPBM after LPS or PMA treatment was demonstrated. This seems to be in contrast with the earlier observation that the human monocyte cell line U937 produces significant amounts of TFPI in response to treatment with LPS and PMA.
Insights
Bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) induce tissue factor (TF) mRNA in human peripheral blood monocytes (HPBM) via gene transcription, not protein synthesis. TF activity follows mRNA accumulation, but TFPI is not induced.
Area of Science:
- Hematology
- Immunology
- Molecular Biology
Background:
- Monocytes are the sole circulating cells initiating blood coagulation via tissue factor (TF) expression.
- Understanding the regulation of TF induction in monocytes is crucial for hemostasis and thrombosis research.
Purpose of the Study:
- To investigate the mechanism and kinetics of TF mRNA and TF activity induction in human peripheral blood monocytes (HPBM).
- To explore the role of protein kinase C pathway in TF gene transcriptional activation.
Main Methods:
- Northern blot analysis to assess TF mRNA accumulation.
- Nuclear run-on assays to determine TF gene transcription.
- Protein kinase inhibitors to study signaling pathways.
- Assays for TF activity and TFPI levels.
Main Results:
- Both LPS and PMA induced transient TF mRNA accumulation in HPBM, requiring de novo gene transcription.
- Transcriptional activation of the TF gene does not necessitate protein synthesis.
- Protein kinase C pathway activation is involved in TF mRNA induction.
- TF activity increased following TF mRNA accumulation in LPS-stimulated HPBM, with delayed appearance after PMA stimulation.
- No induction of TFPI (mRNA, activity, or antigen) was observed in HPBM.
Conclusions:
- TF gene expression in HPBM is transcriptionally regulated and independent of de novo protein synthesis.
- LPS and PMA differentially regulate the kinetics of TF activity despite similar TF mRNA induction.
- HPBM do not appear to upregulate TFPI in response to LPS or PMA, contrasting with U937 cell line behavior.