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Updated: May 14, 2026
Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets
Published on: November 22, 2024
The effects of selective complement and CD14 inhibition on the E. coli-induced tissue factor mRNA upregulation,
O L Brekke1, C Waage, D Christiansen
1Department of Laboratory Medicine, Nordland Hospital, Bodø, N-8092, Norway.
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.
Insights
Complement activation, not CD14, primarily drives E. coli-induced tissue factor (TF) mRNA upregulation in human blood. CD14 plays a minor role in monocyte TF expression and plasma TF activity.
Area of Science:
- Immunology
- Hematology
- Microbiology
Background:
- The complement pathway and CD14 are crucial in inflammation.
- Their specific roles in E. coli-induced tissue factor (TF) mRNA upregulation, monocyte expression, and activity in human blood are not well understood.
Purpose of the Study:
- To investigate the relative contributions of complement activation and CD14 to E. coli-induced TF mRNA upregulation, expression, and activity in human whole blood.
Main Methods:
- Human whole blood with E. coli was treated with complement inhibitors (compstatin, C5aRa) or anti-CD14 antibody.
- TF mRNA levels were quantified via RT-qPCR.
- TF expression and activity were assessed by flow cytometry and plasma microparticle analysis.
Main Results:
- Compstatin significantly reduced E. coli-induced TF mRNA upregulation.
- C5a receptor antagonist (C5aRa) reduced monocyte TF expression and plasma TF activity.
- Anti-CD14 had minimal effect alone but a modest additive effect with complement inhibitors.
- Combined inhibition reduced pro-inflammatory cytokines.
Conclusions:
- E. coli-induced TF mRNA upregulation is predominantly mediated by complement activation.
- CD14 plays a secondary role in monocyte TF expression and plasma TF activity in this context.
