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Published on: February 14, 2017
Generation of tissue factor-rich microparticles in an ex vivo whole blood model
Einar S Breimo1, Bjarne Østerud
1Department of Biochemistry, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway.
Abstract:
The aim of this study was to investigate the role of blood cells in the expression of tissue factor (TF) and P-selectin in platelets and microparticles from blood stimulated with lipopolysaccharide (LPS), with or without the further addition of phorbol myristyl acetate (PMA). TF activity was found to be associated with platelets after 2 h incubation of whole blood with LPS or LPS + PMA, while no TF activity was detected in microparticles from blood subjected to such stimulation. In blood stimulated for 6 and 24 h, addition of PMA to the samples led to a substantial increase in TF activity associated with microparticles, compared with stimulation with LPS alone. Addition of PMA to blood samples also led to a three-fold increase in the amount of P-selectin found in the isolated microparticle fraction, and a 50% reduction in P-selectin measured in platelets, compared with LPS alone used for stimulation. In a different experiment, TF-rich microparticles were shown to be absorbed very efficiently by neutrophils in a calcium-independent reaction. Our results imply that LPS stimulation of whole blood is associated with a direct transfer of TF from monocytes to platelets in the absence of free TF-rich microparticles, which probably is accounted for by the fusion of TF-rich microparticles with activated platelets exposing P-selectin. Further addition of PMA to samples generates both free TF-rich microparticles as well as enhanced transfer of TF from monocytes to platelets.
Insights
Lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) stimulation of blood reveals tissue factor (TF) transfer from monocytes to platelets. PMA enhances TF in microparticles and P-selectin on platelets.
Area of Science:
- Hematology
- Immunology
- Biochemistry
Background:
- Tissue factor (TF) and P-selectin are critical in hemostasis and inflammation.
- Blood cells, including platelets and microparticles, play complex roles in TF and P-selectin expression.
- Lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) are common stimuli used to investigate cellular responses.
Purpose of the Study:
- To investigate the role of blood cells in expressing tissue factor (TF) and P-selectin.
- To examine TF and P-selectin expression in platelets and microparticles following LPS and PMA stimulation.
- To understand the dynamics of TF transfer between monocytes, platelets, and microparticles.
Main Methods:
- Whole blood was stimulated with lipopolysaccharide (LPS) alone or with phorbol myristate acetate (PMA).
- TF and P-selectin expression were measured in platelets and microparticles at different time points (2, 6, and 24 hours).
- Neutrophil absorption of TF-rich microparticles was assessed in a calcium-independent reaction.
Main Results:
- TF activity associated with platelets was observed after 2 hours of LPS or LPS + PMA stimulation.
- PMA addition significantly increased TF activity in microparticles at later time points (6 and 24 hours).
- PMA enhanced P-selectin on microparticles and reduced it on platelets, suggesting altered cellular distribution.
Conclusions:
- LPS stimulation leads to TF transfer from monocytes to platelets, likely via microparticle fusion.
- PMA generates free TF-rich microparticles and promotes TF transfer from monocytes to platelets.
- These findings elucidate mechanisms of TF and P-selectin regulation in stimulated blood.
