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Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells
Published on: December 27, 2017
Synthesis of platelet-activating factor by human monocytes stimulated by platelet-activating factor
1Department of Medicine, University of California, San Francisco.
Abstract:
The capacity of platelet-activating factor (PAF) to stimulate its own synthesis by human monocytes was examined. Adherent human monocytes of greater than 85% purity were incubated with 100 fM to 10 nM of PAF in the presence of 20 microCi of [3H]acetic acid to radiolabel newly synthesized PAF. After incubation for 15 minutes to 15 hours, PAF was purified by high-performance liquid chromatography, and newly synthesized PAF was quantified by its radioactivity. PAF stimulated its own synthesis in a dose-related manner with a maximal twofold to threefold increase in synthesis at 10 pM to 100 pM of PAF. Maximal PAF synthesis occurred after incubation for 6 to 8 hours. There was a good correlation (r = 0.95) between PAF quantified by [3H]acetic acid incorporation and by rabbit platelet aggregation bioassay, indicating that the radioactive material is PAF. The protein synthesis inhibitor, cycloheximide, did not inhibit delayed PAF synthesis, indicating that delayed PAF synthesis does not require protein synthesis. PAF is metabolized rapidly in vivo. The capacity of PAF to stimulate its own synthesis would result in a prolonged effective half-life in vivo. This prolonged half-life could contribute to the capacity of PAF to induce prolonged inflammatory reactions in vivo.
Insights
Platelet-activating factor (PAF) stimulates its own synthesis in human monocytes, prolonging its effects. This self-stimulation, independent of new protein synthesis, may explain sustained inflammatory reactions.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammation.
- Understanding the regulation of PAF synthesis is crucial for comprehending inflammatory processes.
Purpose of the Study:
- To investigate the ability of platelet-activating factor (PAF) to stimulate its own production in human monocytes.
- To determine the kinetics and regulatory mechanisms of PAF self-synthesis.
Main Methods:
- Human monocytes were incubated with varying concentrations of PAF and radiolabeled acetic acid.
- High-performance liquid chromatography (HPLC) was used to purify and quantify newly synthesized PAF.
- The effect of cycloheximide, a protein synthesis inhibitor, on PAF synthesis was assessed.
Main Results:
- PAF stimulated its own synthesis in a dose-dependent manner, with maximal effects observed at 10-100 pM.
- Peak PAF synthesis occurred between 6 and 8 hours of incubation.
- Delayed PAF synthesis was not inhibited by cycloheximide, suggesting it does not require de novo protein synthesis.
Conclusions:
- Human monocytes possess the capacity to amplify PAF production in response to PAF.
- This autocrine stimulation mechanism may contribute to the prolonged half-life of PAF in vivo.
- The sustained presence of PAF could underlie its role in prolonged inflammatory reactions.
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