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Related Experiment Videos

Tissue factor in cultured cells: metabolic control.

J R Maynard, D J Fintel, F A Pitlick

    Laboratory Investigation; a Journal of Technical Methods and Pathology
    |December 1, 1976
    PubMed
    Summary

    WISH amnion cell coagulant activity dramatically increases after subculture, requiring fresh media and serum for maximal development. Protein synthesis inhibitors block this increase, indicating a de novo synthesis process for tissue factor.

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    Area of Science:

    • Cell Biology
    • Biochemistry
    • Coagulation Science

    Background:

    • WISH amnion cells exhibit dynamic changes in tissue factor content.
    • Understanding the regulation of tissue factor expression is crucial for hemostasis research.

    Purpose of the Study:

    • To investigate the regulation of tissue factor expression in WISH amnion cells post-subculture.
    • To elucidate the mechanisms underlying the rapid increase and subsequent decline of coagulant activity.

    Main Methods:

    • Spinner culture of WISH amnion cells.
    • Assessment of coagulant activity over time.
    • Inhibition studies using actinomycin D, cycloheximide, and oxidative phosphorylation inhibitors.
    • Analysis of macromolecular synthesis (3H-leucine, 35SO4=, 3H-thymidine) and enzyme activity (naphthylamidase, alkaline phosphatase).

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    Main Results:

    • Tissue factor content increased 3- to 10-fold within 12 hours post-subculture, returning to basal levels by 30-50 hours.
    • Maximal activity required fresh serum and medium; actinomycin D and cycloheximide inhibited development but not decay.
    • Carbonyl cyanide m-chlorophenylhydrazone inhibited activity increase but not decline, suggesting an energy-dependent synthesis process.
    • Cell division and surface coat/enzyme levels remained unchanged, while 3H-thymidine incorporation peaked near the tissue factor maximum.

    Conclusions:

    • The burst of coagulant activity is dependent on de novo protein synthesis and requires specific culture conditions.
    • The decline in activity appears independent of protein synthesis and oxidative phosphorylation inhibition.
    • Tissue factor expression regulation in WISH cells involves complex signaling pathways not directly linked to cell division or certain enzymatic activities.