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Related Experiment Videos

Human prothrombinase complex assembly and function on isolated peripheral blood cell populations.

P B Tracy, L L Eide, K G Mann

    The Journal of Biological Chemistry
    |February 25, 1985
    PubMed
    Summary

    The prothrombinase complex, crucial for blood clotting, forms a 1:1 stoichiometric complex on activated platelets and various immune cells. This complex exhibits high affinity binding and efficient thrombin generation, with varying activity across cell types.

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    Area of Science:

    • Biochemistry
    • Hematology
    • Immunology

    Background:

    • The prothrombinase complex, comprising Factor Va and Factor Xa, is essential for converting prothrombin to thrombin.
    • Understanding the binding kinetics and stoichiometry of this complex on cell surfaces is critical for elucidating coagulation pathways.
    • Platelets and leukocytes play significant roles in hemostasis and immune responses, often involving protein-protein interactions.

    Purpose of the Study:

    • To determine the binding parameters and stoichiometry of the prothrombinase complex on activated human platelets.
    • To investigate the binding characteristics of Factor Va and Factor Xa on different leukocyte subpopulations.
    • To compare the catalytic efficiency (kcat) of the prothrombinase complex when bound to platelets versus leukocytes.

    Main Methods:

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    • Kinetic analyses of prothrombin activation using isolated thrombin-activated human platelets and leukocyte subpopulations.
    • Factor Va and Factor Xa titrations were performed on platelets from both normal and Factor V-deficient individuals.
    • Binding isotherms were analyzed to calculate apparent dissociation constants (Kd) and catalytic rates (kcat).

    Main Results:

    • Factor Xa binds to approximately 2700 functional sites on activated platelets with high affinity (Kd ≈ 10⁻¹⁰ M).
    • Factor Va and Factor Xa form a 1:1 stoichiometric complex on activated platelets, with similar binding affinities and catalytic rates.
    • Monocytes, lymphocytes, and neutrophils exhibit distinct numbers of prothrombinase binding sites (16,000, 45,000, and 8,000 sites/cell, respectively), with comparable affinities but varying catalytic activities.

    Conclusions:

    • The prothrombinase complex exhibits high-affinity, 1:1 stoichiometric binding to activated platelets and various immune cells.
    • Cell-bound prothrombinase complexes demonstrate efficient thrombin generation, with lymphocytes showing reduced catalytic activity.
    • These findings highlight the significant role of cellular interactions in regulating blood coagulation and thrombin formation.