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Prothrombin fragments. Ca2+ binding and activation kinetics.

S P Bajaj, R J Butkowski, K G Mann

    The Journal of Biological Chemistry
    |March 25, 1975
    PubMed
    Summary
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    Calcium ions bind to specific sites on prothrombin, influencing its activation. Strong Ca2+ binding sites facilitate phospholipid interaction, while weaker sites mediate Factor V binding, crucial for thrombin formation.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Hematology

    Background:

    • Prothrombin activation is a critical step in the coagulation cascade.
    • Calcium ions (Ca2+) play a vital role in modulating prothrombin's interaction with other factors.
    • Understanding Ca2+ binding sites is key to elucidating prothrombin activation mechanisms.

    Purpose of the Study:

    • To investigate the Ca2+ binding characteristics of prothrombin and its activation intermediates.
    • To determine the role of Ca2+ binding sites in the interaction with phospholipid and Factor V.
    • To elucidate the functional significance of Ca2+ binding in the activation pathway.

    Main Methods:

    • Equilibrium dialysis using 45Ca2+ to quantify Ca2+ binding.
    • Scatchard plot analysis to determine binding affinities and site numbers.

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  • Enzymatic assays to study the activation rates of prothrombin and its intermediates using Factor Xa, Ca2+, phospholipid, and Factor V.

    • Main Results:

    • Prothrombin exhibits 10-11 Ca2+ binding sites, with 6 high-affinity sites (log Kassoc = 3.5) in the NH2-terminal region (intermediate 3) and 4-5 lower-affinity sites (log Kassoc = 2.7) in the COOH-terminal region (intermediate 1).
    • Ca2+ binding sites are primarily located in the "pro" fragment (intermediates 3 and 4).
    • Activation rates are enhanced by Ca2+ and phospholipid, with Factor V significantly accelerating intermediate 1 activation in a Ca2+-dependent manner and intermediate 2 conversion when combined with intermediate 4.

    Conclusions:

    • Strong Ca2+ binding sites in intermediate 3 are implicated in phospholipid binding.
    • Weaker Ca2+ binding sites in intermediate 4 are associated with Factor V binding.
    • These findings highlight the differential roles of Ca2+ binding sites in mediating interactions essential for efficient prothrombin activation and thrombin generation.