Activation of Bovine Factor X in the Presence of Calcium, Magnesium, Barium or Manganese ion

Summary

The binding of divalent metal ions to bovine factor X, factor X_a and the coagulant protein in Russell’s viper venom was studied by the technique of fluorescence quenching. Titration of factor X with Ca⁺², Mg⁺² or Ba⁺² revealed that these metal ions can bind to factor X. A tightly binding site(s) was observed with K_d of 79 and 98 μM for Ca⁺² and Mg⁺² respectively. A loosely binding site(s) was evident with K_d of 0.55, 0.50 and 0.35 mM for Ca⁺², Mg⁺² and Ba⁺² respectively. The quenching phenomenon was also observed when Mn⁺² was used as titrant but factor X precipitated out when the concentration of Mn⁺² was 10 mM. The binding of Ca⁺², Mg⁺², Ba⁺² or Mn⁺² to bovine factor X_a or to the purified coagulant fraction of Russell’s viper venom was very weak in each case.

In the absence of Ca⁺², the coagulation fraction of Russell’s viper venom could not activate bovine factor X. Activation of factor X was achieved when Ca⁺² was replaced by either Mg⁺², Ba⁺² or Mn⁺². When the concentration of these ions were 5 mM, the efficiency of factor X_a generation was estimated to be: Ca⁺²> Mg⁺²> Ba⁺²> Mn⁺². Higher concentration of Mg⁺², Ba⁺², or Mn⁺² retarded the activation process. However, Ca⁺², Mg⁺², Ba⁺² or Mn⁺² has little or no influence on the esterase activity of factor X_a or purified Rusell’s viper venom.

The results suggest that complexation of divalent metal ion with factor X is prerequisite in the activation process. The binding of Mg⁺², Ba⁺² or Mn⁺² to these loosely binding sites might have altered the geometrical configuration as well as the electrostatic environment on factor X significantly. Thus, it is more difficult to form the binary complex and a slower generation of factor X_a results. Therefore, divalent metal ion serves as a dual role in the activation of factor X to factor X_a depending upon the ionic concentration.

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