Kinetic Description Of Conversion Of Bovine Prethrombin 2 To Thrombin

Bovine Factor Xa slowly converts Prethrombin 1 to thrombin plus Fragment 2. Inclusion of Ca²⁺ increased the rates of Prethrombin 1 consumption, Prethrombin 2 production and thrombin formation detected by SDS polyacrylamide gel electrophoresis or by thrombin assay. Ca²⁺ also increased the rate of thrombin formation from equimolar mixtures of Prethrombin 2 and Fragment 2 (Prethrombin 2/Fragment 2) by approximately 1.8 fold. Calcium ion thus increases the rate of both proteolyses required to generate thrombin from Prethrombin 1. Studies using Factor Xa (des light chain residues 1-44) indicated that this effect of Ca²⁺ required the region of Factor Xa containing gamma-carboxyglutamic acid.

Factor Va markedly lowered the apparent Km of Factor Xa for Prethrombin 2/Fragment 2, with decreases greater than 20 fold observed under some conditions. The apparent maximum velocity also increased by up to 50 fold. The extent of increase was greater at higher concentrations of Factor Va, and was about 6 fold greater in the presence of Ca²⁺ than in its absence. Factor Va binding to Factor Xa (forming XaVa with enhanced substrate binding and/or catalytic efficiency), and Factor Va binding to Prethrombin 2/Fragment 2 (forming a substrate more readily bound and/or cleaved) must be considered among the possible explanations for these effects. Previous qualitative observations suggest that these effects of Factor Va on activation of Prethrombin 2/ Fragment 2 are important in understanding the activation of prothrombin.