Isolation And Characterization Of The Vitamin K Dependent Domain Of Human Prothrombin

Chymotryptic cleavage of human prothrombin (_hPt) produces two fragments of 68000 and 5000 MW respectively. These two species were separated by Ba citrate adsorption and Sephadex G100 chromatography. The 68000 MW species corresponds to _hPt (des 1-44) and has lost all the vitamin K dependent properties of _hPt : adsorption on Ba citrate, Ca⁺² and phospholipid dependent stimulation of the activation by FXa, presence of strong Ca⁺² binding sites (as shown by dialysis equilibrium) and Ca⁺² induced fluorescence quenshing. The 5000 MW species corresponds to the N-terminal portion of _hPt (residues 1-41). It contains the 10 Gla residues and the 17-22 disulfide bond. This peptide is quantitatively adsorbed on Ba citrate. Activation of _hPt in a mixture containing FXa, Ca⁺² and phospholipid is drastically inhibited by the addition of peptide 1-41 (≃ 50 % inhibition for a 1/1 peptide/Pt ratio, ≃ 7 % for a 40/1 ratio).

Ca⁺² produces a quenshing of the intrinsic fluorescence of peptide 1-41 (λ emission = 355 nm). This quenshing plateausat 40 % of the initial fluorescence at 3 mM Ca⁺². The plot of the fluorescence quenshing versus Ca⁺² concentration however is not cooperative. Mn⁺² and Mg⁺² also induced fluorescence quenshing but to a lesser extent. Hence peptide 1-44 represents a functionnal domain in itself interacting with Ca⁺² and phospholipid. It contains only one Trp (residue 41), showing directly the involvment of this residue in the Ca⁺² induced fluorescence quenshing observed for Pt fragment (FI) and Pt. This isolated vitamin K dependent domain therefore retains many of the vitamin K dependent properties of FI or Pt, but shows differences in the Ca⁺² induced conformational change in that it is no longer a cooperative process.