Interaction of polylysine with bovine factor Xa: Effect of divalent cations

Abstract

Poly- L -lysine has been demonstrated to partially replace biological cofactors in the activation of prothrombin by factor Xa. The present study was initiated to determine if poly- L -lysine has an effect on the enzymatic activity of factor Xa in the absence of prothrombin. At low ionic strength(50 mM Tris-C1, pH 8.0, ambient temperature), poly- L -lysine inhibits amidase activity(S-2222) of bovine factor Xa with high affinity(Ki = 7 nM). The inhibition was readily reversed by 100 mM NaCl 2. The inhibition was also markedly reduced by the addition of 1.0 mM CaCl 2 but not by MnCl 2 or MgCl 2. All three metal ions enhance amidase activity in the absence of poly- L -lysine. Poly- L -lysine also inhibits the amidase activity of factor Xa from which the gamma-carboxyglutamic acid domain has been removed by limited proteolysis with chymotrypsin(factor Xa-GD) but with somewhat lower avidity(Ki = 35 nM). As with native factor Xa, calcium ions reduce the observed inhibition while either manganese or magnesium ions are much less effective. The amidase activity of factor Xa-GD is enhanced with any one of the three divalent cations. These results provide additional support for the existence of a functionally significant binding site for calcium ions outside of the gamma-carboxyglutamic domain of factor Xa.