γ-Carboxyglutamic Acid (Gla)-Domainless Blood Coagulation Factor IXa Species: Preparation and Properties

Abstract

To investigate the function of the γ-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated “factor IXaβ', ” and its corresponding Gla-domainless form, designated “Gla-domainless factor IXaβ', ” were prepared under controlled conditions and characterized. First, bovine factor IXaα was converted by α-chymotrypsin in the presence of calcium ions to factor IXaβ' (M_r 47, 000). Compared with factor IXaβ, factor IXaβ' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXaβ' by additional selective cleavage by α-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXaβ' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXaa, factor IXaα', and Gla-domainless factor IXaβ', with factor IXaβ revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXaβ' was less than 0. 5% of that of factor IXaβ'. In the complete factor X-activating mixture (factor IXaβ', calcium ions, phospholipid, and factor VIIIa), factor X was activated more than ten thousand-fold more rapidly than it was by factor IXaβ' in the absence of phospholipids and factor VIIIa. However, no amplification in the activation of factor X by Gla-domainless factor IXaβ and calcium ions was observed, even in the presence of phospholipids and factor VIIIa. These results indicate that the Gla-domain of factor IXa is essential for the amplification of the activation of factor X by phospholipid and factor VIIIa. Factor IXaβ' and its Gla-domainless derivative prepared in this study are species of new factor IXa with which to investigate in greater detail the functional properties of Gla residues in factor IXa β.