Comparison of lipid binding and kinetic properties of normal, variant, and gamma-carboxyglutamic acid modified human factor IX and factor IXa.

The abilities of normal and three abnormal factor IXa molecules to activate factor X and to bind to phospholipid membranes have been compared to define the contributions of protein-lipid interactions and factor IXa light chain-heavy chain interactions to the functioning of this protein. The abnormal proteins studied had altered amino acid residues in their light chains. The heavy-chain regions, containing the active site serine and histidine residues, were normal in the abnormal proteins on the basis of titration by antithrombin III. The binding constants (Kd) for normal (N), variant [Chapel Hill (CH) and Alabama (AL)], and gamma-carboxyglutamic acid (Gla) modified (MOD) factors IX and IXa to phosphatidylserine (PS)/phosphatidylcholine (PC) small, unilamellar vesicles (SUV) were measured by 90 degrees light scattering. The Kd values for factor IXN binding were quite sensitive to the PS content of the membrane but less sensitive to Ca2+ concentrations between 0.5 and 10 mM. The zymogen and activated forms of both normal and abnormal factor IX bound with similar affinities to PS/PC (30/70) SUV. In the cases of factor IXaN and factor IXaAL, but not factor IXaCH or factor IXaMOD, irreversible changes in scattering intensity suggested protein-induced vesicle fusion. Since the activation peptide is not released from factor IXaCH, the normal interaction of factor IXa with a membrane must require the release of the activation peptide and the presence of intact Gla residues. The rate of factor X activation by normal and abnormal factor IXa was obtained by using a chromogenic substrate for factor Xa in the presence of PS/PC (30/70) SUV and 5 mM Ca2+.