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

Abstract

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+.

Platelet activity of high‐dose factor VIIa is independent of tissue factor

“…This study shows that factor VIIa binds to platelets with a K d of about 100 nM. This value is similar to the affinity of factor IXa for phospholipid vesicles (Jones et al, 1985) and low-affinity factor IXa binding to platelets (Hoffman et al, 1992a) but is tighter than previously observed for factor VIIa binding to phospholipid vesicles (Bom & Bertina, 1990). Hedner et al (1993), using a modified coagulation assay described by Lindley et al (1994), showed that individuals Factor X activation on platelets in the presence of factor VIIa.…”

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

“…The interaction of this factor IX with phospholipids was normal, but the activation of factor X either in the presence or absence of factor VIII was found to occur at a reduced rate, relative to the normal protein (Briet et al, 1982;Jones et al, 1985). The level of (3-hydroxyaspartate was found to be normal in this variant (McGraw et al, 1985).…”

Thrombogenic role of cells undergoing apoptosis

“…The rest of the cascade follows the intrinsic pathway including the formation of a factor IXa–factor VIIIa complex, activation of factor X and then generation of thrombin. In these reactions, the presence of phospholipid is also essential (Jones et al , 1985; Liebman et al , 1987). Either phopholipid vesicle or cell surface is required for the enzymatic activity of factor IXa (Van Dieigen et al , 1981).…”