Role of γ-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Abstract

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of γ-carboxyglutamic acid (Gla) residues. In the presence of factor Villa and factor X, there were 344 (±52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 ± 0.9 nmol/L) and 275(±35)sites/ platelet for factor IXaDES (kdapp = 5.0 ± 0.8 nmol/L) compared with 580 (±65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 ± 0.1 nmol/L) and 300 (±62) sites/ platelet for factor IX (kdapp = 2.9 ± 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (∼0.8 nmol/L · min-1) and factor IXaDES (∼0.4 nmol/L · min-1) were decreased compared with factor IXaN (∼10.5 nmol/L · min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor Villa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX. © 1992 by The American Society of Hematology.