THE ROLE OF THE 2ND GROWTH-FACTOR DOMAIN OF HUMAN FACTOR-IXA IN BINDING TO PLATELETS AND IN FACTOR-X ACTIVATION

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX((Xegf2)), and the wildtype, factor IX(wt), produced in embryonic kidney cells 293 were radiolabelled with I-125 and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXa(wt) in the presence of factor VIIIa (2 units/ml) and factor X (1.5 mu M). However, under similar experimental conditions, factor IXa((Xegf2)) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (K-d) of 1.4 nM, compared with normal factor IXa, factor IXa(N) (558 sites/platelet; K-d 0.67 nM), or factor IXa(wt) (590 sites/platelet; K-d 0.61 nM). The concentrations of factor IXa(N) and factor IXa(wt) required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a dose correspondence of the K-d,K-app. for binding of factor IXa(wt) to the factor-X activating complex on activated platelets to the K-d obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa((Xegf2)) showed a decreased affinity (K-d 1.5 nM), in agreement with results of binding studies. These studies with factor IX((Xegf2)) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.