The second epidermal growth factor-like domain of human factor IXa mediates factor IXa binding to platelets and assembly of the factor X activating complex.
Factor IXa binding to the activated platelet surface is required for efficient catalysis of factor X activation. Platelets possess a specific binding site for factor IXa, occupancy of which has been correlated with rates of factor X activation. However, the specific regions of the factor IXa molecule that are critical to this interaction have not yet been fully elucidated. To assess the importance of the second epidermal growth factor (EGF2) domain of factor IXa for platelet binding and catalysis, a chimeric protein (factor IXa(Xegf2)) was created by replacement of the EGF2 domain of factor IX with that of factor X. Competition binding experiments showed 2 different binding sites on activated platelets (approximately 250 each/platelet): (1) a specific factor IXa binding site requiring the intact EGF2 domain; and (2) a shared factor IX/IXa binding site mediated by residues G(4)-Q(11) within the Gla domain. In kinetic studies, the decreased V(max) of factor IXa(Xegf2) activation of factor X on the platelet surface (V(max) 2. 90 +/- 0.37 pM/min) versus normal factor IXa (37.6 +/- 0.15 pM/min) was due to its decreased affinity for the platelet surface (K(d) 64.7 +/- 3.9 nM) versus normal factor IXa (K(d) 1.21 +/- 0.07 nM), resulting in less bound enzyme (functional complex) under experimental conditions. The hypothesis that the binding defects of factor IXa(Xegf2) are the cause of the kinetic perturbations is further supported by the normal k(cat) of bound factor IXa(Xegf2) (1701 min(-)(1)) indicating (1) an intact catalytic site and (2) the normal behavior of bound factor IXa(Xegf2). The EGF2 domain is not a cofactor binding site since the mutant shows a normal rate enhancement upon the addition of cofactor. Thus, the intact EGF2 domain of factor IXa is critical for the formation of the factor X activating complex on the surface of activated platelets.
Department of Biochemistry, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
M Y
MY
Wong
J A
JA
Gurr
P N
PN
Walsh
D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D001792:Blood Platelets / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000502:physiology*; D015949:Factor IXa / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism; D015539:Platelet Activation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D013552:Swine
D010446:Peptide Fragments; D010743:Phospholipids; D004815:Epidermal Growth Factor; D015944:Factor VIIIa; D015949:Factor IXa; D015951:Factor Xa