Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 6

Factors IX and IXa bind specifically to bovine aortic endothelial cells and compete for the same site. MLID83247428 MLID83178296 MLID85190528  94–96 Binding is half-maximal at 2.1 nM for factor IX and 2.5 nM for factor IXa. MLID83247428  94 About 20,000 molecules of factor IX bind per cell, and a putative receptor protein (Mr 140,000) has been identified. MLID87194814  97 Binding of factors IX or IXa to the receptor is not inhibited by antibodies directed against factor VIII, von Willebrand factor, or the EGF receptor. The interaction of factor IX with endothelial cells is inhibited by a peptide corresponding to the g-carboxyglutamic acid domain (residues 1–42) MLID90062154  98; the Ki for this interaction is about 0.05 mM. Chimeric proteins in which the aromatic amino acid stack or first EGF domain of factor IX are substituted by the comparable domain from factor X compete for factor IX binding to endothelial cells, suggesting that these domains are not important in the interaction of factor IX with endothelial cells. MLID91224974  99 Cheung and colleagues MLID93015940  100 have utilized site-directed mutagenesis of the g-carboxyglutamic acid domain of factor IX to show that residues 5 and 10 are required for the binding of factor IX to endothelial cells. However, the coagulant activity of the mutant proteins remained intact, suggesting distinct interactions between factor IX with endothelial cells and phospholipids. MLID93015940  100

Activated platelets, but not resting platelets, bind factors IX and IXa. MLID90241883 MLID89123446  101,102 The nature of the platelet factor IX binding site is not known, nor are the actual surfaces on factor IX that are required for interaction with platelet membranes. The binding site for factors IX and IXa may be distinct. The interaction of factor IXa with platelets is at least in part mediated by the g-carboxyglutamic acid domain. 103 Factor IXa, in which the first EGF domain of factor IX is substituted by the first EGF domain of factor X, binds normally to platelets, suggesting that either the first EGF domain is not involved in platelet binding or the first EGF domain of factor X is sufficient for this interaction. MLID92235087  104 Factor IXa binds to the factor IX binding site but also binds to additional sites that do not bind factor IX. In the presence of saturating concentrations of factors VIIIa and X, the Kd of factor IXa (Kd = 0.5 nM) binding is five times lower than that of factor IX. MLID90062111 MLID86243664  105,106 The importance of factor VIII for factors IX and IXa binding to cell membranes remains uncertain.

Factor IX is activated by factor XIa in a reaction that is independent of membrane surfaces. The rate of factor IX activation is not affected by the binding of factor XIa to platelets. MLID86243664  106

Formation of the Tenase Complex

The binding of factor IXa to factor VIII has been studied using both porcine and human factor VIII. Using fluorescence anisotropy to monitor the interaction of porcine factor VIIIa and factor IXa derivatized with a fluorescent label within its active site, Duffy et al. MLID92381007  107 have shown that these proteins interact on phospholipid membrane surfaces with a Kd of 2 nM in 1:1 molar stoichiometry. The factor IXa active site is distinct from the membrane surface, and its structure is perturbed on interaction with factor VIIIa during the formation of the tenase complex. MLID92381008  108 Activation of factor IX to factor IXa leads to the expression of a factor VIII-binding site and active enzyme site with full coagulant activity. 109 Reciprocally, activation of factor VIII by thrombin yields factor VIIIa, which binds more tightly than factor VIII to factor IXa. MLID92381007  107 Cleavage of Arg 180-Val 181 is required for at least partial expression of the factor VIII binding site; the cleavage of Arg 145-Ala 146 in addition to Arg 180-Val 181 leads to full enzymatic and cofactor binding activity. 109