Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 8

The activation of factor IX by factor XIa is highly metal selective, with optimal activation only in the presence of Ca2+ and suboptimal activation in the presence of Sr2+. MLID79005660 82,121 The Km for the activation of factor IX by factor XIa without calcium is 18 mM, and the kcat is 2.4 min- 1. In the presence of calcium the Km is decreased to 2 mM, and the kcat is increased to 10.4 min- 1. MLID82167483 MLID88000587  122,123 Although the proteolytic activity of factor XIa is present on the light chain, the heavy chain of factor XIa may be necessary for the Ca2+-dependent acceleration of factor IX activation. MLID88000587 MLID83290881 MLID85289259  123–125 The factor IX-phospholipid interaction is inhibited by anti-factor IX/Ca2+-specific antibodies. The metal requirements for antibody recognition of factor IX parallels that for activation by factor XIa. These data suggest that there may be a surface of factor XIa that interacts with factor IX in the presence of Ca2+. An antibody directed against the heavy chain of factor XIa neutralized the enzyme competitively, but an antibody directed against the light chain neutralized the enzyme noncompetitively. MLID88000587  123 The region of the factor XIa heavy chain that interacts with factor IX has been localized to residues 134–172 in the heavy chain of factor XI. MLID92084730  126 Furthermore, the presence or absence of calcium did not change the rate of activation of factor IX by factor XIa light chain. MLID88000587  123 On the basis of these studies and antibody inhibition studies using antibodies to factor IX, the heavy chain of factor XIa has been implicated as specifically interacting with the phospholipid-binding site of factor IX in the presence of Ca2+.

Activation of Factor IX by the Extrinsic Pathway

Factor IX is also activated by factor VIIa and tissue factor MLID83273589 MLID84204056 MLID78094386 MLID80204333  114,127–129 (Fig. 107-7). This reaction, dependent on calcium ions, is characterized by a Km of 0.3 mM. MLID84000424 MLID84204056  113,127 The kcat for this reaction has been determined to be between 13 and 68 min- 1. MLID84000424 MLID84204056  113,127 The activation of factor IX by factor VIIa and tissue factor is accelerated by the activation of factor IX to factor IXa by factor Xa. MLID91250450  130

The relative importance of factor IX activation by factors XIa or VIIa and tissue factor has been evaluated by measuring the factor IX activation peptide in patients with factors VII and XI deficiency. MLID90345027  131 There was a significant reduction in the baseline levels of the factor IX activation peptide in patients with factor VII deficiency but not in patients with factor XI deficiency when compared with normal controls. Furthermore, the administration of recombinant factor VIIa to chimpanzees results in a significant increase in both the factor IX and factor X activation peptides at the same time point. MLID90345027  131 Infusion of recombinant factor VIIa into patients with factor VII deficiency causes an increase in the abnormally low levels of the factors IX and X activation peptides and prothrombin fragment F1.2. MLID92223384  132 These studies suggest that the activation of factor IX by factor VIIa and tissue factor is important in vivo.

Almus et al. MLID90315433  133 have evaluated the activation of factors IX and X by factor VIIa and tissue factor in an umbilical vein model. In this system the activation of factors IX and X occurs at the same rate. These results further substantiate the importance of both of these reactions in the initiation of the extrinsic pathway of blood coagulation.

Factors IXa Enzymatic Activity

Once generated, factor IXa forms a complex with factor VIIIa on membrane surfaces in an interaction that is calcium dependent. This complex on the membrane is required for the activation of factor X at rates that are physiologically significant. MLID81142359  134 Factor IXa is inhibited by antithrombin III, which forms a 1:1 stoichiometric complex with factor IXa as well as other activated coagulation factors. This reaction is accelerated approximately 1,000-fold by the addition of heparin. MLID76069210 MLID76114804 MLID81046828 MLID81046829  135–138 Under comparable conditions, antithrombin III neutralizes 57% of factor IXa activity within 30 minutes in the absence of heparin but completely inhibits factor IXa within 15 seconds in the presence of heparin. MLID76069210  135 In contrast to the solution-phase activation of factor IX by factor XIa, factor X is converted to its enzyme form, factor Xa, by factor IXa in complex with factor VIIIa on membrane surfaces. This reaction requires calcium ions. Membrane surfaces, including phospholipid vesicles, activated platelets, or endothelial cells, are required for significant factor X activation. Thus, factor IX is a membrane-binding protein, and the presence of g-carboxyglutamic acid is intimately related to these membrane-binding properties. These proteins may have two distinct binding domains, one related to zymogen activation and the other to macromolecular assembly of the enzyme in complex with the specific cofactor.