Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 9

Factor IXa is a serine protease that expresses its activity via the catalytic domain, which resides in the heavy chain. This activity is generated by the classic catalytic triad that in factor IXa is located at His 41, Asp 89, and Ser 185 in the heavy chain.

The activation of factor X by factor IXa has been evaluated under multiple experimental conditions. Calcium by itself accelerates factor IXa activation of factor X, but its more important role is to allow for the formation of the tenase complex on membrane surfaces. The presence of both factor VIIIa and membrane surfaces greatly accelerates the activation of factor X, while the presence of either of these cofactors alone has a minimal effect. MLID89117143 MLID86123587  139,140 The Kcat for factor X activation by factor IXa in the presence of factor VIIIa but without phospholipid vesicles is 0.058 min- 1; the same reaction without factor VIIIa but with phospholipid vesicles (25 mM) has a kcat of 0.095 min- 1, whereas in the presence of factor VIIIa and phospholipid vesicles the reaction is greatly accelerated, with a kcat of 1,740 min - 1. MLID90241883  101 The activation of factor X by factor IXa is also supported by activated platelets. The kcat for this reaction is 1,240 min- 1, which is very close to the kcat for factor X activation in the presence of phospholipid vesicles. These results suggest a physiologic role for platelets in this process.

Factor IXa activates factor X in the presence of both bovine MLID85207678  141 and human umbilical vein MLID87101493  142 endothelial cells. Stern et al. MLID85207678  141 have demonstrated equal binding of factors IX and IXa to bovine aortic endothelial cells. There is an equivalent increase in binding of both factor IX and factor IXa in the presence of activated or unactivated factor VIII. However, when factor X is added to the reaction mixture, a high-affinity binding site with relative specificity for factor IXa is expressed, and the interaction of factor IXa with endothelium is increased 10–40-fold. Factor X does not enhance factor IXa binding without the presence of factor VIII. While similar interactions have been demonstrated for factor IXa, factor VIII, and human umbilical vein endothelial cells, the rate of this reaction did not approach the rate of a control performed with phospholipid vesicles and only reached maximal velocity after a 10–12-minute lag phase. MLID87101493  142

Factor IX Eagle Rock and factor IX Bergamo are both characterized by the substitution of valine for Gly 363 and are functionally normal except for their inability to bind antithrombin III and their inability to activate factor X. MLID90147571  120,143 This may be due to the location of this mutation next to the active site Ser 365, causing distortion of the specificity pocket of the enzyme.

Defects Affecting Enzymatic Activity

Mutations within the catalytic domain of factor IX may decrease enzymatic activity. A mutation of IIe 397 B Thr has been recognized in multiple hemophilia B families, including factor IX Vancouver, MLID89174731  144 factor IX Long Beach, MLID88294349  145 and factor IX Los Angeles. MLID90147571  143 This lesion is near, but not within, the active site of factor IXa; it may alter the extended substrate binding site for factor X. Factor IX Angers (Gly 396 B Arg) has a mutation within the substrate-binding pocket of factor IXa that disrupts enzymatic activity. 146 Factor IX Bm Lake Elsinore (Ala 390 B Val) MLID88273164  147 also possesses a mutation within the substrate binding pocket that interferes with enzymatic activity. Factor IX Eagle Rock (Gly 363 B Val) MLID90147571  143 and factor IX Bergamo MLID90293016  119 both have mutations adjacent to Ser 365, the active site serine, that interfere with enzymatic activity. A mutation of Arg 333 also leads to a defect in the ability to catalyze factor X cleavage.

Molecular Basis of Hemophilia B

Defects in Gene Structure that Cause Hemophilia B

Gross deletions of the factor IX gene causes severe, antigen-negative hemophilia B MLID83192505 MLID89300787  148,149 (Fig. 107-8). These defects have been associated with anti-factor IX antibodies in response to replacement therapy with factor IX. MLID83192505  148 While antibody production may be associated with gross gene deletions, not all such patients have demonstrated such a response. MLID88297711 MLID89065717  150,151 In addition, some patients with anti-factor IX antibodies do not possess gross gene deletions, MLID89305505 MLID88264925  62,152 and in at least one case antibodies developed in an antigen-positive patient. MLID88264925  152 Thus, the lack of the factor IX gene is not the sole factor controlling the development of an immunologic response to factor IX replacement therapy.