Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 7

The binding of factor IXa, factor VIIIa, and phospholipid (the tenase complex) (Fig. 107-5) has been explored with chimeric proteins in which the first and second EGF domains of factor X are substituted for the native domains in factor IX. The chimeras in which the second EGF domain of factor IX has been substituted with the second EGF domain of factor X do not bind significantly in the tenase complex but interact normally with phospholipid. 71 The chimera in which the first EGF domain of factor IX has been substituted with the first EGF domain of factor X has full or nearly full biologic activity, indicating that the first EGF domain is not involved in factor VIIIa binding. MLID90094389  71,72 Furthermore, this form does bind in the tenase complex. 71 This finding suggests that the second EGF domain of factor IX is required for enzymatic activity and that it contains a recognition site for factor VIIIa. 71

Activation of Factor IX by the Intrinsic Pathway

Factor IX is a proenzyme with no catalytic activity that is activated to factor IXa by factor XIa or by factor VIIa and tissue factor in a reaction that depends on the presence of calcium (Fig. 107-6). The kinetics of factor IX activation by factor VIIa and tissue factor or factor XIa are comparable. Factor IX is converted to its enzyme form, factor IXa, by factor XIa. This reaction requires calcium ions, but takes place in solution. In contrast to the other vitamin K-dependent blood clotting proteins, membrane surfaces, including phospholipid vesicles or cell surfaces, do not enhance factor IX activation. The activation of factor IX by factor XIa is abnormally slow in factor IX New London (Pro 50 B Glu). MLID90167273  110 This implicates the EGF domain in factor IX activation. When factor IX is activated by factor XIa, two peptide bonds are cleaved: one bond is located at Arg 145-Ala 146 and the other bond is at Arg 180-Val 181 MLID78194509 MLID78130043  111,112 (Fig. 107-7). Factor IX is first cleaved to form factor IXa, in which the Arg-Ala bond at residues 145–146 is cleaved followed by the cleavage of the Arg-Val bond at residues 180–181. MLID84000424  113 With the release of the internal carbohydrate-rich activation fragment (Mr 11,000), from residues 146–180, the factor IXa light chain (Mr 17,000) and heavy chain (Mr 28,000) remain bound by a single disulfide bond. The enzyme active site is located on the heavy chain. The factor IXa generated by this reaction is designated factor IXab.

Factor IX Chapel Hill (Arg 145 B His), the first factor IX mutation to be characterized at the molecular level, is the prototype of a genetic defect in which activation of a zymogen is impaired due to the mutation of an arginine preceding a sessile bond. MLID83273589  114 It has <20% of the coagulant activity when compared with factor IXab. MLID86131633  76 Factors IX Chicago-2 (Arg 145 B His) MLID89323336  115 and IX Albuquerque (Arg 145 B Cys) 116 have been found to have mutations at the same site. In each of these mutations, the loss of the arginine at position 145 precludes the cleavage of the sessile bond separating the factor IX light chain from the activation peptide. Failure to cleave this bond results in a molecule that cleaves factor X very slowly. The active site of factor IXa is formed, however, as demonstrated by its ability to cleave synthetic substrate normally. MLID82098578  117 Factor IX mutants at position 180 include factor IX Hilo (Arg 180 B Gln), factor IX Milano (Arg 180 B Gln), factor IX Deventer (Arg 180 B Trp), and factor IX Nagoya (Arg 180 B Trp), all resulting in hemophilia BM, a type of hemophilia in which the patient's mutant factor IX causes a marked prolongation of the ox brain thromboplastin time due to increased inhibition of the in vitro activation of factor VII. MLID89135000 MLID90293016  118,119 Factor IX Novara (Val 181 B Phe) results in the hemophilia BM phenotype, while factor IX Kashihara (Val 182 B Phe) results in hemophilia B. MLID90293016  119,120