Maekawa H, Sugo T, Yamashita N, Kamiya K, Umeyama H, Miura N, Naka H, Nishimura T, Yoshioka A, Matsuda M
Institute of Hematology, Jichi Medical School, Tochigi, Japan.
Biochemistry. 1993 Jun 22;32(24):6146-51. doi: 10.1021/bi00075a005.
Utilizing polymerase chain reaction and directly sequencing the amplified exon 6 of the factor IX gene derived from a mild hemophilia Bm patient, we have identified a T to C mutation at nucleotide 20,525. This point mutation predicted a Val182 to Ala substitution in the abnormal factor IX molecule, designated as factor IX Tokyo. The patient manifested a low factor IX activity and a moderately prolonged ox-brain prothrombin time but a normal factor IX antigen level in plasma. Immunopurified factor IX derived from the patient was found to have a normal molecular weight but a reduced specific activity (23% of normal). Limited proteolysis by activated factor XI or by a snake venom-derived factor X-activating enzyme was considerably delayed, indicating the presence of structural alteration(s) most probably at or near the second enzyme-cleavage site. Once activated, however, factor IXa Tokyo was able to activate factor X normally and was inactivated by antithrombin III also in a normal fashion. The structural model of factor IXa and a docking model of factor IX and activated factor VII (factor VIIa) suggested that the Val182 to Ala substitution would not affect the local conformation of the catalytic domain. This mutation would rather loosen the fitness of the molecule into the substrate-binding pocket of factor VIIa due to a shorter side chain of the Ala substitution at the P2' position of the second cleavage site.
通过聚合酶链反应并直接对一名轻度B型血友病患者的因子IX基因扩增外显子6进行测序，我们在核苷酸20525处鉴定出一个从T到C的突变。该点突变预测异常因子IX分子（命名为因子IX东京型）中第182位缬氨酸被丙氨酸取代。该患者表现出低因子IX活性和中度延长的脑凝血酶原时间，但血浆中因子IX抗原水平正常。从该患者体内免疫纯化得到的因子IX分子量正常，但比活性降低（为正常的23%）。被活化因子XI或蛇毒来源的因子X激活酶进行的有限蛋白水解显著延迟，表明最可能在第二个酶切位点或其附近存在结构改变。然而，一旦被激活，因子IXa东京型能够正常激活因子X，并且也能以正常方式被抗凝血酶III灭活。因子IXa的结构模型以及因子IX与活化因子VII（因子VIIa）的对接模型表明，第182位缬氨酸被丙氨酸取代不会影响催化结构域的局部构象。由于在第二个切割位点的P2'位置丙氨酸取代的侧链较短，该突变反而会使分子与因子VIIa底物结合口袋的适配性降低。
