Structural studies on Functional Abnormality of Factor XII and Fibrinogen.

(1)纤维蛋白原名古屋：-链上精氨酸取代谷氨酰胺-329。损害纤维蛋白单体聚合：对一种遗传性异常纤维蛋白原，纤维蛋白原名古屋进行结构研究，以确定导致纤维蛋白单体聚合受损的异常。还原条件下十二烷基硫酸钠-聚丙烯酰胺凝胶电泳研究。名古屋纤维蛋白原显示除了正常的三个亚基链外，还存在一个表观分子量为49,500的额外蛋白带。从名古屋纤维蛋白原cnbr片段的赖氨酸内肽酶酶解中分离得到的肽段的氨基酸序列分析表明，-链上的Gln-329已被精氨酸取代。这种取代可以解释为Gln-329 （CAG - CGG）的结构域发生了单核苷酸变化。我们认为-链上的GLN-329对于纤维蛋白单体的正常聚合是必不可少的。(2)凝血因子IX Niigata：催化区域缬氨酸取代丙氨酸-390：因子IX Niigata是一种突变因子IX，在具有正常水平的因子IX抗原但凝血活性降低（正常水平的1-4%）的患者中负责中重度血友病B。我们之前报道过纯化的突变蛋白可以通过因子XIa/Ca^<2+>以与正常因子IX相似的速率转化为因子IXa形式，但在因子XIII、Ca^<2+>和磷脂存在的情况下，产生的突变因子IXa不能激活因子X。在本研究中，我们在结构水平上分析因子IX Niigata，以阐明导致凝血活性丧失的分子异常。对赖氨酸内肽酶酶切获得的肽段进行氨基酸序列分析，并结合随后的SP-V8酶切，表明在因子IX Niigata分子的催化结构域中，390位的丙氨酸被缬氨酸取代。

(1) Fibrinogen Nagoya, a Replacement of Glutamine-329 by Arginine in the -chain. That Impairs the Polymerization of Fibrin Monomer: Structural studies on a hereditary abnormal fibrinogen, fibrinogen Nagoya were performed to identify the abnormality responsible for the impaired polymerization of fibrin nomomer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. fibrinogen Nagoya showed the presence of an extra protein band with an apparent molecular weight of 49,500 in addition to the normal three subunit chains. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of one of the cnbr fragments derived from fibrinogen nagoya indicated that Gln-329 in the -chain had been replaced by Arg. This substitution can be explained by a single uncleotide chainge in the dodon for Gln-329 (CAG - CGG). We conclude that GLN-329 in the -chain is indispensable for the normal polymerization of fibrin monomer.(2) Blood Clontting Factor IX Niigata: Substitution of Alanine-390 by Valine in the Catalytic Domain: Factor IX Niigata is s mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa form by factor XIa/Ca^<2+> at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa could not activate factor X in the presence of factor XIII, Ca^<2+>, and phospholipids. In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.