Spitzer S G, Warn-Cramer B J, Kasper C K, Bajaj S P
Department of Medicine, St. Louis University School of Medicine, MO 63104.
Biochem J. 1990 Jan 1;265(1):219-25. doi: 10.1042/bj2650219.
Previously, from the plasma of unrelated haemophilia-B patients, we isolated two non-functional Factor IX variants, namely Los Angeles (IXLA) and Long Beach (IXLB). Both variants could be cleaved to yield Factor IXa-like molecules, but were defective in catalysing the cleavage of Factor X (macromolecular substrate) and in binding to antithrombin III (macromolecular inhibitor). In the present study we have identified the mutation of IXLA by amplifying the exons (including flanking regions) as well as the 5' end of the gene by polymerase-chain-reaction (PCR) method and sequencing the amplified DNA by the dideoxy chain-termination method. Comparison of the normal IX and IXLA sequences revealed only one base substitution (T----C) in exon VIII of IXLA, with a predicted replacement of Ile-397 to Thr in the mature protein. This mutation is the same as found recently for IXLB. The observation that IXLB and IXLA have the same mutation is an unexpected finding, since, on the basis of their ox brain prothrombin time (PT, a test that measures the ability of the variant Factor IX molecules to inhibit the activation of Factor X by Factor VIIa-tissue factor complex), these variants have been classified into two different groups and were thought to be genetically different. Our observation thus suggests that the ox brain PT does not reflect the locus of mutation in the coding region of the variant molecules. However, our analysis suggests that the ox brain PT is related to Factor IX antigen concentration in the patient's plasma. Importantly, although the mutation in IXLA or IXLB protein is in the catalytic domain, purified IXaLA and IXaLB hydrolyse L-tosylarginine methyl ester at rates very similar to that of normal IXa. These data, in conjunction with our recent data on Factor IXBm Lake Elsinore (Ala-390----Val mutant), strengthen a conclusion that the peptide region containing residues 390-397 of normal Factor IXa plays an essential role in macromolecular substrate catalysis and inhibitor binding. However, the two mutations noted thus far in this region do not distort S1 binding site in the Factor IXa enzyme.
此前，我们从非血友病B患者的血浆中分离出两种无功能的凝血因子IX变体，即洛杉矶型（IXLA）和长滩型（IXLB）。这两种变体均可被切割产生类似凝血因子IXa的分子，但在催化凝血因子X（大分子底物）的切割以及与抗凝血酶III（大分子抑制剂）结合方面存在缺陷。在本研究中，我们通过聚合酶链反应（PCR）方法扩增外显子（包括侧翼区域）以及基因的5'端，并采用双脱氧链终止法对扩增的DNA进行测序，从而确定了IXLA的突变。正常凝血因子IX与IXLA序列的比较显示，IXLA的外显子VIII中仅有一个碱基替换（T----C），预计成熟蛋白中的Ile-397被Thr取代。此突变与最近在IXLB中发现的相同。IXLB和IXLA具有相同突变这一观察结果是一个意外发现，因为基于它们的牛脑凝血酶原时间（PT，一种测量变体凝血因子IX分子抑制因子VIIa - 组织因子复合物激活因子X能力的试验），这些变体已被分为两个不同的组，并被认为在遗传上是不同的。因此，我们的观察结果表明，牛脑PT不能反映变体分子编码区的突变位点。然而，我们的分析表明，牛脑PT与患者血浆中的凝血因子IX抗原浓度有关。重要的是，尽管IXLA或IXLB蛋白中的突变位于催化结构域，但纯化的IXaLA和IXaLB水解L - 甲苯磺酰精氨酸甲酯的速率与正常IXa非常相似。这些数据，连同我们最近关于埃尔西诺湖凝血因子IXBm（Ala - 390----Val突变体）的数据，强化了一个结论，即正常凝血因子IXa中包含390 - 397位残基的肽区域在大分子底物催化和抑制剂结合中起重要作用。然而，迄今为止在该区域发现的两个突变并未扭曲凝血因子IXa酶中的S1结合位点。
