Molecular Genetic Studies of Thrombosis

1. 蛋白C缺乏的基因异常分析。我们利用聚合酶链反应（PCR）分析了日本遗传性蛋白C缺乏症患者的蛋白C基因，并在两个独立的先证者中发现了两个分子异常。一个是外显子VI的3414T到C突变，该突变将Tyrl24 （ZAC）转化为第二个EGF结构域的His （CAC）。另一种是编码羧基末端的外显子IX的8854G-8857G区域中单个G核苷酸的移码缺失，预计会导致推导出的羧基末端氨基酸序列的改变和延长。家族性蛋白C缺乏的诊断。基于上述结果，我们建立了一种直接诊断第二例移码缺失的家族性蛋白C缺乏症的方法，即利用PCR诱变引物将Ava I或Bal I位点引入扩增产物中。蛋白C基因多态性。我们已经确定了蛋白C基因的两个序列变异。第一个是未翻译外显子1 -1476核苷酸上的a - t序列多态性，第二个是编码Ser-99的三联体（TCT - TCG）上的T-G序列多态性。我们发现蛋白C和活性更强的蛋白C与肝素相互作用，肝素在钙离子存在下增强，并提出了蛋白C的一个特定区域作为肝素结合位点。富组氨酸糖蛋白（HRG）对蛋白C抑制剂活性的调节。我们发现，在血浆中锌离子和钙离子的生理浓度下，HRG可以中和蛋白C抑制剂对活化蛋白C和凝血酶的肝素依赖性抑制，这表明HRG作为蛋白C抑制剂的生理调节剂具有新的功能。HRG基因在3号染色体上的分配。我们已经将HRG基因分配到3号染色体上。

1. Analysis of Genetic Abnormalities for Protein C Deficiency. Makinal use of the polymerase chain reaction (PCR), we analyzed the genes for protein C from Japanese patients with a hereditary protein C deficiency, and identified two molecular abnormalities in two independent probands. One is a 3414T to C mutation in exon VI which converts Tyrl24 (ZAC) to His (CAC) in the second EGF domain. The other is a frameshift deletion of a single G nucleotide in a region of 8854G-8857G in exon IX coding for the carboxy-terminal region, which is predicted to result in an alteration and elongation of the deduced carboxy-terminal amino acid sequence.2. Diagnosis of Familial Protein C Deficiency. Based on a result described above, we have established a direct diagnostic method of familial protein C deficiency in the second case of a frameshift deletion by use of mutacenic primers for PCR to introduce Ava I or Bal I site into the amplified product.3. Polymorphism in the Protein C Gene. We have identified two sequence variations in the protein C gene. The first one is an A-T sequence polymorphism at nucleotide -1476 in the untranslated exon 1, and the second one is a T-G sequence polymorphism in the triplet coding for Ser-99 (TCT - TCG).4. Heparin-Binding Property of Protein C. We found that protein C, and more strongly activated protein C, interacts with heparin, which is enhanced in the presence of calcium ions, and proposed a specific region of protein C as a heparin-binding site.5. Modulation of Protein C Inhibitor Activity by Histidine-Rich Glycoprotein (HRG). We have found that HRG neutralizes the heparin-dependent inhibitions of activated protein C and thrombin by protein C inhibitor at physioloical concentrations of zinc and calcium ions in plasma, suggesting a new function of HRG as a physiological modulator of protein C inhibitor.6. Assignment of the Gene for HRG to Chromosome 3. We have assigned the gene for HRG to chromosome 3.