Chaing S, Clarke B, Sridhara S, Chu K, Friedman P, VanDusen W, Roberts H R, Blajchman M, Monroe D M, High K A
Department of Medicine, University of North Carolina at Chapel Hill.
Blood. 1994 Jun 15;83(12):3524-35.
Factor VII (F.VII) is a vitamin-K-dependent serine protease required in the early stages of blood coagulation. We describe here a patient with severe F.VII deficiency, with a normal plasma F.VII antigen level (452 ng/mL) and F.VII activity less than 1%, who is homozygous for two defects: a G-->A transition at nucleotide 6055 in exon 4, which results in an Arg-->Gln change at amino acid 79 (R79Q); and a G-->A transition at nucleotide 8961 in exon 6, which results in an Arg-->Gln substitution at amino acid 152 (R152Q). The R79Q mutation occurs in the first epidermal growth factor (EGF)-like domain, which has previously been implicated in binding to tissue factor. The R152Q mutation occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated F.VII (F.VIIa). Analysis of purified F.VII from patient plasma shows that the material cannot be activated by F.Xa and cofactors. In addition, in an in vitro binding assay using relipidated recombinant tissue factor, patient plasma showed markedly reduced binding to tissue factor at all concentrations tested. In an effort to separate the contributions of the two mutations, three recombinant variants, wild-type, R79Q, and R152Q, were prepared and analyzed. The R152Q variant had markedly reduced activity in a clotting assay, whereas R79Q showed a milder, concentration-dependent reduction. The R152Q variant exhibited nearly normal binding in the tissue factor binding assay, whereas the R79Q variant had markedly reduced binding. The time course of activation of the R79Q variant was slowed compared with wild-type. Our results suggest that the first EGF-like domain is required for binding to tissue factor and that the F.VII zymogen lacks activity and requires activation for expression of biologic activity.
凝血因子VII（F.VII）是血液凝固早期所需的一种维生素K依赖的丝氨酸蛋白酶。我们在此描述一名严重F.VII缺乏症患者，其血浆F.VII抗原水平正常（452 ng/mL），F.VII活性低于1%，该患者为两个缺陷的纯合子：外显子4中核苷酸6055处的G→A转换，导致氨基酸79处的Arg→Gln变化（R79Q）；以及外显子6中核苷酸8961处的G→A转换，导致氨基酸152处的Arg→Gln替代（R152Q）。R79Q突变发生在第一个表皮生长因子（EGF）样结构域，该结构域先前被认为与组织因子结合有关。R152Q突变发生在一个位点（Arg 152 - Ile 153），该位点通常被切割以产生活化的F.VII（F.VIIa）。对患者血浆中纯化的F.VII进行分析表明，该物质不能被F.Xa和辅因子激活。此外，在使用重组脂化组织因子的体外结合试验中，患者血浆在所有测试浓度下与组织因子的结合均显著降低。为了区分这两个突变的作用，制备并分析了三种重组变体，即野生型、R79Q和R152Q。R152Q变体在凝血试验中的活性显著降低，而R79Q表现出较轻的、浓度依赖性的降低。R152Q变体在组织因子结合试验中表现出几乎正常的结合，而R79Q变体的结合显著降低。与野生型相比，R79Q变体的激活时间进程减慢。我们的结果表明，第一个EGF样结构域是与组织因子结合所必需的，并且F.VII酶原缺乏活性，需要激活才能表达生物学活性。
