O'Brien D P, Kemball-Cook G, Hutchinson A M, Martin D M, Johnson D J, Byfield P G, Takamiya O, Tuddenham E G, McVey J H
Haemostasis Research Group, Clinical Research Centre, Harrow, Middlesex, U.K.
Biochemistry. 1994 Nov 29;33(47):14162-9. doi: 10.1021/bi00251a027.
The blood coagulation cascade is initiated when vessel injury allows factor VII (FVII) to form a complex with tissue factor (TF). Complete deficiency of FVII causes a lethal bleeding diathesis, but individuals with moderately reduced FVII levels are often asymptomatic. Some of these individuals have circulating partially functional FVII, as a result of point missense mutations in critical parts of the molecule. One such mutation has been reported at position 79 in the first epidermal growth factor-like (EGF) domain of FVII, where an arginine residue has been replaced by glutamine. There is controversy as to whether or not this mutation reduces the affinity of the FVII/TF interaction compared to wild-type FVII. To address this problem, we have expressed recombinant FVII-R79Q and subjected it to detailed biochemical analysis. One-stage FVII:C assays show the variant FVII to have reduced activity with respect to the wild type. Rates of autoactivation and activation by FXa to the two-chain molecule were identical for wild-type and variant FVII. The Vmax for FX activation was lower for the mutant as measured using an amidolytic assay for FX activity. In contrast, the Km for FX was lower for the variant than the wild-type molecule. Peptidyl substrate hydrolysis was virtually identical for both variant and normal FVIIa in the presence and absence of TF. The variant has reduced affinity for TF as measured by surface plasmon resonance. FVII-R79Q has an association rate constant (kassoc) one-fifth of that of normal FVII, but a similar kdiss, resulting in a decrease in the affinity of the enzyme for its cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)
当血管损伤使因子VII（FVII）与组织因子（TF）形成复合物时，血液凝固级联反应启动。FVII完全缺乏会导致致命的出血素质，但FVII水平中度降低的个体通常无症状。这些个体中的一些人由于分子关键部位的点错义突变而具有循环的部分功能性FVII。在FVII的第一个表皮生长因子样（EGF）结构域的第79位报道了这样一种突变，其中一个精氨酸残基被谷氨酰胺取代。与野生型FVII相比，这种突变是否降低了FVII/TF相互作用的亲和力存在争议。为了解决这个问题，我们表达了重组FVII-R79Q并对其进行了详细的生化分析。单阶段FVII：C测定表明，变异型FVII相对于野生型具有降低的活性。野生型和变异型FVII的自激活率和被FXa激活为双链分子的激活率相同。使用FX活性的酰胺分解测定法测量，突变体的FX激活Vmax较低。相比之下，变异型的FX Km低于野生型分子。在有和没有TF的情况下，变异型FVIIa和正常FVIIa的肽基底物水解几乎相同。通过表面等离子体共振测量，变异型对TF的亲和力降低。FVII-R79Q的缔合速率常数（kassoc）是正常FVII的五分之一，但kdiss相似，导致该酶对其辅因子的亲和力降低。（摘要截短为250字）
