DETECTION OF FACTOR X ACTIVATION IN HUMANS-猫眼课题宝

人类 X 因子激活的检测

DOI：

发表时间：

1989

期刊：

Thrombosis and Haemostasis

影响因子：

6.7

通讯作者：

中科院分区：

医学2区

文献类型：

作者：

Kasimir ‐ Bauer;B. Kass;H. ten Cate;MA Bednarek;JJ Hawiger;RD Rosenberg

因子X被因子VII/ via -组织因子或因子IXa激活在止血机制中起关键作用。该反应从酶原中释放出一段肽，我们已经开发了一种敏感和特异性的放射免疫测定法（RIA），该天然肽是通过羟基磷灰石色谱和反相高压液相色谱（HPLC）从活化的人因子X中纯化出来的。凝胶过滤实验表明，肽与酶没有物理关联。采用Merrifield固相法合成了具有活化片段cooh末端序列的15个氨基酸的肽段。对牛血清白蛋白与戊二醛偶联的合成类似物进行了兔抗血清培养。获得的抗体群体用于构建双抗体RIA，能够测量该成分的小至0.02 nM。抗体对因子X酶原的反应性可以忽略不计（摩尔基础上小于激活肽的1/36,000）。然而，由于其他血浆成分在RIA中贡献了非特异性基础信号，我们开发了一种利用高氯酸提取天然肽的方法。正常人血浆肽水平为~ 0.1 nM，弥散性血管内凝血患者血浆肽水平升高至0.8 nM。长期使用香豆素衍生物抗凝的个体血浆中该肽的水平被抑制到~ 0.02 nM。我们在体内测量因子X激活的有效性得到了以下事实的支持：免疫反应信号以与天然激活肽相同的方式在反相HPLC上迁移，并且可以定量恢复。这一分析应该是有用的研究病理生理学血栓以及出血性疾病在人类。

The activation of factor X by factor VII/VIIa-tissue factor or factor IXa plays a pivotal role in the hemostatic mechanism. This reaction results in the liberation of a peptide from the zymogen for which we have developed a sensitive and specific radioimmunoassay (RIA), The native peptide was purified from activated human factor X by hydroxylapatite chromatography and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration experiments demonstrated that the peptide was not physically associated with the enzyme. A 15 amino acid peptide with the COOH-terminal sequence of the activation fragment was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used to construct a double antibody RIA and was able to measure as little as 0.02 nM of this component. The antibody reactivity toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However because other plasma constituents contributed to a nonspecific basal signal in the RIA, we developed an extraction procedure for the native peptide utilizing perchloric acid. Plasma peptide levels in normal individuals were ∼0.1 nM, and elevations up to 0.8 nM were observed in patients with evidence of disseminated intravascular coagulation. Individuals chronically anticoagulated with coumarin derivatives had plasma levels of this peptide suppressed to ∼0.02 nM. The validity of our measurements of factor X activation in vivo is supported by the fact that the immunoreactive signal migrates on reverse-phase HPLC in a manner identical to that of the native activation peptide and can be quantitatively recovered. This assay should be useful for studying the pathophysiology of thrombotic as well as bleeding disorders in humans.