Chiu H C, Whitaker E, Colman R W
J Clin Invest. 1983 Aug;72(2):493-503. doi: 10.1172/jci110997.
Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG(4)lambda) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M alpha-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (M(r), 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity. The isolated Factor V neutralized the inhibitory activities of the monoclonal antibody that was used to purify it, as well as the rabbit antibodies produced by immunizing the animals with the purified Factor V. Immunoelectrophoresis of purified Factor V against the polyclonal rabbit antiserum resulted in a single precipitin arc of identical mobility to the Factor V in normal human plasma. Analysis by double immunodiffusion showed a line of identity between plasma and purified Factor V and crossed immunoelectrophoresis showed a single species in normal plasma.A competitive enzyme-linked immunosorbent assay using the rabbit antibody against Factor V was applied to quantify Factor V antigen level in human plasma. Reconstitution of congenitally deficient or immunodepleted plasma with normal plasma or purified Factor V gave parallel dose-response curves. In 14 normal plasma the coagulant activity was 0.98+/-0.02 U/ml (mean+/-SEM) and antigen concentration was 11.1+/-0.4 mug/ml. A pool of 14 patients with congenital Factor V deficiency were studied. 10 patients had Factor V antigen ranging from 1.0 to 2.4 mug/ml with corresponding coagulant activities (0-0.17 U/ml) indicating a low concentration of normal Factor V, presumably due to decreased synthesis or increased degradation. When these patient plasmas and the normal plasmas were analyzed together an excellent correlation (r = 0.97, P < 0.01) was obtained. However, four patients with coagulant activity (0-0.08 U/ml) had Factor V antigen concentrations ranging from 4.4 to 6.1 mug/ml, indicating the presence of a reduced concentration of abnormal Factor V protein. The presence of patients with antigen similar in concentration to coagulant activity and antigen in excess of Factor V activity indicates the heterogeneity of congenital Factor V deficiency.
功能性人凝血因子Ⅴ已通过一种快速免疫亲和方法纯化。在血浆经柠檬酸钡吸附后，用浓度为5%至14%的聚乙二醇沉淀凝血因子Ⅴ。将所得制剂应用于含有固定化免疫吸附剂的柱上，该免疫吸附剂由含有天然存在的具有抗人凝血因子Ⅴ抑制活性的人单克隆（IgG(4)λ）抗体的IgG组分组成。固相免疫球蛋白定量结合人血浆中的凝血因子Ⅴ。用含有1.2M氯化钠和1Mα-甲基-D-甘露糖苷的pH7.2 Tris缓冲液有效洗脱结合态的凝血因子Ⅴ。分离得到的具有高比活性（92U/mg）的天然凝血因子Ⅴ在还原和非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上均显示出一条带（相对分子质量，350,000）。凝血因子Ⅴ比血浆纯化了5100倍，总产率为77%。纯化的凝血因子Ⅴ经凝血酶激活后，凝血活性增加了18倍。分离得到的凝血因子Ⅴ中和了用于纯化它的单克隆抗体以及用纯化的凝血因子Ⅴ免疫动物产生的兔抗体的抑制活性。用纯化的凝血因子Ⅴ对兔多克隆抗血清进行免疫电泳，得到一条沉淀弧，其迁移率与正常人血浆中的凝血因子Ⅴ相同。双向免疫扩散分析显示血浆和纯化的凝血因子Ⅴ之间有一条同一线，交叉免疫电泳显示正常人血浆中有一个单一成分。使用抗凝血因子Ⅴ的兔抗体的竞争性酶联免疫吸附测定法用于定量人血浆中凝血因子Ⅴ抗原水平。用正常血浆或纯化的凝血因子Ⅴ重建先天性缺乏或免疫耗竭的血浆，得到平行的剂量-反应曲线。在14份正常血浆中，凝血活性为0.98±0.02U/ml（平均值±标准误），抗原浓度为11.1±0.4μg/ml。研究了14例先天性凝血因子Ⅴ缺乏患者的血浆池。10例患者的凝血因子Ⅴ抗原范围为1.0至2.4μg/ml，相应的凝血活性为（0至0.17U/ml），表明正常凝血因子Ⅴ浓度低，推测是由于合成减少或降解增加。当将这些患者血浆和正常血浆一起分析时，得到了极好的相关性（r = 0.97，P < 0.01）。然而，4例凝血活性为（0至0.08U/ml）的患者，其凝血因子Ⅴ抗原浓度范围为4.4至6.1μg/ml，表明存在浓度降低的异常凝血因子Ⅴ蛋白。存在抗原浓度与凝血活性相似且抗原超过凝血因子Ⅴ活性的患者，表明先天性凝血因子Ⅴ缺乏具有异质性。
