PURIFICATION OF A FIBRINOLYSIS ENHANCING PROTEIN

在我们的纤溶检测系统的开发过程中，我们注意到 无法计算的血浆纤溶活性的增强 因为任何已知的纤溶因子。使用肝素亲和力 柱层析，0.5M氯化钠洗脱，免疫亲和柱 已经能够部分提纯这一因素。免疫亲和柱 使用固定的MCab-3，一种针对血浆洗脱液的单抗 肝素亲和柱。我们已经证明了它的增强活性 酶联纤溶试验(ELFA)中的蛋白质，一种凝块溶解 添加t-PA的检测和使用可转移固体的生物免疫检测 相酶标记纤维蛋白。使用我们已经估计的生物免疫分析 血浆水平约为4.5个单位。高血压病患者血浆蛋白水平 辅因子似乎低于50ug/ml。高效液相凝胶渗透 层析显示MCab-3结合蛋白为78kD。这种蛋白质可以 在高浓度的外源物质存在时不会被激活 纤溶激活剂，排除纤溶酶原存在的可能性 对活动负责。该蛋白不能激活纤溶酶原， 没有可测量的纤溶活性。免疫扩散分析 完整的蛋白质显示与富含组氨酸的Vitronectin不同 糖蛋白，其他与肝素结合的血浆蛋白 纤溶调节。我们建议将该蛋白质提纯至均一。 并通过N端序列分析对其进行正向鉴定。我们建议 提高针对该蛋白的多克隆抗体以用于发育 用于测定血浆中蛋白质的免疫测定法。

During the development of our fibrinolytic assay system we noted an enhancement of fibrinolytic activity in plasma which could not be accounted for by any known fibrinolytic factor. Using heparin affinity chromatography, with a 0.5M NaCl elution and an immuno affinity column we have been able to partially purify this factor. The immuno affinity column uses immobilized MCab-3, a monoclonal antibody against a plasma eluate from a heparin affinity column. We have demonstrated the enhancing activity of the protein in the enzyme linked fibrinolytic assay (ELFA), a clot lysis assay with added t-PA, and a bio immunoassay using a transferable solid phase enzyme labelled fibrin. Using the bio immunoassay we have estimated plasma levels to be approximately 4.5 units. Plasma protein levels for the cofactor appear to be less than 50ug/ml. HPLC gel permeation chromatography reveals a MCab-3 binding protein of 78KD. The protein could not be activated in the presence of high concentrations of extrinsic fibrinolytic activators, precluding the possibility of plasminogen being responsible for the activity. The protein did not activate plasminogen and had no measurable fibrinolytic activity. Immunodiffusion analysis of the intact protein revealed non identity with vitronectin or histidine rich glycoprotein, other heparin binding plasma proteins implicated in fibrinolysis modulation. We propose to purify the protein to homogeneity and positively identify it by N-terminal sequence analysis. We propose raising a polyclonal antibody against the protein to use in the development of immunoassays for the measurement of the protein in plasma.