Detection of the three Kunitz-type single domains of membrane-bound tissue factor pathway inhibitor (TFPI) by flow cytometry.

C Tiemann, T Brinkmann, K Kleesiek
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引用次数: 11

Abstract

Tissue factor pathway inhibitor, a natural anticoagulant in the extrinsic pathway of blood coagulation, is associated with the endothelial membrane and presumed to be released by heparin. For flow cytometric detection of membrane-bound tissue factor pathway inhibitor we synthesized polyclonal monospecific antibodies directed against each of the three Kunitz-type domains. Antisera were obtained by immunisation of rabbits with synthetic oligopeptides representing the reactive site of each domain. Kunitz-domain delta 1: 26CAFKDDGPCKAIMKR41, domain delta 2: 101EDPGICRGYITR112 and domain delta 3: 192PADRGLCRANENR204. Different cell lines (chondrosarcoma, synovial sarcoma, synovial cells, leukaemic monocytes) and endothelial cells were investigated by flow cytometric analysis using these antibodies. The three tissue factor pathway inhibitor domains were detected on the surface of all cells by the corresponding antisera. Similar results were obtained by immuno-histochemical staining. Since domain delta 3 was recognised by the appropriate antibody, it would seem that this third domain is not the membrane binding site. To investigate the cellular tissue factor pathway inhibitor release, endothelial cells were cultivated with heparin. Protein resynthesis and translocation were inhibited by puromycin and monensin, respectively. After heparin incubation an increased tissue factor pathway inhibitor concentration was determined in the cell culture medium by a chromogenic substrate assay. However, the tissue factor pathway inhibitor density on the cell surface was not influenced by heparin, as shown by flow cytometry using the three tissue factor pathway inhibitor antisera. Our results suggest that functionally active tissue factor pathway inhibitor is not released from the cell surface. Therefore, the effect of heparin appears to be mediated by secretion of tissue factor pathway inhibitor from intracellular stores.

流式细胞术检测膜结合组织因子通路抑制剂(TFPI)的三个kunitz型单结构域。
组织因子途径抑制剂(Tissue factor pathway inhibitor)是一种天然抗凝剂,存在于凝血的外源性途径中,与内皮膜有关,可能由肝素释放。为了流式细胞术检测膜结合组织因子途径抑制剂,我们合成了针对三个kunitz型结构域的多克隆单特异性抗体。用合成的代表每个结构域活性位点的寡肽免疫家兔获得抗血清。Kunitz-domain 1: 26CAFKDDGPCKAIMKR41, domain 2: 101EDPGICRGYITR112, domain 3: 192PADRGLCRANENR204。用这些抗体对不同细胞系(软骨肉瘤、滑膜肉瘤、滑膜细胞、白血病单核细胞)和内皮细胞进行了流式细胞分析。通过相应的抗血清在所有细胞表面检测到三种组织因子途径抑制域。免疫组织化学染色也得到了类似的结果。由于结构域δ 3被适当的抗体识别,这第三个结构域似乎不是膜结合位点。用肝素培养内皮细胞,观察细胞组织因子通路抑制剂的释放情况。嘌呤霉素和莫能菌素分别抑制了蛋白质的再合成和易位。肝素孵育后,通过显色底物测定细胞培养基中组织因子途径抑制剂浓度的增加。然而,三种组织因子途径抑制剂抗血清流式细胞术显示,细胞表面的组织因子途径抑制剂密度不受肝素的影响。我们的研究结果表明,功能活跃的组织因子途径抑制剂不会从细胞表面释放。因此,肝素的作用似乎是由细胞内储存的组织因子途径抑制剂的分泌介导的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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