Riesbeck K, Dorling A, Kemball-Cook G, McVey J H, Jones M, Tuddenham E G, Lechler R I
Dept. of Immunology, MRC Clinical Sciences Centre, London, UK.
Thromb Haemost. 1997 Dec;78(6):1488-94.
Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.
组织因子途径抑制剂（TFPI）是凝血组织因子（TF）途径的主要调节因子之一。为了将人TFPI连接到细胞表面，将缺乏第三个Kunitz结构域的全长或截短的TFPI与人CD4的第三个和第四个结构域以及羧基末端序列融合。构建体被转染到小鼠成纤维细胞系中，并使用针对前两个TFPI Kunitz结构域和CD4的单克隆抗体检查各个克隆的表达情况。使用抗FX多克隆抗体通过流式细胞术检测特异性人FXa结合，并使用S-2765通过发色底物测定法验证FXa蛋白水解活性的抑制作用。此外，用FXa预孵育的表达TFPI-CD4的细胞，用抗人TF多克隆抗体显示，特异性结合人TF-FVIIa复合物。全长或截短的TFPI-CD4之间未观察到功能差异。这些结果表明，功能完整的TFPI可以连接到细胞表面。例如，对内皮细胞进行基因操作以导致TFPI的稳定表达，可能会抑制心脏同种异体移植后冠状动脉心脏病的发展，并可能抑制异种移植中的血栓形成。
