Ott Ilka, Vukovich Ruth, Schömig Albert, Neumann Franz-Josef
Medizinische Klinik und Deutsches Herzzentrum, Technische Universität München, Lazarettstr. 36, 80636 München, Germany.
Blood Coagul Fibrinolysis. 2003 Sep;14(6):539-44. doi: 10.1097/00001721-200309000-00004.
The cellular initiation of coagulation by the tissue factor (TF)-activated factor VII complex is transiently inhibited by endogenous tissue factor pathway inhibitor-1 (TFPI-1), whereas exogenously added TFPI-1 is targeted to a degradation pathway. This study investigates the relevance of glycosyl phosphatidylinositol (GPI) anchoring for the anticoagulant properties of TFPI-1. Experiments were performed with the human cell line ECV304 using liposomal gene transfer. For GPI anchoring of TFPI-1 we used a fusion protein of TFPI-1 and the GPI attachment sequence of decay-accelerating factor (GPI-TFPI-1), and compared it with wild-type TFPI-1. We measured TF and TFPI-1 surface expression by flow cytometry and TF proteolytic activity by a chromogenic assay for activated factor X generation. After transfection of GPI-TFPI-1, surface expression of TFPI-1 increased to 134 +/- 9% of mock transfected cells (mean +/- SEM, P = 0.004), and transfection with wild-type TFPI-1 did not significantly alter TFPI-1 surface expression. After transfection with GPI-TFPI-1, TF activity was reduced by 18 +/- 9% compared with mock transfections (P = 0.003), whereas after transfection with TFPI-1 wild type no significant inhibition was observed. This effect was not due to altered TF expression. GPI anchoring is an essential prerequisite for surface expression of TFPI-1 and inhibition of TF activity. Gene transfer of GPI-anchored TFPI, therefore, may be an efficient tool to inhibit local TF-induced coagulation.
组织因子（TF）激活的因子VII复合物对凝血的细胞起始作用会被内源性组织因子途径抑制物-1（TFPI-1）短暂抑制，而外源性添加的TFPI-1则靶向降解途径。本研究探讨糖基磷脂酰肌醇（GPI）锚定与TFPI-1抗凝特性的相关性。使用脂质体基因转移技术在人细胞系ECV304上进行实验。为了使TFPI-1进行GPI锚定，我们使用了TFPI-1与衰变加速因子的GPI附着序列的融合蛋白（GPI-TFPI-1），并将其与野生型TFPI-1进行比较。我们通过流式细胞术测量TF和TFPI-1的表面表达，并通过发色法检测活化因子X生成来测定TF的蛋白水解活性。转染GPI-TFPI-1后，TFPI-1的表面表达增加至模拟转染细胞的134±9%（平均值±标准误，P = 0.004），而转染野生型TFPI-1并未显著改变TFPI-1的表面表达。转染GPI-TFPI-1后，与模拟转染相比，TF活性降低了18±9%（P = 0.003），而转染野生型TFPI-1后未观察到显著抑制作用。这种效应并非由于TF表达改变所致。GPI锚定是TFPI-1表面表达和抑制TF活性的必要前提条件。因此，GPI锚定的TFPI的基因转移可能是抑制局部TF诱导的凝血的有效工具。
