Ott I, Miyagi Y, Miyazaki K, Heeb M J, Mueller B M, Rao L V, Ruf W
Deutsches Herzzentrum, Munich, Germany.
Arterioscler Thromb Vasc Biol. 2000 Mar;20(3):874-82. doi: 10.1161/01.atv.20.3.874.
Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-1), which regulates tissue factor (TF) function by TF. VIIa. Xa. TFPI-1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF. VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF. VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.
内皮细胞和肿瘤细胞合成组织因子途径抑制剂（TFPI-1），它通过TF·VIIa·Xa·TFPI-1四元复合物的形成（其中VIIa和Xa是凝血因子）以及这些复合物转位到细胞膜富含糖鞘脂的微结构域中来调节组织因子（TF）的功能。外源添加到细胞中的重组TFPI-1靶向一条降解途径。本研究分析了与内源性TFPI-1形成四元复合物是否也会导致内化和降解。我们证明内源性TFPI-1和重组TFPI-1在细胞表面的分布不同。重组TFPI-1存在于富含磷脂和糖鞘脂的膜结构域中，而内源性TFPI-1优先定位于富含糖鞘脂的微结构域。在形成四元复合物时，内源性TFPI-1对蛋白酶仍敏感，并且在完整细胞上可被抗体识别，这表明它没有明显内化。相反，TFPI-1对TF的调节在12小时内恢复，这与细胞表面四元复合物的解离一致。内源性TFPI-1可通过磷脂酶处理从细胞表面释放，这表明TFPI-1要么是糖基磷脂酰肌醇（GPI）锚定蛋白，要么与GPI连接的受体结合。我们证明重组GPI锚定形式的TFPI-1的表达将TF·VIIa复合物靶向富含糖鞘脂的膜组分。因此，TFPI-1的GPI锚定足以通过可逆抑制途径而非内化和降解来调节TF·VIIa复合物的功能。
