Ruf W, Rehemtulla A, Morrissey J H, Edgington T S
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Biol Chem. 1991 Feb 5;266(4):2158-66.
Membrane anchoring of tissue factor (TF), the cell receptor for coagulation factor VIIa (VIIa), exemplifies an effective mechanism to localize proteolysis at the cell surface. A recombinant TF mutant (TF1-219), deleted of membrane spanning and intracellular domains, was used to evaluate the role of phospholipid interactions for assembly of substrate with the catalytic TF.VIIa complex. TF1-219 was secreted by cells rather than expressed as a cell membrane protein. Unlike free VIIa, TF1-219 as well as the TF1-219.VIIa complex demonstrated no stable association with phospholipid. In the absence of lipid, kinetic evaluation of substrate factor X cleavage by free VIIa, TF.VIIa, and TF1-219.VIIa suggests that the catalytic function of VIIa rather than substrate recognition is enhanced by complex formation. Furthermore, compared with free factor X, factor X on phospholipid was preferentially cleaved as a substrate by TF1-219.VIIa. TF-dependent initiation of the coagulation protease cascades thus involves an enhancement of the activation of factor X on the cell surface by a crucial role of the TF transmembrane domain to membrane anchor the reaction, by the TF extracellular domain to provide protein-protein interactions with VIIa to enhance the activity of the catalytic domain of VIIa, and the preferential presentation of factor X as a substrate when associated with phospholipid surfaces.
组织因子（TF）是凝血因子VIIa（VIIa）的细胞受体，其膜锚定是在细胞表面定位蛋白水解的一种有效机制。一种缺失跨膜和细胞内结构域的重组TF突变体（TF1-219）被用于评估磷脂相互作用在底物与催化性TF.VIIa复合物组装中的作用。TF1-219由细胞分泌，而非作为细胞膜蛋白表达。与游离的VIIa不同，TF1-219以及TF1-219.VIIa复合物与磷脂没有稳定的结合。在没有脂质的情况下，对游离VIIa、TF.VIIa和TF1-219.VIIa切割底物因子X的动力学评估表明，复合物形成增强了VIIa的催化功能而非底物识别功能。此外，与游离因子X相比，磷脂上的因子X作为底物被TF1-219.VIIa优先切割。因此，TF依赖的凝血蛋白酶级联反应的启动涉及通过TF跨膜结构域对反应进行膜锚定、TF细胞外结构域与VIIa提供蛋白质-蛋白质相互作用以增强VIIa催化结构域的活性以及因子X与磷脂表面结合时作为底物的优先呈现，从而增强细胞表面因子X的激活。
