Liu L W, Rezaie A R, Carson C W, Esmon N L, Esmon C T
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City.
J Biol Chem. 1994 Apr 22;269(16):11807-12.
Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.
血栓调节蛋白（TM）与凝血酶结合形成一种复合物，该复合物可激活血浆抗凝酶原蛋白C。TM是一种整合膜糖蛋白，含有硫酸软骨素部分。与凝血酶的相互作用涉及TM的蛋白质成分，特别是生长因子样重复序列4-6（TM 4-6）以及硫酸软骨素。去除硫酸软骨素会使对凝血酶的亲和力降低约10倍，并将蛋白C激活的Ca2+依赖性从在≥500μM Ca2+时的简单饱和状态转变为在约100μM Ca2+时的明显最佳状态。凝血酶具有两个高正电荷区域，即阴离子结合外位点1和2。阴离子结合外位点1与TM的生长因子区域相互作用，而外位点2则参与凝血酶原激活片段2或肝素的结合。我们证明，在跨膜结构域处截断的重组TM或TM 4-6在片段2共价连接（中凝血酶去片段1）或可逆结合时能够结合凝血酶。对于中凝血酶去片段1，蛋白C激活的Ca2+依赖性与TM上硫酸软骨素的存在无关。在0.27 mM Ca2+时，含有硫酸软骨素的TM结合凝血酶（Kd(app)=0.3 nM）的紧密程度约为中凝血酶去片段1（Kd(app)=14 nM）的45倍。然而，不含硫酸软骨素的形式结合凝血酶（Kd(app)=2.4 nM）的紧密程度仅约为中凝血酶去片段1（Kd(app)=9.4 nM）的4倍。这些研究表明，硫酸软骨素或片段2占据阴离子结合外位点2会改变凝血酶的构象，从而导致蛋白C激活的Ca2+依赖性发生改变。
