Suzuki K, Hayashi T, Nishioka J, Kosaka Y, Zushi M, Honda G, Yamamoto S
Department of Laboratory Medicine, Mie University School of Medicine, Japan.
J Biol Chem. 1989 Mar 25;264(9):4872-6.
Thrombomodulin, an endothelial thrombin receptor, acts as a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. The extracellular region of human thrombomodulin consists of three tentative domains, a NH2-terminal domain (D1), a domain involving six consecutive epidermal growth factor-like structures (D2), and an O-glycosylation-rich domain (D3). To identify the domain onto which thrombin binds, a series of recombinant proteins corresponding to the entire protein, D1, D2, D1 + D2, D1 + D2 + D3, and D2 + D3 were expressed in simian COS-1 cells. The proteins were partially purified by rabbit anti-thrombomodulin-F(ab')2-agarose chromatography. Western blotting analysis showed the expression of the respective recombinant proteins. All proteins involving D2, as well as D2 alone, had cofactor activity that allowed binding directly to thrombin, but D1 did not. The cofactor activity of the entire protein but not the mutants is increased in the presence of phospholipids and this is the only protein that binds to the phospholipid layer. These results indicate that the domain involving the epidermal growth factor-like structures of thrombomodulin is essential for thrombin binding and expression of the cofactor activity for protein C activation and that none of the extracellular domains interact with phospholipids.
血栓调节蛋白是一种内皮凝血酶受体，作为凝血酶催化的抗凝蛋白C活化的辅因子。人血栓调节蛋白的细胞外区域由三个暂定结构域组成，一个氨基末端结构域（D1）、一个包含六个连续表皮生长因子样结构的结构域（D2）和一个富含O-糖基化的结构域（D3）。为了确定凝血酶结合的结构域，在猴COS-1细胞中表达了一系列与完整蛋白、D1、D2、D1+D2、D1+D2+D3和D2+D3相对应的重组蛋白。通过兔抗血栓调节蛋白-F(ab')2-琼脂糖层析对这些蛋白进行了部分纯化。蛋白质印迹分析显示了各个重组蛋白的表达。所有包含D2的蛋白以及单独的D2都具有辅因子活性，能够直接与凝血酶结合，但D1没有。在磷脂存在的情况下，完整蛋白而非突变体的辅因子活性增加，并且这是唯一与磷脂层结合的蛋白。这些结果表明，血栓调节蛋白中包含表皮生长因子样结构的结构域对于凝血酶结合以及蛋白C活化的辅因子活性表达至关重要，并且细胞外结构域均不与磷脂相互作用。
