Nawa K, Sakano K, Fujiwara H, Sato Y, Sugiyama N, Teruuchi T, Iwamoto M, Marumoto Y
Research Institute, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.
Biochem Biophys Res Commun. 1990 Sep 14;171(2):729-37. doi: 10.1016/0006-291x(90)91207-9.
We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.
我们使用劳斯肉瘤病毒（RSV）启动子构建了人可溶性血栓调节蛋白（sTM）表达载体。重组sTM（rsTM）在CHO细胞中表达，并通过离子交换色谱法从培养基中回收。检测到两个活性组分，分别命名为rsTMα（低盐洗脱）和rsTMβ（高盐洗脱），并通过免疫亲和色谱法进一步纯化。通过对软骨素酶ABC和AC I消化产物2-乙酰氨基-2-脱氧-3-O-（β-D-葡萄糖-4-烯吡喃糖醛酸）-4-O-磺基-D-半乳糖的HPLC检测判断，纯化后的rsTMβ含有结合的硫酸软骨素-4。α和β对凝血酶的表观解离常数（Kd）值分别为7.4和1.4 nM。RsTMβ在抑制凝血酶凝血活性方面更有效，并且具有rsTMα所不具备的抗凝血酶III依赖性抗凝活性。软骨素酶处理rsTMβ后，两种抗凝活性均丧失。
