A study on human thrombomodulin-immobilized antithrombogenic biomaterials

血栓调节蛋白(TM)是一种新发现的内皮细胞相关蛋白，通过将凝血酶从一种蛋白酶转化为一种抗凝剂而发挥强大的天然抗凝剂的作用。近年来，利用基因技术成功地制备了重组人血栓调节蛋白(HTM)，含有HTM的生物材料引起了人们的兴趣。在本项目中，我们用水溶性碳二亚胺将HTM固定在各种聚合物表面，如聚丙烯酸或聚乙烯胺接枝聚醚氨酯尿素或聚乙二亚胺薄膜。结果表明，HTM的活性被保留到足以显示其抗血栓形成的程度。然后将HTM固定在由纤维素膜组成的小型透析器上进行血流测试。此外，将HTM片段(E456)固定在聚乙烯膜上，并与天然hTM的固定量、活性和抗凝血性进行了比较。从固定量来看，HTM片段的固定量明显高于天然hTM。最后，为了比较天然hTM及其片段的活性，用表面等离子体共振法测定了f-HTM与凝血酶亲和作用的解离常数。

Thrombomodulin (TM) is a newly described endothelial cell associated protein that functions as a potentnatural anticoagulant by converting thrombin from a protease to an anticoagulant. Recently, the preparation of reconbinant human thrombomodulin (hTM) was achieved by using gene technology, and since then biomaterials containing hTM have been of interest. In this project, we have done the hTM-immobilization onto various polymer surfaces such as poly (acrylic acid) or poly (vinylamine) grafted poly (ether urethane urea) or poly (ethylene) film using water soluble carbodiimide. As the results, it was apparent that hTM activity was retained enough to show its antithrombogenicity. And then hTM was immobilized onto small dialyzer consisting of cellulose film to carry out blood flow test. Moreover, hTM fragment (E456) was immobilized onto poly (ethylene) film and compared its amount of immobilized, activity and antithrombogenicity with native hTM.With regard to the amount of immobilized, it was apparent that the amount of immobilized of hTM fragment is higher than that of native hTM.Finally, as to compare the activity of native hTM and its fragment, the dissociation constants of f-hTM and thrombin by affinity interaction was evaluated using a surface plasmon resonance.