Immobilization of human thrombomodulin onto polymeric thinfilms for biomedical material

人血栓调节蛋白(HTM)是一种内皮细胞相关蛋白，通过将凝血酶从促凝血酶转化为抗凝血剂而具有很强的天然抗凝活性。我们以前报道过通过与基片表面形成共价键将HTM固定在聚合物薄膜表面。我们发现，HTM固定化材料具有良好的抑制人体血液凝血和血小板聚集的性能。然而，通过共价固定化过程降低了生物活性蛋白质的活性，化学固定化也有留下缩合试剂和化学连接物等残留化学污染物的风险。利用石英晶体微天平(QCM)定量分析了HTM在聚砜(PSF)膜上的吸附情况，研究了HTM在聚合物生物材料表面的物理吸附行为，因为物理吸附可以防止变性，不会留下残留的化学试剂。根据朗缪尔型吸附的假设计算了HTM在PSF膜上的吸附常数和最大吸附量，结果表明HTM在PSF膜上的吸附作用较弱。物理吸附的HTM对蛋白C具有较高的辅酶活性和抗凝血活性。此外，PSF膜的表面润湿性很容易通过疏水和亲水生物活性蛋白的物理吸附来控制。体外动物实验也证实了吸附在透析器表面的HTM的抗凝血活性。通过层层组装的方法制备了多种生物相容的纳米膜，并将其应用于HTM的吸附，通过聚合物薄膜对HTM的物理吸附，成功地开发出了血液相容的生物医学材料，阐明了HTM的吸附机理，获得了对血液相容的生物医学材料的重要认识。

Human thrombomodulin (hTM) is an endothelial cell-associated protein with potent natural anticoagulant activity by converting thrombin from a procoagulant protease to an anticoagulant. We previously reported the immobilization of hTM onto polymeric film surface by covalent bond formation with the surface of the substrates. We discovered that hTM-immobilized materials had excellent properties for inhibiting both the coagulation and platelet aggregation of human blood. However, the activity of bioactive proteins is decreased through the covalent immobilization process, and chemical immobilization also has the risk of leaving remnant chemical contaminants such as condensation reagents and chemical linkers. In this study, we focused on the physical adsorption of hTM onto a polymeric biomaterial surface because physical adsorption can prevent denaturation and leave no remnant chemical reagents.The adsorption of hTM onto polysulfone (PSF) films was analyzed quantitatively by quartz crystal microbalance (QCM) analysis. The adsorption constant and the maximum adsorption amount, calculated by the assumption of a Langmuir-type adsorption, showed that hTM adsorbed with a relatively weak interaction onto the PSF film. The physically adsorbed hTM showed high co-enzymatic activity for the activation of protein C, as well as anticoagulant activity. Furthermore, the surface wettability of the PSF film was easily controllable by the physical adsorption of hydrophobic and hydrophilic bioactive proteins. The anticoagulant activity of hTM adsorbed on the surface of dialyzer was also confirmed by ex vivo animal studies. The various biocompatible nanofilms were fabricated by layer-by-layer assembly, which can be applied for hTM adsorption.We have successfully developed the blood-compatible biomedical materials by physical adsorption of hTM onto polymeric thinfilms, clarified the adsorption mechanism of hTM, and obtained significant knowledge about blood-compatible biomedical materials.