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Immobilization of human thrombomodulin onto polymeric thinfilms for biomedical material

将人血栓调节蛋白固定在用于生物医学材料的聚合物薄膜上

基本信息

批准号：
16300156
负责人：
AKASHI Mitsuru
金额：
$ 9.22万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

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.

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蛋白酶转化为抗凝血剂，具有有效的天然抗凝血活性。我们之前报道过通过与基材表面形成共价键将 hTM 固定到聚合物膜表面。我们发现hTM固定化材料具有优异的抑制人体血液凝固和血小板聚集的特性。然而，生物活性蛋白的活性通过共价固定化过程而降低，并且化学固定化还存在留下残留化学污染物（例如缩合试剂和化学接头）的风险。在本研究中，我们重点研究了 hTM 在聚合生物材料表面的物理吸附，因为物理吸附可以防止变性并且不会留下化学试剂残留。通过石英晶体微天平 (QCM) 分析对 hTM 在聚砜 (PSF) 膜上的吸附进行定量分析。通过朗缪尔型吸附假设计算的吸附常数和最大吸附量表明，hTM 以相对较弱的相互作用吸附到 PSF 膜上。物理吸附的 hTM 显示出用于激活蛋白 C 的高辅酶活性以及抗凝血活性。此外，PSF薄膜的表面润湿性很容易通过疏水性和亲水性生物活性蛋白的物理吸附来控制。吸附在透析器表面的hTM的抗凝活性也得到离体动物研究的证实。通过层层组装的方式制备了各种生物相容性纳米薄膜，可用于hTM吸附。我们通过将hTM物理吸附到高分子薄膜上，成功开发了血液相容性生物医用材料，阐明了hTM的吸附机理，获得了关于血液相容性生物医用材料的重要知识。