Smith Jason D, Chen Andrew, Ernst Lauren A, Waggoner Alan S, Campbell Phil G
Institute for Complex Engineered Systems and Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Bioconjug Chem. 2007 May-Jun;18(3):695-701. doi: 10.1021/bc060265o. Epub 2007 Apr 14.
The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.
(1)保持其功能;(2)控制纤维蛋白降解。通过鸡胚绒毛尿囊膜(CAM)试验,我们发现血管不会直接侵入含有固定化成纤维细胞生长因子-2的纤维蛋白构建体。由于纤维蛋白在大约5天内迅速降解,我们推测控制纤维蛋白溶解可能会改善血管的直接侵入。抑肽酶是一种通常添加用于减缓纤维蛋白溶解的蛋白酶抑制剂,它是一种小蛋白,会从凝胶中扩散出来,导致失去对纤维蛋白溶解的保护作用。因此,采用一种新颖的合成策略,将抑肽酶和荧光报告分子Cy3化学结合到纤维蛋白原上。体外微孔板吸光度测定表明,结合的抑肽酶能够抑制纤溶酶介导的纤维蛋白降解,其活性与等摩尔水平的可溶性、未结合的抑肽酶相当。此外,我们发现通过改变凝胶中结合的抑肽酶水平可以调节纤维蛋白溶解速率。结合的抑肽酶在体内也表现出功能。在鸡胚绒毛尿囊膜试验中,含有结合抑肽酶的纤维蛋白凝胶在4天后比含有可溶性抑肽酶的凝胶大约大5倍。同样,为支持我们的假设,我们发现纤维蛋白凝胶中固定化的抑肽酶显示出大量血管侵入。