Sheffield William P, Gataiance Sharon, Eltringham-Smith Louise J
McMaster University, Department of Pathology and Molecular Medicine, HSC 4N66, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
Thromb Res. 2007;119(2):195-207. doi: 10.1016/j.thromres.2006.01.003. Epub 2006 Feb 14.
There are continuing needs for new antithrombotic agents and procedures. We hypothesized that the slowly cleared recombinant fusion proteins barbourin--albumin (BLAH6) and hirudin--albumin (HLAH6) would be effective in limiting fibrin(ogen) and/or platelet deposition in a rabbit model of arterial injury.
Recombinant fusion proteins were expressed in Pichia pastoris fermenter cultures and purified by nickel-chelate affinity chromatography. They were injected intravenously into rabbits prior to blood sampling and platelet aggregometry, assessment of deposition of 125I-fibrin(ogen) and 51Cr-platelet onto the balloon-injured thoracic aorta, electron microscopy (EM) and immunohistochemistry of aortic sections, and determination of bleeding time following a standardized ear incision.
BLAH6 administration elicited a dose- and time-dependent inhibition of platelet aggregation in post-injection whole blood samples, and reduced both fibrin(ogen) and platelet deposition on the injured aorta, although the former effect was both more durable and more significant than the latter. In contrast, HLAH6 injection reduced fibrin(ogen) but not platelet deposition. Doses of the two proteins ineffective in preventing fibrin(ogen) deposition when given alone were effective when combined, suggesting at least additive effects. Immunohistochemistry and EM supported the radioactive deposition studies, while bleeding times were decreased with combined BLAH6 and HLAH6 administration compared to HLAH6 alone in a rabbit ear bleeding model. The data show that these fusion proteins exert an antithrombotic effect in vivo and may indicate that combined low-dose administration of antiplatelet and antithrombin agents could offer safety advantages in the treatment of thrombosis.
对新型抗血栓药物和治疗方法的需求持续存在。我们推测，清除缓慢的重组融合蛋白巴伯林 - 白蛋白（BLAH6）和水蛭素 - 白蛋白（HLAH6）在兔动脉损伤模型中可有效限制纤维蛋白（原）和/或血小板沉积。
重组融合蛋白在毕赤酵母发酵罐培养物中表达，并通过镍螯合亲和层析纯化。在进行血液采样和血小板聚集测定、评估125I - 纤维蛋白（原）和51Cr - 血小板在球囊损伤胸主动脉上的沉积、主动脉切片的电子显微镜（EM）和免疫组织化学分析以及标准化耳切口后出血时间测定之前，将它们静脉注射到兔子体内。
给予BLAH6后，注射后全血样本中的血小板聚集受到剂量和时间依赖性抑制，并且损伤主动脉上的纤维蛋白（原）和血小板沉积均减少，尽管前者的作用比后者更持久且更显著。相比之下，注射HLAH6可减少纤维蛋白（原）沉积，但不减少血小板沉积。单独使用时对预防纤维蛋白（原）沉积无效的两种蛋白剂量联合使用时有效，表明至少具有相加作用。免疫组织化学和EM支持放射性沉积研究，并且在兔耳出血模型中，与单独给予HLAH6相比，联合给予BLAH6和HLAH6可缩短出血时间。数据表明这些融合蛋白在体内发挥抗血栓作用，并且可能表明联合低剂量使用抗血小板和抗凝血酶药物在血栓形成治疗中可能具有安全性优势。
