Modification of rattle snake venom derived blood clot dissolver, Alfimeprase, by fusion with C-terminal domain of habu-HR1a and fibrin-targeting peptide

Alfimeprase(A)能直接消化纤维蛋白，不产生与tPA （Activase）不同的纤溶酶。由于A能被α -2巨球蛋白（alpha- 2m）快速捕获并在静脉内灭活，因此A在二期临床的效果一直不太好，无法推进其发展。我们设计了A的c端侧与HR1a的c端结构域（胶原结合结构域）融合的基因。考虑到AH基因靶向于纤维蛋白凝块，我们将AH基因的c端与人纤溶酶原衍生的kringle结构域1区基因融合，这种融合蛋白被称为AHP。将A、AH和AHP基因插入pE-SUMO载体，在大肠杆菌BL21（DE3）星中表达。无论是否存在n端sumo肽，HR1a c端结构域与A的融合在避免α - 2m失活方面没有任何影响。

Alfimeprase(A) can digest fibrin directly, and do not produce any plasmin in different from tPA (Activase). Because A can be rapidly trapped by alpha-2 macroglobulin (alfpha-2M) and inactivated in the blood vein, the clinical effect of A in the phase 2 has not been so good to advance its development. We designed the gene in which the C-terminal side of the A was fused with the C-terminal domain (collagen-binding domain) of the HR1a. Considering targeting to fibrin clots, the C-terminal side of the AH gene was fused with the gene for the human plasminogen-derived kringle domain 1 region, which fusion protein is called AHP. The A, AH and AHP genes were inserted into the pE-SUMO vectors and were expressed in the E.coli BL21(DE3)star. Fusion of the HR1a C-teriminal domain with A did not have any effect in terms of avoiding of inactivation by alpha-2M regardless of the presence or absence of the N-terminal SUMO-peptide.