Tait J F, Engelhardt S, Smith C, Fujikawa K
Department of Laboratory Medicine, University of Washington, Seattle 98195, USA.
J Biol Chem. 1995 Sep 15;270(37):21594-9. doi: 10.1074/jbc.270.37.21594.
Annexin V is a human protein that binds with high affinity to the abundant phosphatidylserine molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. We designed two chimeras that use annexin V as a means to target thrombolytic agents to platelet-containing thrombi: prourokinase (1-411)-annexin V (1-320); and prourokinase (144-411)-annexin V (1-320) (amino acid numbers of parent proteins given in parentheses). Chimeras were produced by cytoplasmic expression in Escherichia coli, refolded, and purified in single-chain form. Both chimeras had the same specific activity as annexin V in binding to cell membranes containing exposed phosphatidylserine. After activation with plasmin, both chimeras had specific amidolytic activity similar to that of urokinase. Both chimeras activated plasminogen in vitro with kinetic parameters similar to those for urokinase, and both showed full activity compared to urokinase in an assay of clot lysis in vitro. This study shows the feasibility of producing chimeric plasminogen activators in which annexin V provides the thrombus-targeting component; although not yet tested in vivo, such chimeras may have advantages over antibody-based targeting agents.
膜联蛋白V是一种人类蛋白质，它能与活化血小板上大量暴露的磷脂酰丝氨酸分子高亲和力结合，并在动脉血栓形成的动物模型中静脉给药后选择性地在血栓中积聚。我们设计了两种嵌合体，利用膜联蛋白V将溶栓剂靶向含血小板血栓：单链尿激酶原（1-411）-膜联蛋白V（1-320）；以及单链尿激酶原（144-411）-膜联蛋白V（1-320）（括号内为亲本蛋白的氨基酸编号）。嵌合体通过在大肠杆菌中的细胞质表达产生，重新折叠，并以单链形式纯化。两种嵌合体在与含有暴露磷脂酰丝氨酸的细胞膜结合方面具有与膜联蛋白V相同的比活性。用纤溶酶激活后，两种嵌合体都具有与尿激酶相似的特异性酰胺水解活性。两种嵌合体在体外激活纤溶酶原的动力学参数与尿激酶相似，并且在体外凝块溶解试验中与尿激酶相比都显示出完全活性。这项研究表明了生产嵌合纤溶酶原激活剂的可行性，其中膜联蛋白V提供血栓靶向成分；尽管尚未在体内进行测试，但这种嵌合体可能比基于抗体的靶向剂具有优势。
