Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.
Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.
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DOI：
10.1016/j.biomaterials.2017.04.048
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发表时间：
2017-08
期刊：
影响因子：
14
通讯作者：
Hubbell JA
中科院分区：
文献类型：
--
作者：
Briquez PS;Lorentz KM;Larsson HM;Frey P;Hubbell JA
抑酶蛋白是一种广谱丝氨酸蛋白酶抑制剂，在临床中用作纤维蛋白基组织密封剂中的抗纤溶剂。然而，在再次接触后，一些患者会出现过敏免疫反应，可能与抑蛋白蛋白的牛源性有关。在这里，我们的目标是开发一种人类衍生的抑肽蛋白替代品。基于序列同源性分析，我们确定了人类淀粉样蛋白-β A4前体蛋白的kunitz型蛋白酶抑制剂（KPI）结构域是一个潜在的候选区域。虽然KPI的内在抗纤溶活性低于抑蛋白蛋白，但我们认为，它的功效也受到纤维蛋白材料快速释放的限制，就像抑蛋白蛋白一样。因此，我们设计了KPI变体，以控制纤维蛋白生物材料中的保留，通过结合凝血转谷氨酰胺酶因子XIIIa底物的共价结合或通过设计细胞外基质蛋白超亲和结构域将其隔离到纤维蛋白中。我们发现，这两种工程KPI变体在体外显著减缓了纤溶酶介导的纤维蛋白溶解，优于抑酶蛋白。在体内，我们设计的最佳KPI变体（包含转谷氨酰胺酶底物）在抑蛋白蛋白没有表现出功效的剂量下，将纤维蛋白基质的寿命延长了50%，从而使其成为纤维蛋白密封剂中抑蛋白蛋白的竞争性替代品。
Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin’s is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants.
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