Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology & Emory University, USA.
School of Chemical, Materials, and Biomedical Engineering, University of Georgia, USA.
Biochim Biophys Acta Gen Subj. 2018 Sep;1862(9):1925-1932. doi: 10.1016/j.bbagen.2018.06.015. Epub 2018 Jun 23.
Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described.
Fibrin gels were polymerized and incubated with recombinant human cathepsins (cat) K, L, or S, or plasmin, for dose-dependent and time-dependent studies. Dissolution of fibrin gels was imaged. SDS-PAGE was used to resolve cleaved fragments released from fibrin gels and remnant insoluble fibrin gel that was solubilized prior to electrophoresis to assess fibrin α, β, and γ polypeptide hydrolysis by cathepsins. Multiplex cathepsin zymography determined active amounts of cathepsins remaining.
There was significant loss of α and β fibrin polypeptides after incubation with cathepsins, with catS completely dissolving fibrin gel by 24 h. Binding to fibrin stabilized catL active time; it associated with cleaved fibrin fragments of multiple sizes. This was not observed for catK or S. CatS also remained active for longer times during fibrin incubation, but its association/binding did not withstand SDS-PAGE preparation.
Human cathepsins K, L, and S are fibrinolytic, and specifically can degrade the α and β fibrin polypeptide chains, generating fragments unique from plasmin.
Demonstration of cathepsins K, L, and S fibrinolytic activity leads to further investigation of contributory roles in disrupting vascular hemostasis, or breakdown of fibrin-based engineered vascular constructs where non-plasmin mediated fibrinolysis must be considered.
纤维蛋白的形成和溶解归因于蛋白酶级联激活，最终导致凝血酶激活和纤维蛋白降解的纤溶酶蛋白水解。半胱氨酸组织蛋白酶是内皮细胞和其他细胞在心血管疾病和糖尿病期间分泌的强大蛋白酶。它们的纤溶活性及其在止血中的潜在作用尚未得到很好的描述。
纤维蛋白凝胶聚合后，用重组人组织蛋白酶（cat）K、L 或 S 或纤溶酶孵育，进行剂量依赖性和时间依赖性研究。凝胶溶解的图像。SDS-PAGE 用于解析从纤维蛋白凝胶释放的被切割片段和残余不溶性纤维蛋白凝胶，该凝胶在电泳前溶解以评估组织蛋白酶对纤维蛋白α、β和γ多肽的水解。多通道组织蛋白酶酶谱测定法确定剩余组织蛋白酶的活性量。
与组织蛋白酶孵育后，α 和 β 纤维蛋白多肽明显丢失，catS 在 24 小时内完全溶解纤维蛋白凝胶。与纤维蛋白结合稳定了 catL 的活性时间；它与多种大小的切割纤维蛋白片段结合。在 catK 或 S 中未观察到这种情况。CatS 在纤维蛋白孵育过程中也保持更长时间的活性，但它的结合/绑定不能承受 SDS-PAGE 准备。
人组织蛋白酶 K、L 和 S 具有纤维蛋白溶解活性，并且可以特异性降解 α 和 β 纤维蛋白多肽链，生成不同于纤溶酶的片段。
组织蛋白酶 K、L 和 S 的纤溶活性的证明导致进一步研究其在破坏血管止血或基于纤维蛋白的工程血管构建体的破坏中的贡献作用，在这些构建体中必须考虑非纤溶酶介导的纤维蛋白溶解。
