武汉云克隆科技股份有限公司

Abstract:

Adequate cleavage of von Willebrand factor (VWF) prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM) was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3), cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

摘要：

充分裂解血管性血友病因子(VWF)可以防止血栓形成。ADAMTS13是主要的VWF裂解蛋白酶，其缺乏将导致血栓形成性微血管病变。其他蛋白酶也可能具有VWF裂解活性，但在预防血栓形成中发挥的生理作用尚不清楚。本研究探讨了在肾小球中是否存在具有VWF裂解活性的蛋白酶以及种类。以肾小球基底膜(GBM)的含量作为肾小球内皮下间隙过程的反映。用人的GBMs孵育VWF，采用多聚体结构分析、免疫印迹和质谱分析。通过GBM将VWF裂解为最小的多聚体，其中含有ADAMTS13、中性粒细胞蛋白酶、弹性蛋白酶、蛋白酶3(PR3)、组织蛋白酶G和基质金属蛋白酶9。研究发现GBM中最有效的组分是丝氨酸蛋白酶或金属蛋白酶类，而不是ADAMTS13。中性粒细胞丝氨酸蛋白酶的中和作用抑制了GBM介导的VWF裂解活性，表明弹性蛋白酶和/或PR3发挥重要作用。在灌流系统中，弹性蛋白酶和GBM酶解原代肾小球内皮细胞表面聚集的VWF-血小板，弹性蛋白酶抑制剂可以阻断此过程。对人肾脏的超微结构研究表明，中性粒细胞向GBM释放弹性蛋白酶。中性粒细胞蛋白酶可能参与调控血管内皮细胞分裂，与GBM共同作用，从而调节血栓大小。这种抗血栓机制可以在炎症发生时保护肾脏免受损害，也解释了为什么大多数ADAMTS13缺乏的患者未出现严重的肾功能衰竭。

使用试剂原文信息: Levels of ADAMTS13 (Cloud-CloneCorp,Houston,TX),elastase (Hycult Biotech, Uden, Holland), cathepsin G (Nordic BioSite, Täby, Sweden) and MMP9 (R&D Systems, Minneapolis, MN) were measured by ELISA kits according to the manufacturers' protocols. The level of PR3 was measured by ELISA as previously described (Kahn et al., 2009).