Laboratory of Protein Chemistry, Department of Pharmaceutical Sciences, University of Padova, Padua, Italy.
Biochem J. 2012 Mar 1;442(2):423-32. doi: 10.1042/BJ20111798.
CKD (chronic kidney disease) is a life-threatening pathology, often requiring HD (haemodialysis) and characterized by high OS (oxidative stress), inflammation and perturbation of vascular endothelium. HD patients have increased levels of vWF (von Willebrand factor), a large protein (~240 kDa) released as UL-vWF (ultra large-vWF polymers, molecular mass ~20000-50000 kDa) from vascular endothelial cells and megakaryocytes, and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by LSPs (leucocyte serine proteases), released by activated PMNs (polymorphonuclear cells) during bacterial infections. Previous studies have shown that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF that are not only more pro-thrombotic than shorter vWF oligomers, but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD patients have increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In the present study, we first purified vWF from HD patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers that are also more resistant to ADAMTS-13. Using TMS (targeted MS) techniques, we estimated that HD-vWF contains >10% of Met1606 in the sulfoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate platelets more efficiently and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.
慢性肾脏病（CKD）是一种危及生命的疾病，常需接受血液透析（HD）治疗，其特征为氧化应激（OS）水平升高、炎症反应和血管内皮功能紊乱。HD 患者的血管性血友病因子（von Willebrand factor，vWF）水平升高，vWF 是一种从血管内皮细胞和巨核细胞中释放的大型蛋白（~240 kDa），其前体形式为 UL-vWF（超大 vWF 多聚体，分子量约 20000-50000 kDa），在初次止血中发挥作用。vWF 的促止血潜能与其长度相关，其长度由 ADAMTS-13（一种含金属蛋白酶基序的解整合素金属蛋白酶 13）和白细胞丝氨酸蛋白酶（leucocyte serine proteases，LSPs）进行蛋白水解调节。ADAMTS-13 可在 vWF 的 Tyr1605-Met1606 键处将其特异性切开，LSPs 则由细菌感染时被激活的多形核白细胞（polymorphonuclear cells，PMNs）释放。既往研究显示，Met1606 的氧化可抑制 ADAMTS-13 对 vWF 的剪切，导致 UL-vWF 的蓄积，与较短的 vWF 寡聚物相比，这些 UL-vWF 不仅更具促血栓形成作用，而且在脓毒症时与细菌黏附因子的结合能力更强。值得注意的是，HD 患者发生严重心血管和脓毒症并发症的风险增加，但其潜在机制尚不清楚。在本研究中，我们首先从 HD 患者中纯化 vWF，然后对其氧化状态进行化学表征。有趣的是，HD-vWF 含有高水平的羰基，且含有更多的 UL-vWF 多聚体，其对 ADAMTS-13 的抵抗性也更强。通过靶向 MS（targeted MS）技术，我们估计 HD-vWF 中有超过 10%的 Met1606 处于亚砜形式。我们的结论为，Met1606 的氧化会抑制 ADAMTS-13 的剪切，导致 UL-vWF 多聚体的蓄积，这会更有效地募集和激活血小板，并与细菌黏附因子更紧密地结合，从而导致 CKD 患者血栓形成和脓毒症并发症的发生。
