From the Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia (J.B., J.X., Y.M., X.L.Z.); The University of Pennsylvania Perelman School of Medicine, Philadelphia (X.L.Z.); and the Department of Obstetrics and Gynecology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China (J.X., X.L.Z.).
Arterioscler Thromb Vasc Biol. 2014 Feb;34(2):397-407. doi: 10.1161/ATVBAHA.113.302547. Epub 2013 Dec 19.
ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, 13) cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface-exposed free sulfhydryls on ADAMTS13 that seemed to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow.
A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2 to 8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5 to 8 Thrombospondin type 1 (TSP1) repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging analysis showed that the carboxyl-terminal fragment of ADAMTS13 (T2C or T5C) was capable of inhibiting the formation and elongation of platelet-decorated ultra large (UL) VWF strings and the adhesion of platelets/leukocytes on endothelium in mesenteric venules after oxidative injury. The inhibitory activity of T2C and T5C on platelet aggregation and ULVWF string formation were dependent on the presence of their surface free thiols; pretreatment of T2C and T5C or full-length ADAMTS13 with N-ethylmaleimide that reacts with free sulfhydryls abolished or significantly reduced its antithrombotic activity.
Our results demonstrate for the first time that the carboxyl terminus of ADAMTS13 has direct antithrombotic activity in a free-thiol-dependent manner. The free thiols in the carboxyl-terminal domains of ADAMTS13 may also contribute to the overall antithrombotic function of ADAMTS13 under pathophysiological conditions.
ADAMTS13（解整合素和金属蛋白酶与血小板反应素 1 型重复，13）切割血管性血友病因子（VWF），从而抑制血栓形成。蛋白水解切割依赖于氨基末端（MDTCS）结构域，但 ADAMTS13 的更远端羧基末端结构域的作用尚不完全清楚。先前的研究表明，ADAMTS13 上存在多个表面暴露的游离巯基，这些巯基似乎在剪切下与 VWF 上的巯基相互作用。在这里，我们确定了这种相互作用在流动状态下抗血栓反应中的生理相关性。
微流控分析表明，ADAMTS13 的羧基末端片段，包括 2 至 8 个血小板反应素 1 型（TSP1）重复和 CUB 结构域（T2C）或 5 至 8 个血小板反应素 1 型（TSP1）重复和 CUB 结构域（T5C），可直接抑制动脉剪切下胶原表面上的血小板黏附/聚集。此外，活体显微镜成像分析表明，ADAMTS13 的羧基末端片段（T2C 或 T5C）能够抑制血小板修饰的超大（UL）VWF 串的形成和延伸，以及氧化损伤后肠系膜静脉内皮上血小板/白细胞的黏附。T2C 和 T5C 对血小板聚集和 ULVWF 串形成的抑制活性依赖于其表面游离巯基的存在；用与游离巯基反应的 N-乙基马来酰亚胺预处理 T2C 和 T5C 或全长 ADAMTS13 会消除或显著降低其抗血栓活性。
我们的结果首次证明，ADAMTS13 的羧基末端以游离巯基依赖的方式具有直接的抗血栓活性。ADAMTS13 的羧基末端结构域中的游离巯基也可能有助于 ADAMTS13 在病理生理条件下的整体抗血栓功能。
