Hematology-Oncology Division, Department of Medicine, University of California, San Diego, La Jolla, CA 92093;
Hematology-Oncology Division, Department of Medicine, University of California, San Diego, La Jolla, CA 92093.
Proc Natl Acad Sci U S A. 2019 Mar 12;116(11):4983-4988. doi: 10.1073/pnas.1819156116. Epub 2019 Feb 25.
Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.
血小板介导体液止血，最近的研究强调了血小板在免疫和炎症中的作用。血小板特异性整合素αIIbβ3 作为纤维蛋白原受体在止血中的功能已得到明确界定，但αIIbβ3 或整合素相关蛋白在非止血性血小板功能中的作用仍知之甚少。本文作者展示了人血小板表达整合素相关蛋白 SHARPIN 及其具有功能意义。在白细胞中，SHARPIN 与整合素α 胞质尾相互作用，它也是线性泛素链组装复合物（LUBAC）的必需成员，该复合物介导蛋白 Met1 线性泛素化，从而导致经典 NF-κB 激活。SHARPIN 在下拉和共沉淀实验中与αIIb 相互作用。SHARPIN 与αIIbβ3 部分定位于血小板边缘，凝血酶刺激诱导更多的中央 SHARPIN 定位。SHARPIN 也与 LUBAC 的另外两个蛋白，E3 连接酶 HOIP 和 HOIL-1 从血小板中共沉淀。血小板用凝血酶或炎症激动剂（包括脂多糖或可溶性 CD40 配体（sCD40L））刺激诱导 NF-κB 通路蛋白 NEMO 的 Met1 线性泛素化和 NF-κB 的 p65RelA 亚基丝氨酸-536 磷酸化。在人巨核细胞和/或源自诱导多能干细胞（iPS）细胞的血小板中，SHARPIN 敲低导致基础和激动剂诱导的纤维蛋白原与αIIbβ3 的结合增加，以及 Met1 泛素化和 RelA 磷酸化减少。此外，这些 SHARPIN 敲低细胞表现出 MHC Ⅰ类分子的表面表达增加和 sCD40L 的释放增加。这些结果表明，SHARPIN 通过整合素和免疫/炎症信号交汇点的蛋白相互作用在人巨核细胞/血小板谱系中发挥作用。
