Department of Pharmacology, University of Illinois College of Medicine, Chicago (J.L., K.K., S.-Y.J, B.X., X. Du, J. Cho).
Korean Medicine-Application Center, Korea Institute of Oriental Medicine, Daegu (K.K.).
Circulation. 2019 Mar 5;139(10):1300-1319. doi: 10.1161/CIRCULATIONAHA.118.036323.
Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions.
Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions.
Deletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage.
Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.
血小板-中性粒细胞相互作用导致血栓炎症性疾病中的血管闭塞和组织损伤。血小板糖蛋白 Ibα（GPIbα）是细胞间相互作用的关键受体，被认为对配体结合具有组成性活性。在这里，我们确定了血小板源性蛋白二硫键异构酶（PDI）在减少血栓炎症条件下 GPIbα 中的变构二硫键并增强配体结合活性中的作用。
生物信息学分析鉴定了 GPIbα 中的 2 个潜在变构二硫键。聚集测定、流式细胞术、表面等离子体共振分析、蛋白质-蛋白质对接模型、临近连接测定和质谱用于证明 PDI 与 GPIbα 之间的直接相互作用，并确定 PDI 在调节 GPIbα 功能和血小板-中性粒细胞相互作用中的作用。此外，实时显微镜和动物疾病模型用于研究血栓炎症条件下 PDI-GPIbα 信号的病理生理作用。
血小板 PDI 的缺失或抑制显著降低了 GPIbα 介导的血小板聚集。使用 PDI 缺陷型血小板和重组 PDI 或 Anfibatide（一种临床阶段的 GPIbα 抑制剂）的研究表明，血小板表面结合的 PDI 的氧化还原酶活性是 GPIbα 配体结合功能所必需的。PDI 直接结合到血小板表面 GPIbα 的细胞外结构域，并减少 Cys4-Cys17 和 Cys209-Cys248 二硫键。血小板特异性 PDI 条件性敲除和镰状细胞病小鼠的实时显微镜研究表明，PDI 调节的 GPIbα 功能对于血栓炎症条件下的血小板-中性粒细胞相互作用和血管闭塞至关重要。使用缺血/再灌注诱导的中风小鼠模型的研究表明，PDI-GPIbα 信号在组织损伤中起关键作用。
我们的结果表明，PDI 促进的变构二硫键的切割紧密调节 GPIbα 功能，促进血栓炎症条件下的血小板-中性粒细胞相互作用、血管闭塞和组织损伤。
