The Centenary Institute, University of Sydney, Camperdown, NSW, Australia.
Research School of Chemistry, Australian National University, Canberra, ACT, Australia; and.
Blood. 2021 Oct 14;138(15):1359-1372. doi: 10.1182/blood.2021012441.
The αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.
αIIbβ3 整合素受体协调血栓形成和止血过程中的血小板黏附、激活和机械感知。使用差异半胱氨酸烷基化和质谱分析，我们在 αIIb 亚基中鉴定出一个二硫键，连接着静息和激活的人血小板表面上约 1/3 的整合素分子中缺失的 490 位和 545 位半胱氨酸。这种 αIIbβ3 的替代共价形式是预先确定的，因为它也存在于重组整合素转染的人巨核母细胞和幼仓鼠肾成纤维细胞中。通过共免疫沉淀实验，该替代形式选择性地分配到激活血小板表面的焦点黏附上。我们在表达一种 C490-C545 二硫键缺失突变整合素的幼仓鼠肾成纤维细胞中评估了其功能。该二硫键突变整合素具有功能性的内-外信号转导，但由于网格蛋白介导的整合素内化和再循环的速率降低，其在焦点黏附中的居留时间延长，这与 αIIb 亚基对网格蛋白衔接蛋白 2 的亲和力增强有关。分子动力学模拟表明，替代的共价形式的 αIIb 需要更高的力才能从弯曲到开放的构象状态转变，这与对纤维蛋白原的亲和力降低以及锰离子的激活相一致。这些发现表明，αIIbβ3 整合素受体以不同的细胞表面分布和功能产生各种共价形式。C490、C545 半胱氨酸对在所有 18 种整合素 α 亚基中都保守存在，并且在培养细胞中的 αV 和 α2 亚基中二硫键也同样缺失，这表明替代的整合素形式和功能也具有保守性。
