Platelet GpIba binding to von Willebrand Factor under fluid shear:contributions of the D′D3-domain, A1-domain flanking peptide and O-linked glycans.
Platelet GpIba binding to von Willebrand Factor under fluid shear:contributions of the D′D3-domain, A1-domain flanking peptide and O-linked glycans.
复制标题
DOI：
10.1161/jaha.114.001420
复制
发表时间：
2014-10-23
影响因子：
5.4
通讯作者：
Neelamegham S
中科院分区：
文献类型：
--
作者：
Madabhushi SR;Zhang C;Kelkar A;Dayananda KM;Neelamegham S
血管性血友病因子 (VWF) A1 结构域与血小板受体 GpIbα 的结合是一种重要的流体剪切依赖性相互作用，可调节可溶性 VWF 与血小板的结合以及血小板与固定 VWF 的结合。我们评估了 A1 结构域 N 端不同结构元件在调节剪切依赖性血小板结合中的作用。具体来说，重点是 VWF D'D3 结构域、A1 结构域 N 末端侧翼肽 (NFP) 以及该肽上的 O 聚糖。表达了全长二聚体 VWF (ΔPro-VWF)、缺乏 D'D3 结构域的二聚体 VWF (ΔD'D3-VWF) 和缺乏 NFP (ΔD'D3NFP─-VWF) 或仅在该肽上的 O-聚糖 (ΔD'D3OG─-VWF) 的 ΔD'D3-VWF 变体。还产生了单体 VWF-A1 和 D'D3-A1。在 ELISA 中，可溶性 ΔPro-VWF 与固定化 GpIbα 结合的表观解离常数 (KD) (KD≈100 nmol/L) 比缺乏 D'D3 结构域的其他蛋白 (KD~0.7 至 2.5 nmol/L) 高 50 至 100 倍。此外，在表面等离子共振研究中，与单个VWF-A1结构域（kon=5.1±0.4×104（mol/L）−1·s−1；KD=1.2）相比，D′D3-A1与固定化GpIbα结合的结合率（kon=1.8±0.4×104（mol/L）−1·s−1；KD=1.7 μmol/L）降低。微摩尔/升）。因此，VWF-D'D3 主要控制可溶性 VWF 与 GpIbα 的结合。相反，在 VWF 固定后，所有分子特征都调节 A1-GpIbα 结合。在这里，在 ELISA 中，ΔPro-VWF 上可用于结合 GpIbα 的表观 A1 结构域位点的数量约为 ΔD'D3-VWF 变体的 50%。在基于微流体的固定 VWF 血小板粘附测量和胶原蛋白血栓形成测定中，人类血小板募集变化为 ΔPro-VWF<ΔD'D3-VWF<ΔD'D3NFP─-VWF<ΔD'D3OG─-VWF。尽管 VWF-D'D3 是可溶性 VWF 与血小板 GpIbα 结合的主要调节因子，但 D'D3 结构域和 N 末端肽均调节血小板易位和血栓形成。
Von Willebrand Factor (VWF) A1‐domain binding to platelet receptor GpIbα is an important fluid‐shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N‐terminus of the A1‐domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D′D3‐domain, A1‐domain N‐terminal flanking peptide (NFP), and O‐glycans on this peptide. Full‐length dimeric VWF (ΔPro‐VWF), dimeric VWF lacking the D′D3 domain (ΔD′D3‐VWF), and ΔD′D3‐VWF variants lacking either the NFP (ΔD′D3NFP─‐VWF) or just O‐glycans on this peptide (ΔD′D3OG─‐VWF) were expressed. Monomeric VWF‐A1 and D′D3‐A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro‐VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50‐ to 100‐fold higher than other proteins lacking the D′D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on‐rate of D′D3‐A1 binding to immobilized GpIbα (kon=1.8±0.4×104 (mol/L)−1·s−1; KD=1.7 μmol/L) was reduced compared with the single VWF‐A1 domain (kon=5.1±0.4×104 (mol/L)−1·s−1; KD=1.2 μmol/L). Thus, VWF‐D′D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1‐GpIbα binding. Here, in ELISA, the number of apparent A1‐domain sites available for binding GpIbα on ΔPro‐VWF was ≈50% that of the ΔD′D3‐VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro‐VWF<ΔD′D3‐VWF<ΔD′D3NFP─‐VWF<ΔD′D3OG─‐VWF. Whereas VWF‐D′D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D′D3‐domain and N‐terminal peptide regulate platelet translocation and thrombus formation.