Madabhushi Sri R, Zhang Changjie, Kelkar Anju, Dayananda Kannayakanahalli M, Neelamegham Sriram
Department of Chemical and Biological Engineering and The NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY (S.R.M., C.Z., A.K., K.M.D., S.N.).
J Am Heart Assoc. 2014 Oct 23;3(5):e001420. doi: 10.1161/JAHA.114.001420.
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±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.7 μmol/L) was reduced compared with the single VWF-A1 domain (kon=5.1±0.4×10(4) (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.
血管性血友病因子（VWF）的A1结构域与血小板受体糖蛋白Ibα（GpIbα）的结合是一种重要的流体剪切力依赖性相互作用，它既调节可溶性VWF与血小板的结合，也调节血小板与固定化VWF的栓系。我们评估了A1结构域N端不同结构元件在调节剪切力依赖性血小板结合中的作用。具体而言，重点关注VWF的D'D3结构域、A1结构域N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）、缺失D'D3结构域的二聚体VWF（ΔD'D3-VWF）以及缺失该肽上NFP（ΔD'D3NFP(─)-VWF）或仅缺失O-聚糖（ΔD'D3OG(─)-VWF）的ΔD'D3-VWF变体。还制备了单体VWF-A1和D'D3-A1。在酶联免疫吸附测定（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）比其他缺失D'D3结构域的蛋白（KD~0.7至2.5 nmol/L）高50至100倍。此外，在表面等离子体共振研究中，D'D3-A1与固定化GpIbα结合的结合速率（kon=1.8±0.4×10(4) （mol/L）(-1)·s(-1)；KD=1.7 μmol/L）与单个VWF-A1结构域相比有所降低（kon=5.1±0.4×10(4) （mol/L）(-1)·s(-1)；KD=1.2 μmol/L）。因此，VWF-D'D3主要控制可溶性VWF与GpIbα的结合。相反，当VWF固定化时，所有分子特征均调节A1-GpIbα的结合。在此，在ELISA中，ΔPro-VWF上可用于结合GpIbα的表观A1结构域位点数量约为ΔD'D3-VWF变体的50%。在基于微流控技术的固定化VWF上的血小板黏附测量以及胶原上的血栓形成试验中，人血小板募集情况为ΔPro-VWF<ΔD'D3-VWF<ΔD'D3NFP(─)-VWF<ΔD'D3OG(─)-VWF。
虽然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端肽均调节血小板移位和血栓形成。
