Platelet gpiba binding to von willebrand factor under fluid shear: Contributions of the D'D3-domain, A1-domain flanking peptide and O-linked glycans

Background-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. Methods and Results-Full-length dimeric VWF (ΔPro-VWF), dimeric VWF lacking the D'D3 domain (DD'D3-VWF), and DD'D3-VWF variants lacking either the NFP (DD'D3NFP^--VWF) or just O-glycans on this peptide (DD'D3OG^--VWF) were expressed. Monomeric VWF-A1 and D'D3-A1 were also produced. In ELISA, the apparent dissociation constant (K_D) of soluble ΔPro-VWF binding to immobilized GpIbα (K_D≈100 nmol/L) was 50-to 100-fold higher than other proteins lacking the D'D3 domain (K_D~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on-rate of D'D3-A1 binding to immobilized GpIbα (k_on=1.8±0.4×10⁴ (mol/L)^-1·s^-1; K_D=1.7 μmol/L) was reduced compared with the single VWF-A1 domain (k_on=5.1±0.4×10⁴ (mol/L)^-1·s^-1; K_D=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 DD'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<DD'D3-VWF<DD'D3NFP^--VWF<DD'D3OG^--VWF. Conclusions-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.