Platelet GpIbα 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 (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 (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±0.4×10(4) (mol/L)(−1)·s(−1); K(D)=1.7 μmol/L) was reduced compared with the single VWF‐A1 domain (k(on)=5.1±0.4×10(4) (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 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. 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.