Platelet GpIba binding to von Willebrand Factor under fluid shear:contributions of the D′D3-domain, A1-domain flanking peptide and O-linked glycans

Abstract

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 (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.

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.

Keywords: blood platelets; cell adhesion; microfluidics; thrombosis; von Willebrand factor.