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: Von Willebrand Factor (VWF) A1-domain binding to platelet receptor GpIb? 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 (?Pro-VWF), dimeric VWF lacking the D'D3 domain (?D'D3-VWF), and ?D'D3-VWF variants lacking either the NFP (?D'D3NFP(?)-VWF) or just O-glycans on this peptide (?D'D3OG(?)-VWF) were expressed. Monomeric VWF-A1 and D'D3-A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ?Pro-VWF binding to immobilized GpIb? (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 GpIb? (kon=1.8±0.4×10(4) (mol/L)(-1)·s(-1); KD=1.7 ?mol/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 ?mol/L). Thus, VWF-D'D3 primarily controls soluble VWF binding to GpIb?. In contrast, upon VWF immobilization, all molecular features regulated A1-GpIb? binding. Here, in ELISA, the number of apparent A1-domain sites available for binding GpIb? on ?Pro-VWF was ?50% that of the ?D'D3-VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ?Pro-VWF

SUBMITTER: Madabhushi SR 

PROVIDER: S-EPMC4323794 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature