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 (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±0.4×104 (mol/l)-1·s-1; kd=1.7 μmol/l) was reduced compared with the single vwf-a1 domain (kon=5.1±0.4×104 (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 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. © 2014 the authors.