title: 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.
  
  
  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.
authors.affiliation: Department of Chemical and Biological Engineering and The NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY (S.R.M., C.Z., A.K., K.M.D., S.N.).
authors.forename: Sri R
authors.initials: SR
authors.lastname: Madabhushi
authors.affiliation: Department of Chemical and Biological Engineering and The NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY (S.R.M., C.Z., A.K., K.M.D., S.N.).
authors.forename: Changjie
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Chemical and Biological Engineering and The NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY (S.R.M., C.Z., A.K., K.M.D., S.N.).
authors.forename: Anju
authors.initials: A
authors.lastname: Kelkar
authors.affiliation: Department of Chemical and Biological Engineering and The NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY (S.R.M., C.Z., A.K., K.M.D., S.N.).
authors.forename: Kannayakanahalli M
authors.initials: KM
authors.lastname: Dayananda
authors.affiliation: Department of Chemical and Biological Engineering and The NY State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY (S.R.M., C.Z., A.K., K.M.D., S.N.).
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D018495:Fluid Shifts / Q000502:physiology; D006439:Hemodynamics / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D044085:Microfluidics / Q000379:methods; D010455:Peptides / Q000378:metabolism; D010974:Platelet Aggregation / Q000502:physiology*; D010979:Platelet Function Tests; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013927:Thrombosis / Q000503:physiopathology*; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; D011134:Polysaccharides; C094990:adhesion receptor; C063515:bioactive peptide A1, Rana esculenta; D014841:von Willebrand Factor
keywords: blood platelets; cell adhesion; microfluidics; thrombosis; von Willebrand factor