title: Heparin modulates the conformation and signaling of platelet integrin αIIbβ3.
abstract: BACKGROUND
  The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.
  
  
  METHODS
  We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance.
  
  
  CONCLUSIONS
  Heparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.
authors.affiliation: Research & Development, Veterans Affairs Puget Sound Health Care System, Seattle, WA 98108, USA. myagi@uw.edu
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yagi
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Murray
authors.forename: Kurt
authors.initials: K
authors.lastname: Strand
authors.forename: Scott
authors.initials: S
authors.lastname: Blystone
authors.forename: Gianluca
authors.initials: G
authors.lastname: Interlandi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Suda
authors.forename: Michael
authors.initials: M
authors.lastname: Sobel
mesh_terms: D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D006493:Heparin