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.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.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.
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