Abstract
Integrins are heterodimeric cell-adhesion receptors comprising α and β subunits that transmit signals allosterically in both directions across the membrane by binding to intra- and extracellular components. The human platelet antigen-1 (HPA-1) polymorphism in αIIbβ3 arises from a Leu → Pro exchange at residue 33 in the genu of the β3 subunit, resulting in Leu33 (HPA-1a) or Pro33 (HPA-1b) isoforms. Although clinical investigations have provided conflicting results, some studies have suggested that Pro33 platelets exhibit increased thrombogenicity. Under flow-dynamic conditions, the Pro33 variant displays prothrombotic properties, characterized by increased platelet adhesion, aggregate/thrombus formation, and outside-in signaling. However, the molecular events underlying this prothrombotic phenotype have remained elusive. As residue 33 is located >80 Å away from extracellular binding sites or transmembrane domains, we hypothesized that the Leu → Pro exchange allosterically shifts the dynamic conformational equilibrium of αIIbβ3 toward an active state. Multiple microsecond-long, all-atom molecular dynamics simulations of the ectodomain of the Leu33 and Pro33 isoforms provided evidence that the Leu → Pro exchange weakens interdomain interactions at the genu and alters the structural dynamics of the integrin to a more unbent and splayed state. Using FRET analysis of fluorescent proteins fused with αIIbβ3 in transfected HEK293 cells, we found that the Pro33 variant in its resting state displays a lower energy transfer than the Leu33 isoform. This finding indicated a larger spatial separation of the cytoplasmic tails in the Pro33 variant. Together, our results indicate that the Leu → Pro exchange allosterically shifts the dynamic conformational equilibrium of αIIbβ3 to a structural state closer to the active one, promoting the fully active state and fostering the prothrombotic phenotype of Pro33 platelets.