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 α IIb β 3 arises from a Leu→Pro exchange at residue 33 in the genu of the β3 subunit, resulting in or Pro-33 (HPA1b) isoforms. Although clinical investigations have provided conflicting results, some studies have suggested that Pro-33 platelets exhibit increased thrombogenicity. Under flow-dynamic conditions, the Pro-33 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, allatom molecular dynamics simulations of the ectodomain of the Leu-33 and Pro-33 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 Pro-33 variant in its resting state displays a lower energy transfer http://www.jbc.org/cgi