The F153Sβ3 mutation exerts functional effect on αIIbβ3 surface expression and ligand binding. (A) Superimposition of crystal structures of normal αIIbF153β3 globular head region, highlighting the structural changes of β3 I domain from resting (in green) to active state (in blue). The metal ions of MIDAS and ADMIDAS are shown as spheres. The red side chain localizes 153aa in the core of βI-domain in the central part of β-sheet number 2, which is underneath the α2-helix. An inward movement of α2-helix is noted along with the inward movement of β1-α1 loop and α1/α1′-helix, and the downward movement of β6-α7 loop and α7-helix. The structural comparison was made using PDB 2vdo and 3fcs with PyMol. (B) In silico mutagenesis of F153β3. The hydrophobic interaction between F153 and the residues V207 and F203 of α2-helix in the WT form is shown (top). The S153 mutation losses the bulky hydrophobic contact with the α2-helix is shown (bottom). The F153S mutation may render αIIbβ3 constitutively active by facilitating an unrestricted inward movement of α2- and α1-helices during the conformational transition of the β3 integrin from a resting to an active state, because bulky/nonpolar to small/polar amino acid side chain provide space for structural change to facilitate increased movement. Enlarged images of the β2-strand βI-domain also predict the effect on Van der Waal force (red cloud) resulting from the change in structure of the β3 153aa side-chain going from bulky F to small S. Changes induced by F153Sβ3 are depicted in the smaller and hydrophilic serine residue, which also may introduce disorganization by permitting entrance of water molecules that may competitively form H-bonds with oxygen atoms or NH₂-groups of constituent amino acid of the core and disrupt structure. In silico mutagenesis were made in PyMol using PDB 2vdo and 3fcs. (C) Immunofluorescent cytometric analysis detection of level of cell surface expression of αIIbβ3 using distinct mAbs: 10E5 and HIP8 specific for αIIb, AP3 and VIPL2 recognizing β3, and AP2 and 7E3 against the αIIbβ3 complex. Integrin αIIb and F153β3 or S153β3 subunits plus GFP were cotransfected into HEK293FT cells. Harvested cells were incubated with indicated mAb and then GFP⁺ cells were analyzed for MFI of mAb binding on the cell surface via immunocytometry. The S153β3 mutant is observed to be expressed at ∼10% to 20% of F153β3 (WT). Data are presented as percent of WT control, MFI arbitrarily set at 100% (n = 3). (D) Measurement of αIIb-F153Sβ3 ability to bind activation-dependent mAb “PAC-1” in the absence (−Fg) or presence (+Fg) of unlabeled fibrinogen. αIIb and F153β3 or S153β3 subunits were cotransfected into HEK293FT cells and harvested cells then incubated with or without unlabeled fibrinogen in the presence of Ca²⁺/Mg²⁺ (Ca/Mg) or an extracellular αIIbβ3 agonist Ca²⁺/Mn²⁺ (Ca/Mn) before the addition of PAC-1. Integrin-positive cells were analyzed for PAC-1 MFI binding. The bar graph demonstrates PAC-1 binding that was normalized to the total MFI in percent of each form of F153β3 or S153β3 expression in panel C . Data are presented as MFI in percentage + SEM (n ≥ 3) and unpaired 2-tailed Student t test was performed to compare the mutants with WT under the same condition or as indicated. ∗P < .05, ∗∗P < .01.