Platelet studies and structural in silico modeling showing effects of the novel β3 L718del. (A) Family pedigree. Blue symbols indicate MTP, and red symbols indicate a normal platelet count and function. The heterozygous presence of the ITGB3 variant is shown (+/–). (B) Light transmission aggregometry performed in citrated platelet-rich plasma (PRP) compared typical response of platelets from the proband (P1) and her affected daughter (P2) to that of a control donor. P1 and P2 were studied on 3 and 2 occasions respectively. Aggregation with high doses of ADP, collagen (Col), or arachidonic acid (AA) was of much lower intensity and was slow. Reduced although somewhat better responses were seen with TRAP, whereas ristocetin-induced platelet agglutination was normal. (C) Flow cytometry with the monoclonal antibodies (mAbs) AP-2 and Bx-1 show αIIbβ3 and GPIb content for P1 platelets (left panel, typical histograms; mean fluorescence intensity [MFI]). Binding of the immunoglobulin M (IgM) mAb PAC-1 showed much reduced activation of αIIbβ3 for P1 after stimulation of citrated PRP with ADP and TRAP (center panel). Binding of fluorescein isothiocyanate–Fg to stimulated washed platelets from P1 confirmed a reduced activation of the integrin (right panel). (D) In silico PyMOL modeling shows how β3 Leu718del changes the synchronization of the αIIb and β3 cytoplasmic tails. Left panel: cartoon representation of the normal nonactivated αIIb (purple) and β3 (green) transmembrane and cytoplasmic tail segments highlighting amino acids engaged in their clasp. Aromatic amino acids in π interactions are shown as spheres. The salt bridge involves polar amino acids of opposite charge; the positive αIIb-R995 and the negative β3-D723 are represented as sticks. Center panel: upper view schematic representations of the αIIb and β3 transmembrane α helix showing the distribution of consecutive amino acids on the helix’s circumference. (b1) Normal distribution with a β3-D723 localizing face to αIIb-R995. (b2) Configuration with the L718del. β3-D723 is now displaced by a quarter turn away from αIIb-R995. A positively charged Arg (R) now faces αIIb-R995; in addition to the repulsive charge effect, Arg is larger than Asp (arm of 7 atoms compared with 3), and this encumbrance will also push the cytoplasmic tails apart. Right panel: Cartoon representation of αIIb and β3 transmembrane segments, with superimposed β3 α helices with and without the mutation (colored orange and white, respectively). The arrow shows the twist. All methods and details of PymMOL modeling have been previously described in Nurden et al.⁴