A phosphoserine GPIbα 580-590 peptide inhibits GPIb-IX-14-3-3ζ association. (A) Triton X-100 lysates of resting human platelets were immunoprecipitated with the mAb RAM.1 in the presence or absence of the nonphosphorylated ØP (LVAGRRPSALS) or diphosphorylated P₁P₂ (LVAGRRPpSALpS) GPIbα 580-590 synthetic peptide. Ctrl represents incubation with an unrelated peptide (TNRQPRDKNVKK) from the P2Y₁₂ receptor sequence. RAM.1 coprecipitation of 14-3-3ζ was reduced in the presence of P₁P₂ and to a lesser extent in the presence of ØP, compared with incubation with no peptide or the Ctrl peptide. NI indicates nonimmune serum. (B) The coprecipitated 14-3-3ζ band was quantified with Quantity One Software (Bio-Rad, Hercules, CA) in 3 separate experiments, and results were expressed as the mean percentage (± SEM) of the band obtained in the absence of peptide (^*P < .05). (C)Alignment of the amino acid sequences of the GPIbα 580-610 region in the human, mouse, and dog species. Ser residues are in boldface. The 580-590 domain (underlined) contains fully conserved serine residues with a spacing that corresponds to a consensus 14-3-3ζ binding motif.²²  X indicates any amino acid; S, serine; pS, phosphorylated serine; R, arginine; and ^*, the pS609 residue. (GPIbα human, GPIbα mouse, and GPIbα canis have the GenBank accession numbers PO7359, AAC53320, and AAC14361, respectively.)