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Integrin αIIb/β3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the αIIb/β3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of αIIb. Using αIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin α2 tail, β3 tail and lamin, but showed a weak binding to the αV tail which shares the highest homology with αIIb tail among the integrin α family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and αIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to αIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with αIIb and may play a critical role in αIIb/β3-mediated platelet function.