Abstract 5880: Localization of the Binding Site for Fibrin-Derived Peptide P3 in the αIIb β-Propeller of Integrin αIIbβ3
The currently available anti-thrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIbIIIa) with fibrinogen during platelet aggregation, an initial step of thrombus formation. However, platelets also bind fibrin deposited at the later stages of thrombus growth. It was proposed that inhibition of platelet interactions with fibrin may be a necessary and importantproperty of αIIbβ3 antagonists. We have previously identified the γ370 –383 sequence (P3) in the γC-domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. We have also demonstrated that the mechanism by which P3 binds αIIbβ3 is different from that involved in binding of AGDV, the binding site for αIIbβ3 in soluble fibrinogen. Here, using surface plasmon resonance, we demonstrated that P3 can bind to multiple binding sites within the αIIb β-propeller of αIIbβ3. To localize these sites, we performed comprehensive screening of overlapping and substitutional peptide libraries spanning the entire sequence (residues 1– 451) of αIIb β-propeller and identified several segments and amino acid residues as possible candidates for P3 binding. Guided by the data from peptide libraries, we generated chimeric receptors expressed on the surface of model HEK293 cells in which 8 residues identified as critical for P3 binding in the αIIb β-propeller were substituted for homologous residues in the αM β-propeller of the I-less form of integrin αMβ2, a receptor that does not bind fibrin(ogen). Mutations of 6 candidate residues resulted in the loss (40 – 60%) of cell adhesion to immobilized fibrinogen, a mimic of fibrin. Inspection of the three-dimensional structure of the αIIb β-propeller demonstrated that the residues are situated within the W2:4 –1/1–2 and W4:4 –1/1–2 loops of its ‘W’ repeats. The P3-binding sites are clustered at the edge of αIIb β-propeller which is distant from the interface with the I-like domain of the β3 subunit where the binding site for RGD was previously localized. These data define specific molecular requirements for binding of fibrin-specific peptide P3 to αIIbβ3 and further indicate that the recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen.
This research has received full or partial funding support from the American Heart Association, National Center.