αIIbβ3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible

Abstract

AbstractPlatelet integrin receptor αIIbβ3 supports platelet aggregation by binding fibrinogen. The interaction between the fibrinogen C-terminal γ-chain peptide composed of residues γ-404-411 (GAKQAGDV) and the Arg-Gly-Asp (RGD) binding pocket on αIIbβ3 is required for fibrinogen-mediated platelet aggregation, but data suggest that other ancillary binding sites on both fibrinogen and αIIbβ3 may lead to higher-affinity fibrinogen binding and clot retraction. To identify additional sites, we analyzed the ability of platelets and cells expressing normal and mutant αIIbβ3 to adhere to an immobilized fibrinogen plasmin fragment that lacks intact γ-404-411 ('D98'). We found the following: (1) Activated, but not unactivated, platelets adhere well to immobilized 'D98.' (2) Cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3 or αVβ3, adhere well to 'D98.' (3) Monoclonal antibodies 10E5 and 7E3 inhibit the adhesion to 'D98' of activated platelets and cells expressing constitutively active αIIbβ3, as do small-molecule inhibitors that bind to the RGD pocket. (4) EDTA paradoxically induces normal αIIbβ3 to interact with 'D98.' Because molecular modeling and molecular dynamics simulations suggested that the αIIb L151-D159 helix may contribute to the interaction with 'D98,' we studied an αIIbβ3 mutant in which the αIIb 148-166 loop was swapped with the corresponding αV loop; it failed to bind to fibrinogen or 'D98.' Our data support a model in which conformational changes in αIIbβ3 and/or fibrinogen after platelet activation and the interaction between γ-404-411 and the RGD binding pocket make new ancillary sites available that support higher-affinity fibrinogen binding and clot retraction.