Recognition of Fibronectin by the Platelet Integrin αIIbβ3 Involves an Extended Interface with Multiple Electrostatic Interactions

Normal platelet function is dependent on the ability of integrin αIIbβ3 (glycoprotein IIb/IIIa) to interact with components of the subendothelial matrix, such as fibronectin (Fn), exposed at sites of vascular injury. Studies using synthetic peptides derived from human Fn sequences Asp1373−Thr1383 and Arg1493−Asp1495 have suggested a role for both the 9th (3fn9) and 10th (3fn10) type III repeats of this ligand in binding to αIIbβ3. In this study, we have taken a charge-to-alanine mutagenesis approach to evaluate the importance of these sites, and other charged residues, within the context of recombinant 3fn9−10 modules for binding to αIIbβ3. To identify residues that are involved in Fn binding to αIIbβ3, recombinantly expressed 3fn9−10 module pairs with alanine substitutions introduced into each of the 38 charged residues were individually assayed for the ability to inhibit Fn binding to purified αIIbβ3. Substitutions at Fn residues Arg1493 and Asp1495 of the RGD sequence were found to have the greatest effect on αIIbβ3 binding, as expected. However, Fn residues Arg1369, Arg1371, Arg1379, Arg1445, and Arg1448 were needed for optimal interaction of the 3fn9−10 module pair with αIIbβ3. All Fn residues found to affect binding of 3fn9−10 to αIIbβ3 are located on the same face and extend from the surface of the molecule. Additionally, the epitopes for two anti-Fn monoclonal antibodies that inhibit binding of this ligand to αIIbβ3 were found to overlap the sites identified. These results demonstrate that αIIbβ3−Fn binding involves multiple electrostatic interactions.