Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat alpha IIb beta 3 molecules, we found that this difference in Arg-Gly-Asp-Ser ( RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the alpha IIb beta 3 propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human alpha IIb beta 3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the alpha IIb beta 3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with alpha IIb in a different manner than with these small molecules. None of these species based substitutions affected the ability of alpha IIb beta 3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the alpha IIb beta-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins. (Blood. 2009;113:902-910)