Detailed Mapping of the Binding Site within Platelet Glycoprotein Ibα for Leukocyte Integrin Mac-1 (αMβ2).

We have shown that the platelet counter-receptor for integrin Mac-1 (αMβ2, CD11b/CD18) is glycoprotein (GP) Ibα, the largest component of the GP Ib-IX-V complex, better known for its role in mediating platelet attachment to sites of vessel wall injury. The GP Ibα-Mac-1 interaction mediates the firm adhesion of leukocytes on platelet thrombi, enabling their subsequent transmigration into the vessel wall. Our previous studies showed that the I-domain of Mac-1 binds the C-terminal flanking sequences (Phe201–Gly268) of GP Ibα, as demonstrated by the fact that binding is almost completely inhibited by the I domain ligands fibrinogen and heparin, the I-domain monoclonal antibody LPM19c, and the GP Ibα-specific monoclonal antibody AP1. The latter antibody is of interest because it also inhibits the binding of von Willebrand factor and thrombin. We have mapped the AP1 epitope to a 10-amino acid sequence spanning Arg218 to Tyr228. In the current investigation, we constructed a series of cell lines expressing mutants of human GP Ibα, either by replacement of the human sequence with the corresponding dog sequence (dog GP Ibα does not bind human Mac-1) or by targeted mutagenesis, and tested their ability to bind the recombinant Mac-1 I domain. The GP Ibα region Phe201-Asn223 was crucial for Mac-1 binding, with residues Arg218, Asp222 and Asn223 playing vital roles. In addition, a peptide containing the AP1 epitope (Leu214-Val229) bound Mac-1 I-domain specifically and saturably. Peptide binding was blocked by LPM19c and soluble GP Ibα, and by the M2 peptide, which corresponds to the GP Ibα-binding site in the Mac-1 I domain (Phe201-Lys217). Peptide binding was also blocked by an antibody against the M2 sequence. The AP1 peptide inhibited the attachment of GP Ib-IX-V complex-expressing CHO cells to immobilized Mac-1 I domain, and the adhesion of THP-1 cells—a monocytoid line expressing Mac-1—to immobilized GP Ibα. This peptide did not inhibit attachment of the cells VWF surfaces, or thrombin-induced platelet aggregation. In summary, we have defined the GP Ibα sequence Arg214 to Val229 as a critical binding site for Mac-1. Because a peptide corresponding to this region inhibits GP Ibα binding to Mac-1 but blocks neither platelet adhesion to immobilized VWF nor thrombin-induced platelet aggregation, it has potential to guide the development of agents that will specifically inhibit leukocyte-platelet complexes that promote vascular inflammation.