Mapping of the Binding Site within Glycoprotein Ibα for the Leukocyte Integrin Mac-1 (αMβ2).
Authors: Munday Adam D , Peng Yuandong , Wang Yunmei , Simon Daniel I , Lopez Jose A. 
Published: October 2019

Abstract 472

To maintain hemostasis, the human body uses more that 7,000 platelets/μl of blood a day; 3.5 billion platelets a day in a 70 kg adult male. Lack of fully functional platelets results in bleeding disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia and platelet-type von Willebrand disease. It is becoming increasingly well appreciated that in addition to their hemostatic role, platelets play important roles in inflammation and wound healing. The initial step of platelet adhesion is mediated by the glycoprotein (GP) Ib-IX-V complex on the platelet surface, which binds von Willebrand factor (VWF). This interaction leads to activation of the integrin αIIbβ3, platelet arrest, and spreading and aggregation. The GPIb-IX-V complex also has a key role in inflammation, mediating a key interaction of platelets with leukocytes by binding the integrin Mac-1 (αMβ2, CD11b/CD18). This interaction mediates the firm adhesion of leukocytes on platelet thrombi, enabling their migration through the thrombus into the vessel wall. Interestingly, the insert domain (I-domain) of the αM subunit of Mac-1 has a similar 3-dimensional structure to the A1 domain of VWF. Our previous studies showed that the I-domain of Mac-1 binds the C-terminal flanking sequence of GPIbα (Phe201-Gly268), demonstrated by the ability of the anti-GPIbα monoclonal antibody AP1 to inhibit the interaction. The epitope of AP1 has been mapped 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 GPIbα, either by replacement of the human sequence with the corresponding dog sequence (dog GPIbα does not bind human Mac-1) or by targeted mutagenesis, and tested their ability to bind the recombinant αM I domain. TheGPIbα 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 αM I-domain specifically and saturably. Peptide binding was blocked by LPM19c, a monoclonal anti-αM I-domain antibody, and soluble GPIbα, and by the M2 peptide, which corresponds to the GPIbα–binding site in the αM I domain (Phe201–Lys217). Peptide binding was also blocked by an antibody against the M2 sequence. The AP1 peptide inhibited the attachment of GPIb-IX complex–expressing CHO cells to immobilized αM I domain, and the adhesion of THP-1 cells—a monocytic cell line expressing Mac-1—to immobilized GPIbα. In summary, we have defined the GPIbα sequence Arg218 to Ala224 as a critical binding site for Mac-1. Because a peptide corresponding to this region inhibits GPIbα 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.

Disclosures:

No relevant conflicts of interest to declare.