Platelet-Leukocyte Conjugation Mediated By GPIbα-αXβ2 (CD11c/CD18) Interaction (2013)

Abstract Background The formation of platelet-leukocyte conjugates has been implicated in a variety of pathologies, including thrombosis, atherosclerosis, glomerulonephritis, multiple sclerosis and other inflammatory diseases. In prior studies (Simon et al., J. Exp Med, 2000, 193-204), platelet GPIbα and leukocyte αMβ2 (CD11b/CD18) had been implicated as receptor:counter-receptor pairing in mediating formation of platelet:leukocyte complexes. A specific segment (αM201-217) in the I domain of αM (CD11b) subunit of αMβ2 engaged GPIbα, and a peptide (M2) corresponding to this segment blocked neutrophil recognition of platelets and anti-M2 blocked conjugate formation (Ehlers et al., J. Exp. Med, 2003, 1077-88). The I-domains of αM and αX are highly conserved (74% homology), but αX has different amino acids at two positions shown to be critical for recognition of GPIbα by the αM I-domain. This study considers the role of a particular receptor:counter-receptor pairing, GPIb on platelets and αXβ2 (CD11c/CD18) on leukocytes, in mediating this heterotypic cell interaction. Methods and Results To directly address whether αXβ2 recognizes GPIbα, αXβ2 was expressed in HEK cells and its corresponding I-domain was expressed and purified. αXβ2 cells adhered specifically to GPIbα, and purified αX I-domain bound to platelets by flow cytometry and to GPIbα by surface plasmon resonance. However, this interaction was readily distinguished from that of αM I-domain binding to GPIbα. αX binding to GPIbα was not inhibited by: 1) M2 or anti-M2; 2) a peptide from a region of GPIbα implicated in vWF interaction and that blocked adhesion of αMβ2 cells to GPIbα; 3) small molecules that blocked αM I-domain binding; and 4) mutations in GPIbα that blocked αM I-domain binding. Furthermore, by mutational analyses, a region of αX I domain was implicated binding GPIbα that is structurally distant and unrelated to the αM I domain binding site. Next, we examined the role of αXβ2 in vivo in a cytokine- and neutrophil-dependent inflammatory response. The local Shwartzman-like reaction (LSR), induced by successive LPS and cytokine injections into the skin, produces a thrombo-hemorrhagic vasculitis, which depends on leukocytes and their interaction with activated platelets and endothelial cells. In prior work, we have shown that the LSR is dependent upon leukocyte αM and platelet GPIba. Leukocyte-derived microparticles promote the accumulation of fibrin, generation of occlusive thrombi, and eventual hemorrhage from inflamed blood vessels. The LSR produces hemorrhage in the intact skin 24 hours after TNF-a injection. CD11c-/- mice subjected to LSR exhibited a significant reduction in the development of hemorrhagic lesions. In analysis blinded to genotype, CD11c-/- mice had a 76% reduction in lesion severity (lesion hemorrhage score: WT, 3.4 ± 1.0, n=12 vs. CD11c-/-, 0.8 ± 1.0, n=11; P&lt;0.001). Histopathology of vasculitic lesions demonstrated substantial reductions in erythrocyte extravasation, edema, leukocyte accumulation, and occlusive thrombi in CD11c-/- mice. Conclusions Leukocyte αXβ2 (CD11c/CD18) recognizes GPIbα on platelets in a way distinct from αMβ2 (CD11bCD18). This interaction appears to be functionally significant in vivo and could be targeted for suppressing adverse inflammatory responses. Disclosures: Simon: Cordis/J&amp;J: Consultancy; Janssen/J&amp;J: Consultancy; Medtronic Vascular: Consultancy; Merck: Consultancy; Medtronic Foundation: Research Funding.