Abstract 523: Expression, Activation and Function of Integrin α M β 2 on Neutrophil-derived Microparticles

Abstract

The levels of circulating leukocyte-derived microparticles (MPs) are elevated in thrombosis and most cardiovascular diseases, and it has been suggested that MPs are not only markers of disease but also active contributors to pathogenesis via their interaction with various cell types. Such interactions will depend on the presence and activity of molecules expressed on the MP surface. In our study we have considered the presence and activation state of a major adhesion receptor on the surface of leukocytes, integrin α M β 2 (CD11b/18, Mac-1) on MPs derived from human neutrophils (PMN). MPs were obtained from resting or agonist-stimulated PMNs, and the presence of α M β 2 was assessed by FACS. α M β 2 expression was significantly enhanced on MPs derived from stimulated PMN (e.g., resting MPs: MFI = 3.5 ± 0.7 vs PMA-MPs: MFI = 20 ± 4, and fLMP-MPs: MFI = 24 ± 5). PSGL-1 was expressed by MPs but levels were not affected by stimulation. Of note, α M β 2 expressed on MPs obtained from stimulated PMNs was in active conformation as assessed using mAb (CBRM1/5) to activation-dependent epitope of this integrin. Recognition of a protein ligand, fibrinogen, by activated α M β 2 on the MPs was also demonstrable. With evidence of a functionally active integrin, we sought to evaluate its role in mediating MP interaction with platelets. MP binding to isolated, resting platelets was dose and time dependent. However, MPs expressing activated α M β 2 bound to more platelets, and only MPs obtained from stimulated PMNs induced platelet activation as assessed P-selectin expression (resting-MPs: MFI = 25.1 ± 6.5 vs PMA-MPS: MFI = 293 ± 60, n = 4). MP binding and platelet activation were reduced by ~40–50% by function-blocking mAbs to α M β 2 and GPIbα, implicated as a major counter-receptor for α M β 2 . Complete abrogation of these events was achieved by the combination of blocking reagents to both α M β 2 /GPIbα and to PSGL-1/P-selectin. In conclusion, MPs released by stimulated PMNs express activated α M β 2 . This integrin cooperates with PSGL-1 to mediate interaction with and activation of platelets.