Receptor-mediated activation of murine peritoneal macrophages by antithrombin III acts as a costimulatory signal for nitric oxide synthesis

We evaluated the effect of antithrombin III (ATIII), a serine protease inhibitor (SERPIN), on induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Incubation of macrophages with ATIII plus interferon- γ (IFN-γ) but not ATIII alone induced nitrite accumulation (a metabolite of NO) in a dose-dependent manner. Expression of the inducible nitric oxide synthase isoform was confirmed by Western blot. NO synthesis was inhibited by N(G)-monomethyl-L-arginine, by complexing ATIII with thrombin or by rabbit anti-human ATIII antiserum. Addition of polymyxin B to macrophage cultures failed to inhibit ATIII/IFN-γ-induced NO synthesis, excluding lipopolysaccharide contamination. ¹²⁵I-ATIII bound to macrophages in a dose-dependent, specific, and saturable manner, with a K(m) of ~7.1 nM. Our results demonstrate that ATIII, but not ATIII/thrombin complex, acts to costimulate macrophage activation and NO synthesis via a novel receptor mediated mechanism, which may indicate a role for SERPINs in macrophage activation.