title: Unraveling thrombin's true microglia-activating potential: markedly disparate profiles of pharmaceutical-grade and commercial-grade thrombin preparations.
abstract: Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long-term phenotypic changes. We and others have recently reported that commercial-grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re-investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical-grade recombinant human thrombin (rh-thr) and compared it with a commercial-grade plasma-derived bovine thrombin (pb-thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin-6 and tumour necrosis factor-alpha release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb-thr induced robust responses in all variables tested. In contrast, rh-thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species-specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co-stimulatory effects. In addition, these results suggest that previous studies examining thrombin's activation of microglia should be cautiously re-interpreted.
authors.affiliation: Department of Neurology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Weinstein
authors.forename: Soyon
authors.initials: S
authors.lastname: Hong
authors.forename: John D
authors.initials: JD
authors.lastname: Kulman
authors.forename: Caroline
authors.initials: C
authors.lastname: Bishop
authors.forename: Jon
authors.initials: J
authors.lastname: Kuniyoshi
authors.forename: Henrik
authors.initials: H
authors.lastname: Andersen
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Ransom
authors.forename: Uwe-Karsten
authors.initials: UK
authors.lastname: Hanisch
authors.forename: Thomas
authors.initials: T
authors.lastname: Möller
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D019013:CD40 Antigens / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects*; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D012697:Serine Endopeptidases / Q000145:classification / Q000494:pharmacology*; D013917:Thrombin / Q000145:classification / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D019013:CD40 Antigens; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D009569:Nitric Oxide; D012697:Serine Endopeptidases; D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone