Prothrombin Kringle-2 Activates Cultured Rat Brain Microglia

Abstract

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-κB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-κB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.

The Microglia-activating Potential of Thrombin

“…Such "impurities" could also account for variable findings on other serum proteins (see Table I for factor Xa). We were also unable to demonstrate microglial effects for prothrombin, which was recently reported to be a factor for microglial NO release and cytokine mRNA induction (42). Attributing a function to the "dispensable" N-terminal prothrombin portion, in particular the Kringle-2 region, is as intriguing as it has been for the primary N terminus of PAR (43).…”

Modulation of the Purinergic P2X7Receptor Attenuates Lipopolysaccharide-Mediated Microglial Activation and Neuronal Damage in Inflamed Brain

“…Our results suggest that p38 MAPK and NFB regulate a proliferative microglial response rather than cellular activation. Proliferation of microglia has been associated with phosphorylation of p38 MAPK (Tikka et al, 2001) and activation of NFB (Ryu et al, 2002;Suo et al, 2003).…”

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

“…Total RNA (2 g) was used as a template for the reverse transcription (RT) reaction. For the semiquantitative polymerase chain reaction (PCR), the primers were synthesized according to the previously reported sequences for rat iNOS cDNA, 5Ј-GCA GAA TGT GAC CAT CAT GG-3Ј (forward) and 5Ј-ACA ACC TTG GTG TTG AAG GC-3Ј (reverse) and for rat TNF-␣ cDNA, 5Ј-GTA GCC CAC GTC GTA GCA AA-3Ј (forward) and 5Ј-CCC TTC TCC AGC TGG GAG AC-3Ј (reverse), respectively (Ryu et al, 2002b). The PCR cycles consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s (TNF-␣) or 60°C for 30 s (iNOS), and extension at 72°C for 90 s for 30 cycles.…”