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Prothrombin kringle-2 activates cultured rat brain microglia
Jooyoung Ryu1, Kyoung-Jin Min, Tai Youn Rhim
1Department of Pharmacology, Neuroscience Graduate Program, and Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea.
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-1beta, and TNF-alpha 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-1beta, and TNF-alpha. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-kappaB 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-kappaB (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.
Insights
Prothrombin activates microglia, immune cells in the brain, by inducing nitric oxide (NO) release. A specific domain, kringle-2, independently triggers this activation, highlighting a novel pathway in neuroinflammation.
Area of Science:
- Neuroimmunology
- Molecular Biology
- Cellular Signaling
Background:
- Microglia are key immune cells in the central nervous system (CNS) that activate upon brain injury.
- Prothrombin is a precursor to thrombin, an enzyme involved in blood clotting.
- Understanding microglial activation pathways is crucial for addressing neuroinflammatory conditions.
Purpose of the Study:
- To investigate the role of prothrombin in activating rat brain microglia.
- To determine if prothrombin's protease activity is necessary for microglial activation.
- To identify specific domains of prothrombin responsible for inducing microglial responses.
Main Methods:
- Treatment of rat brain microglia with prothrombin and its domain, kringle-2.
- Measurement of nitric oxide (NO) release and mRNA expression of inflammatory markers (iNOS, IL-1beta, TNF-alpha).
- Inhibition studies using hirudin (thrombin inhibitor) and pathway-specific inhibitors (MAPK, NF-kappaB, PKC, PLC).
Main Results:
- Prothrombin induced NO release and inflammatory gene expression in microglia.
- This effect was independent of thrombin's protease activity.
- The kringle-2 domain of prothrombin mimicked prothrombin's effects.
- Both prothrombin and kringle-2 activated similar intracellular signaling pathways, including MAPK and NF-kappaB.
Conclusions:
- Prothrombin is capable of activating microglia, independent of its thrombin activity.
- The kringle-2 domain is sufficient to activate microglia and induce inflammatory responses.
- Prothrombin and kringle-2 activate microglia via conserved intracellular signaling cascades, offering potential therapeutic targets.