Persistent Protease-activated Receptor 4 Signaling Mediates Thrombin-induced Microglial Activation

Abstract

We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-␣ (TNF-␣) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-␣. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca 2؉ ] i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFB activation may be responsible for PAR4AP-induced TNF-␣ production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations.

Lipopolysaccharide is a frequent and significant contaminant in microglia‐activating factors

“…In the case of pb-thr, there is strong evidence for the combination (Hanisch et al, 2004;Weinstein et al, 2005). Microglia are known to express PARs (Balcaitis et al, 2003;Suo et al, 2002Suo et al, ,2003 and as seen here (see Fig. 1) and previously (Weinstein et al, 2005), pharmaceutical-grade rh-thr induces small increases in CD95(Fas) and CD40 expression in the N9 cell line.…”

The Microglia-activating Potential of Thrombin

“…Moreover the cells responded with proliferation and the release of cytokines, a major executive feature of activated microglia. Induction of cytokines by thrombin would agree with an assumed profile as a proinflammatory signal (5)(6)(7)(8)10).…”

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).…”