The Microglia-activating Potential of Thrombin

Abstract

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non-proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous ␣-and ␥-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact ␣-and ␥-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease.

Adjunctive Minocycline in Clozapine-Treated Schizophrenia Patients With Persistent Symptoms

“…Liaury et al [27] found that microglial activation was attenuated with related improvements in memory related to inflammation during minocycline treatment. Microglial cells are known to contribute to synaptic modulation, learning and memory processes [55, 56]. Many other animal studies have found minocycline to reduce memory and working memory deficits in animal models [57–61].…”

CXCR3 promotes plaque formation and behavioral deficits in an Alzheimer’s disease model

“…Cortical murine microglia were prepared from brains of pups at postnatal day 1 as previously described (89). Briefly, meninges of isolated brains were removed mechanically, and brains were dissociated by trituration to culture cells in high-glucose (4.5 mg/ml) DMEM (Gibco, Life Technologies) supplemented with 10% fetal calf serum (PAN Biotech) and 1% penicillin/streptomycin (PAA) for up to 14 days.…”

Thrombin-Induced Regulation of CD95(Fas) Expression in the N9 Microglial Cell Line: Evidence for Involvement of Proteinase-Activated Receptor1 and Extracellular Signal-Regulated Kinase 1/2

“…The current data is consistent with the former, but not the latter. This discrepancy could reflect differences in the cell preparations used, the specific activation parameters examined or the purity of reagents [14][15][16].…”

“…We have previously shown that pharmaceutical-grade recombinant human thrombin (rh-thr) induces only a restricted set of proteolysis-dependent responses from microglia including induction of intracellular calcium signals and regulation of selected surface antigens [14,15]. In this study, we characterize the dose-dependence and kinetics of the rh-thr-induced CD95(Fas) response and address the mechanism of this regulation in the N9 mouse microglial cell line.…”

“…In addition to its hemostatic effects, thrombin can induce numerous effects on neural cells through activation of proteinase-activated receptors (PARs). Recently, we demonstrated that a number of previously reported microglial activation responses induced by commercial-grade preparations of thrombin were due to either proteinacious [14,15] or lipopolysaccharide/ endotoxin (LPS) [16] contamination. However, several proteolysis-dependent responses were seen in microglia following stimulation with a pharmaceutical-grade, endotoxin-free, recombinant human a-thrombin preparation (rh-thr) including regulation of surface antigen expression and generation of intracellular calcium transients [15].…”