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Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
Thrombin activates mast cells to release inflammatory mediators
through a mechanism involving protease-activated receptor-1 (PAR-1). We
hypothesized that PAR-1 activation would induce mast cell adhesion to
fibronectin (FN). Fluorescent adhesion assay was performed in 96-well
plates coated with FN (20 µg/ml). Murine bone marrow cultured mast
cells (BMCMC) were used after 3–5 wk of culture (>98% mast cells by
flow cytometry for c-Kit expression). Thrombin induced
-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from
BMCMC. Thrombin and the PAR-1-activating peptide
AparafluoroFRCyclohexylACitY-NH2 (cit) induced BMCMC
adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive
peptide FSLLRY-NH2 had no effect. Thrombin and cit induced
also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was
inhibited by anti-
5 integrin Ab (51.1 ± 6.7%;
n = 5). The combination of
anti-5 and anti-4 Abs induced
higher inhibition (65.7 ± 7.1%; n = 5).
Unlike what is known for Fc
RI-mediated adhesion, PAR-1-mediated
adhesion to FN did not increase mediator release. We then explored the
signaling pathways involved in PAR-1-mediated mast cell adhesion.
Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis
toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 ± 7.3%
(n = 4), indicating that Gi proteins
are involved. Wortmannin and calphostin almost completely inhibited
PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and
protein kinase C are involved. Adhesion was partially inhibited by the
mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5
± 3.3%; n = 3) and the p38 inhibitor SB203580
(25.1 ± 10.4%; n = 3). The two inhibitors
had additive effects. Therefore, thrombin mediates mast cell adhesion
through the activation of Gi proteins, phosphoinositol
3-kinase, protein kinase C, and mitogen-activated protein kinase
pathways.
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