Thrombin Induces Mast Cell Adhesion to Fibronectin: Evidence for Involvement of Protease-Activated Receptor-1

摘要

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 β-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-α5 integrin Ab (51.1 ± 6.7%; n = 5). The combination of anti-α5 and anti-α4 Abs induced higher inhibition (65.7 ± 7.1%; n = 5). Unlike what is known for FcεRI-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.