J-S Bae1, A R Rezaie. 1. Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO 63104, USA.
Abstract
SUMMARY BACKGROUND: Recent studies have indicated that antithrombin (AT) possesses both anti-inflammatory and antiangiogenic properties. OBJECTIVES: The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild-type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin-binding site. METHODS: Direct cellular effects of the AT derivatives were compared in the LPS-stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan-4 or sphingosine 1-phosphate receptor 1 (S1P(1)). Furthermore, the roles of prostacyclin and nuclear factor (NF)-kappaB in modulating these effects were investigated. RESULTS: Both wild-type and the RCL mutant, AT/Proth-2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF-kappaB pathway. Indomethacin abrogated both activities. The heparin-binding site mutants, AT-K114E and AT-K125E, did not exhibit any protective activity in either one of these assays, but a potent pro-apoptotic activity was observed for the AT-K114E in endothelial cells. Both PTX and siRNA for syndecan-4 inhibited the protective effect of AT, but the siRNA for S1P(1) was inconsequential. CONCLUSIONS: The interaction of AT with syndecan-4 is required for its prostacyclin-dependent protective effect through a PTX-sensitive and non-S1P(1)-related G(i)-protein coupled receptor. The RCL mutant, AT/Proth-2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti-inflammatory drug without increasing the risk of bleeding.
SUMMARY BACKGROUND: Recent studies have indicated that antithrombin (AT) possesses both anti-inflammatory and antiangiogenic properties. OBJECTIVES: The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild-type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin-binding site. METHODS: Direct cellular effects of the AT derivatives were compared in the LPS-stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan-4 or sphingosine 1-phosphate receptor 1 (S1P(1)). Furthermore, the roles of prostacyclin and nuclear factor (NF)-kappaB in modulating these effects were investigated. RESULTS: Both wild-type and the RCL mutant, AT/Proth-2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF-kappaB pathway. Indomethacin abrogated both activities. The heparin-binding site mutants, AT-K114E and AT-K125E, did not exhibit any protective activity in either one of these assays, but a potent pro-apoptotic activity was observed for the AT-K114E in endothelial cells. Both PTX and siRNA for syndecan-4 inhibited the protective effect of AT, but the siRNA for S1P(1) was inconsequential. CONCLUSIONS: The interaction of AT with syndecan-4 is required for its prostacyclin-dependent protective effect through a PTX-sensitive and non-S1P(1)-related G(i)-protein coupled receptor. The RCL mutant, AT/Proth-2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti-inflammatory drug without increasing the risk of bleeding.
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