Antiphospholipid antibodies (aPL) have been shown to induce tissue factor (TF) expression in monocytes and endothelial cells. However, the underlying signal transduction has been more or less elusive in the past. We have recently shown that aPL enter the lysosomal route in monocytes and dendritic cells, and subsequently activate endosomal NADPH-oxidase (NOX). The generation of superoxide which is dismutated to hydrogen peroxide upregulates the intracellular toll like receptors (TLR) 7 and 8, and leads to robust production of inflammatory cytokines. Here we show that induction of TF by aPL follows the same signaling pathway. Inhibition of endosomal NOX by the anion channel blocker niflumic acid or capture of superoxide by the radical scavenger N-acetylcysteine blocks TF induction by aPL. Furthermore, monocytes from mice deficient in NOX2 do not increase TF surface expression in response to aPL, while cells from mice deficient in glutathione peroxidase-1 (GPx-1) show an increased response. Unexpectedly, also induction of TF by tumour necrosis factor (TNF)α and lipopolysaccharide (LPS) was strongly dependent on the activation of endosomal NOX. While TNFα apparently depends alm ost fully on endosomal NOX, signalling of LPS is only partially dependent on this pathway. These data provide further insight into the well-known role of reactive oxygen species in the induction of TF expression and suggest that endosomal signalling may represent a central coordinating point in this process.
In eukaryotes, proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family are believed to have a general role for the fusion of intracellular transport vesicles with acceptor membranes. Arabidopsis thaliana PEN1 syntaxin resides in the plasma membrane and was previously shown to act together with its partner SNAREs, the adaptor protein SNAP33, and endomembrane-anchored VAMP721/722 in the execution of secretory immune responses against powdery mildew fungi. We conducted a structure-function analysis of PEN1 and show that N-terminal phospho-mimicking and non-phosphorylatable variants neither affected binary nor ternary SNARE complex formation with cognate partners in vitro. However, expression of these syntaxin variants at native protein levels in a pen1 mutant background suggests that phosphorylation is required for full resistance activity in planta. All tested site-directed substitutions of SNARE domain or "linker region" residues reduced PEN1 defense activity. Two of the variants failed to form ternary complexes with the partner SNAREs in vitro, possibly explaining their diminished in planta activity. However, impaired pathogen defense in plants expressing a linker region variant is likely because of PEN1 destabilization. Although Arabidopsis PEN1 and SYP122 syntaxins share overlapping functions in plant growth and development, PEN1 activity in disease resistance is apparently the result of a complete functional specialization. Our findings are consistent with the hypothesis that PEN1 acts in plant defense through the formation of ternary SNARE complexes and point to the existence of unknown regulatory factors. Our data indirectly support structural inferences that the four-helical coiled coil bundle in ternary SNARE complexes is formed in a sequential order from the N- to C-terminal direction.
It has been shown that stimulation of endothelial cells and monocytes by antiphospholipid antibodies leads to a prothrombotic state involving upregulation of tissue factor (TF). We examined the in vitro effects of IgG fractions from patients with antiphospholipid syndrome (APS) and of a beta-2-glycoprotein 1-independent human monoclonal antiphospholipid antibody (HL-5B) on human umbilical vein endothelial cells (HUVEC) in comparison to untreated cell controls and to exposure to monoclonal IgG control antibody. We also examined the effect of recombinant monocyte chemoattractant protein-1 (MCP-1) on peripheral blood monocytes. Stimulation of endothelial cells with APS IgG fractions or HL-5B resulted in time-dependent upregulation of MCP-1 mRNA and protein expression. Stimulation with HL-5B also led to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 protein expressions. Stimulation of monocytes with recombinant MCP-1 resulted in an upregulation of TF mRNA and TF protein. In conclusion these results might represent a mechanism for antiphospholipid antibody-mediated thrombosis in APS patients.
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