Microvascular endothelium is major target for antibodies directed against HLA and non-HLA antigens. Functional antibodies targeting G-protein coupled receptors (GPCRs) such as AT1R or ETAR are recently emerging as important mediators of antibody mediated rejection. Beside AT1R and ETAR, high expression of protease-activated receptor-1 (PAR-1) was detected in the systematic screen for GPCR expression in quiescent and activated microvascular endothelium. Structural analysis of extracellular domains of screen positive GPCRs confirmed striking homology between second extracellular loops of AT1R, ETAR and PAR-1. Subsequently, solid phase assay using membrane extracts from PAR-1 overexpressing cells was developed for detection of PAR-1 antibodies. PAR-IgG was isolated from sera of patients with acute rejection with microvascular pathology and used for stimulation of human microvascular endothelial cells. Transcriptional regulation of interleukin 6 (IL-6) was studied by promoter deletion assay, transcription factor activation and binding by qRT-PCR, western blot, EMSA, cFOS knockdown and site directed mutagenesis. IL-6 secretion was determined by ELISA. PAR-1-IgG enhanced IL-6 release via increased IL-6 promoter activity which was dependent on cFos protein expression via its binding to the IL-6 promoter. Pretreatment with AP-1 inhibitor, cFos siRNA induced knockdown or site directed mutagenesis decreased IL-6 levels and IL-6 promoter activity. IL-6 secretion could be normalized by pretreatment with specific PAR-1 inhibitor. Specificity was further confirmed by peptide targeting of the 2nd extracellular loop of the PAR-1 which significantly decreased IL-6 release. PAR-1 is a new target for functional antibodies in the context of microvascular rejection with deregulated IL-6 levels. Targeting PAR-1 and/or IL-6 could offer new therapeutic possibilites to withstand microvascular inflammation.

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