apc derived mp in vitro

Background-The endothelial protein C receptor (EPCR) plays an important role within the protein C (PC) pathway in regulating coagulation and inflammation. Recently, we described a novel mechanism of EPCR release from the surface of primary physiological cells. Induced by exogenous activated protein C (APC), EPCR is released in microparticulate form. Its bound APC retains proteolytic anticoagulant activity and we now hypothesise that this microparticulate EPCR-APC complex can also cleave endothelial protease activated receptor 1 (PAR1) to modulate inflammation and cytoprotection. Methods & Results-The gene profiling effect on human endothelial cells by 40nM APC, in free or microparticulate form, was assessed. Transcript profile results showed upregulation of anti-apoptotic and inflammatory genes by APC in either form, which were confirmed by RT-PCR. These translated into increased GM-CSF and interleukin 8 secretion and cytoprotection against staurosporine-induced apoptosis. PC or PAR1 antagonism reversed these results to demonstrate that induced effects by microparticles were APC specific and PAR1-dependent. Further analysis of human plasma from septic patients, by confocal microscopy and ELISA, showed evidence of circulating microparticle-associated EPCR during recombinant APC treatment. Functional coagulation and cellular studies demonstrate that these express APC-specific effects on anticoagulation with PAR1-mediated gene induction and anti-apoptotic function. Conclusions-APC induction of microparticle-associated EPCR release can occur in vivo. These microparticles could potentially disseminate the function of the EPCR-APC complex to PAR1 on different cells and vascular sites. Keywords: free apc versus mp-apc MP were isolated from huvec after estimulation with apc; the apc content of the mp was estimated by chromogenic substrate and a standard curve. 40nM apc on mp was used for estimulation of huvec for 4 hours for study of gene expression. APC relevance was studied by blocking experiments with antibody and par1 involvement was studied by including a par1 antagonist. All experiments were done in triplicate. A basline control was obtained by looking at the profile of non-estimulated cells under the same conditions.