A. Morales Allende2, K. De Ceunynck2, S. Chaudhry2, C. Peters2, A. Jain2, S. Higgins2, O. Aisiku2, J. Fitch-Tewfik2, C. Dockendorff2, S. Parikh2, D. Ingber2, R. Flaumenhaft2 2Beth Israel Deaconess Medical Center,Vascular Surgery,Boston, MA, USA
Introduction:
Protease-activated receptor 1 (PAR1) is a member of a subfamily of G protein-coupled receptors, PARs, and is highly expressed on platelets and endothelial cells. PARs are unique in that they are activated through proteolytic cleavage. On endothelial cells, PAR1 exhibits distinct responses when cleaved by different proteases: thrombin induces apoptotic and inflammatory signaling, whereas the activated form of the anticoagulant protein C (APC) elicits cytoprotective and anti-inflammatory pathways. We have discovered small molecules, termed parmodulins (PM1 and PM2), which like APC induce a cytoprotective pathway in endothelial cells.
Methods:
We monitored apoptosis in cells incubated with parmodulins (PM1 or PM2) or APC for 4 hours. In addition, we assessed whether parmodulins also act via the PAR1 receptor by knocking out PAR1. Finally, we assessed the effect of parmodulins on thrombin generation in Human Umbilical Vein Endothelial Cells (HUVECs). Cells were incubated with inflammatory mediators (either TNF-α or LPS) for 3 hours in media, then replaced with plasma and 2.5 micromole calcium chloride for 20 minutes, and thrombin activity was measured.
Results:
Apoptosis induced by TNF, thrombin or staurosporine was reduced when cells were pre-incubated with PM1, PM2, or APC for 4 hours. PMs or APC were no longer capable of protecting from TNF-induced apoptosis when PAR1 was silenced using siRNA. PAR1 knockdown was confirmed using qRT-PCR and Western Blot, showing >90% reduction in gene and protein expression. This demonstrates that the cytoprotective effects of PMs, like APC, are mediated through PAR1.
Unlike the enzyme APC that cleaves PAR1 at its extracellular side, parmodulins act at the cytosolic face of the receptor. PM2 blocked thrombin generation on HUVECs in response to inflammatory mediators TNF-α and LPS. Our data showed that APC also inhibited thrombin generation upon LPS or TNF stimulation. The difference is that PM2 required prolonged exposure indicating activation of a cytoprotective pathway, whereas APC inhibition occurred due to its anti-coagulant activities.
Conclusion:
Parmodulin activation of the cytoprotective pathway, without affecting coagulation, may provide a new approach for treatment of thromboinflammatory diseases.