Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells.

It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.