Thrombin responses in human endothelial cells - Contributions from receptors other than Par1 include the transactivation of Par2 by thrombin-cleaved Par1

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin, Hum an umbilical vein endothelial cells (HUVEC) are known to express PAR1 and t he trypsin/tryptase receptor, PAR2, Northern blots detected both of those r eceptors and, to a lesser extent, PAR3, but PAR4 message was undetectable a nd there was no response to PAR4 agonist peptides. To determine whether PAR 3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cl eavage was blocked with two selective antibodies and PAR1 activation was in hibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibit s PAR1 signaling at the concentrations used. Since peptides mimicking the P AR1 tethered ligand domain can also activate PAR2, we asked whether the rem aining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1, Evidenc e that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors, Conversely, in HUVEC, complete blockade of the thrombin response by the PAR 1 antagonist occurred only when signaling through PAR2 was also blocked. Fr om these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelia l cell responses to thrombin; 2) although PAR3 may be expressed, there is s till no evidence that it mediates thrombin responses; 3) PAR4 is not expres sed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling thr ough PAR1 is blocked. Such transactivation may limit the effectiveness of P AR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.