Proteolysis and phosphorylation-mediated regulation of thrombin receptor activity in in situ endothelial cells.

The regulatory mechanism of thrombin receptor responsiveness in in situ endothelial cells was investigated by evaluating elevations of cytosolic Ca(2+) concentration (Ca(2+)) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the second thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused Ca(2+) elevation, which was smaller than that seen without preceding stimulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of responsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in thrombin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by staurosporine-sensitive kinase, and restored the responsiveness through dephosphorylation by type 1 phosphatase. The mechanism of regulation of thrombin receptor activity in in situ endothelial cells differed from those reported in cultured endothelial cells. We suggest that the cell-specific regulatory mechanism may be altered by culture conditions.