THROMBIN RECEPTOR 14-AMINO-ACID PEPTIDE BINDS TO ENDOTHELIAL-CELLS AND STIMULATES CALCIUM TRANSIENTS

We examined the binding characteristics of the recently described thrombin receptor amino-terminal peptide, SFLLRNPNDKYEPF (T. K H. Vu, D. T. Hung, V. 1. Wheaton, and S. R. Coughlin. Cell 64: 1057-1068, 1991), termed TRP-14, and its effect in activating intracellular calcium transients in pulmonary vascular endothelial cells. Binding of I-125-labeled TRP-14 was found to be saturable with a affinity constant of 2 muM and maximum binding of 41 pmol/mg of cell protein. The I-125-labeled TRP-14 also interacted with bovine pulmonary microvessel endothelial cells, human umbilical vein endothelial cells, and porcine pulmonary artery smooth muscle cells. Binding of I-125-labeled diisopropylphosphoryl (DIP)-alpha-thrombin, which is catalytically inactive but binds to thrombin receptors, was not inhibited by TRP-14 or vice versa, indicating that TRP-14 did not compete for the alpha-thrombin binding site(s) on the endothelial cell surface. TRP-14 (>1 muM) increased the concentration of intracellular calcium ([Ca2+]i) in endothelial cells with kinetics similar to the increase in [Ca2+]i triggered by alpha-thrombin. In contrast, DIP-alpha-thrombin did not increase [Ca2+]i and also did not prevent the rise in [Ca2+]i induced by the subsequent challenge with either TRP-14 or alpha-thrombin. Because the generation of TRP-14 by the proteolytically active forms of thrombin stimulated a rise in endothelial [Ca2+]i, TRP-14 may be the agonist responsible for the activation of the alpha-thrombin receptor in pulmonary vascular endothelial cells. The finding that TRP-14 did not compete for alpha-thrombin binding on endothelial cells suggests that TRP-14 recognizes the thrombin receptor site distinct from the thrombin binding domain of the receptor.