TRAP-14

Thrombin, a potent activator of cellular responses, proteolytically cleaves, and thereby activates its receptor. In the present study, we compared the effects of the thrombin receptor 14-amino acid peptide (TRP-14; SFLLRNPNDKYEPF), which comprises the NH2 terminus after cleavage of the thrombin receptor, and of the native alpha-thrombin on endothelial monolayer permeability. Addition of TRP-14 (1-200 microM) to bovine pulmonary artery endothelial cells increased [Ca2+]i in a dose-dependent manner. The peak increase in [Ca2+]i in response to 100 microM TRP-14 or 0.1 microM alpha-thrombin was similar (i.e., 931 +/- 74 nM and 1032 +/- 80 nM, respectively), which was followed by a slow decrease with t1/2 values of 0.73 and 0.61 min, respectively. Extracellular Ca2+ chelation with 5 mM EGTA abolished the sustained increases in [Ca2+]i induced by either TRP-14 or alpha-thrombin. alpha-thrombin (0.1 microM) increased transendothelial [125I]albumin permeability, whereas TRP-14 (1-100 microM) had no effect. Coincubation of 100 microM TRP-14 with 1 microM DIP-alpha-thrombin also did not increase permeability over control values. Stimulation of BPAEC with 0.1 microM alpha-thrombin induced translocation of protein kinase C (PKC) from the cytosol to the plasma membrane indicative of PKC activation, whereas TRP-14 had no effect at any concentration.

This study investigates the role of the transcription factor NFkappaB in thrombin- and thrombin receptor activating peptide (TRAP, SFLLRNPNDKYEPYF)-induced mitogenesis of cultured bovine coronary artery smooth muscle cells (SMC). Stimulation of resting cells by thrombin (10 nM) or TRAP (10-100 microM) resulted in a comparable time-dependent activation of NFkappaB as detected by Western blotting and electrophoretic mobility shift assay (EMSA) of nuclear extracts. The NFkappaB activation was antagonized by N-acetyl-L-cysteine (20 mM) and pentoxifylline (0.5 mM). Thrombin caused a 3-4-fold increase in [3H]thymidine incorporation within 24 h which was prevented by inhibitors of NFkappaB activation. In contrast, TRAP did not cause any mitogenic response. These results demonstrate that activation of NFkappaB is an essential but not a sufficient signal for SMC mitogenesis.

Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRAP)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC). Here, we have compared the ability of these synthetic peptide ligands and thrombin to stimulate platelet-derived growth factor (PDGF) production by, and monocyte adhesion to, HUVEC. Thrombin (50 units/ml) and TRAP (25 microM) maximally stimulated monocyte adhesion. Furthermore, the stimulation of E-selectin cell surface expression and the steady-state E-selectin mRNA levels by thrombin and TRAP were comparable. Thrombin (50 units/ml) stimulated PDGF production 400% above the basal level in 24 h, whereas the 6-mer and 14-mer TRAP, even at 200 microM, did not significantly stimulate PDGF production. Northern analysis, however, revealed that TRAP at 100 microM stimulated PDGF-A and -B chain mRNA expression to a level similar to that induced by thrombin. These results suggest that activation of cell signaling by TRAP can mimic thrombin and is sufficient for the stimulation of monocyte adhesion to HUVEC; however, thrombin-stimulated PDGF production by HUVEC may require mechanisms in addition to the signaling events initiated by TRAP or may require the participation of a novel thrombin receptor.