Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-proteincoupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca 2؉. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca 2؉ . These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Thrombin-mediated changes in endothelial cell adherens junctions modulate vascular permeability. We demonstrate that the nonreceptor protein-tyrosine phosphatase SHP2 co-precipitates with VE-cadherin complexes in confluent, quiescent human umbilical vein endothelial cells. Ligand-binding blots using a SHP2-glutathione S-transferase fusion peptide established that SHP2 associates selectively with ␤-catenin in VEcadherin complexes. Thrombin treatment of human umbilical vein endothelial cells promotes SHP2 tyrosine phosphorylation and dissociation from VE-cadherin complexes. The loss of SHP2 from the cadherin complexes correlates with a dramatic increase in the tyrosine phosphorylation of ␤-catenin, ␥-catenin, and p120-catenin complexed with VE-cadherin. We propose that thrombin regulates the tyrosine phosphorylation of VEcadherin-associated ␤-catenin, ␥-catenin, and p120-catenin by modulating the quantity of SHP2 associated with VE-cadherin complexes. Such changes in adherens junction complex composition likely underlie thrombin-elicited alterations in endothelial monolayer permeability.Confluent, quiescent endothelial and epithelial cell monolayers form semi-permeable barriers that regulate the transit of agents across the monolayer. This barrier function depends, in part, on the cadherin complexes that form the intercellular junctions. The cadherins are transmembrane proteins that draw neighboring cells together through calcium-dependent, homophilic associations. The stability of these cell-cell junctions is modulated by the catenins, cytoplasmic proteins that bind directly (␤-catenin, ␥-catenin, and p120-catenin) or associate indirectly (␣-catenin) with the intracellular tail of the cadherin (1-3). The catenins regulate tethering of the cadherin complexes to the actin cytoskeleton and lateral cadherin multimer formation (4, 5). VE-cadherin, an endothelial cell-specific cadherin, localizes at the intercellular adhesion junctions formed by endothelial cells and appears to be responsible for the exclusion of N-cadherin, the other highly expressed cadherin in endothelial cells, from these junctions (6, 7). In proliferating endothelial cells, as in epithelial cells, significant tyrosine phosphorylation of ␤-catenin, ␥-catenin, and p120-catenin can be detected (8, 9). As the cells reach confluence and undergo contact inhibition of proliferation and stabilization of cell-cell junctions, tyrosine phosphorylation of the catenins decreases dramatically. This correlates with the density-dependent increase in protein-tyrosine phosphatases (PTP), 1 especially at cell-cell junctions where they associate with cadherin complex proteins or platelet endothelial cell adhesion molecule (PECAM-1) (10 -12).Thrombin, a potent activator of endothelial cells, causes increased permeability and intercellular gap formation in confluent endothelial cell monolayers (13-15). Such endothelial barrier dysfunction can be elicited by protein-tyrosine phosphatase inhibitors and can be attenuated by protein-tyrosine kinase inhibitors (16 -20). The...

Cytoskeletal alterations in endothelial cells have been linked to nitric oxide generation and cell-cell interactions. Transforming growth factor (TGF)-beta has been described to affect cytoskeletal rearrangement in numerous cell types; however, the underlying pathway is unclear. In the present study, we found that human umbilical vein endothelial cells (HUVEC) have marked cytoskeletal alterations with short-term TGF-beta treatment resulting in filipodia formation and F-actin assembly. The cytoskeletal alterations were blocked by the novel TGF-beta type I receptor/ALK5 kinase inhibitor (SB-505124) but not by the p38 kinase inhibitor (SB-203580). TGF-beta also induced marked stimulation of reactive oxygen species (ROS) within 5 min of TGF-beta exposure. TGF-beta stimulation of ROS was mediated by the NAPDH oxidase homolog Nox4 as DPI, an inhibitor of NADPH oxidase, and dominant-negative Nox4 adenovirus blocked ROS production. Finally, inhibition of ROS with ROS scavengers or dominant-negative Nox4 blocked the TGF-beta effect on cytoskeleton changes in endothelial cells. In conclusion, our studies show for the first time that TGF-beta-induced ROS production in human endothelial cells is via Nox4 and that TGF-beta alteration of cytoskeleton in HUVEC is mediated via a Nox4-dependent pathway.

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HU-VEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence 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 PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.

Activated thrombin receptors on human umbilical vein endothelial cells rapidly undergo homologous desensitization, leaving the cells unable to respond to thrombin. The present studies examine the fate of activated thrombin receptors on endothelial cells and the mechanisms that restore intact receptors to the cell surface. The results show that: 1) at biologically relevant concentrations, thrombin rapidly cleaves all of its receptors on the cell surface. 2) The cleaved receptors are cleared from the cell surface in a two-phase process, with 60% being internalized within 10 min, the remainder requiring several hours. 3) The restoration of intact, thrombin-responsive receptors on the cell surface initially occurs from an intracellular pool of receptors in a process that is independent of protein synthesis. 4) Recycling of cleaved receptors either does not occur on endothelial cells or is masked by receptor clearance. 5) Subconfluent endothelial cells re-express intact receptors on the cell surface at a slower rate than confluent cells. 6) The agonist peptide, SFLLRN, also causes receptor internalization, although at concentrations greater than those required for receptor activation and desensitization. These results are distinctly different from those observed with megakaryoblastic cell lines, where > 90% of the activated thrombin receptors are internalized rapidly, up to 40% of the cleaved receptors are recycled, and no intracellular pool of intact receptors has been detected. Since the primary structure of the thrombin receptor is the same in all the cell types studied, these results demonstrate that there can be substantial differences between cell types in the mechanisms involved in the clearance of activated receptors and the re-expression on the cell surface of intact receptors capable of responding to thrombin.