VE-cadherin phosphorylation at tyrosine 685 was not affected by thrombin stimulation, independent of angiopoietin treatment. The main finding of the present study is that the angiopoietins Ang-2 and Ang-1 had opposing effects on the very initial, but not on the prolonged late phase of the thrombin-induced hyperpermeability response of cultured human pulmonary microvascular endothelial cells. Specifically, Ang-2 enhanced the initial hyperpermeability, while Ang-1 reduced the initial hyperpermeability by attenuation of thrombin-induced reorganization of the adherence junctions. The limited effect of angiopoietins on basal permeability as compared to thrombin-stimulated permeability suggests that in the adult endothelium the angiopoietin-Tie2 system is a sensitizer of the activated endothelium in the presence of other inflammatory or coagulation mediators, rather than an independent actor of the permeability response. This is in line with previous findings that Ang-2 alone did not affect the adhesion of leukocytes to quiescent endothelium, while it promoted adhesion of leukocytes to endothelial cells activated by tumor necrosis factor-a. The permeability enhancing effect of thrombin in endothelial monolayers in culture can be separated in two phases. During the initial phase the TEER decreases FG-4592 rapidly, while the passage of HRP starts increasing. During this phase the endothelial junctions become instable and locally small gaps between the cells are formed. After 15 min stress fibers have become formed reflecting a major change in the F-actincytoskeleton. In the subsequent phase the rate of HRP passage becomes maximal. This phase includes continued actin-myosin interaction within the cells and cell contraction. However, the TEER starts to recover during this period suggesting that junctional complexes and focal adhesion sites are locally recovering, although still relatively large gaps between cells remain. After 90 min a full recovery of the GSI-IX monolayer is observed both with regard to TEER and HRP passage. In the context of this dual effect of thrombin, the effect of angiopoietins only on the initial thrombin response is of interest and points to an effect at the junctional level in particular. Indeed, we observed changes in VE-cadherin localization that reflected unstable junctions and intracellular gap formation. While similar alterations in adherence junctions and VE-cadherin relocalization are induced by VEGF via Src phosphorylation at Tyr685 and subsequent activities , thrombin did not affect this phosphorylation. This is accordance with Kinney et al. , who showed that thrombin has no effect on Src and Yes, but only on the Srclike protein Fyn, which has less permeability enhancing properties. Apparently another mechanism induces the dissociation of VE-cadherins in adherence junctions.