Alteration in endothelial permeability occurs in response to the activation of PAR2 by factor Xa but not directly by the TF-factor VIIa complex

Abstract

Alterations in the endothelial permeability occur in response to the activation of coagulation mechanisms in order to control clot formation. The activation of the protease activated receptors (PAR) can induce signals that regulate such cellular responses. PAR2 is a target for the coagulation factor Xa (fXa) and tissue factor-factor VIIa (TF-fVIIa) complex. By measuring the permeability of dextran blue across endothelial monolayer, we examined the mechanisms linking coagulation and endothelial permeability. Activation of PAR2 using the agonist peptide (PAR2-AP) resulted in increased permeability across the monolayer and was comparable to that obtained with VEGF at 60 min. Incubation of cells with activated factor Xa (fXa) resulted in an initial decrease in permeability by 30 min, but then significantly increased at 60 min. These responses required fXa activity, and were abrogated by incubation of the cells with a PAR2-blocking antibody (SAM11). Activation of PAR2 alone, or inhibition of PAR1, abrogated the initial reduction in permeability. Additionally, inclusion of Rivaroxaban (0.6 µg/ml) significantly inhibited the response to fXa. Finally, incubation of the endothelial monolayers up to 2 h with TF-containing microvesicles derived from MDA-MB-231 cells, in the presence or absence of fVIIa, did not influence the permeability across the monolayers.In conclusion, fXa but not TF-fVIIa is a noteworthy mediator of endothelial permeability. The rapid initial decrease in permeability requires PAR2 and PAR1 which may act to constrain bleeding. The longer-term response is mediated by PAR2 with increased permeability, presumably to enhance clot formation at the site of damage.

The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells

“…Microvesicle TF-fVIIa activities were measured by modification of previously described procedures. 42 43 To measure TF activity, microvesicle samples were incubated with fVIIa (10 nM) in HEPES-buffered saline (HBS) pH 7.4, containing 1% (w/v) bovine serum albumin (BSA) and 5 mM CaCl 2 , together with fX (100 nM) and a fXa chromogenic substrate (0.2 mM; Hyphen) diluted in the same buffer (200 µL). The samples were incubated for 60 minutes to develop the color.…”

Beyond thrombosis: the impact of tissue factor signaling in cancer

“…The release of flTF-MVs by tumors can also promote metastasis via paracrine signaling within the tumor microenvironment and at distal sites. flTF-MVs are elevated in the plasma of cancer patients [ 171 , 172 ] and can activate PAR1 and PAR2 on nonmalignant cells [ 173 , 174 ]. Endothelial cells that are stimulated with flTF-MVs express increased adhesion molecules and secrete pro-inflammatory molecules that can recruit pro-tumor monocytes, establishing a pre-metastatic niche for circulating cancer cells [ 173 , 175 ].…”

Coagulation Factor Xa Promotes Solid Tumor Growth, Experimental Metastasis and Endothelial Cell Activation

“…Moreover, morphological and functional observations in vivo and in vitro suggest that FXa, independently of Thrombin, have anti-angiogenic properties over vein-derived (HUVEC) endothelial cells and this is mediated by PAR-1 but not PAR-2 [48]. Recently, it has been proposed that alterations in the permeability of the endothelium occur in response to the activation of PAR-2 by FXa in arterial and microvascular cells in vitro and this is independent of the TF/FVIIa complex [49]. While we clearly demonstrate specific actions of FXa in in vitro/in vivo models and we show an effect of the predominantly FXa inhibitor dalteparin over metastasis, our study has caveats.…”