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

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

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p

Paroxysmal and cognitive phenotypes in Prrt2 mutant mice

Mutations in proline-rich transmembrane protein 2 (PRRT2) cause a range of episodic disorders that include paroxysmal kinesigenic dyskinesia and benign familial infantile epilepsy. Mutations are generally loss of function and include the c649dupC frameshifting mutation that is present in around 80% of affected individuals. To investigate how Prrt2 loss of function mutations causes disease, we performed a phenotypic investigation of a transgenic Prrt2 knockout (Prrt2 KO) mouse. We observed spontaneous paroxysmal episodes with behavioural features of both seizure and movement disorders, as well as unexplained deaths in KO and HET animals. KO mice showed spatial learning deficits in the Morris water maze, as well as gait abnormalities in the quantitative Digigait analysis; both of which may be representative of the more severe phenotypes experienced by homozygous patients. These findings extend the described phenotypes of Prrt2 mutant mice, further confirming their utility for in vivo investigation of the role of Prrt2 mutations in episodic diseases.Louise Robertson, Travis Featherby, Stuart Howell, James Hughes, Paul Thoma