Thrombin‐induced increase in albumin permeability across the endothelium

Abstract

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.

Neutrophil-derived Glutamate Regulates Vascular Endothelial Barrier Function

“…Paracellular flux was calculated by linear regression of sample fluorescence. Consistent with previous reports, control experiments demonstrated decreased permeability with forskolin and 8-bromo-cAMP (11) and increased permeability with thrombin and hydrogen peroxide (data not shown) (12). Paracellular flux was also determined across monolayers treated with glutamate, 3,5-dihydrophenylglycine (DHPG; selective group I mGluR agonist), L-2-amino-4-phosphonobutyrate (L-AP4; selective group III mGluR agonist), or glutamate (1 M) plus N-phenyl-7-(hydroxyamino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; selective group I mGluR antagonist), or (RS)-␣-cyclopropyl-4-phosphonophenylglycine (CPPG; selective group III mGluR antagonist) (Tocris Cookson Inc., Ellisville, MO).…”

Role of G protein-coupled receptors in inflammation

“…This process is reversible, and focal adhesion kinase plays a role in the reversal of the increased vascular permeability [64] . Thrombin-induced increase in vascular endothelial permeability contributes to the edema seen in inflammatory disorders such as acute lung injury [65,66] . Both the receptor and its downstream signaling pathways are targets for therapeutic intervention [67,68] .…”

Signaling pathways involved in OxPAPC-induced pulmonary endothelial barrier protection

“…A number of agonists including phospholipid products (Zhang, Magnusson et al 1997;Huang, Subbaiah et al 2005) and bioactive peptides (Garcia, Siflinger-Birnboim et al 1986;van Nieuw Amerongen, Draijer et al 1998;Petrache, Verin et al 2001;Moy, Blackwell et al 2002;Birukova, Adyshev et al 2005) increase vascular endothelial permeability, which my lead to alveolar flooding and pulmonary edema. However, much smaller number of bioactive molecules capable of enhancing endothelial barrier properties have been reported so far (Garcia, Liu et al 2001;Liu, Schaphorst et al 2002;VouretCraviari, Bourcier et al 2002;Birukov, Bochkov et al 2004;Kolosova, Mirzapoiazova et al 2005), and signaling mechanisms underlying these barrier protective effects have been a major focus of ongoing studies (Lee, Lee et al 2000;Garcia, Liu et al 2001;Vouret-Craviari, Bourcier et al 2002;Birukov, Leitinger et al 2004;Dudek, Jacobson et al 2004;Kolosova, Mirzapoiazova et al 2005;Mehta, Konstantoulaki et al 2005;Singleton, Dudek et al 2005).…”