Fibrinolytic activity of human mesothelial cells is counteracted by rapid uptake of tissue-type plasminogen activator

Background. Human mesothelial cells (HMCs) have an important role in mainta ining an adequately functioning fibrinolytic system in the peritoneal cavit y by secreting the fibrinolytic enzymes tissue-type and urokinase-type plas minogen activator (t-PA and u-PA), as well as a specific PA inhibitor, PA i nhibitor type 1 (PAI-1). In this study, we investigated whether the fibrino lytic capacity of HMCs is further counterbalanced by rapid uptake of I-PA a nd u-PA from the medium. Methods. Cultured HMCs were used to study the uptake and degradation of rad iolabeled t-PA and u-PA in the absence or presence of an inhibitor of cellu lar protein degradation, chloroquine, and of specific receptor antagonists. Northern blotting and ligand-blotting techniques were applied to demonstra te the presence of specific receptors for binding of t-PA and u-PA. Results. At 37 degrees C, HMCs rapidly internalized and degraded I-125-t-PA and I-125-u-PA, which could be inhibited by an excess of unlabeled t-PA an d u-PA, respectively, and by the lysosomotropic agent chloroquine. Northern blot analysis showed the expression of low-density lipoprotein (LDL) recep tor-related protein (LRP), very low density lipoprotein (VLDL) receptor, an d u-PA receptor. The addition of recombinant 39 kDa receptor-associated pro tein (RAP; an inhibitor of LRP and VLDL receptor) almost completely blocked the degradation of t-PA and partly that of u-PA. RAP ligand blotting demon strated predominantly the presence of LRP, suggesting a major role for the LRP in mediating uptake and degradation of t-PA in HMCs. Endocytosis of u-P A occurs via two different pathways. After binding to u-PA receptor, a RAP- inhibitable and a non-RAP-inhibitable route for u-PA degradation was demons trated. Tumor necrosis factor alpha (TNF alpha) diminished the fibrinolytic activity of HMCs by decreasing t-PA and increasing PAI-1 synthesis. The fa ll in t-PA levels could be counteracted by inhibiting t-PA degradation by e ither RAP or chloroquine. Interestingly, chloroquine also quenched the TNF alpha-induced changes in t-PA and PAI-1 mRNA levels. Using TNF alpha mutant s and agonistic or blocking monoclonal antibodies specific for the TNF rece ptors p55 and p75, we found evidence that chloroquine interfered with the a ctivation of the TNF receptor p55 and/or its intracellular signaling route. Conclusions. Receptor-mediated endocytosis plays a crucial role in regulati ng the fibrinolytic capacity of HMCs by its participation in the degradatio n of t-PA and u-PA, and in the TNF alpha-induced decrease in t-PA and the i ncrease in PAI-1 expression.