Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C

Highlights •A range of thiol isomerase enzymes were expressed by HMEC-1 endothelial cells. •Inhibition of thiol isomerases reduced plasminogen activation on HMEC-1 cells. •Exogenous bovine protein disulphide isomerase increased plasminogen activation in HMEC-1 cells. •Inhibition of thiol isomerases also reduced fibrin clot lysis, but not via direct effects on tPA. •HMEC-1 mediated protein C activation was unaffected by thiol isomerase inhibition. Abstract Introduction Cell surface thiol isomerase enzymes, principally protein disulphide isomerase (PDI), have emerged as important regulators of platelet function and tissue factor activation via their action on allosteric disulphide bonds. Allosteric disulphides are present in other haemostasis-related proteins, and we have therefore investigated whether thiol isomerase inhibition has any influence on two endothelial activities relevant to haemostatic regulation, namely activation of protein C and activation of plasminogen, with subsequent fibrinolysis. Materials and Methods The study was performed using the human microvascular endothelial cell line HMEC-1. Thiol isomerase gene expression was measured by RT-PCR and activation of protein C and plasminogen by cell-based assays using chromogenic substrates S2366 and S2251, respectively. Cell mediated fibrinolysis was measured by monitoring absorbance at 405 nm following fibrin clot formation on the surface of HMEC-1 monolayers. Results and Conclusions A variety of thiol isomerase enzymes, including PDI, were expressed by HMEC-1 cells and thiol reductase activity detectable on the cell surface was inhibited by both RL90 anti-PDI antibody and by the PDI inhibitor quercetin-3-rutinoside (rutin). In cell-based assays, activation of plasminogen, but not of protein C, was inhibited by RL90 antibody and, to a lesser extent, by rutin. Fibrin clot lysis occurring on a HMEC-1 monolayer was also significantly slowed by RL90 antibody and by rutin, but RL90-mediated inhibition was abolished in the presence of exogenous tissue plasminogen activator (tPA). We conclude that thiol isomerases, including PDI, are involved in fibrinolytic regulation at the endothelial surface, although not via a direct action on tPA. These findings broaden understanding of haemostatic regulation by PDI, and may aid in development of novel anti-thrombotic therapeutic strategies targeted via the fibrinolysis system

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 μM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) (100 μM) or inhibiting PDI with bacitracin (5 mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 μM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 μM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain

Rapid S-nitrosothiol metabolism by platelets and megakaryocytes

RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs. Exposure of platelets to GSNO (S-nitrosoglutathione) for as little as 5 s inhibited thrombin-induced platelet aggregation by >95%; however, AlbSNO (S-nitrosoalbumin) was much less effective over these short time periods. Incubation of 1 mM GSNO or AlbSNO with platelets and megakaryocytes resulted in a 25-34% loss of RSNO recoverable from the supernatant (P<0.02) within 30 s. This rapid cell-mediated RSNO decay did not progress further over 5 min, and could not be accounted for by release of free NO. The g-glutamyl transpeptidase inhibitor acivicin (100 mM) partially decreased GSNO decay, whereas the membrane-impermeable thiol-blocking agent 5.5'-dithiobis-(2-nitrobenzoic acid) (100 mM) completely blocked cell-mediated GSNO decay and partially blocked AlbSNO decay. Our results highlight differences between high- and low-molecular-mass RSNOs with regard to their rapid metabolism/uptake and subsequent cellular responses, and indicate a critical role for extracellular thiols in RSNO metabolism by platelets and megakaryocytes