Inhibition of protein disulfide isomerase with PACMA-31 regulates monocyte tissue factor through transcriptional and posttranscriptional mechanisms

Protein disulfide isomerase (PDI) contributes to tissue factor (TF) regulation in monocytes. While bacitracin and quercetin-3-rutinoside mitigate myeloid TF production, the effect of PACMA-31, a more specific PDI inhibitor with distinct pharmacologic properties, remains unclear. Lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMCs) or citrate-anticoagulated whole blood was carried out in the presence of PACMA-31 or DMSO vehicle before monocytes were analyzed for TF expression, including antigen, procoagulant activity (PCA) and mRNA, release of IL-6 and TNFα, and LPS-induced signaling pathways. While PACMA-31 alone had no effect, coincubation with LPS and PACMA-31 (25 μM) enhanced LPS-induced monocyte TF production in whole blood. The effect was at least partially regulated on the transcriptional level and could not be explained by increased phosphatidylserine membrane exposure. In contrast, the same PACMA-31 concentrations were cytotoxic in isolated PBMCs. A lower dose of PACMA-31, however, restored the stimulating effect by enhancing IκB-NFκB signaling that also increased the release of IL-6 and TNFα. The protease-activated receptor 2 (PAR2) inhibitor ENMD547 but not TF antibody 10H10 or factor Xa inhibitor rivaroxaban prevented the stimulatory effect of PACMA-31 on inflammatory monocytes. In sharp contrast, short time incubation of LPS-stimulated PBMCs with 25 μM PACMA-31 was non-cytotoxic and significantly inhibited cellular TF PCA but not surface antigen expression. PACMA-31 regulates monocyte TF in a concentration-dependent manner by opposing transcriptional and posttranscriptional mechanisms. While low concentrations of PACMA-31 augment monocyte TF production by amplifying LPS-dependent PAR2 signaling, high concentrations convert monocyte TF into its non-coagulant state. Proposed mechanisms of PACMA-31-mediated regulation of LPS-induced monocyte TF production LPS stimulation induces TF production by monocytes in an NFκB- and PAR2-dependent manner (left upper panel). Co-stimulation with LPS and PACMA-31 at low, non-cytotoxic concentrations amplifies LPS-induced monocyte TF production through a PAR2-dependent signaling pathway (right upper panel). In contrast, high concentrations of PACMA-31 convert surface-located TF into its cryptic, non-coagulant form and downregulate LPS-induced TF production through cytotoxic effects (left lower panel). [Display omitted] •Protein disulfide isomerase (PDI) contributes to TF regulation i