Aberrant expression of tissue factor (TF) by transformed myeloblasts and inflammatory monocytes drives coagulation activation in acute myeloid leukemia (AML). Although regulation of TF procoagulant activity (PCA) involves thiol-disulfide exchange reactions, the specific role of protein disulfide isomerase (PDI) and other thiol isomerases in AML-associated TF biology is unclear. THP1 cells and peripheral blood mononuclear cells (PBMCs) from healthy controls or AML patients were analyzed for thiol isomerase-dependent TF production under various experimental conditions. Total cellular and membrane TF antigen, TF PCA and TF mRNA were analyzed by ELISA, flow cytometry, clotting or Xa generation assay and qPCR, respectively. PBMCs and THP1 cells showed significant insulin reductase activity, which was inhibited by bacitracin or rutin. Co-incubation with these thiol isomerase inhibitors prevented LPS-induced TF production by CD14-positive monocytes and constitutive TF expression by THP1 cells and AML blasts. Downregulation of the TF antigen was mainly restricted to the cryptic pool of TF, efficiently preventing phosphatidylserine-dependent TF activation by daunorubicin, and at least partially regulated on the mRNA level in LPS-stimulated monocytes. Our study thus delineates a complex role of thiol isomerases in the regulation of myeloid TF PCA, with PDI being a promising therapeutic target in the management of AML-associated coagulopathies.

Myeloperoxidase (MPO) is a cationic heme enzyme stored in neutrophilic polymorphonuclear leukocytes (PMNs) that has recently been implicated in inflammatory cell signaling and tissue damage. Although PMNs play a critical role in both innate immunity and vascular thrombosis, no previous study has systematically investigated the effect of MPO on blood coagulation. Here, we show that PMN-derived MPO inhibits the procoagulant activity (PCA) of lipidated recombinant human tissue factor (rhTF) in a time- and concentration-dependent manner that involves, but is not entirely dependent on the enzyme's catalytic activity. Similarly, MPO together with its substrate, H2O2, inhibited the PCA of plasma microvesicles isolated from lipopolysaccharide (LPS)-stimulated whole blood, an effect additive to that of a function blocking TF antibody. Treatment of whole blood with LPS or phorbol-myristate-acetate dramatically increased MPO plasma levels, and co-incubation with 4-ABAH, a specific MPO inhibitor, significantly enhanced the PCA in plasma supernatants. MPO and MPO/H2O2 also inhibited the PCA of activated platelets and purified phospholipids (PLs), suggesting that modulation of negatively charged PLs, i.e., phosphatidylserine, rather than direct interference with the TF/FVIIa initiation complex was involved. Consistently, pretreatment of activated platelets with MPO or MPO/H2O2 attenuated the subsequent binding of lactadherin, which specifically recognizes procoagulant PS on cell membranes. Finally, endogenously released MPO regulated the PCA of THP1 cells in an autocrine manner dependent on the binding to CD11b/CD18 integrins. Collectively, these findings indicate that MPO is a negative regulator of PL-dependent coagulation and suggest a more complex role of activated PMNs in haemostasis and thrombosis.

No abstract