Thiol isomerase inhibition with bacitracin or rutin interferes with tissue factor production in myelomonocytic HL-60 cells and isolated AML blasts

Introduction: Thiol isomerases, especially protein disulfide isomerase (PDI), are involved in regulation of tissue factor (TF) procoagulant activity (PCA) and its synthesis in myeloid cells [1] [2]. The effects of thiol isomerase inhibition critically depend on the specific inhibitor and individual reaction conditions [1] [2]. Since aberrant TF expression on transformed myeloblasts contributes to coagulation activation in acute myeloid leukemia (AML) [3] [4] [5], we now aimed to analyze the effects of thiol isomerase inhibition by bacitracin and rutin on TF production and PCA in myelomonocytic HL-60 cells and AML blasts.

Method: As a surrogate for AML blasts, peripheral blood mononuclear cells (PBMCs) were isolated from 65 untreated AML patients by density gradient centrifugation. In typical experiments, either PBMCs (2 x 10⁶ cells/mL) or HL-60 cells (1 x 10⁶ cells/mL) were incubated with 10 mM bacitracin, 100 µM quercetin-3-rutinoside (rutin) or PBS buffer control in RPMI medium supplemented with 20% fetal calf serum for 24 h. Subsequently, cells were analyzed for TF PCA by single-stage clotting assay and TF antigen by flow cytometry and ELISA. PDI expression was measured by flow cytometry and a fluorogenic assay. mRNA levels of TF and thiol isomerases were recorded by quantitative PCR.

Results: In sharp contrast to a previous report using THP1 cells [1], bacitracin and rutin significantly increased both TF antigen and PCA in HL-60 cells with less specific bacitracin showing the more prominent effects. Upregulation of TF was time- and concentration-dependent, accompanied by elevated phosphatidylserine (PS) membrane exposure and mediated by an increased TF de-novo production. Neither addition of a PAR2 inhibitor (ENMD547 or I 191) nor inhibition of the TF/FVIIa/FXa signaling complex using rivaroxaban prevented enhanced TF production by bacitracin. Compared to THP1 cells, HL-60 cells showed less cell surface-associated thiol isomerase activity in a fluorogenic assay and a markedly different thiol isomerase expression profile with the most prominent difference in PDIA2 expression. In our AML cohort, leukemic blasts from all patients showed relevant PDI expression. Incubation with bacitracin and rutin slightly but significantly reduced viability of leukemic blasts. In most patients (55/65, 84.6%), both inhibitors exerted similar effects on cellular TF PCA. While bacitracin and rutin increased cellular TF PCA in 63.6% (35/55) of AML patients, a reduction was observed in 36.4% (20/55).

Conclusion: In HL-60 cells and AML blasts, bacitracin and rutin regulate TF synthesis in a similar way. Both inhibitors either amplify or reduce TF synthesis, an effect that likely depends on the specific cellular expression pattern of thiol isomerases.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025

• References

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