Arsenic Trioxide (ATO) and All-Trans Retinoic Acid (ATRA) Differently Affect the Thrombin Generation Potential of Acute Promyelocytic Leukemia (APL) Cells.

A deregulated expression of procoagulant and anticoagulant activities characterises the phenotype of APL cells and contributes to the coagulopathy of this disease. The APL molecular remission induced by differentiation therapy with ATRA or ATO significantly affects specific cellular hemostatic properties of APL blasts. It is not known whether these variations translate into a comparable reduction of the overall procoagulant activity of APL cells. In this study we characterize the overall APL cell hemostatic potential by the calibrated automated method (CAT), a standardized global assay which reflects the net results of pro- and anti-coagulant forces. The endogenous thrombin potential (ETP), measured as the area under the thrombin generation (TG) curve, is a good indicator of overall plasma prothrombotic and hemorrhagic tendency. We evaluated 1) the sensitivity of NB4 cell TG potential to treatment with ATRA or ATO; 2) the correlation of CAT parameters to the levels of two known procoagulants, i.e. tissue factor (TF) and cancer procoagulant (CP), and anticoagulants (i.e. thrombomodulin, TM) and 3) the association of global TG to cell differentiation, proliferation, and apoptosis/necrosis.

NB4 cells were incubated 24h with either 0.1 μM ATO, or 1 μM ATRA, or the combination 0.1 μM ATO/1 μM ATRA, or the vehicle (control). The TG potential of NB4 cells was evaluated in normal pool plasma (NPP) by CAT assay; TF by chromogenic, ELISA, and cytofluorimetric assays; TFmRNA by RT-PCR; CP activity by chromogenic assay; TM by ELISA, cell differentiation by cytofluorimetric analysis of surface CD11b expression; and cell apoptosis/necrosis by annexin V/propidium iodide staining.

TG potential of control NB4 cells (1350±70 nM*min) was significantly higher compared to normal granulocytes (p<0.05). Lower levels of ETP were generated when TG was induced in plasma deficient of factor VII (FVII), confirming a major role to global TG for TF, but also suggesting the contribution for other procoagulants (i.e. CP, phospholipids) to the remaining FVII-independent activity. The TG of NB4 cells treated with ATRA (980±100 nM*min) and ATRA/ATO (1090±90 nM*min) was significantly lower compared to control cells, whereas ATO alone had a minor effect (1200±120 nM*min). Results of specific assays to identify TF and CP confirmed a greater effect of ATRA and the combination ATO+ATRA in reducing both procoagulants compared to ATO alone. The observed reduction of TG potential by the two drugs paralleled the reduction of cell proliferation due to induction of apoptosis and cell differentiation (ATRA and ATO+ATRA) or to cell necrosis (ATO).

ATRA alone or in combination with ATO is more effective in reducing the NB4-associated TG potential compared to ATO alone. While the ATRA±ATO-induced reduction of NB4-TG potential is associated to a downregulation of procoagulant factors (i.e. TF and CP) and to a parallel increase of the anticoagulant TM, differently, the ATO-induced decrease of NB4-TG potential is associated with the downregulation of TF and CP, without TM modification. CAT is a reliable method to characterize the TG potential of APL cells, and is sensitive to cell pharmacological treatments. Studies are needed to confirm the utility of the CAT to understand the role of different TG phenotypes in predicting the APL coagulopathy.

No relevant conflicts of interest to declare.