Promyelocytic Extracellular Chromatin Exacerbates Coagulation Disorder in Acute Promyelocytic Leukemia
Authors: Cao Muhua , Ma Ruishuang , Wu Xiaoming , Wang Lixiu , Zhao Lu , Zhang Yan , Yao Zhipeng , Li Tao , Kou Junjie , Bi Yayan , Zhou Jin , Shi Jialan 
Published: November 2019

Abstract
Introduction:Despite treatment with all-trans-retinoic acid, the early death rate in unselected acute promyelocytic leukemia (APL) due to hemorrhage still remains unacceptably high. It is attractive to speculate whether other uncovered procoagulants exist which are not attenuated by ATRA. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin (Ma R et al, Cell Death Dis 2016). However, the role of promyelocytic extracellular chromatin in APL-associated coagulation disorder remains unclear. The aims of this study were to identify the novel role of extracellular chromatin in induction of the hypercoagulable state in APL, and to evaluate its interaction with fibrin and endothelial cells (ECs).
Methods:Twenty-two newly diagnosed APL patients were included. Fresh APL blasts from bone marrow specimens were treated with 1 μM ATRA or phosphate buffered saline (PBS). ETosis was distinguished by rounded cells whose nuclei stained with PI and whose nuclear contents diffused throughout the cell. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Elastase-DNA complexes and TAT (thrombin-antithrombin) complexes were detected by ELISA. ECs were incubated in growth media containing 20% pooled serum obtained from healthy donors in the presence or absence of 20-fold concentrated extracellular chromatin. Procoagulant activity (PCA) of ECs and APL cells was evaluated by one-stage recalcification time assay, pro-thrombinase assay and fibrin formation assay. DNase I or anti-TF were included in the inhibition assays.
Results: ATRA treatment induced markedly increased cf-DNA release in a time-dependent manner compared with no ATRA group. Furthermore, ETosis was the major cell death pattern in the ATRA-treated group while apoptosis was predominant in the no-treatment group until the third day, indicating that the increased cell-free DNA triggered by ATRA was mainly from ETosis. NE-DNA, defined as marker of ETosis, peaked on day 3 and showed no significant elevation to day 5, indicating that increased part of cf-DNA from day 3 to day 5 was mainly from apoptosis. Additionally, thrombin generation was found to parallel the change in the releasing of promyelocytic extracellular chromatin induced by ATRA. Pretreatment with DNase I inhibited thrombin generation by 47%, diminished PCA by 35%, prolonged coagulation time, and attenuated fibrin formation by 50%, while neutralizing anti-TF antibody produced no effect. Confocal microscopy showed that fibrin was preferentially deposited on promyelocytic chromatin from ETosis or apoptosis and exposed PS. Lastly, we found that extracellular chromatin from the ATRA group significantly triggered PS exposure on ECs, converting them to a pro-coagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C (APC) by 31% indicating that DNA scaffold and histones were both necessary for the cytotoxic effect of extracellular chromatin.
Conclusions:ATRA promotes procoagulant promyelocytic extracellular chromatin mainly through ETosis. Extracellular chromatin fosters excess thrombin generation, increases fibrin deposition, and causes endothelium damage. To improve the remaining coagulation disturbance in APL patients of high risks during ATRA administration, therapeutic strategies focusing on combined application of DNase I and APC to accelerate the degradation of overwhelmed promyelocytic extracellular chromatin would be of great interest in the future.

Disclosures
No relevant conflicts of interest to declare.