The Role of Prothrombinase Complex Assembly and in Situ Fibrin Deposition on the Surface of Acute Promyelocytic Leukemia Cells in Hemorrhage
Authors: Li Tao , Yang Xiaoyan , Zhang Yan , LI Baorong , Liu Yinmiao , Zhang Cong , Kou Yan , Zou Lili , Liang Hui , Hou Li , Wang Chunxu , Yu Muxin , Du Jingwen , Jing Haijiao , Li Yueyue , Zhou Jin , Shi Jialan 
Published: October 2019

Abstract
Background: Despite dramatic improvement in treatment for acute promyelocytic leukemia (APL), early death due to hemorrhage remains a major obstacle to achieving a complete cure. In contrast to classical disseminated intravascular coagulation, APL-associated coagulopathy is characterized by rare microvascular fibrin thrombi. Thus, it is attractive to speculate whether other unknown mechanisms depleting coagulation factors and unrecognized fibrin-deposition location exist. Procoagulant activity associated with APL cells plays a direct role in bleeding complicationsin. We have shown that exposed phosphatidylserine (PS) on APL cells supports purified prothrombinase (Zhou J et al, JTH 2010) and fibrin preferentially deposits on promyelocytic chromatin from ETosis or apoptosis (Cao M et al, Blood 2017). However, relatively little is known about the PS-driven prothrombinase complex assembly and in situ fibrin deposition on APL cells.
Aims: Our objectives were to determine how APL cells promote thrombin generation and modulate fibrin formation and distribution, as well as to explore the relationship between in situ fibrin deposition and consumptive hemorrhage in APL patients.
Methods: Twenty-seven newly diagnosed APL patients were included. Fresh APL blasts were obtained from bone marrow specimens by centrifugation through Ficoll-Hypaque. Lactadherin was used as a probe for PS exposure on the fresh APL blasts and on an immortalized APL cell line (NB4). PS exposure and fluorescein-labeled FV/X binding were evaluated by flow cytometry. Thrombin generation was measured by modifed thrombin generation test. Fibrin production was quantified by turbidity. The distribution of PS, prothrombinase complex and in situ fibrin deposition were imaged by confocal microscopy. For the inhibition assay, APL cells were pre-treated with lactadherin, DNase I or anti-TF antibody for 10 min at 37 °C before incubation with plasma.
Results: Thrombin generation and fibrin formation supported by NB4 and APL cells increased approximately 1.5-fold after exposure to daunorubicin and decreased 80% after treatment with all-trans retinoic acid (ATRA) or arsenic trioxide (ATO). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure increased approximately 2.7-fold after treatment with daunorubicin, while ATRA and ATO initially led to a 70% reduction in PS exposure, which rose again on day 3 and 5 (P<0.001), respectively. Levels of externalized PS on APL cells paralleled levels of FV/FX binding, lag time, peak thrombin, endogenous thrombin potential and fibrin formation. Lactadherin inhibited the above parameters by approximately 80%, while anti-tissue factor antibody or DNase I produced no effect. Interestingly, confocal imaging showed that fibrin preferentially deposited on the surface of APL cells, which we defined as "in situ fibrin". Untreated viable APL and NB4 cells displayed discrete or occasionally annular fibrin deposition on the membrane. Moreover, fibrin formation supported by apoptotic APL cells displayed the following progression: (i) patchy deposition, (ii) diffuse rim, (iii) "fibrin coat", and (iv) network. The dynamic changes in fibrin formation paralleled the kinetics of PS exposure and prothrombinase assembly. Furthermore, initial percentage of PS-positive fresh APL cells was negatively correlated with plasma levels of fibrinogen and factor II, V, VIII, X in APL patients on admission (all P<0.01).
Conclusion: PS-driven prothrombinase complex assembly and in situ fibrin deposition on the surface of APL cells consume massive coagulation factors, providing a novel explanation for consumptive hemorrhage in APL patients. Blockade of PS might be a novel therapeutic approach for preventing bleeding in APL via inhibiting invisible "in situ coagulation", especially in high-risk APL.

Disclosures
No relevant conflicts of interest to declare.