promyelocytic extracellular chromatin exacerbates coagulation and

11

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute

promyelocytic leukemia

Short title for running head: Procoagulant ETosis in APL

Muhua Cao,1,2 Tao Li,1,2 Zhangxiu He,1 Lixiu Wang,4 Xiaoyan Yang,1 Yan Kou,1 Lili Zou,5 Xue Dong,1

Valerie A Novakovic,3 Yayan Bi,4 Junjie Kou,5 Bo Yu,2 Shaohong Fang,2 Jinghua Wang,6 Jin Zhou,1 and

Jialan Shi1,7

1Department of Hematology of the First Hospital, Harbin Medical University, Harbin, China; 2The Key

Laboratory of Myocardial Ischemia, Ministry of Education, Heilongjiang Province, Harbin, China;

3Department of Research, VA Boston Healthcare System, Boston, MA, USA; 4Department of Cardiology of

the First Hospital, Harbin Medical University, Harbin, China; 5Departments of Cardiology of the Second

Hospital, Harbin Medical University, Harbin, China; 6Department of Hematology of the Second Hospital,

Harbin Medical University, Harbin, China; 7Department of Surgery, Brigham and Women’s Hospital, VA

Boston Healthcare System, and Harvard Medical School, Boston, MA, USA.

Correspondence:

Jialan Shi, or Jin Zhou, Department of Hematology, The First Hospital, Harbin Medical University,

Harbin 150001, China; Tel: +1 857 203 5914; Fax: +1 857 203 5592; e-mail: [email protected] or

[email protected]. Jinghua Wang, or Shaohong Fang, Department of Medicine, the Second Hospital,

Harbin Medical University, Harbin 150086, PR China; e-mail: [email protected] or

[email protected].

Text word count: 3896

Abstract word count: 234

Figure count: 6

Table count: 2

Reference count: 45

Scientific category: Thrombosis and Hemostasis

Key Points

Blood First Edition Paper, prepublished online January 4, 2017; DOI 10.1182/blood-2016-09-739334

Copyright © 2017 American Society of Hematology

For personal use only.on April 11, 2018. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

22

� ATRA promotes ETosis leading to procoagulant promyelocytic extracellular chromatin.

� Extracellular chromatin fosters excess thrombin production and fibrin deposition, increases plasmin and

causes endothelium damage.

Abstract

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid

(ATRA), early death due to hemorrhage remains unacceptably common and the mechanism underlying this

complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death

program, termed ETosis, which involves release of extracellular chromatin. However, the role of

promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were

to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and

hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results

from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases

thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases

fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence

staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide

platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, co-incubation

assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a

procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C (APC) by

31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL

coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA

administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin

degradation.

Introduction

Acute promyelocytic leukemia (APL) is characterized by life-threatening coagulopathy consisting of

thrombotic and bleeding complications.1-3 Thanks to the application of all-trans-retinoic acid (ATRA) during

the last two decades, APL has been considered a highly curable disease with more than a 90% remission

rate.4,5 Disappointingly, the early death (ED) rate still remains at 17-29% in population-based registries (with

coagulopathy accounting for 40-65% of these cases) despite ATRA administration.1-3 Thus, further




33

understanding of the pathogenesis of APL coagulopathy is urgently needed.

ATRA has been shown to rapidly reverse signs of coagulation, reduce blood product consumption, and

reduce bleeding severity through down-regulation of well-known procoagulants such as tissue factor (TF),

cancer procoagulant (CP), and annexin II on both APL cells and human APL cell line NB4.6-8 However,

severe bleeding or thrombotic complications still occur even when rapid administration of ATRA is given at

the first suspicion of APL.3,9 Furthermore, previous trials have reported that clotting activation markers and

fibrinogen often did not return to normal even when the clinical bleeding diathesis is resolved.8,10 It is

attractive to speculate whether other unknown procoagulants exist which can not be attenuated by ATRA.

Previous studies suggested that cell free-DNA (cf-DNA), from apoptotic cells or in the form of NETs,

may activate coagulation via the contact pathway11-15 and thereby enhance the pathology of various

coagulation-associated diseases such as sepsis,16 acute myocardial infarction,17 and acute venous

thromboembolism (VTE).18 Recently, we have shown that APL blasts undergo ETosis, a novel cell death

pathway distinct from apoptosis or necrosis, which releases intact chromatin into the extracellular space in

response to stimulation.19 However, relatively little is known about the role of promyelocytic extracellular

chromatin in APL coagulopathy or its response to ATRA treatment.

Here we continued our previous study by evaluating how extracellular chromatin influences the

fibrinolytic and procoagulant activity (PCA) of APL cells and observing the distribution of fibrin on APL

cells undergoing ETosis or apoptosis after ATRA treatment. Moreover, since endothelium damage can initiate

differentiation syndrome and exacerbate coagulopathy, the interaction between promyelocytic extracellular

chromatin and endothelial cells (ECs) was also explored. Our study may help identify novel targets for

coagulopathy intervention and prevention of early death for APL patients following ATRA administration.

Materials and methods

Patients

Forty newly diagnosed APL patients admitted to the First and Second Affiliated Hospital of Harbin Medical

University from October 2014 to November 2016 were studied after informed consent. The diagnosis was

based on clinical data, morphology, cytochemistry, immunology, cytogenetics, and molecular pathology

testing or alternatively confirmation of the presence of the t (15; 17) (PML-RARA) fusion gene.20 This study

was approved by Ethics Committee of Harbin Medical University and conducted according to the




44

Declaration of Helsinki. The main characteristics of the patients on the day of bone marrow aspiration are

shown on Table 1.

Reagents

Human APL NB4 cell line was a gift from Dr James O’kelly (Los Angeles, CA). Human umbilical vein cells

(HUVECs), ECs medium, and Poly-L-Lysine were from ScienCell (San Diego, CA, USA). RPMI 1640

medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Ficoll-Hypaque,

bovine serum albumin (BSA), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, interleukin-6, ATRA,

daunorubicin (DNR)，EDTA, Triton X-100 and DNase I were all obtained from Sigma-Aldrich (St Louis,

MO, USA). Human recombinant activated protein C (APC) was obtained from Eli Lilly (Hoosier state,

USA). Propidium iodide (PI) was from Shanghai DobioCO, LTD. (Shanghai, China). Spectrozyme-PL

(H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide), tissue type plasminogen activator (t-PA), polyclonal

anti-human TF (4502) and FITC-conjugated anti-human TF (4508CJ) were all obtained from American

Diagnostica (Stamford, CT). Human alpha-thrombin (IIa)，human factor X, Xa, IXa, VIIa, VIII and thrombin

were obtained from Enzyme Research Laboratories (South Bend, IN). Human factors V, Va, fluorescein

EGR-Chloromethylketone and lactadherin were all obtained from Haematologic Technologies (Burlington,

VT). Chromogenic substrates S-2765 and S-2238 were obtained from Instrumentation Laboratory Company

(MA, USA). Alexa Fluor 488 or 647-conjugated lactadherin, fluorescein-labeled fibrinogen, FVa and FXa

were prepared in our laboratory.

Promyelocytic extracellular traps stimulation, quantification, and isolation

Isolated APL cells and NB4 cells were resuspended in RPMI 1640 and 1×106 cells were seeded per well in

6-well plates. Cells were primed with cytokine mixture of TNF-α (10 ng/mL), IL-1β (10 ng/mL), and IL-6

(10 ng/mL) for 1 h at 37 °C. The media was removed and wells were washed with RPMI. Cells were then

treated with 1μM ATRA or phosphate buffered saline (PBS) for the indicated time points (0, 1, 3, 5 days) at

37 °C. Extracellular chromatin isolation was performed as previously described17,21. To characterize cell

death, APL or NB4 cells were incubated with PI and FITC-labeled lactadherin.22 Cells were washed and

analyzed immediately on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss Jena GmbH, Jena,

Germany). Samples were excited with the 488 nm emission line of a krypton-argon laser. Cells undergoing

ETosis were identified by rounded morphology, PI staining, and the presence of nuclear content diffused

throughout the cell.19 Cells were counted from six random fields in triplicate wells for each condition and




55

expressed as percentage of total number of cells in the field.23

Determination of cell-free DNA, MPO-DNA complex and thrombin-antithrombin complex levels in the

supernatant

Cf-DNA was quantified in the supernatant and plasma of newly diagnosed APL patients using the Quant-iT

PicoGreen dsDNA Assay Kit (Invitrogen) according to the manufacturer’s instructions. Extracellular

chromatin from ATRA-treated APL cells on day 3 was about 800 ng/mL. 20 and 50 fold concentrated

extracellular chromatin from ATRA-treated APL/NB4 cells used in the indicated experiments were about 16

μg/mL and 40 μg/mL, respectively. Myeloperoxidase–DNA (MPO-DNA) complex was detected in the

supernatant and plasma using a capture ELISA as previously described.17,19,24,25 TAT complex was detected

by ELISA in control plasma incubated with isolated extracellular chromatin in vitro as previously

described.17

ECs stimulation assays

ECs were incubated in RPMI in the presence or absence of concentrated extracellular chromatin (20-fold

concentrated) derived from NB4 cells treated by PBS or ATRA (day 3) at room temperature for 24 h. For

inhibition assays, isolated extracellular chromatin was pre-treated with DNase I (100 U/mL)26 for 20 min or

APC (100 nM)21 for 1h at 37 °C prior to its introduction into culture supernatant of HUVECs. At designated

time points, ECs were centrifuged and washed twice with PBS for the following experiments.

Phosphatidylserine (PS) exposure was detected by flow cytometer. Pro-thrombinase, intrinsic FXa, and

extrinsic FXa assays were performed as previously described.26

Procoagulant activity and fibrin formation assays

PCA of APL cells after ATRA treatment was evaluated by one-stage recalcification time assay in a

KC4A-coagulometer (Amelung, Labcon, Heppenheim, Germany).27 Fibrin formation on APL cells was

quantified by turbidity as described.28 Briefly, cell-containing suspensions (25 μL 1×106 cells were washed

twice and resuspended in 75 μL of Tyrode’s buffer) were incubated with pre-warmed microparticle-depleted

plasma (MDP) (20%) from healthy controls in the presence of 3 mM calcium. For the inhibition assays,

isolated extracellular chromatin was pre-treated with lactadherin (128 nM), anti-TF (40 μg/mL) for 10 min or

DNase I (100 U/mL) for 20 min at 37°C before incubation with plasma. After incubation for 3 minutes, 100

μL of pre-heated 25 mM CaCl2 was added and the time to fibrin strand formation was recorded.

After incubation with isolated extracellular chromatin, HUVECs were rinsed with Tyrode’s buffer and

then were overlaid with pre-warmed MDP (15%) from healthy controls in the presence of 3 mM calcium.




66

Fibrin production was measured by turbidity at 405 nm in a SpectraMax 340PC plate reader. All clotting

assays were performed in triplicate.

Confocal microscopy

PS exposure on cultured ECs was determined by incubation of HUVECs with Alexa Fluor 488-labeled

lactadherin and Alexa Fluor 647-labeled CD31. To observe FXa and FVa binding, stimulated ECs and NB4

cells were co-stained with factor Va-fluorescein-maleimide and factor Xa-EGRck-biotin (complexed to

Alexa 647-streptavidin). Fibrin networks on stimulated ECs and NB4 cells were stained by Alexa Fluor

647-conjugated fibrinogen and Alexa Fluor 488-labeled lactadherin. All above samples were excited with

488 or 568 nm emission lines of a krypton-argon laser, and narrow band pass filters were used for restricting

emission wavelength overlap. Images were obtained using an LSM 510 SYSTEM. Background signal was

calculated by using a similarly labeled isotype matched control antibody.

Plasminogen activation assay

Plasminogen activation assay was performed as previously described.29

Plasma clot lysis assay

Plasma clot lysis assay was performed as previously described.30

Clot permeability assays

Clots were prepared from 8 μM fibrinogen supplemented with 20 mM Ca2+ ± extracellular chromatin

(50-fold concentrated) and were clotted with 16 nM thrombin in plastic pipette tips. After 70 min of

incubation at 37°C, PBS was permeated through the clots. Pressure was kept constant by maintaining a fixed

head volume (pressure drop was 0.056 N cm-2). Permeability coefficient (Ks) was calculated as previously

described.31

The methods for “Blood collection”, “Cell culture”, “Plasminogen activation assay”, and “Plasma clot

lysis assay” are presented in detail in the “Supplementary Methods”.

Statistical analysis

Numerical variables were tested for normal distribution with the Kolmogorov-Smirnov test. Normally

distributed variables were summarized as mean ± standard deviation (SD) and statistical analysis was made

by t-test or ANOVA as appropriate. Non normally distributed variables were summarized as medians with

interquartile ranges (IQR) and Mann-Whitney U-test was conducted. Correlations between two continuous

variables were performed using Pearson correlation coefficients, and Spearman rank correlation were used to




77

detect discrete variables. Categorical variables were compared using the χ2-test. P<0.05 was considered

statistically significant.

Results

ATRA potentiates procoagulant extracellular chromatin release from APL and NB4 cells

ATRA treatment induced markedly increased cf-DNA release in a time-dependent manner compared with the

untreated group (Figure 1A) (NB4 data not shown). MPO-DNA, a marker of ETosis,24,25 was higher in the

ATRA-treated cells than in controls. Additionally, MPO-DNA showed no significant increase from day 3 to

day 5, indicating that the increase in cf-DNA during this time was mainly from apoptosis (Figure 1B). To

determine the predominant cell death pattern which is responsible for the increased extracellular chromatin,

APL/NB4 cells were stained with lactadherin and PI and analyzed by confocal microscopy (Figure 1C).

Results showed that ETosis was the major cell death pattern seen in the ATRA-treated group up to the third

day，indicating that the increase in cell-free DNA triggered by ATRA was mainly from ETosis. Apoptosis

was predominant in the no-treatment group on day 3, consistent with our previous study19. Extracellular

chromatin showed no significant degradation when incubated with control plasma for up to 45 min

(supplemental Figure 1). Thrombin generation in the presence of extracellular chromatin isolated from all

cells progressively increased over time. Extracellular chromatin isolated from the ATRA treatment group

increased thrombin generation approximately 2-fold on day 3 compared with that from the untreated group

and 1.5-fold on day 5 (Figure 1D). Degrading cell-free DNA using DNase I decreased thrombin generation

by 42% on day 3 and 51% on day 5 for APL cells. For NB4 cells, DNase inhibited thrombin generation by

36% on day 3 and 47% on day 5, indicating a relatively strong procoagulant effect of promyelocytic cell-free

DNA. Neutralizing anti-TF antibody had no effect (Figure 1E). Cf-DNA and MPO-DNA complexes were

measured in the peripheral blood of newly diagnosed APL patients. Patients had markedly increased levels of

cf-DNA and MPO-DNA complexes compared to controls. Further, baseline WBC counts were positively

correlated to both plasma cf-DNA and MPO-DNA complexes (supplemental Figure 2A).

Effect of ATRA on integrated PCA of APL cells

Having identified a procoagulant effect of isolated soluble chromatin, we next tested the overall effect of

ATRA treatment for various incubation times on PCA and fibrin formation when APL cells were mixed with

plasma. ATRA differentiation resulted in a marked decrease in PCA and fibrin formation of cytokine-primed

APL cells on day 1-3 compared with day 0 followed by an increase on day 5. In contrast, the PCA and fibrin




88

formation of PBS-treated APL cells progressively increased during the five days. In comparison with PBS

treated group, ATRA-treated APL cells exhibited lengthened coagulation time and decreased fibrin formation

during the five days (Figure 2A, B). For inhibition assays, cells were mixed with either DNase I, anti-TF, or

lactadherin prior to coagulation time or fibrin formation assays. Lactadherin caused a significant reduction in

PCA and fibrin formation at all time points for both ATRA and PBS treated cells. DNase I reduced PCA and

fibrin formation for ATRA-treated cells on day 3 and day 5 (Figure 2C, D) while having no effect on

PBS-treated cells. Anti-TF had minimal effect on ATRA-treated cells, but dramatically decreased PCA and

fibrin formation on untreated cells (Figure 2E, F).

Distribution of fibrin on NB4 cells undergoing apoptosis or ETosis after ATRA treatment

Since extracellular chromatin causes elevated fibrin generation, we then explored how fibrin is distributed

during ETosis or apoptosis using confocal microscopy. For cells undergoing ETosis, the nuclei expanded and

filled with cytoplasm. Then the cells expanded into a single bubble which was larger than the quiescent cells,

with a diffuse rim weakly stained by lactadherin. When the thinner membrane of the bubble was damaged,

the chromatin spilled out into the extracellular space (Figure 3A). Fibrin deposited on the same position of

expanded chromatin inside the single bubble. Furthermore, fibrin (red) also deposited around the bubble,

co-localized with exposed PS on the thin intact membrane and the remaining broken membrane fragments

(Figure 3B). On day 5, most cells underwent apoptosis, with clear staining of the nuclear and externalized

phosphatidylserine (PS) on the cell membrane (Figure 3C). We observed fibrin deposition at the same

position as the compromised chromatin inside the apoptotic cells. Moreover, fibrin also co-localized with

externalized PS on the membrane which was strongly stained by lactadherin (Figure 3D). Staining of FXa

(red) and FVa (green) showed patchy distributions that overlapped (yellow) on apoptotic NB4 cell

membranes (Figure 3E), indicating Xa/Va (prothrombinase) complex assembly. ET-releasing cells also

showed substantial FVa deposition on the thin membrane around the bubble. Both FXa and FVa share a

preference for convex surfaces, similar to lactadherin. For some adjacent late apoptotic NB4 cells, a large

amount of fibrin (red) strands deposited on the membranes with exposed PS (green), forming a net structure

among NB4 cells (Figure 3F).

Promyelocytic extracellular traps impair fibrinolysis

Because fibrin bound to chromatin (extracellular and intracellular) in the microscopy experiments, we

investigated whether promyelocytic chromatin influences the structure of fibrin and the process of

fibrinolysis such as plasminogen activation and clot digestion. Plasma clots formed in the presence of




99

concentrated chromatin from ATRA-treated or PBS-treated APL cells were mixed with tPA and the

generation of plasmin was measured. A titration curve of plasmin generation using different concentrations

of extracellular chromatin was performed to find an appropriate experimental concentration (supplemental

Figure 3). Concentrated chromatin from the ATRA-treated cells markedly enhanced plasmin generation

while chromatin from the PBS-treated cells had little effect compared to control clots formed without

chromatin. Although the amount of chromatin peaked on day 5 (Figure 1A), plasmin generation from the

ATRA-treated cells on day 5 was not significantly different than seen on day 3 (Figure 4A), suggesting

different effects from chromatin resulting from ETosis versus apoptosis. Clot lysis assays were performed on

control plasma supplemented with isolated chromatin. Addition of chromatin from the ATRA-treated group

to control plasma increased maximum absorbance by about 1.64-fold (Figure 4B) and caused a 3.03-fold

prolongation of the clot lysis time compared with PBS-treated groups (Figure 4C). In the clot permeability

assays, chromatin from the ATRA-treated groups increased the permeability of clots by approximately

3.3-fold while chromatin from PBS-treated groups had no significant effect (Table 2). No significant

difference in maximum absorbance, clot lysis or clot permeability assays were seen between chromatin

isolated from APL cells on day 3 and day 5 of ATRA treatment. For newly diagnosed APL patients, plasma

cf-DNA was negatively correlated with fibrinogen. No correlation was found between cf-DNA or

MPO-DNA complexes with D-dimer (supplemental Figure 2B).

Cytotoxic effect of promyelocytic extracellular chromatin on HUVECs

In response to lung injury, neutrophil extracellular traps and histones induce activation and apoptosis of

endothelial cells.21,32 We therefore wondered whether isolated extracellular chromatin from NB4 cells have a

cytotoxic effect on HUVECs. Titration curve of %PS+ HUVECs triggered by different concentration of

isolated extracellular chromatin for 24 h was performed to find an appropriate experimental concentration

(supplemental Figure 4). ECs treated with ATRA-derived extracellular chromatin lost their normal

morphology and retracted from the cell-cell junctions obviously (Figure 5B), while changes of

PBS-extracellular chromatin treated ECs was minimal (Figure 5A). Filopodia and localized regions on the

EC margins co-stained with Alexa Fluor 647-lactadherin and Alexa Fluor 488-annexin, indicating PS

exposure (Figure 5C). Results showed that ATRA-derived chromatin triggered an increase in PS exposure on

ECs in a time-dependent manner compared with PBS-derived chromatin (Figure 5D). Inhibition studies were

performed using DNase I to degrade the DNA scaffold and APC to dissect histones. PS exposure on ECs was

inhibited by 20%, 31%, and 37.2% when DNase I, APC, or both respectively, were included in the assay,




1010

indicating that the DNA scaffold and histones are both necessary for the cytotoxic effect of extracellular

chromatin. Additionally, the combined inhibitory effect of DNase I and APC was significantly stronger than

DNase I alone (P < .05) (Figure 5E).

Promyelocytic extracellular traps convert HUVECs to a procoagulant phenotype.

Since ATRA-derived extracellular chromatin induced increased PS exposure on ECs, we then speculated

whether the increased PS exposure can support elevated tenase and prothrombinase activity. Data showed

that compared with those treated by PBS-derived chromatin, ECs incubated with ATRA-derived chromatin

increased production of intrinsic and extrinsic FXa complexes and thrombin generation (Figure 6A). For the

inhibition assays: thrombin, intrinsic and extrinsic FXa production were reduced by 38%, 31.1% and 29.5%,

respectively, in the presence of DNase I; by 46％, 44.7% and 40.7% with APC; and by 57.5%, 57.6% and

59.4% with both DNase I and APC (Figure 6B). Next, the integrated PCA of ECs was evaluated using

coagulation time and fibrin generation assays. As expected, treatment of ECs with ATRA-derived chromatin

triggered rapid and massive fibrin formation, which accelerated clotting (Figure 6C). Additionally,

combination of DNase I and APC produced a significantly stronger inhibitory effect than DNase I alone,

indicating that while the DNA scaffold is necessary, the cytotoxicity of extracellular chromatin was mainly

due to histones (Figure 6D). Using confocal microscopy, a significant co-localized fraction of bound FVa and

FXa was observed, indicating that ECs treated with ATRA-extracellular chromatin were able to offer a

biological surface for binding coagulation factors, most likely through externalized PS (Figure 6E).

Furthermore, large fibrin strands were radially distributed along the filopodia of ECs that were pretreated

with ATRA-extracellular chromatin (Figure 6F).

Discussion

In this study, we made four significant observations. First, thrombin generation paralleled the release of

promyelocytic extracellular chromatin induced by ATRA. Moreover, on the third day of ATRA treatment,

DNase I reduced PCA and fibrin generation, while anti-TF antibody produced no effect. For untreated

APL/NB4 cells, anti-TF antibody significantly inhibited PCA and fibrin generation, while the effect of

DNase I was minimal. Second, confocal microscopy showed that fibrin preferentially deposits on

promyelocytic chromatin from ETosis or apoptosis and exposed PS. Notably, extracellular chromatin from

ETosis, but not apoptosis, promotes plasmin generation while at the same time impairing clot lysis, thus

highlighting the involvement of extracellular chromatin in fibrinolysis. Fourth, isolated promyelocytic

extracellular chromatin exerted a strong cytotoxic effect on ECs, converting them to an increased




1111

pro-coagulant phenotype.

NETosis can be triggered by many factors including bacteria infection, inflammatory cytokines, and

immune disorder. Particular emphasis has been placed on the harmful effects of increased extracellular

chromatin in various disease states. For APL cells, ETosis appears to be the major source of the increased

extracellular chromatin in the early period of ATRA treatment, although it is difficult to determine whether

ETosis is promoted directly from the drug treatment or indirectly through induced differentiation. However,

this effect seems predominantly dependent on increased cytokines from differentiating myeloid cells during

ATRA administration, as evidenced by our previous study.19 Patients with infection, disseminated

intravascular coagulation (DIC), a high number of leukocytes,33 and differentiation syndrome all present with

high systematic inflammatory responses, where enhanced ETosis occurred. Increased cf-DNA and decreased

DNase I activity are associated with many coagulopathy-associated diseases such as thrombotic

microangiopathies34 and systemic lupus erythematosus.35 Although ATRA successfully suppresses the

extrinsic pathway by down-regulating the expression of TF and annexin-II, it also triggers the release of

promyelocytic extracellular chromatin, a potential dangerous factor. Extracellular chromatin may further

promote intrinsic coagulation processes, cause excess consumption of coagulation factors such as

plasminogen and fibrinogen, enhance fibrinolytic resistance, and damage ECs, contributing to increased

incidence of induction failure in high-risk APL patients.

Recent evidence suggests that fibrin, chromatin, and von Willebrand factor form a co-localized network

within the thrombus in many thromboembolism diseases such as VTE and acute myocardial infarction.17,36

Here, we provided visible evidence that APL cells undergoing ETosis or apoptosis supported fibrin

deposition around chromatin (extracellular or intracellular) and accessible PS. These results are supported by

a recent study showing high affinity binding of cf-DNA to fibrinogen and fibrin in a purified system in

vitro.30 Of particular interest, we observed that FVa and FXa bind to the PS from APL cells undergoing

ETosis or apoptosis, indicating prothrombinase complex assembly. We have previously reported that PS

exposure on apoptotic APL cells supports increased generation of intrinsic FXase complex and

prothrombinase complex.20 This study goes beyond the prior reports, correlating the release of promyelocytic

chromatin and PS exposure from ETosis and apoptosis with increases in prothrombinase complex assembly

and fibrin deposition. The combination of increased chromatin with accessible PS could cause excess

consumption of coagulation factors and fibrinogen, leading to increased risk of hemorrhage and DIC.

Another interesting finding was that extracellular chromatin produces two competing effects in the




1212

fibrinolytic system; it directly facilitates t-PA mediated plasmin generation while also impairing clot lysis.

Delayed fibrin lysis could in turn cause continued stimulation of the fibrinolytic system, leading to

exacerbated fibrinolysis disorder. Importantly, chromatin from day 5 did not exert a stronger effect on

fibrinolysis than that from day 3. It seems that chromatin from apoptotic cells (day3-5) does not have the

same effects on fibrinolysis as chromatin from ETosis (before day 3). This may be due to the special

characteristics of the high molecular weight fragments of extracellular chromatin from APL cells undergoing

ETosis. The size of cf-DNA is known to be dependent on the cellular process by which it was liberated;

apoptotic cells release a ladder pattern of DNA at ≈150-bp interval,37 whereas necrotic cells and NET

releasing cells release high molecular weight fragments >10 000 bp.38,39 Our results thus reveal the distinct

functions of chromatin originating from different death patterns in fibrinolysis. Additionally, extracellular

chromatin caused increased cell permeability. Previous research has shown that high level of cf-DNA

decreased the density of clots while low and intermediate level of cf-DNA increased clot density.30

Furthermore, different concentrations of DNA, histones, or complexes have different effects on fibrin

structure.29 Further studies are needed to identify the exact mechanism behind these differences.

Differentiation syndrome is a relatively common and serious complication that can occasionally be

life-threatening in patients with acute promyelocytic leukemia (APL) undergoing induction therapy with

all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO). Endothelium damage is the major

pathophysiologic process of DS, leading to local hypoxia, ischemia, and tissue edema, leading to multiple

organ failure (MOF) and DIC.40 A previous study suggested that ATRA enhances the potential of NB4 cells

to stimulate the expression of TF and PCA in endothelium, though the mechanism is still not fully known.7 In

our study, we demonstrate that extracellular chromatin from ATRA treatment triggered PS exposure on ECs

and converted them to a pro-coagulant phenotype. In addition, our photomicrographs provide visible

evidence that ECs treated with ATRA-derived chromatin bound FVa and FXa primarily on the filopods and

fibrils of retracted ECs. The selective distribution of fibrin is similar to the preferential binding of

prothrombinase to PS. It has been confirmed that NETs derived from neutrophils can cause tissue damage in

severe acute pancreatitis41 and sterile inflammatory liver injury.42 However, controversy still remains over

whether the DNA scaffold influences the cytotoxic effect of NETs. Saffarzadeh et al. showed that

NET-mediated cytotoxicity occurs primarily through histones21 and is not influenced by DNA digestion. This

controversy may be due to (a) different processes of ETosis occurring in APL/NB4 cells versus PMNs19 or (b)

interactions between histones and other enzymes (elastase, MPO)43 since enzymes inside the granules were




1313

different between promyelocytic cells and PMNs.44 Moreover, many previous studies have shown that

DNase I breakdown of NETs partially attenuates tissue injury, consistent with our results that indicate a

cytotoxic role for both histones and the DNA scaffold.32,41,42,45 Our results show that DNase I and APC

protects endothelial cells from the cytotoxic effects of promyelocytic extracellular chromatin and decreases

the resulting PCA.

We have previously reported that APL plasma triggered enhanced promyelocytic ETosis than control

plasma.19 In this study, we found a statistically significant positive correlation between baseline WBC counts

and both cf-DNA and MPO-DNA. Moreover, cf-DNA was negatively correlated with fibrinogen. Previous

studies reported that total white cell count have emerged as good general predictors of hemorrhagic death. 46

Since prompt treatment with all-trans retinoic acid, with or without arsenic trioxide, has been the most

important step in preventing bleeding complications, future therapeutic strategies could focus on combined

application of DNase I and APC with ATRA to accelerate degradation of promyelocytic extracellular

chromatin to further decrease risk of coagulopathy, especially in high-risk APL patients.

Acknowledgments

We thank Yanming Xue for the sample collection, and Jiangtian Tian, Ji Li, Hulun Li for excellent technical

assistance. We thank James O’Kelly (Los Angeles, CA, USA) for providing NB4 cells. This work was

supported by grants from the National Science Foundation of China (81270588, 81670128, 81670298，

81470301).

Authorship Contributions

Contribution: M.C. designed the research, performed experiments, analyzed results, made the figures and

wrote the paper; J.S. obtained funding, designed the study, performed experiments, analyzed results, made

the figures, and revised the manuscript; J.Z. provided partial funding support; T.L., Z.H., L.W., X.Y., Y.K.,

L.Z., X.D. performed some experiments; V.N., Y.B., J.K., B.Y., S.F., J.W. analyzed data and revised the

manuscript.

Conflict of Interest Disclosure: The authors declare no competing financial interests.

References

1. Park JH, Qiao B, Panageas KS, et al. Early death rate in acute promyelocytic leukemia remains high

despite all-trans retinoic acid. Blood. 2012;118(5):1248-1254.




1414

2. Lehmann S, Ravn A, Carlsson L, et al. Continuing high early death rate in acute promyelocytic

leukemia: a population-based report from the Swedish Adult Acute Leukemia Registry. Leukemia.

2011;25(7):1128-1134.

3. McClellan JS, Kohrt HE, Coutre S, et al. Treatment advances have not improved the early death rate in

acute promyelocytic leukemia. Haematologica. 2012;97(1):133-136.

4. de Thé H, Chen Z. Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat Rev

Cancer. 2010;10(11):775-783.

5. Sanz MA, Montesinos P, Rayón C, et al. Risk-adapted treatment of acute promyelocytic leukemia based

on all-trans retinoic acid and anthracycline with addition of cytarabine in consolidation therapy for

high-risk patients: further improvements in treatment outcome. Blood. 2010;115(25):5137-5146.

6. Watts JM, Tallman MS. Acute promyelocytic leukemia: What is the new standard of care? Blood Rev.

2014;28(5):205-212.

7. Zhu J, Guo WM, Yao YY, et al. Tissue factors on acute promyelocytic leukemia and endothelial cells are

differently regulated by retinoic acid, arsenic trioxide and chemotherapeutic agents. Leukemia. 1999;

13(7):1062-1070.

8. Tallman MS, Lefèbvre P, Baine RM, et al. Effects of all-trans retinoic acid or chemotherapy on the

molecular regulation of systemic blood coagulation and fibrinolysis in patients with acute promyelocytic

leukemia. J Thromb Haemost. 2004;2(8):1341-1350.

9. Rashidi A, Riley M, Goldin TA, et al. Delay in the administration of all-trans retinoic acid and its effects

on early mortality in acute promyelocytic leukemia: Final results of a multicentric study in the United

States. Leuk Res. 2014;38(9):1036-1040.

10. Mi JQ, Li JM, Shen ZX, et al. How to manage acute promyelocytic leukemia. Leukemia.

2012;26(8):1743-1751.

11. Longstaff C, Varjú I, Sótonyi P, et al. Extracellular nucleosome networks, neutrophils, and thrombosis. J

Biol Chem. 2013;288(10):6946-6956.

12. Geddings JE, Mackman N. New players in haemostasis and thrombosis. Thromb Haemost. 2014;

111(4):570-574.

13. Kannemeier C, Shibamiya A, Nakazawa F, et al. Extracellular RNA constitutes a natural procoagulant

cofactor in blood coagulation. Proc Natl Acad Sci U S A. 2007;104(15):6388-6393.

14. Swystun LL, Mukherjee S, Liaw PC. Breast cancer chemotherapy induces the release of cell-free DNA,

a novel procoagulant stimulus. J Thromb Haemost. 2011;9(11):2313-2321.

15. Gould TJ, Vu TT, Swystun LL, et al. Neutrophil Extracellular Traps Promote Thrombin Generation

Through Platelet-Dependent and Platelet-Independent Mechanisms. Arterioscler Thromb Vasc Biol.

2014;34(9):1977-1984.




1515

16. Luo L, Zhang S, Wang Y, et al. Proinflammatory role of neutrophil extracellular traps in abdominal

sepsis. Am J Physiol Lung Cell Mol Physiol. 2014;307(7):L586-596.

17. Stakos DA, Kambas K, Konstantinidis T, et al. Expression of functional tissue factor by neutrophil

extracellular traps in culprit artery of acute myocardial infarction. Eur Heart J. 2015;36(22):1405-1414.

18. Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis.

Arterioscler Thromb Vasc Biol. 2012;32(8):1777-1783.

19. Ma R, Li T, Cao M, et al. Extracellular DNA traps released by acute promyelocytic leukemia cells

through autophagy. Cell Death Dis. 2016;30;7(6):e2283.

20. Zhou J, Shi J, Hou J, et al. Phosphatidylserine exposure and procoagulant activity in acute

promyelocytic leukemia. J Thromb Haemost. 2010;8(4):773-782.

21. Saffarzadeh M, Juenemann C, Queisser MA, et al. Neutrophil extracellular traps directly induce

epithelial and endothelial cell death: a predominant role of histones. PLoS One. 2012;7(2):e32366.

22. Shi J, Shi Y, Waehrens LN, et al. Lactadherin detects early phosphatidylserine exposure on immortalized

leukemia cells undergoing programmed cell death. Cytometry A. 2006;69(12):1193-201.

23. Demers M, Krause DS, Schatzberg D, et al. Cancers predispose neutrophils to release extracellular DNA

traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A.

2012;109(32):13076-13081.

24. Kessenbrock K, Krumbholz M, Schönermarck U, et al. Netting neutrophils in autoimmune small-vessel

vasculitis. Nat Med. 2009;15(6):623-625.

25. Yoo DG, Floyd M, Winn M, et al. NET formation induced by pseudomonas aeruginosa cystic fibrosis

isolates measured as release of myeloperoxidase–DNA and neutrophil elastase–DNA complexes. Immunol

Lett. 2014;160(2):186-194.

26. He Z, Si Y, Jiang T, et al. Phosphotidylserine exposure and neutrophil extracellular traps enhance

procoagulant activity in patients with inflammatory bowel disease. Thromb Haemost.

2016;115(4):738-751.

27. Xie R, Gao C, Li W, et al. Phagocytosis by macrophages and endothelial cells inhibits procoagulant and

fibrinolytic activity of acute promyelocytic leukemia cells. Blood. 2012;119(10):2325-2334.

28. Campbell RA, Overmyer KA, Selzman CH, et al. Contributions of extravascular and intravascular cells to

fibrin network formation, structure, and stability. Blood. 2009;114(23):4886-4896.

29. Varjú I, Longstaff C, Szabó L, et al. DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic

effects in a plasma environment. Thromb Haemost. 2015;113(6):1289-1298.

30. Gould TJ, Vu TT, Stafford AR, et al. Cell-free DNA modulates clot structure and impairs fibrinolysis in

sepsis. Arterioscler Thromb Vasc Biol. 2015;35(12):2544-2553.

31. Woodhead JL, Nagaswami C, Matsuda M, et al. The ultrastructure of fibrinogen Caracas II molecules,




1616

fibres, and clots. J Biol Chem. 1996;271(9):4946-4953.

32. Caudrillier A, Kessenbrock K, Gilliss BM, et al. Platelets induce neutrophil extracellular traps in

transfusion-related acute lung injury. J Clin Invest. 2012; 122(7):2661-2671.

33. Röllig C, Ehninger G. How I treat hyperleukocytosis in acute myeloid leukemia. Blood.

2015;125(21):3246-3252.

34. Jiménez-Alcázar M, Napirei M, Panda R，et al. Impaired DNase1-mediated degradation of neutrophil

extracellular traps is associated with acute thrombotic microangiopathies. J Thromb Haemost. 2015;

13(5):732-742.

35. Yasutomo K, Horiuchi T, Kagami S, et al. Mutation of DNASE1 in people with systemic lupus

erythematosus. Nat Genet. 2001;28(4):313-314.

36. Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood. 2014;123(18):2768-2776.

37. Nagata S, Nagase H, Kawane K, et al. Degradation of chromosomal DNA during apoptosis. Cell Death

Differ. 2003;10(1):108-116.

38. Jahr S, Hentze H, Englisch S, et al. DNA fragments in the blood plasma of cancer patients: quantitations

and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001;61(4):1659-1665.

39. Fuchs TA, Abed U, Goosmann C, et al. Novel cell death program leads to neutrophil extracellular traps.

J Cell Biol. 2015;176(2):231-241.

40. Sanz MA, Montesinos P. How we prevent and treat differentiation syndrome in patients with acute

promyelocytic leukemia. Blood. 2014;123(18):2777-2782.

41. Merza M, Hartman H, Rahman M, et al. Neutrophil Extracellular Traps Induce Trypsin Activation,

Inflammation, and Tissue Damage in Mice with Severe Acute Pancreatitis. Gastroenterology.

2015;149(7):1920-1931.

42. Huang H, Tohme S, Al-Khafaji AB, et al. Damage-Associated Molecular Pattern–Activated Neutrophil

Extracellular Trap Exacerbates Sterile Inflammatory Liver Injury. Hepatology. 2015;62(2):600-614.

43. Papayannopoulos V, Metzler KD, Hakkim A, et al. Neutrophil elastase and myeloperoxidase regulate

the formation of neutrophil extracellular traps. J Cell Biol. 2010; 191(3):677-691.

44. Guimarães-Costa AB, Nascimento MT, Wardini AB, et al. ETosis: A Microbicidal Mechanism beyond

Cell Death. J Parasitol Res. 2012;2012:929743.

45. Kolaczkowska E, Jenne CN, Surewaard BG, et al. Molecular mechanisms of NET formation and

degradation revealed by intravital imaging in the liver vasculature. Nat Commun. 2015;6:6673.

46. Mantha S, Tallman MS, Soff GA. What's new in the pathogenesis of the coagulopathy in acute

promyelocytic leukemia? Curr Opin Hematol. 2016;23(2):121-6.




1717

Tables

Table 1. APL patients’ laboratory characteristics

Characteristics Control

(n=28)

APL

(n=40)

Age (yr) 42.4±5.12 40.6±10.8

Male sex, n(%) 12 (42.9%) 17 (42.5%)

Diagnosis

M3/bcr1, n(%) － 23 (57.5%)

M3/bcr2, n(%) － 7 (17.5%)

M3/bcr3, n(%) － 10 (25%)

WBC (× 109) 6.5 (5.2-7.9) 10.8 (4.3-24.8)

Hb (g/L) 132.5 (118-138) 68.6 (60.6-80.8) *

PLTs (× 109) 230 (195.8-256.8) 21.2 (12.0-31.1) *

Blasts (BM%) － 86.3 (82.6-90.0)

PT (sec)

(r.v.10-15) 12.7 (11.5-13.6) 16.1 (13.8-18.7) *

APTT (sec)

(r.v.20-40) 33.0 (30.1-35.6) 27.5 (23.8-32.9) *

Fibrinogen (g/L) 2.9 (2.5-3.5) 1.4 (1.2-1.9) *

D-dimer (µg/mL) 0.25 (0.17-0.30) 2.50 (0.99-3.53) *

Hemorrhage (+), n(%) － 31 (77.5%)

The main clinical and laboratory features of 28 healthy controls and 40 newly diagnosed APL patients at the

moment of bone marrow aspiration are reported. WBC, white blood cells; Hb, hemoglobin; PLTs, platelets;

Blasts, promyelocytes + blasts; BM, bone marrow; bcr, breakpoint cluster region (bcr1=intron 6, bcr2=exon

6, bcr3=intron 3). Hemorrhage was manifested as mucosal bleeding, spontaneous ecchymoses, petechiae,

hematemesis, hematuria, melena, or menorrhagia. Data are presented as numbers (percentages) or median ±

SD or median values (25th and 75th percentiles). *P<0.001 vs. healthy control.

Table 2. Permeability of clots containing promyelocytic extracellular chromatin

None PBS+APL (3d) PBS+APL (5d) ATRA+APL (3d) ATRA+APL (5d)

Ks (10-9 cm) 0.61 ± 0.23 0.59 ± 0.18 0.68 ± 0.22 1.92 ± 0.31* 2.03 ± 0.18

*

Clots were prepared from either 8 μM fibrinogen supplemented with 20 mM CaCl2, the indicated additives,

and 16 nM thrombin. The permeability coefficient (Ks) was calculated as described in Materials and




1818

methods. Ks values and standard deviation were calculated from at least six samples originating from three

independent experiments. *P<0.05 vs. control containing no additives.




1919

Figure legends

Figure 1 ATRA induces APL/NB4 cells to release procoagulant extracellular chromatin.

Fresh APL cells were primed with a mix of cytokines (10 ng/mL TNF-α, 10 ng/mL IL-1β and 10 ng/mL IL-6)

for 1 hour and then were incubated with 1 μM ATRA. Levels of cf-DNA (A) and MPO-DNA complexes (B)

in the supernatant were measured at the indicated time points. (C) Representative confocal microscopy

images of APL/NB4 cells stained by lactadherin (green) and PI (red). Cells with expanded nuclei that lost

shape and filled most of the cytoplasm were counted as ETs releasing cells (arrow). Those with condensed

and fragmented nuclei were counted as apoptotic cells (arrowhead). Bars represent 10 μm. One out of six

independent experiments is shown. (D) Promyelocytic extracellular chromatin was isolated and incubated

with 20% plasma from healthy controls. TAT complexes were measured by ELISA. (E) For inhibition assays,

isolated extracellular chromatin was pre-treated with DNase I or anti-TF antibody before incubation with

plasma. Data are from six independent experiments and presented as means ± SD. *P < .05, **P < .01, ***P

< .001 versus day 0; #P < .05, ##P < .001 versus no inhibitor group in F.

Figure 2 Effect of ATRA on integrated PCA of CK-primed APL cells

Cytokines-primed APL cells were treated with or without ATRA for the indicated time and then incubated

with MP depleted plasma (MDP) from healthy controls. (A) Coagulation time was measured using a

recalcification-time assay and (B) fibrin formation was measured using turbidity. (C, D) For inhibition

assays, ATRA-differentiated APL cells were treated with DNase I, anti-TF antibody, or lactadherin before

incubation with plasma. Coagulation time and fibrin formation were then evaluated. (E, F) Inhibition assays

were also performed using APL cells treated with PBS as a control. Data are representative of four

independent experiments and are displayed as mean ± SD. *P < .05, **P < .01versus day 0; #P < .05, ##P < .01

versus ATRA treated group in A and B; *P < .01, **P < .001 versus no inhibitor treated group in C to F.

Figure 3 Different topographical distribution of fibrin on ATRA-treated NB4 cells undergoing

apoptosis and ETosis

(A) On the third day of ATRA treatment, NB4 cells undergoing ETosis were stained with lactadherin (green)

and PI (red). Decondensed extracellular chromatin could be seen enclosed by a thin layer of membrane

(arrowhead) or spilled out of the ruptured cell (arrow). (B) Fibrin deposited on the diffuse decondensed

chromatin within the bubble and also along the thin membrane around the bubble (arrowhead) and on the

remaining broken cell membrane (arrow), similar to the binding sites for lactadherin. (C) On day 5, ATRA




2020

treatment led to diffuse rim staining by lactadherin on most cells undergoing early apoptosis (arrowhead).

Late apoptotic cells stained with lactadherin and had condensed nuclei stained with PI (arrow). (D) Fibrin

deposited at the same position as condensed chromatin inside the apoptotic cells (arrow) and also

co-localized with externalized PS on the membrane that could be identified by strong staining with

lactadherin (arrowhead). (E) FXa (red) and FVa (green) had patchy coherent distributions that overlapped on

apoptotic NB4 cell membrane (arrow). FVa staining was observed on the thin membrane around ETs

releasing cells (arrowhead). (E) A large amount of fibrin (red) strands deposited on the adjacent late

apoptotic APL cells (arrow) and formed a net of fibrin (arrowhead). Representative images of six

independent experiments are shown. Bars represent 10 μm in A, C, E; 20 μm in B, D, F.

Figure 4 Promyelocytic extracellular chromatin enhances plasmin generation and fibrin structure

(A) Plasma containing plasminogen and isolated concentrated extracellular chromatin were prepared. tPA

and the plasmin substrate Spectrozyme-PL were then added and the absorbance of the liberated

p-nitroaniline was continuously measured at 405 nm (OD405). The figure shows mean values of triplicate

measurements from six independent experiments. Clot lysis assays were performed in normal plasma

supplemented with isolated concentrated extracellular chromatin in the presence of 1 nM tPA. Changes in

clot structure were measured by observing clot turbidity at 405 nm (B), lysis time was recorded (C). Each

point represents mean ± SD for triplicate samples of independent experiments. *P < .05, **P < .01.

Figure 5 Promyelocytic extracellular chromatin triggers PS exposure on HUVECs

On the third day of PBS or ATRA treatment, extracellular chromatin from NB4 cells was isolated. HUVECs

were incubated with or without isolated extracellular chromatin (50-fold concentrated) for 24h. Cells were

stained with CD31-Alexa Fluor 488 (A, B) and with Alexa 647-lactadherin and FITC-annexin V (C). (A)

Representative confocal microscopy image showed morphology of HUVECs incubated with culture medium

without extracellular chromatin. (B, C) Stimulation with ATRA-derived chromatin led to the retraction of

cell margins and extension of filopods (arrows) and PS exposure on the filopods (arrows) which co-stained

with lactadherin (red) and annexin V (green). The inset bar equals 5 μm in A-C. One of six independent

experiments is shown. (D) Kinetics of PS reversal on ECs with different treatment. (E) For inhibition assays

HUVECs were incubated with isolated ATRA-derived chromatin in the presence or absence of DNase I or

APC. One of six independent experiments is shown. Each point represents mean ± SD. *P < .001 versus PBS

treated group in D; *P < .01, **P < 0.001 versus no inhibitor group and #P < .05 versus DNase treated group




2121

in E.

Figure 6 Promyelocytic extracellular chromatin converts HUVECs into a procoagulant phenotype.

(A) Thrombin, intrinsic Xa and extrinsic Xa production were measured on HUVECs treated with NB4

extracellular chromatin from the different treatment groups. (B) Inhibition assays of protein production were

performed using DNase I or APC to degrade extracellular chromatin before incubation with ECs.

Coagulation time (C) and fibrin formation (D) of ECs stimulated with ATRA- or PBS-derived NB4

chromatin for 24 hours in the presence of DNase I or APC were measured. Data shown from three

independent experiments and presented as mean ± SD. (E) FXa (red) and FVa (green) co-staining (yellow)

was observed on filopods near the retracted edges of ECs and on newly-formed thin filaments (arrow),

similar to the binding sites for lactadherin. (F) ECs pretreated with ATRA-derived chromatin and incubated

with healthy plasma showed considerable fibrin strand formation arranged radially along with filopodia

(arrow) which formed a fibrin network. The inset bar equals 5 μm in E and F. One of six independent

experiments is shown. *P < .01, **P < .001versus no inhibitor group and #P < .05 versus DNase I (+) APC (+)

in B; **P < .001 versus PBS treated NB4 cells in A, C and D; #P < .05, ##P < .01, ###P < .001 versus DNase I

(-) APC (-) in C and D; &P < .05 versus DNase I (+) APC (+) in D.




doi:10.1182/blood-2016-09-739334Prepublished online January 4, 2017;

Novakovic, Yayan Bi, Junjie Kou, Bo Yu, Shaohong Fang, Jinghua Wang, Jin Zhou and Jialan ShiMuhua Cao, Tao Li, Zhangxiu He, Lixiu Wang, Xiaoyan Yang, Yan Kou, Lili Zou, Xue Dong, Valerie A. fibrinolysis in acute promyelocytic leukemiaPromyelocytic extracellular chromatin exacerbates coagulation and

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

digital object identifier (DOIs) and date of initial publication. indexed by PubMed from initial publication. Citations to Advance online articles must include final publication). Advance online articles are citable and establish publication priority; they areappeared in the paper journal (edited, typeset versions may be posted when available prior to Advance online articles have been peer reviewed and accepted for publication but have not yet

Copyright 2011 by The American Society of Hematology; all rights reserved.Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of


http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





promyelocytic extracellular chromatin exacerbates coagulation and

Documents