# DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma

Dayana Barbosa Da Cruz, Julie Helms, Lluvia Ramírez Aquino, Laure Stiel, Lucas Cougourdan, Cedric Broussard, Philippe Chafey, Madeleine Riès-Kautt, Ferhat Meziani, Florence Toti, et al.

► To cite this version:

Dayana Barbosa Da Cruz, Julie Helms, Lluvia Ramírez Aquino, Laure Stiel, Lucas Cougourdan, et al. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma. FASEB Journal, 2019, Quantitative Biology, 33 (12), pp.14270-14280. 10.1096/fj.201901363RRR. inserm-05368569

HAL Id: inserm-05368569

https://inserm.hal.science/inserm-05368569v1

Submitted on 17 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# DNA-bound elastase of Neutrophil Extracellular Traps degrades plasminogen, reduces plasmin formation and decreases fibrinolysis: proof of concept in septic shock plasma

Dayana Barbosa da Cruz,<fnref n="1" /> Julie Helms,<fnref n="2" />,<fnref n="3" />* Lluvia Ramírez Aquino,<fnref n="1" />* Laure Stiel,<fnref n="2" />,<fnref n="4" /> Lucas Cougourdan,<fnref n="1" /> Cedric Broussard,<fnref n="5" />,<fnref n="6" /> Philippe Chafey,<fnref n="5" />,<fnref n="6" /> Madeleine Riès-Kautt,<fnref n="5" />,<fnref n="6" /> Ferhat Meziani,<fnref n="2" />,<fnref n="4" />** Florence Toti,<fnref n="4" />** Pascale Gaussem<fnref n="1" />,<fnref n="7" />** and Eduardo Anglés-Cano<fnref n="1" />**

**Corresponding author:** Eduardo Anglés-Cano (Eduardo.Angles-Cano@inserm.fr)

Address Inserm UMR_S1140, Faculté de Pharmacie de Paris
4 Avenue de l'Observatoire, 75006 PARIS, FRANCE

Phone: + 33 (0)1 53 73 95 07 Fax: + 33 (0)1 44 07 17 72

**Running Title:** Plasminogen cleavage by NETs dampens fibrinolysis

<footnote n="1">¹ Université de Paris, Innovative Therapies in Haemostasis, INSERM UMR_S 1140, F-75006 Paris, France.</footnote>

<footnote n="2">² Service de Réanimation Médicale, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.</footnote>

<footnote n="3">³ ImmunoRhumatologie Moléculaire, INSERM UMR_S 1109, Fédération de Médecine Translationnelle de Strasbourg, Faculté de Médecine, Université de Strasbourg, Strasbourg, France</footnote>

<footnote n="4">⁴ Université de Strasbourg, Regenerative Nanomedicine, INSERM UMR_S 1260, F-67100, Strasbourg, France</footnote>

<footnote n="5">⁵ Université de Paris, Plateforme Protéomique 3P5, F-75006, Paris, France</footnote>

<footnote n="6">⁶ Université de Paris, Institut Cochin, INSERM, CNRS, F-75006 Paris, France</footnote>

<footnote n="7">⁷ Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique, F-75015 Paris, France.</footnote>

<footnote>(*) J.H. and Ll. R. A. contributed equally to this study.</footnote>

<footnote>(**) F.M., F.T., P.G. and E.A-C. are senior authors.</footnote>
Nonstandard Abbreviations:

ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

AEBSF, aminoethyl-benzene-sulfonyl fluoride

α₁-PI, α₁-proteinase inhibitor

DIC, disseminated intravascular coagulation

HBSS, Hank's balanced salt solution (HBSS)

HNE, human neutrophil elastase

K, kringle domain of plasminogen

NETs, neutrophil extracellular traps

MeO-Suc-AAPV-CMK, N-(Methoxysuccinyl)-L-alanyl-L-alanyl-L--prolyl-L-valine
chloromethylketone

MeO-Suc-AAPV-pNA, N-(Methoxysuccinyl)-L-alanyl-L-alanyl-L--prolyl-L-valine p-nitroanilide

MM-H-Pro-Arg-pNA, methyl(malonyl)hydroxyprolylarginine -p-nitroanilide, CBS0065

NETs, neutrophil extracellular traps

PMA, phorbol 12-myristate 13-acetate

PMN, polymorphonuclear leukocyte

PMSF, phenylmethylsulphonyl fluoride

tPA, tissue plasminogen activator

uPA, urokinase plasminogen activator

uPAR, urokinase plasminogen activator receptor
ABSTRACT

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface, and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustain its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains ($K_{1+2+3}$, $k_{1+2+3+4}$) and the serine protease (SP) region ($K_5$-SP). We further demonstrate that septic shock patients with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin and localized plasmin formation.

KEY WORDS

Proteolysis. Neutrophil proteases. Elastase inhibitors. Multi-organ dysfunction. Fibrinolytic failure. Disseminated intravascular coagulation.
INTRODUCTION

Thrombus lysis, a major line of defense against thrombosis, is triggered by plasmin generated
upon cleavage-activation of plasminogen bound to clot components, mostly fibrin or platelet
membranes.(1) Plasminogen is a single-chain molecule consisting of an N-terminal region, 5
kringle (K) domains and the serine protease (SP) region.(2) K₁ and K₄ contain each a lysine-
binding site that ensures binding of plasminogen to carboxy-terminal lysine residues of
fibrin.(3) Plasminogen thus bound is cleaved at Arg<sub>561</sub>-Val<sub>562</sub> and transformed into plasmin by
the tissue-type or the urokinase-type plasminogen activators, tPA and uPA respectively, in a
surface and localized dependent manner. Purposely, tPA is fully active once bound to fibrin
whereas uPA preferentially operates when bound to its membrane-anchored receptor uPAR.
Proteases like human neutrophil elastase (HNE) degrade plasminogen without generating
plasmin.(4, 5) In the circulation, HNE is rapidly inhibited by the α₁-proteinase inhibitor, (α₁-
PI, ka = 6.5 x 10⁷ M⁻¹ s⁻¹)(6) that circulates at a high concentration (20 to 40 µM). HNE-α₁PI
complexes thus formed preclude proteolytic degradation of circulating plasminogen.
However, experimental evidence indicates that HNE bound to negatively charged membrane
proteoglycans or other macromolecules escapes inhibition and remains active.(7, 8)
Interestingly, Belorgey and Bieth (1995, 1998)(9, 10) demonstrated that HNE forms a tight
complex with DNA that strongly impairs its inhibition by α₁-PI. Besides this protective action
on HNE, the group of Bieth fully documented the protective action of DNA on other bound
proteases.(11, 12) The extrusion into the extracellular space of functional scaffolds combining
intact DNA with added-on HNE by neutrophils in a process termed neutrophil extracellular
traps (NETs) formation or netosis, was discovered in 2004.(13) PMA and bacterial infections
use a related pathway for NET induction: activation of protein kinase C and generation of
reactive oxygen species.(14)

NETs are decondensed nuclear DNA fibers with bound nuclear proteins (histones), and
cytoplasmic and granular proteases including HNE, cathepsin G and myeloperoxidase.(15)
HNE is the most abundant (5.24 µmoles per g NET-DNA),(16) characteristic component of
NETs that is bound to DNA with nanomolar affinity.(17) NETs are proteolytically active and
were originally described as beneficial (16) extracellular killers of microorganisms.(18)
Recent data indicate that NETs are also of pathophysiological relevance in a variety of
inflammatory disorders, including autoimmune diseases and thrombosis.(19, 20) The
combined action of NETs and the clotting system characterizes immunothrombosis.(20) It has
indeed been shown that NETs are released during thrombus formation and are integrated
together with von Willebrand factor into the fibrin scaffold.(21, 22) The presence of NETs in
human coronary and cerebral thrombi has actually been demonstrated.(23-25) Furthermore, it
has been suggested that formation of an intricate network between fibrin and NETs results in
a modified fibrin clot structure that is resistant to thrombolysis.(26-28) In addition, the NETs'
DNA-backbone insensitivity to plasmin and the formation of non-fibrinolytic plasmin-DNA-
fibrin complexes were also suggested as possible causes of fibrinolytic resistance.(27, 29)
Since NETs may compromise thrombus lysis, our studies were designed to investigate the
role of HNE-DNA complexes on fibrinolysis. We hypothesized that the reservoir of these
inhibitor-insensitive complexes of NETs directly interferes with fibrinolysis via plasminogen
proteolysis. We demonstrate that in the presence of NETs containing active HNE-DNA
complexes plasminogen is reduced to fragments that inhibit plasminogen binding and plasmin
formation onto fibrin resulting in impaired fibrinolysis. Our proof-of-concept study provides
new mechanistic insights into septic shock thrombosis based on the capacity of HNE-DNA
complexes to trigger a new unconventional mechanism of fibrinolytic insufficiency.

MATERIALS AND METHODS

Plasma from septic shock patients and healthy subjects

Seven patients (mean age 74.5±10 years) referred for septic shock with multiple organ failure (mean SOFA score 13±2)(30) and presenting disseminated intravascular coagulation (DIC) diagnosed during the first 24 h after admission according to the JAAM 2016 score(31) were enrolled, as well as ten healthy individuals (Trial Registration: Clinicaltrial.gov identifier NCT #02391792). The Strasbourg University Hospital Ethics Committee approved this study. Informed consent was obtained from the patient or relatives at admission and confirmed by the patient as soon as possible. Platelet-free plasma was prepared from blood collected in 0.13 M sodium citrate (Vacutainer™, Becton Dickinson) by a double centrifugation step (2500 g for 15 minutes), aliquoted and immediately frozen at -80 °C. Regular haemostasis studies, and detection of HNE-α₁PI and plasmin-α₂-antiplasmin complexes were assessed in human plasmas as indicated in the Supplemental file.

Human proteins and antibodies

Human Glu-plasminogen was purified as described and was over 99% pure as assessed by
SDS/PAGE and by amino-terminal sequence analysis(3, 32). Plasminogen elastase-derived
fragments of plasminogen were prepared by incubating for 30 min at 37 °C, 10 µM of the purified protein with 250 nM of purified HNE. The reaction was stopped by addition of 5 mM PMSF. Fragments were then isolated by affinity chromatography on Lysine-Sepharose. Fibrinogen was purified from fresh-frozen human plasma and characterized as described.(33) Human tPA (>95% single-chain) was obtained from Biopool (Uppsala, Sweden). HNE and the elastase inhibitor α₁PI were from Calbiochem (Merck KGaA, Darmstadt, Germany). A sheep polyclonal antibody directed against plasminogen and a monoclonal IgG1 antibody directed against plasminogen kringle 1 (CPL15) were produced and characterized previously.(34, 35) Horseradish peroxidase (HRP)-conjugated CPL15 mAb was obtained using a peroxidase labelling kit according to the manufacturer's instructions (Roche, Mannheim, Germany). The polyclonal rabbit anti-sheep (HRP)-conjugated IgG was from DakoCytomation (Glostrup, Denmark).

## Neutrophil Isolation and generation of neutrophil extracellular traps

Venous blood from healthy volunteers was collected on Acid-Citrate-Dextrose by the Blood Donor Center (Etablissement Français du Sang). Neutrophils were isolated from the anticoagulated blood as described.(36) Briefly, leukocytes were separated from red cells on a separating medium containing 9% Dextran T-500 in Radioselectan. After red cell sedimentation, the leukocyte suspension was centrifuged on Pancoll 1077. The cell pellet was washed with phosphate-buffered saline and contaminating erythrocytes were removed by hypotonic lysis. Polymorphonuclear neutrophils (PMN) were then resuspended in RPMI-1640 or Hank's balanced salt solution (HBSS) (see below) and used immediately after isolation. Flow cytometry showed the absence of CD14+, CD3+, CD19+ cells, confirming the recovery of highly purified PMN (CD16+) that were identified by nuclear morphology on May-Grünwald-Giemsa staining (not shown). Routinely, the purity of neutrophil preparations and cell viability (Trypan blue) were ≥ 98%. Isolated neutrophils were re-suspended in RPMI-1640 medium or HBSS at a concentration allowing distribution of 200 000 cells per well. Experiments were performed on 96-well flat-bottomed plates containing or not a well-defined fibrin surface prepared and characterized as described below.

Since PMA and bacteria use related pathway (protein kinase C activation and generation of reactive oxygen species for induction of proteolytically active NETs), we used the well standardized technique of PMA stimulation.(14) After 1 hour, plated neutrophils were treated with 50 nM PMA in a humidified 5% CO₂ atmosphere at 37 °C, during 4 hours. NETs were
identified by (1) DNA staining with a 10 µg/ml solution of Hoechst 33342 and (2) measuring
the activity of DNA-bound elastase.

Stained nuclei of non-stimulated neutrophils and PMA-induced extracellular traps were detected in optic fields of three wells for each condition using the X20 objective of a Zeiss AxioObserver D1 fluorescence microscope equipped with a CCD Imaging camera using the Histolab software from Microvision Instruments (Evry, France).

To detect elastase bound to NETs, the substrate MeO-Suc-AAPV-pNA (100 µl per well, 1.5 mM final concentration in HBSS) was incubated with NETs after two gentle washes with HBSS. DNA-bound HNE activity was detected in a multi-well plate counter at 37 °C by measuring the release of p-nitroaniline (pNA) at A<sub>405nm</sub>. The elastase inhibitors MeO-Suc-AAPV-CMK (100 µM final) or α<sub>1</sub>-PI (10 µM final) and AEBSF (1 mM) were used to authenticate preservation of DNA-bound elastase activity.

Preparation and characterization of fibrin matrices

Fibrin matrices were prepared as described previously. (34, 37, 38) Briefly, a monolayer of fibrinogen was immobilised onto poly gluta'aldehyde)-activated flat-bottomed 96-well plates. The immobilised fibrinogen (410±4 fmol/cm²) was transformed into fibrin using a 10 N.I.H. u/ml solution of thrombin containing 2 mM CaC12. The release of fibrinopeptide A was followed using the monoclonal antibody Y18. (39) Fibrin thus obtained specifically interacts with the finger domain of tPA whereas plasminogen binds to newly exposed carboxy-terminal lysine residues unveiled during on-going fibrin degradation. (3, 33, 37) Fibrin degradation was followed with mAb DD3B6 specific for D-dimer structures unveiled by plasmin on the immobilised fibrin. (40)

Study of plasminogen activation/fragmentation on fibrin-NETs matrices

Fibrin-NETs matrices were achieved by activation with PMA of neutrophils seeded onto
fibrin surfaces prepared as described above. The neutrophils were seeded onto the fibrin
matrix at 200 000 cells per well and were stimulated with PMA after 1 h plating as indicated
above. After 4 h of stimulation a fibrin-NETs matrix was obtained as visualized by Hoechst
staining and elastase activity detection (Figure 1). Following the formation of NETs, the
supernatant solution was removed, the wells were carefully washed once with HBSS, and the
following experiments were performed:

1. Plasminogen activation onto the fibrin-NET matrices. After washing with HBSS, tPA at 50 i.u./ml was incubated with the surfaces for 1 h at 37°C to allow binding of tPA to fibrin.
Unbound tPA was carefully discarded and a solution of plasminogen at varying concentrations was added to trigger plasmin formation by tPA bound to the fibrin-NETs lattice. In parallel experiments the effect of elastase on plasminogen fragmentation was blocked with the elastase inhibitor MeO-Suc-AAPV-CMK. Kinetics of plasminogen transformation into plasmin was followed during 1 h by measuring the release of pNA at A<sub>405nm</sub> in a microplate counter. Initial velocities of the reaction were calculated using an ad hoc computer program.

2. Plasminogen fragmentation by NETs-bound Elastase. Following the formation of NETs and a wash with HBSS, plasminogen at 1 µM was incubated with NETs at 37 °C for 15 min to 4 hours. When indicated the NETs were pre-treated with the elastase inhibitor MeO-Suc-AAPV-CMK or α₁-PI before addition of plasminogen. Post incubation, the plate was centrifuged at 3000 g for 5 min and the supernatants from each well were collected and stored at -20 °C for further analysis. Material remaining at the bottom onto the fibrin surface was recovered by scraping using 100 µl of sample buffer (60 mM Tris-HCl pH 6.8, 2% SDS) or 100 µl of HBSS, and used for Western blotting or detection of HNE activity, respectively. HNE activity in samples collected in HBSS was measured with the chromogenic substrate MeO-Suc-AAPV-pNA (100 µl per well, 1.5 mM final concentration in HBSS) as indicated above. Plasminogen proteolysis was investigated in samples collected in Tris-SDS by SDS-10% PAGE followed by Western blotting using a mAb directed against plasminogen K₁ (CPL15-HRP, 100 ng/ml)(35) or a polyclonal sheep IgG (125 µg/ml) followed by a secondary anti-sheep IgG-HRP (1:20 000). Signal was detected using the ECL kit from Amersham (Arlington Heights, U.K.) and the Curix-60-AGFA for development.

Detection of plasminogen, plasminogen fragments and plasmin formation in septic shock plasmas

Plasminogen was quantified with a functional activity assay as described with modifications.(41) Briefly, plasmas were diluted 1:50 in assay buffer (Phosphate 80 mM, NaCl 50 mM, BSA 2 mg/ml), supplemented with 75 i.u./ml urokinase, 1 mM tranexamic acid and 0.75 mM of the plasmin-selective chromogenic substrate in a final volumen of 50 µl. Kinetics of the reaction was followed by measuring the release of pNA at A<sub>405nm</sub> in a microplate photometer in kinetics mode. Results were given in nM concentration by reference to a standard curve prepared with plasminogen-depleted plasma supplemented with known concentrations of purified plasminogen (0.2 to 1 µM).

Plasminogen fragments present in plasma were initially detected by Western blot as indicated
above. Further identification was obtained by mass spectrometric (see below).

Plasmin formation on fibrin matrices was measured using the euglobulin fraction from healthy control and patient plasmas as source of plasminogen as indicated under 1. Plasminogen activation onto the fibrin-NET matrices. Kinetics of plasminogen transformation into plasmin was followed during 1 h by measuring the release of pNA at A<sub>405nm</sub> and the initial velocity of the reactions were calculated as indicated above.

## Neutrophil extracellular traps detection in human plasmas

Plasma HNE-DNA complexes of NETs were identified using an ELISA capture assay as described (19) with the following modifications. Since HNE is the enzyme involved in plasminogen fragmentation and is also the most abundant granular component of NETs, we choose to capture HNE-DNA complexes using an anti-HNE antibody. The antibody (10 µg/ml) was immobilized on poly gluta'aldehyde)-activated U 96-well polyvinyl chloride plates as described above for fibrinogen. A volume of 50 µl of diluted human plasma was added per well in combination with the peroxidase-labeled anti-DNA monoclonal antibody (component No.2 of the commercial cell death detection ELISA kit;) according to the manufacturer's instructions (Roche Diagnostic, Mannheim, Germany). After 1 h incubation at 37 °C, the samples were washed three times with 100µl PBS per well and 100µl/well of the peroxidase substrate ABTS at 1 mg/ml were added. The absorbance at 405 nm wavelength was measured at 37 °C in the dark using a multi-well plate photometer as indicated above.

## Identification of plasminogen fragments by Mass Spectrometry

From Western blot images, samples were selected for proteomic analysis using mass spectrometry as indicated in the Supplemental Data file. Samples used were purified plasminogen, plasmin or HNE-derived plasminogen fragments, and patients' plasma protein fractions of interest depleted of highly abundant proteins as obtained by euglobulin precipitation or lysine-affinity separation.

## RESULTS

### NETs released onto a fibrin matrix form a dense lattice

NETs were identified as extracellular fibrillar networks primarily composed of DNA stained by Hoechst dye. Further identification was achieved by detecting HNE bound to DNA using
an activity assay (Figure 1). Non-stimulated neutrophils presented typical intracellular
lobulated nuclei easily recognized either before or after 4 h incubation at 37 °C on plates
containing or not a fibrin matrix (Figure1A, Neutrophils). Extracellular DNA could not be
observed in non-stimulated neutrophils indicating that fibrin alone was not competent to boost
the formation and release of NETs. NETs released under similar conditions in the absence of
fibrin appeared isolated and did not knit (Figure 1A, NETs) In contrast, extracellular DNA
released by stimulated neutrophils seeded on fibrin formed a dense interlace of fibers clearly
visible after Hoechst-staining (Figure 1A, NETs-Fibrin), suggesting that DNA spreading on
fibrin was favored by the regular distribution of positive charges along the fibrin fibers.(42)
Of note that fibrin alone display no autofluorescence (not shown) that may influence the
dense appearance of NETs. The presence of HNE coupled to NETs was detected with the
elastase-selective chromogenic substrate MeO-Suc-AAPV-pNA (Figure 1B). The proteolytic
activity of HNE-DNA complexes was completely blocked by the peptidyl chloromethyl
ketone elastase inhibitor MeO-Suc-AAPV-CMK (Figure 1B). In contrast, α₁-PI, which
efficiently inhibits soluble HNE in supernatants or added to plasma (Figure 1C), produced
only around 20% decrease in the activity of DNA-associated HNE (Figure 1B). These data
are in agreement with previous reports on protection of the activity of HNE by DNA.(8-12,
43) Aprotinin, an unrelated inhibitor, was without effect on the activity of HNE when added
to NETs or cell supernatants at a similar concentration than α₁-PI (40 µM) (Figure 1B).
These data indicate that HNE-DNA lattices spread onto the fibrin matrix is fully active.

NETs' HNE•DNA complexes cleave plasminogen into well-defined fragments

It is well-known that HNE in solution (purified or released by neutrophils) produces
plasminogen fragments of known structure.(4, 44) However, it remains unknown whether
HNE bound to NETs is able to cleave plasminogen into fragments. To investigate this
hypothesis, we incubated plasminogen with lattices of NETs spread on the fibrin matrix.
Figure 2A, clearly shows that plasminogen fragments were already present after 30 min and 1
h of incubation with NETs. Three main bands with a relative molecular mass of 92 kDa, 47
kDa and 32 kDa were observed as well as an additional small band of around 50 kDa on top
of the 47 kDa band. Longer incubation times did not modify this pattern (Fig 2B). Proteomic
analysis identified the 92 kDa band as human plasminogen, the 47 and 50 kDa bands as
fragment K<sub>1+2+3</sub>, and the 32 kDa band as K<sub>5</sub>-SP (Supplemental figure S1). K4 was clearly
identified by proteomic analysis in NETs-HNE-treated plasminogen (Supplemental figure S1)
and in purified samples of HNE-treated plasminogen (Supplemental figure S2). We then
verified that HNE-DNA complexes specifically cleave plasminogen by pre-incubating NETs with selective proteinase inhibitors. Pre-incubation with MeO-Suc-AAPV-CMK, an elastase-specific inhibitor, completely inhibited HNE and abolished the conversion of plasminogen into fragments confirming that HNE was active on NETs (Figure 2C). In contrast, aprotinin, a plasmin and kallikrein inhibitor, was unable to modify the activity of elastase or the appearance of plasminogen fragments (Figure 1B). Because, α₁-PI the specific inhibitor of elastase had only a minor reducing effect on the activity of HNE-DNA complexes (Figure 1B), we concluded that the observed plasminogen fragments are generated by the proteolytic activity of HNE-DNA complexes.

## NETs HNE-derived plasminogen fragments interfere with fibrinolysis

Having demonstrated that HNE•DNA complexes of NETs cleave plasminogen into fragments K<sub>1+2+3</sub>, K<sub>4</sub> and K<sub>5</sub>-SP, we sought to answer the critical question of their role in fibrinolysis. For that purpose, neutrophils seeded onto a fibrin matrix were prompted to release NETs with PMA as above. Conversion of plasminogen into plasmin by tPA bound to the fibrin-NET lattice was then assessed using a plasmin-selective chromogenic substrate. The time- and concentration-dependent conversion of plasminogen into plasmin at the fibrin-NETs lattice was reduced as compared to fibrin alone (Figure 3A-B). Plasmin formation was restored to control values when MeO-Suc-AAPV-CMK, an elastase-selective inhibitor, was pre-incubated with the fibrin-NET lattice, indicating that HNE proteolytic activity prevents plasminogen conversion into plasmin. The amount of plasmin formed and remaining bound to the fibrin-NETs lattices was decreased by around 50% as compared to similar conditions in the presence of MeO-Suc-AAPV-CMK (Figure 3C). This decrease in the amount of plasmin formed onto fibrin was related to plasminogen fragmentation by HNE•DNA complexes as indicated by Western blot identification of the molecular species eluted from the fibrin surfaces (Figure 3D). Altogether, data in figure 3 show that plasminogen fragments were identified in fibrin matrices containing HNE•DNA complexes and low fibrinolytic activity. In contrast, plasminogen fragments were absent and the fibrinolytic activity was similar to control fibrin when fibrin-NETs matrices were pre-treated with MeO-Suc-AAPV-CMK.

## NETs, plasminogen fragments, HNE•α₁-PI and fibrinolytic activity in septic shock plasma

Results of the parameters tested are shown in Table 1. NETs were detected by measuring HNE•DNA complexes in samples from patients with septic shock and disseminated
intravascular coagulation (patients 0.276±0.043; healthy controls: 0.086±0.014, A405nm).
The concentration of plasminogen as measured with a functional assay was 520±180 nM
(healthy controls n=31: 1.69±0.33 µM). Circulating HNE•α₁-PI complexes were increased 8-
fold in patients as compared to healthy controls, 437±274 versus 43±19 ng/ml, respectively.
Patients had elevated D-dimers (12±8 mg/l) and plasmin-α₂-antiplasmin (range 4814-159
ng/ml, median 1207 ng/ml), without signs of hepatic dysfunction, although moderate
cytolysis. The presence of HNE-derived plasminogen fragments was evidenced by SDS-
PAGE and Western blotting. Figure 4 shows the pattern of plasminogen fragments detected in
7 selected patients as compared to healthy controls. Besides plasminogen, three main
plasminogen fragments were identified by mass spectrometric analysis of amino-acid
sequence of protein bands from samples depleted of highly abundant proteins (euglobulin
fractions or lysine-affinity fractions): plasminogen fragments containing K<sub>1+2+3</sub>, K<sub>1+2+3+4</sub> and
plasminogen fragments composed of K<sub>5</sub>-SP (Supplemental file Fig S1). We were unable,
however, to detect K4 in plasma samples of septic shock patients despite the use of
acrylamide gels of small pore size or shorter migration time. In contrast K4 was clearly
identified in purified plasminogen treated by NETs or HNE alone (Supplemental figures S1
and S2).

To investigate the effect of NETs and plasminogen fragments on fibrinolysis, the euglobulin
fraction of septic shock plasma samples were incubated with tPA bound to fibrin surfaces and
the amount of plasmin formed was quantified with a chromogenic substrate. The generation
of plasmin was decreased (>50%) when comparing different septic shock plasma to a pool of
normal plasma (Figure 5). To compensate for the rather low plasminogen concentration of
these plasmas, purified plasminogen was added to a concentration similar to that of the
normal plasma pool. As a result, the amount of plasmin formed was increased with a trend to
normality in only three (S1, S3, S5) of the plasma supplemented with plasminogen. An
insufficient or scant response was observed in the remaining plasma samples S2, S4, S6),
suggesting a competitive effect towards added plasminogen.

DISCUSSION

Netosis, a major antimicrobial mechanism, has alternative roles in both infectious and non-
infectious diseases. In septic shock, for instance, activated neutrophils emit extracellular traps
that both ensnare pathogens and activate coagulation leading to fibrin formation. The
microvascular clots that ensue, a mechanism referred to as immunothrombosis, favor pathogens circumscription and killing by NETs enzymes. (20, 45, 46) Several actors of this interconnected mechanism between coagulation and innate immunity may also nurture uncontrolled thrombosis and thereby hinder the initial benefit of the response.(47) As a consequence, NETs may substantially contribute to septic shock-associated intravascular coagulation. Actually, we detected circulating NETs in septic shock patients with intravascular coagulation, (48) a finding that was confirmed in recent studies.(49-52) Since the contribution of NETs to promote vascular occlusion has been the object of previous studies by different groups (critically reviewed in (53)), our objective was to focus on the role of NETs on the other side of the hemostatic equilibrium: the fibrinolytic response. We sought to define how the activity of NETs could affect clot dissolution.

Using a fluorescent DNA binder, we notice that NETs formed in vitro onto a fibrin surface show a denser and more reticulated appearance than NETs released in the absence of fibrin. The positive charges regularly distributed along the fibrin fibers interact with negatively charged DNA yielding a more dense appearance of DNA fibers in the entangled fibrin-NETs matrix, as previously shown for other molecules.(42) These findings agree with data previously reported indicating that cell-free DNA incorporated into a clot modifies the structure of fibrin rendering it resistant to plasmin-mediated degradation.(26, 28) The entangled assembly of DNA and fibrin within the thrombus may be implicated in NETs-mediated resistance to tPA-induced fibrinolysis in vitro (21, 29) and thrombolysis in patients with acute ischemic stroke.(54) Such fibrinolytic resistance may contribute to persisting thrombotic occlusion due to sustained platelet activation by histones(21) and inactivation of tissue factor pathway inhibitor (TFPI) by HNE.(45) Actually, we show that NETs entangled with fibrin contain active HNE in complex with DNA that is insensitive to inhibition by α₁-PI. Similarly, NETs-associated murine NE was found to be proteolytically active within the liver vasculature.(55) Contrastingly, elastase bound to the plasma membrane of neutrophils is easily released to form soluble irreversible complexes with circulating α₁-PI.(7) The protective function of DNA on HNE activity is in agreement with previous findings indicating that leukocyte granular serine proteases bind to DNA with nanomolar affinity and endow HNE with resistance to plasma inhibitors thus ensuring localized degradation of protein substrates such as plasminogen.(9, 11, 12, 17) Since an efficient fibrinolytic system is key to clearance of thrombi in the microcirculation, we sought to investigate if a failure of fibrinolysis via plasminogen proteolysis could be demonstrated in vitro in the presence of NETs bearing active HNE-DNA complexes.
We demonstrate that in the presence of NETs containing active HNE-DNA complexes plasminogen is reduced to fragments of well-defined size: K<sub>1+2+3</sub>, K<sub>1+2+3-4</sub>, K<sub>4</sub> and K<sub>5</sub>-SP that have lost their capacity to generate plasmin by fibrin-bound tPA. These plasminogen fragments were detected when plasminogen was added to NETs released by activated neutrophils in vitro. The nature of the fragments was identified with certainty by mass spectrometric analysis of amino-acid sequences. Since plasminogen fragments bearing K<sub>1</sub> have been previously shown to be strong inhibitors of plasminogen binding to fibrin, plasmin generation and fibrinolysis,(56-58) we sought to investigate the role of NETs on these mechanisms. Our experimental fibrin-NETs lattice is an outstanding set-up suitable to detect both the NETs-HNE-derived plasminogen fragments and the competitive effect of K<sub>1</sub>-containing fragments on plasminogen binding and activation. As expected, when plasminogen was activated by fibrin-bound tPA on the NETs-fibrin lattice, a decrease in the amount of plasmin generated by tPA occurred concomitantly with the appearance of such fragments. These and our previous data indicate that these plasminogen fragments compete with plasminogen for binding to fibrin decreasing thereby its capacity to generate plasmin.(56, 57) At variance with the antifibrinolytic effect described here, there are other protein components of hemostasis that could be a substrate for HNE-some of which may have direct or indirect effects on thrombus formation and lysis. In particular, HNE may exert either a procoagulant activity by inactivating TFPI(45) or a profibrinolytic activity by direct cleavage of fibrin,(59) by degrading α<sub>2</sub>-antiplasmin or PAI-1(60, 61) or by producing mini-plasminogen (K<sub>5</sub>-SP).(5) Although mini-plasminogen (K<sub>5</sub>-SP) may be activated by urokinase in solution, it does not bind to fibrin and is therefore not activated by tPA at the fibrin surface; as a consequence, the impact of K<sub>5</sub>-SP on tPA-dependent clot lysis is negligible.(5, 62) A limitation of the present study is that it does not directly address the cross-talk between these other regulators. We purposely focused on fragmentation of plasminogen by HNE-DNA complexes because its activation is central to the acceleration and amplification of fibrinolysis.(3, 34, 63)

Taken all together, our data indicate that HNE-DNA complexes exert an antifibrinolytic effect by generating plasminogen fragments and decreasing the plasminogen concentration. To explore whether this mechanism could be related to the pathogenesis of microthrombosis within a clinical setting we investigated if indicators of NET-induced fibrinolytic failure in vitro could be present in plasma samples from septic shock patients with DIC and circulating NETs.

We demonstrate that this group of patients have a low plasminogen concentration, circulating plasminogen fragments and a failure to generate plasmin on fibrin (less than 50% when
compared to a pool of normal plasmas) that may be responsible for the observed intravascular coagulation and multiple organ dysfunction. Protein bands detected by Western blot and identified by mass spectrometric amino-acid analysis in plasma fraction of the selected patients corresponded to intact plasminogen and plasminogen fragments: K<sub>1+2+3</sub>, K<sub>1+2+3+4</sub> and K<sub>5</sub>-SP. These fragments were identical to those derived from plasminogen cleaved in vitro by purified HNE.(44) We were unable, however, to identify isolated K<sub>4</sub>. The absence of K<sub>4</sub> in the plasma of these patients is probably related to a fast clearance from the circulation as compared to fragments of bigger size.

Since α₁-PI circulates at µM concentration and inhibits all free HNE with an extremely high association constant (ka = 6.5 × 10⁷ M⁻¹ s⁻¹),(6) elastase released by neutrophils was readily found as HNE·α₁-PI complexes in plasma from septic shock patients. HNE-α₁PI complexes are therefore a marker of neutrophil activation in sepsis. We propose that in the absence of active elastase in the plasma of these patients, the plasminogen fragments found in plasma were produced by active HNE-DNA complexes. These data, strongly support a mechanistic pathway where plasminogen fragments found in patients with septic shock were produced locally on the fibrin-NETs matrix of the clot. Nevertheless, besides fibrin, histone H2B bearing carboxyterminal lysines may capture plasminogen in the vicinity of HNE bound to the DNA fibers.(64) Circulating NETs containing active HNE-DNA complexes insensitive to α₁-PI could also participate in plasminogen fragmentation.

The proteolysis of plasminogen by HNE-DNA complexes may be in part responsible for the low concentration, less than 1µM, of full-length plasminogen that we detected in the plasma of septic shock patients, in agreement with a previous report.(65) The low concentration of plasminogen in conjunction with the anti-fibrinolytic activity of plasminogen fragments may result in insufficient fibrinolysis in septic shock leading to microvascular thrombosis. These parameters, plasminogen concentration, plasminogen fragments and failure to form plasmin could be relevant new prognostic markers to assess the role of NETs in sepsis-DIC and may serve as predictors of mortality and organ failure in septic shock patients.

Since the low plasminogen concentration of these plasmas could explain in part the low fibrinolytic activity, the plasmas were supplemented with plasminogen before testing. In spite of reaching a plasminogen concentration similar to that of the normal plasma pool, 3 out of the 6 plasmas tested failed to restore their fibrinolytic activity to normal values. These results suggest a competitive effect towards plasminogen most probably related to the presence of circulating HNE-derived plasminogen fragments containing K₁.

The limited number of patients included in this proof-of-concept study constitutes a limitation
to establish causality between NETs-septic shock-DIC and fibrinolysis abnormalities induced
by HNE-DNA. To reach this objective, a larger cohort of patients should be studied.

The originality and novelty of our study is the demonstration that a severe decrease in
fibrinolysis induced by HNE-DNA complexes of NETs could contribute to the persistence
and stabilization of thrombi in the microcirculation during septic shock.

We propose new mechanistic insights into septic shock thrombosis in the microcirculation in
connection with the capacity of HNE-DNA complexes to trigger fibrinolytic insufficiency.
Identification of this reduction in the fibrinolytic potential in plasma of septic shock patients,
as shown in figure 5, might have practical implications in the search for a feasible alternative
to improve the fibrinolytic response in these patients. For instance, the active enzyme i.e.
HNE-DNA complexes, and the substrate, i.e. plasminogen, may represent new targets in the
therapy of sepsis-DIC. These new targets should be added to the armamentarium in the
therapy of sepsis/DIC.

Our studies clearly pointed out that NETs or fragments of NETs contain active elastase
responsible for the production of plasminogen fragments with inhibitory activity (competitors
of plasminogen binding to fibrin), which act to decrease thereby both the concentration of
plasminogen and plasmin formation. Altogether, the low plasminogen and the inhibitory
activity of the elastase-derived plasminogen fragments contribute to abrogate the fibrinolytic
system, thus favoring microthrombosis and organ failure.

In summary, the main findings of our study are the fragmentation of plasminogen by active
HNE-DNA complexes and a concomitant decrease in both the concentration of plasminogen,
and plasmin formation required for efficient fibrinolysis (Supplemental Figure S3). We show
that the low capacity to generate plasmin is related to a decrease in the amount of
plasminogen bound to fibrin resulting from both the low plasminogen concentration and a
competitive effect due to the elastase-derived plasminogen fragments. Our data clearly
indicate that HNE-DNA complexes are at the center of a mechanism resulting in severe
fibrinolytic failure via the consumption of plasminogen by proteolysis leading to the
production of antifibrinolytic plasminogen fragments. This fibrinolytic insufficiency may be
responsible for impaired dissolution of microthrombi thereby fostering their stabilization in
the microcirculation and contributing to organ failure during septic shock.

**Acknowledgements.** The authors would like to thank Fatiha El Ghazouani, Keiththana Kamaleswaran and Samir Chenaf for experimental support. Adèle Barrière, Sophie Fouyet
and Kevin Ricard were Master students that contributed to the work. Dayana Barbosa da Cruz was a recipient of an International Mobility Grant from the University of Paris to the Jequitinhonha and Mucuri Valleys Federal University, Brazil. Elsa Bianchini (Inserm, U1176, Bicêtre, France) provided assistance to DBdC with the isolation of neutrophils from blood. Sonia Poirault-Chassac (Inserm, U1140, Paris, France) provided assistance with fluorescence microscopy. We acknowledge the generous provision of MM-H-Pro-Arg-pNA by Geneviève Contant (Stago, Gennevilliers, France). This research was supported by the Institut National de la Santé et de la Recherche Médicale (Inserm) and the Agence National pour la Recherche grants COCERP (ANR-16-CE29-0009-02) and ENDOPAROMP (ANR-17-CE17-0024-03).

**Author Contributions.** D.B.C., Ll.R.A. and LC performed research and analysed data. J.H. and L.S. substantially contributed to patient's recruitment, collected and analysed data. M. R-K designed, analysed and interpreted proteomic data. C.B. performed research and collected data. P.C. designed MS experiments and performed research. E.A-C. P.G., F.M. and F.T. designed research, analysed data and participated in manuscript writing.
REFERENCES

1. Longstaff, C., and Kolev, K. (2015) Basic mechanisms and regulation of fibrinolysis. *J Thromb Haemost* **13 Suppl 1**, S98-105

2. Xue, Y., Bodin, C., and Olsson, K. (2012) Crystal structure of the native plasminogen reveals an activation-resistant compact conformation. *J Thromb Haemost* **10**, 1385-1396

3. Fleury, V., and Angles-Cano, E. (1991) Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. *Biochemistry-Us* **30**, 7630-7638

4. Scapini, P., Nesi, L., Morini, M., Tanghetti, E., Belleri, M., Noonan, D., Presta, M., Albini, A., and Cassatella, M. A. (2002) Generation of biologically active angiostatin kringle 1-3 by activated human neutrophils. *J Immunol* **168**, 5798-5804

5. Moroz, L. A. (1981) Mini-plasminogen: a mechanism for leukocyte modulation of plasminogen activation by urokinase. *Blood* **58**, 97-104

6. Beatty, K., Bieth, J., and Travis, J. (1980) Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin. *J Biol Chem* **255**, 3931-3934

7. Korkmaz, B., Attucci, S., Jourdan, M. L., Juliano, L., and Gauthier, F. (2005) Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils. *J Immunol* **175**, 3329-3338

8. Owen, C. A., Campbell, M. A., Sannes, P. L., Boukedes, S. S., and Campbell, E. J. (1995) Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. *J Cell Biol* **131**, 775-789

9. Belorgey, D., and Bieth, J. G. (1998) Effect of polynucleotides on the inhibition of neutrophil elastase by mucus proteinase inhibitor and alpha 1-proteinase inhibitor. *Biochemistry-Us* **37**, 16416-16422

10. Belorgey, D., and Bieth, J. G. (1995) DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity. *FEBS Lett* **361**, 265-268

11. Duranton, J., Belorgey, D., Carrere, J., Donato, L., Moritz, T., and Bieth, J. G. (2000) Effect of DNase on the activity of neutrophil elastase, cathepsin G and proteinase 3 in the presence of DNA. *FEBS Lett* **473**, 154-156

12. Duranton, J., Boudier, C., Belorgey, D., Mellet, P., and Bieth, J. G. (2000) DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor. *J Biol Chem* **275**, 3787-3792

13. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y., and Zychlinsky, A. (2004) Neutrophil extracellular traps kill bacteria. *Science* **303**, 1532-1535

14. Kenny, E. F., Herzig, A., Kruger, R., Muth, A., Mondal, S., Thompson, P. R., Brinkmann, V., Bernuth, H. V., and Zychlinsky, A. (2017) Diverse stimuli engage different neutrophil extracellular trap pathways. *Elife* **6**

15. Fuchs, T. A., Abed, U., Goosmann, C., Hurwitz, R., Schulze, I., Wahn, V., Weinrauch, Y., Brinkmann, V., and Zychlinsky, A. (2007) Novel cell death program leads to neutrophil extracellular traps. *J Cell Biol* **176**, 231-241

16. Urban, C. F., Ermert, D., Schmid, M., Abu-Abed, U., Goosmann, C., Nacken, W., Brinkmann, V., Jungblut, P. R., and Zychlinsky, A. (2009) Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. *Plos Pathog* **5**, e1000639
17. Thomas, M. P., Whangbo, J., McCrossan, G., Deutsch, A. J., Martinod, K., Walch, M., and Lieberman, J. (2014) Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins. *J Immunol* **192**, 5390-5397

18. Brinkmann, V., and Zychlinsky, A. (2007) Beneficial suicide: why neutrophils die to make NETs. *Nat Rev Microbiol* **5**, 577-582

19. Kessenbrock, K., Krumbholz, M., Schonermarck, U., Back, W., Gross, W. L., Werb, Z., Grone, H. J., Brinkmann, V., and Jenne, D. E. (2009) Netting neutrophils in autoimmune small-vessel vasculitis. *Nat Med* **15**, 623-625

20. Engelmann, B., and Massberg, S. (2013) Thrombosis as an intravascular effector of innate immunity. *Nat Rev Immunol* **13**, 34-45

21. Fuchs, T. A., Brill, A., Duerschmied, D., Schatzberg, D., Monestier, M., Myers, D. D., Jr., Wrobleski, S. K., Wakefield, T. W., Hartwig, J. H., and Wagner, D. D. (2010) Extracellular DNA traps promote thrombosis. *Proc Natl Acad Sci U S A* **107**, 15880-15885

22. Grassle, S., Huck, V., Pappelbaum, K. I., Gorzelanny, C., Aponte-Santamaria, C., Baldauf, C., Grater, F., Schneppenheim, R., Obser, T., and Schneider, S. W. (2014) von Willebrand factor directly interacts with DNA from neutrophil extracellular traps. *Arterioscler Thromb Vasc Biol* **34**, 1382-1389

23. de Boer, O. J., Li, X., Teeling, P., Mackaay, C., Ploegmakers, H. J., van der Loos, C. M., Daemen, M. J., de Winter, R. J., and van der Wal, A. C. (2013) Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction. *Thromb Haemost* **109**, 290-297

24. Riegger, J., Byrne, R. A., Joner, M., Chandraratne, S., Gershlick, A. H., Ten Berg, J. M., Adriaenssens, T., Guagliumi, G., Godschalk, T. C., Neumann, F. J., Trenk, D., Feldman, L. J., Steg, P. G., Desmet, W., Alfonso, F., Goodall, A. H., Wojdyla, R., Dudek, D., Philippi, V., Opinaldo, S., Titova, A., Malik, N., Cotton, J., Jhagroe, D. A., Heestermans, A. A., Sinnaeve, P., Vermeersch, P., Valina, C., Schulz, C., Kastrati, A., Massberg, S., and Prevention of Late Stent Thrombosis by an Interdisciplinary Global European Effort. I. (2016) Histopathological evaluation of thrombus in patients presenting with stent thrombosis. A multicenter European study: a report of the prevention of late stent thrombosis by an interdisciplinary global European effort consortium. *Eur Heart J* **37**, 1538-1549

25. Laridan, E., Denorme, F., Desender, L., Francois, O., Andersson, T., Deckmyn, H., Vanhoorelbeke, K., and De Meyer, S. F. (2017) Neutrophil extracellular traps in ischemic stroke thrombi. *Ann Neurol* **82**, 223-232

26. Longstaff, C., Varju, I., Sotonyi, P., Szabo, L., Krumrey, M., Hoell, A., Bota, A., Varga, Z., Komorowicz, E., and Kolev, K. (2013) Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. *J Biol Chem* **288**, 6946-6956

27. Gould, T. J., Vu, T. T., Stafford, A. R., Dwivedi, D. J., Kim, P. Y., Fox-Robichaud, A. E., Weitz, J. I., and Liaw, P. C. (2015) Cell-Free DNA Modulates Clot Structure and Impairs Fibrinolysis in Sepsis. *Arterioscler Thromb Vasc Biol* **35**, 2544-2553

28. Varju, I., Longstaff, C., Szabo, L., Farkas, A. Z., Varga-Szabo, V. J., Tanka-Salamon, A., Machovich, R., and Kolev, K. (2015) DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment. *Thromb Haemost* **113**, 1289-1298

29. Fuchs, T. A., Brill, A., and Wagner, D. D. (2012) Neutrophil extracellular trap (NET) impact on deep vein thrombosis. *Arterioscler Thromb Vasc Biol* **32**, 1777-1783

30. Singer, M. (2016) The new sepsis consensus definitions (Sepsis-3): the good, the not-so-bad, and the actually-quite-pretty. *Intensive Care Med* **42**, 2027-2029
31. Iba, T., Di Nisio, M., Thachil, J., Wada, H., Asakura, H., Sato, K., Kitamura, N., and Saitoh, D. (2016) Revision of the Japanese Association for Acute Medicine (JAAM) disseminated intravascular coagulation (DIC) diagnostic criteria using antithrombin activity. *Crit Care* **20**, 287

32. Holvoet, P., Lijnen, H. R., and Collen, D. (1985) A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo. *J Biol Chem* **260**, 12106-12111

33. Grailhe, P., Nieuwenhuizen, W., and Angles-Cano, E. (1994) Study of tissue-type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen. *Eur J Biochem* **219**, 961-967

34. Fleury, V., Loyau, S., Lijnen, H. R., Nieuwenhuizen, W., and Angles-Cano, E. (1993) Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface. *Eur J Biochem* **216**, 549-556

35. Montes, R., Paramo, J. A., Angles-Cano, E., and Rocha, E. (1996) Development and clinical application of a new ELISA assay to determine plasmin-alpha2-antiplasmin complexes in plasma. *Br J Haematol* **92**, 979-985

36. Grenier, A., Dehoux, M., Boutten, A., Arce-Vicioso, M., Durand, G., Gougerot-Pocidalo, M. A., and Chollet-Martin, S. (1999) Oncostatin M production and regulation by human polymorphonuclear neutrophils. *Blood* **93**, 1413-1421

37. Angles-Cano, E., and Sultan, Y. (1984) A solid-phase fibrin immunoassay for the specific detection of monoclonal antibodies against different epitopic determinants of tissue-plasminogen activators. *J Immunol Methods* **69**, 115-127

38. Dominguez, M., Montes, R., Paramo, J. A., and Angles-Cano, E. (2002) Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation. *Biochim Biophys Acta* **1598**, 165-176

39. Koppert, P. W., Huijsmans, C. M., and Nieuwenhuizen, W. (1985) A monoclonal antibody, specific for human fibrinogen, fibrinopeptide A-containing fragments and not reacting with free fibrinopeptide A. *Blood* **66**, 503-507

40. Rylatt, D. B., Blake, A. S., Cottis, L. E., Massingham, D. A., Fletcher, W. A., Masci, P. P., Whitaker, A. N., Elms, M., Bunce, I., Webber, A. J., and et al. (1983) An immunoassay for human D dimer using monoclonal antibodies. *Thromb Res* **31**, 767-778

41. Bangert, K., and Thorsen, S. (2000) Assay of functional plasminogen in rat plasma applicable to experimental studies of thrombolysis. *Thromb Haemost* **84**, 299-306

42. Pouit, L., Hudry-Clergeon, G., and Suscillon, M. (1973) Electron microscopy study of positive charges on fibrin fibres. *Biochim Biophys Acta* **317**, 99-105

43. Campbell, E. J., and Owen, C. A. (2007) The sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans in neutrophil plasma membranes are novel binding sites for human leukocyte elastase and cathepsin G. *J Biol Chem* **282**, 14645-14654

44. Warejcka, D. J., and Twining, S. S. (2005) Specific conformational changes of plasminogen induced by chloride ions, 6-aminohexanoic acid and benzamidine, but not the overall openness of plasminogen regulate, production of biologically active angiostatins. *Biochem J* **392**, 703-712

45. Massberg, S., Grahl, L., von Bruehl, M. L., Manukyan, D., Pfeiler, S., Goosmann, C., Brinkmann, V., Lorenz, M., Bidzhekov, K., Khandagale, A. B., Konrad, I., Kennerknecht, E., Reges, K., Holdenrieder, S., Braun, S., Reinhardt, C., Spannagl, M., Preissner, K. T., and Engelmann, B. (2010) Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. *Nat Med* **16**, 887-896
46. Clark, S. R., Ma, A. C., Tavener, S. A., McDonald, B., Goodarzi, Z., Kelly, M. M., Patel, K. D., Chakrabarti, S., McAvoy, E., Sinclair, G. D., Keys, E. M., Allen-Vercoe, E., Devinney, R., Doig, C. J., Green, F. H., and Kubes, P. (2007) Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. *Nat Med* **13**, 463-469

47. McDonald, B., Davis, R. P., Kim, S. J., Tse, M., Esmon, C. T., Kolaczkowska, E., and Jenne, C. N. (2017) Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. *Blood* **129**, 1357-1367

48. Delabranche, X., Stiel, L., Severac, F., Galoisy, A. C., Mauvieux, L., Zobairi, F., Lavigne, T., Toti, F., Angles-Cano, E., Meziani, F., and Boisrame-Helms, J. (2017) Evidence of Netosis in Septic Shock-Induced Disseminated Intravascular Coagulation. *Shock* **47**, 313-317

49. Maruchi, Y., Tsuda, M., Mori, H., Takenaka, N., Gocho, T., Huq, M. A., and Takeyama, N. (2018) Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock. *Crit Care* **22**, 176

50. Li, X., and Ma, X. (2018) A brief comment on the predictive value of myeloperoxidase-conjugated DNA level in patients with septic shock. *Crit Care* **22**, 294

51. Kumar, S., Gupta, E., Kaushik, S., Srivastava, V. K., Saxena, J., Mehta, S., and Jyoti, A. (2019) Quantification of NETs formation in neutrophil and its correlation with the severity of sepsis and organ dysfunction. *Clin Chim Acta* **495**, 606-610

52. Abrams, S. T., Morton, B., Alhamdi, Y., Alsabani, M., Lane, S., Welters, I. D., Wang, G., and Toh, C. H. (2019) A Novel Assay for Neutrophil Extracellular Traps (NETs) Formation Independently Predicts Disseminated Intravascular Coagulation and Mortality in Critically Ill Patients. *Am J Respir Crit Care Med*

53. Noubouossie, D. F., Reeves, B. N., Strahl, B. D., and Key, N. S. (2019) Neutrophils: back in the thrombosis spotlight. *Blood* **133**, 2186-2197

54. Ducroux, C., Di Meglio, L., Loyau, S., Delbosc, S., Boisseau, W., Deschildre, C., Ben Maacha, M., Blanc, R., Redjem, H., Ciccio, G., Smajda, S., Fahed, R., Michel, J. B., Piotin, M., Salomon, L., Mazighi, M., Ho-Tin-Noe, B., and Desilles, J. P. (2018) Thrombus Neutrophil Extracellular Traps Content Impair tPA-Induced Thrombolysis in Acute Ischemic Stroke. *Stroke* **49**, 754-757

55. Kolaczkowska, E., Jenne, C. N., Surewaard, B. G., Thanabalasuriar, A., Lee, W. Y., Sanz, M. J., Mowen, K., Opdenakker, G., and Kubes, P. (2015) Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature. *Nat Commun* **6**, 6673

56. Rouy, D., Koschinsky, M. L., Fleury, V., Chapman, J., and Angles-Cano, E. (1992) Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments. *Biochemistry-Us* **31**, 6333-6339

57. Lamanuzzi, L. B., Mtairag el, M., Pepe, G., and Angles-Cano, E. (2004) Neutrophils stimulated by apolipoprotein(a) generate fragments that are stronger inhibitors of plasmin formation than apo(a). *Thromb Haemost* **92**, 1066-1075

58. Thorsen, S., Clemmensen, I., Sottrup-Jensen, L., and Magnusson, S. (1981) Adsorption to fibrin of native fragments of known primary structure from human plasminogen. *Biochim Biophys Acta* **668**, 377-387

59. Plow, E. F., and Edgington, T. S. (1975) An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte proteases at physiologic pH. *J Clin Invest* **56**, 30-38

60. Machovich, R., and Owen, W. G. (1989) An elastase-dependent pathway of plasminogen activation. *Biochemistry-Us* **28**, 4517-4522

61. Wu, K., Urano, T., Ihara, H., Takada, Y., Fujie, M., Shikimori, M., Hashimoto, K., and Takada, A. (1995) The cleavage and inactivation of plasminogen activator inhibitor type
1 by neutrophil elastase: the evaluation of its physiologic relevance in fibrinolysis.
*Blood* **86**, 1056-1061

62. Duboscq, C., Genoud, V., Parborell, M. F., and Kordich, L. C. (1997) Impaired clot lysis by rt-PA catalyzed mini-plasminogen activation. *Thromb Res* **86**, 505-513

63. Suenson, E., Lutzen, O., and Thorsen, S. (1984) Initial plasmin-degradation of fibrin as the basis of a positive feed-back mechanism in fibrinolysis. *Eur J Biochem* **140**, 513-522

64. Herren, T., Burke, T. A., Das, R., and Plow, E. F. (2006) Identification of histone H2B as a regulated plasminogen receptor. *Biochemistry-Us* **45**, 9463-9474

65. Duboscq, C., Quintana, I., Bassilotta, E., Bergonzelli, G. E., Porterie, P., Sassetti, B., Haedo, A. S., Wainsztein, N., Kruithof, E. K., and Kordich, L. (1997) Plasminogen: an important hemostatic parameter in septic patients. *Thromb Haemost* **77**, 1090-1095
FIGURE LEGENDS

**Figure 1. Neutrophil Extracellular Traps (NETs) generated from neutrophils seeded onto a fibrin matrix: DNA and HNE activity.** Neutrophils isolated from human blood and seeded on wells of a 96-wells microtitre plate containing or not a fibrin matrix, were activated by 50 nM PMA in HBSS. After 4 hours incubation at 37°C in a humidified 5% CO₂ incubator, the plate was centrifuged at 3000 g for 5 min, the supernatant was collected and cells and NETs in the well stained with a 10µg/ml solution of Hoechst 33342 a minor-groove DNA binder. Elastase activity bound to NETs was detected with the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. **A.** Fluorescence microscopy images (X20) of nuclei and extracellular DNA fibers stained with Hoechst. Scale bars: 20 µm. **Neutrophils:** non stimulated neutrophils incubated in the absence of PMA show typical polymorphonuclear appearance. **NETs:** neutrophil extracellular traps generated in the absence of fibrin. **NETs-fibrin:** NETs generated onto a fibrin matrix had a denser and reticulated appearance than NETs extruded in the absence of fibrin. **B.** Detection of HNE activity on HNE-ADN complexes of NETs (black bars) and in supernatants (grey bars) in the absence of inhibitor (No Inh) or in the presence of α₁-proteinase inhibitor (α₁-PI, 10 µM), Aprotinin (10 µM) or the synthetic inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (AAPV-cmk, 100µM). **C.** Detection of soluble HNE activity in buffer (③) and its inhibition when added to normal human plasma (■). Purified HNE was incubated (15 min) at different concentrations in buffer or plasma before activity measurements with 1.5 mM of the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.

**Figure 2. NETs-associated elastase digests plasminogen into fragments.** Plasminogen (1 µM) was incubated with NETs generated by neutrophils treated with 50 nM PMA for 4 hours as indicated in figure 1. Samples were analyzed by SDS-PAGE and Western blot using a specific sheep antibody directed against human plasminogen. **A-B.** Plasminogen fragmentation by NETs HNE•DNA complexes after 30 to 240 min (A) and 2 to 10 hours (B) of incubation. **C.** Western blot of purified plasminogen (Pg), plasmin (Pn) (left panel) and HNE-DNA-derived plasminogen fragments (middle panel). Plasminogen fragments produced by PMA stimulated neutrophils (middle panel) were not observed (right panel) in the presence of the elastase-specific inhibitor (EI) N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone.
**Figure 3. NETs formed on fibrin decrease tPA-mediated plasmin formation.** Neutrophils isolated from human blood were seeded onto fibrin in a 96-wells microtiter plate and activated by 50 nM PMA in HBSS. NETs released by activated neutrophils were identified as indicated in figure 1. After binding of tPA to fibrin, plasminogen at varying concentrations was added to the interlaced NETs-fibrin structure. **A-B.** Plasmin formation from plasminogen (Pg) at 50 nM (A) and 500 nM (B) was monitored by measuring the released of pNA the plasmin-selective chromogenic substrate at 0.75 mM. ☐ Plasmin formation on fibrin. ● Plasmin formation on fibrin-NETs. ■ Plasmin formation on fibrin-NETs treated with the elastase inhibitor MeO-Suc-AAPV-CMK. **C.** After plasminogen activation the wells were carefully washed and the amount of fibrin-bound plasmin detected by adding 0.75 mM of the plasmin-selective chromogenic substrate. Grey bars: plasmin bound to the fibrin-NETs surface. White bars: plasmin bound to the fibrin-NETs surface treated with the elastase inhibitor MeO-Suc-AAPV-CMK. **D.** Analysis of plasminogen fragmentation in protein extracts recovered from the fibrin-NETs surface after 2 hours of incubation. Western blot of non-reduced samples using a plasminogen-specific monoclonal antibody. 1: plasminogen activated by fibrin-bound tPA in the absence of NETs (in non-reduced gels plasminogen and plasmin migrates at a similar position). 2 and 4: Plasminogen incubated with the fibrin-NETs lattice; generation of plasminogen fragments and traces of plasmin. 3, 5. The generation of plasmin was recovered and the formation of plasminogen fragments inhibited in the presence of the elastase inhibitor MeO-Suc-AAPV-CMK.

**Figure 4. Plasminogen fragments circulate in patients with septic shock-induced disseminated intravascular coagulation.** Plasma samples from patients and controls were diluted 1:5 in 125mM Tris-HCl buffer pH 6.8 containing 20% glycerol and 4% SDS. Proteins were separated by SDS-PAGE (10%) followed by Western blot using a specific sheep antibody directed against human plasminogen or the horseradish peroxidase-labelled monoclonal antibody CPL15-PO directed against kringle 1. C1-C4: Plasma from healthy donors. S1-S7: Samples from patients with septic shock-induced disseminated intravascular coagulation. Pg: plasminogen in samples from patients and healthy donors. Bands between 49 kDa and 38 kDa in S1-S7 represent plasminogen fragments (Pg frg). Plasma samples treated to eliminate high-abundant proteins by euglobulin precipitation or lysine-affinity fractionation were used for proteomic analysis of plasminogen fragments.
**Figure 5. Fibrinolytic activity of septic shock plasma.** The fibrinolytic activity of plasma was tested by incubating the corresponding euglobulin fraction with fibrin surfaces to which tPA (50 iu/ml) was previously bound. The euglobulin fraction contains plasminogen and plasminogen fragments bearing K1, K4 and K5 as identified by mass spectrometry (see text and Supplemental file). The amount of plasmin formed was quantified by measuring the change in absorbance at A405nm using a chromogenic substrate selective for plasmin as indicated in figure 3. C (open bar): pool of control plasmas ((n=18, plasminogen concentration: 1.5 µM). S1 to S6: samples from septic shock patients [Plasminogen] (µM): S1 (0.546), S2 (0.863), S3 (0.415), S4 (0.491), S5 (0.372), S6 (0.504). Grey bars: activity of S1 to S6 plasma samples at their native plasminogen concentration. Closed bars: activity of S1 to S6 plasma samples upon normalization of the plasminogen concentration to 1.5 µM.
<table><caption>Table 1. Fibrinolytic parameters and NETs in septic shock and healthy control plasmas</caption><thead><tr><th>Patient's samples (S)</th><th>HNE•α₁-PI ng/ml</th><th>plasmin-α₂- antiplasmin ng/ml</th><th>NETs HNE-DNA A405nm</th><th>Plaminogen μM</th></tr></thead><tbody><tr><td>S1</td><td>223</td><td>1245</td><td>0,211</td><td>0,546</td></tr><tr><td>S2</td><td>230</td><td>366</td><td>0,222</td><td>0,863</td></tr><tr><td>S3</td><td>320</td><td>1654</td><td>0,302</td><td>0,415</td></tr><tr><td>S4</td><td>765</td><td>4814</td><td>0,284</td><td>0,491</td></tr><tr><td>S5</td><td>212</td><td>1207</td><td>0,284</td><td>0,372</td></tr><tr><td>S6</td><td>177</td><td>159</td><td>0,325</td><td>0,504</td></tr><tr><td>S7</td><td>935</td><td>720</td><td>0,302</td><td>0,689</td></tr><tr><td>Mean±SD</td><td>437±274</td><td>1534±1517</td><td>0,276±0,043</td><td>0,554±0,170</td></tr><tr><td>Range</td><td>177-935</td><td>159-4814</td><td>0,211-0,325</td><td>0,372-0,863</td></tr><tr><td>Median</td><td>320</td><td>1207</td><td>0,284</td><td>0,504</td></tr><tr><td>Healthy Controls</td><td>n=31</td><td>n=24</td><td>N=5</td><td>N=31</td></tr><tr><td>Mean±SD</td><td>43±19</td><td>89±76</td><td>0,086±0,014</td><td>1,69±0,33</td></tr><tr><td>Range</td><td>7-85</td><td>10-240</td><td>0,065-0,100</td><td>1,23-2,28</td></tr><tr><td>Median</td><td>45</td><td>59</td><td>0,086</td><td>1,65</td></tr><tr><td>Pool 91</td><td>45</td><td>96</td><td>ND</td><td>1,72±0,13</td></tr></tbody></table>

Pool 91: pool constituted with plasma separated from blood obtained from 18 healthy controls. ND: not done.
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 5</figcaption></figure>
# DNA-bound elastase of Neutrophil Extracellular Traps degrades plasminogen, reduces plasmin formation and decreases fibrinolysis: proof of concept in septic shock plasma

Barbosa da Cruz D¹, *Helms J²,³, *Ramirez Aquino Ll¹, Stiel L²,³, Cougourdan L¹, Broussard C⁴, Chafey P⁴, Riès-Kautt M⁴, Toti F⁴, Meziani F²,³, Gaussem P¹,⁵, Anglés-Cano E¹.

## SUPPLEMENTAL MATERIAL AND METHODS

### Reagents

The chromogenic substrate selective for plasmin (methylmalonyl)hydroxyprolylarginine -p-nitroanilide (CBS0065) was kindly provided by G. Contant (Diagnostica Stago, Asnières, France). Phorbol 12-myristate 13-acetate (PMA), the serine protease inhibitors aminoethyl-benzene-sulfonyl fluoride (AEBSF) and phenylmethylsulphonyl fluoride (PMSF), the elastase-specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (MeO-Suc-AAPV-pNA) and the elastase-specific inhibitor N-methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-valine-chloromethyl ketone (MeO-Suc-AAPV-CMK) were from Sigma-Aldrich (L'Isle d'Abeau-Chenes, France). Hank's balanced salt solution (HBSS) was from Gibco. Dextran T-500 from Serva. Radioselectan was from Schering, France. Pancoll 1.077 mg/ml was from PAN BIOTECH GmbH (distributed by Dutscher, Brumath, France). Hoechst 33342, a minor groove binder of dsDNA was from BioProbes (distributed by ThermoFisher, France).

### Detection of HNE-α₁-PI and plasmin-α₂-antiplasmin complexes

Human neutrophil elastase (HNE) was detected as a complex with the α₁-proteinase inhibitor (α₁-PI) using the Human PMN Elastase Platinum ELISA kit from Affymetrix (Bender MedSystems GmbH, Vienna, Austria). Plasmin-α₂-antiplasmin complexes were detected by ELISA (Technozym, Technoclone GmnH, Vienna, Austria).

### Identification of plasminogen and its fragments by Mass Spectrometry

#### Database search and interpretation of MS datasets

Samples used were either purified proteins (human plasminogen, plasmin and HNE-derived plasminogen fragments) or plasma protein fractions of interest (euglobulins and lysine-affinity fractions) depleted in highly abundant proteins. The euglobulin fraction from 100 µl of plasma (patients or control) was precipitated at low ionic strength and pH 5.8 in the presence of protease inhibitors and resuspended in 2.5% SDS, 62.5 mM
Tris-HCl pH 6.8 sample buffer; this fraction is depleted in albumin and α-globulins but contains all β- and γ-globulins including plasminogen and its fragments. Proteins bound to lysine-Sepharose from 3 ml of plasma (pool of plasma from 6 patients) were eluted with 30 mM ε-aminocaproic acid as previously described.(1) The lysine-affinity fraction specifically contains proteins bearing a lysine-binding site.

Proteins in samples were separated on either 8% or 15% SDS-PAGE gels. Bands were manually excised from gels and were cut into cubes. Then, in-gel digestion was carried out with trypsin, according to a published procedure with minor adjustments(2): sample were destained twice with a mixture of 100 mM ammonium bicarbonate (ABC) and 50% (vol/vol) acetonitrile (ACN) for 30 min at room temperature and then dehydrated using 100% ACN for 20 min, before being reduced with 25 mM ABC containing 10 mM DTT for 1 h at 57 °C and alkylated with 55 mM iodoacetamide in 25 mM ABC for 30 min in the dark at room temperature. Gel pieces were washed twice with 25 mM ABC and dehydrated (twice, 20 min) with 100% ACN. Gel cubes were incubated with sequencing grade-modified trypsin (Promega, USA; 12.5 ng/µl in 40 mM ABC with 10% ACN, pH 8.0) overnight at 37 °C. After digestion, peptides were extracted twice from gel pieces with a mixture of 50% ACN – 5% formic acid (FA) and then with 100% ACN. Extracts were dried using a vacuum centrifuge concentrator plus (Eppendorf).

Mass spectrometry analyses were performed using an Ultimate 3000 Rapid Separation Liquid Chromatographic (RSLC) system (Thermo Fisher Scientific) online with a Q Exactive hybrid quadrupole Orbitrap mass spectrometer (ThermoFisher Scientific). Briefly, peptides were solubilized in 3.5 µL of 10% ACN - 0.1% TriFluoroAcetic acid (TFA). Then, peptides were loaded and washed on a C₁₈ reverse-phase precolumn (3 µm particle size, 100 Å pore size, 75 µm i.d., 2 cm length). The loading buffer contained 98% H₂O, 2% ACN and 0.1% TFA. Peptides were then separated on a C₁₈ reverse-phase resin (2 µm particle size, 100 Å pore size, 75 µm i.d., 25 cm length) with a 30 min gradient from 99% A (0.1% FA and 100% H₂O) to 40% B (80% ACN, 0.085% FA and 20% H₂O).

The mass spectrometer acquired data throughout the elution process and operated in a data-dependent scheme with full MS scans acquired with the Orbitrap, followed by up to 10 MS/MS HCD spectra in the Orbitrap. Mass spectrometer settings were: full MS (AGC: 3 × 10⁶, resolution: 7 × 10⁴, m/z range 400–2000, maximum ion injection time: 100 ms) and MS/MS (AGC: 1 × 10⁵, maximum injection time: 100 ms, isolation window: 4m/z, dynamic exclusion time setting: 15 s). The fragmentation was permitted for
precursors with a charge state of +2, 3 and 4. For the spectral processing, the software
used to generate .mgf files was Proteome discoverer 1.4 (ThermoFisher Scientific).
Database searches were carried out using Mascot version 2.5.1 (Matrix Science, London,
UK) on 'human' proteins (20,273 sequences) from the SwissProt databank containing
550,552 sequences (196,472,675 residues) (February 2016). The search parameters
were as follows: the enzyme specificity as semiTrypsin, carbamidomethylation as a fixed
modification for cysteins and oxidation as a variable modification for methionines. Up to
1 missed cleavage was tolerated, and mass accuracy tolerance levels of 4 ppm for
precursors and 20 mmu for fragments were used for all tryptic mass searches. Positive
identification was based on a Mascot score above the significance level (i.e. 5%). The
reported proteins were always those with the highest number of peptide matches.

References

1. Lamanuzzi, L. B., Mtairag el, M., Pepe, G., and Angles-Cano, E. (2004) Neutrophils stimulated by apolipoprotein(a) generate fragments that are stronger inhibitors of plasmin formation than apo(a). *Thromb Haemost* **92**, 1066-1075

2. Shevchenko, A., Loboda, A., Ens, W., Schraven, B., Standing, K. G., and Shevchenko, A. (2001) Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry. *Electrophoresis* **22**, 1194-1203

3. Warejcka, D. J., and Twining, S. S. (2005) Specific conformational changes of plasminogen induced by chloride ions, 6-aminohexanoic acid and benzamidine, but not the overall openness of plasminogen regulate, production of biologically active angiostatins. *Biochem J* **392**, 703-712
SUPPLEMENTAL FIGURES

Figure S1. NETs DNA-HNE complexes digest plasminogen into fragments of known structure identified by proteomic analysis.

Plasminogen and its fragments were identified in purified samples and plasma protein fractions by mass spectrometry as indicated above. Schematic representation of plasminogen and its main fragments, modified from the plasminogen structure sequence (http://www.chem.cmu.edu/groups/Llinas/res/structure/kringle-big.html).

A. Full-length plasminogen consists of an N-terminal region, 5 kringle (K) domains and the serine protease (SP) region. Arrows indicate the main HNE cleavage sites in plasminogen.(3)

B. B. 27kDa K1-K3 Leu74-Val338

C. C. 34kDa K1-K3 Leu74-Val354

D. D. 39kDa K1-K3 Leu74-Val354

E. E.14kDa K4 Val355-Val443

F. F. 38kDa K5-SP Ala444 -Asp791

Differences in molecular mass for K<sub>I+2+3</sub> are related to the site of cleavage and glycosylation.

<figure><img src="image_12.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /><figcaption>Figure S2. Human Neutrophil Elastase-derived fragments of Glu-plasminogen</figcaption></figure>

Fragments were prepared by incubating for 30 min at 37 °C, 10 µM of the purified protein with 250 nM of purified HNE sufficient to fully transform plasminogen into fragments.

Corresponding fragments were isolated by affinity chromatography on Lysine-Sepharose and were identified by proteomic analysis using mass spectrometry. M. Molecular markers of reference. 1. K5-SP. 2. First band K1+2+3, second and third bands K1+2+3. 3. K1+2+3+4.

4. K4
<figure><img src="image_19.png" /><figcaption>Figure S2. Graphic abstract</figcaption></figure>

Plasminogen is reduced to fragments by active HNE-DNA complexes of NETs. HNE-DNA complexes are at the center of a mechanism resulting in severe fibrinolytic failure via the consumption of plasminogen by proteolysis leading to the production of antifibrinolytic plasminogen fragments. This fibrinolytic insufficiency may be responsible for impaired dissolution of microthrombi thereby fostering their stabilization in the microcirculation and contributing to organ failure during septic shock.