OCCLUSIONS

FIELD OF THE INVENTION:

The present invention relates to methods and kits for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions.

BACKGROUND OF THE INVENTION:

Extracellular genomic DNA has been identified in human blood and in cell cultures ^1_3 .

Circulating extracellular DNA in plasma (cirDNA) benefits from renewed interest, as high affinity fluorescent nucleotide-intercalating probes facilitate its reliable quantification in body fluids. CirDNA was quantified in human pathology and animal models. In healthy humans, cirDNA may occur between 1 and 27 ng/ml according to sample processing and DNA extraction techniques ⁴ ' ⁵ . CirDNA levels are increased during deep vein thrombosis ^6"9 , infection and septic shock ^10~12 , transfusion-related acute lung injury ^13~15 , after severe trauma ^11,16 , or in pre-eclamptic women ¹¹ . Moreover, cirDNA levels increase up to 200 ng/ml in gastric and breast cancer ¹⁸ ' ¹⁹ .

Sickle cell disease (SCD) is a single gene disorder characterized by mutant hemoglobin- S (HbS) and chronic intravascular hemolysis. It is characterized chronic anemia, increased levels of cell-free hemoglobin (Hb) and red blood cell (RBC) membrane microvesicles (MV), also called microp articles (MP), in plasma ^{20 23} . Painful vaso-occlusive crises (VOC) are typical of SCD and often associated to a further rise in hemolysis ^20<24 · ²⁵ . Several studies showed that cirDNA levels are upregulated in SCD, during VOC ⁵ ' ²⁶ ' ²⁷ ,, and associated to high nucleosome levels in plasma ²⁷ ' ²⁸ . Conversely, hydroxyurea treatment of SCD patients, which reduces the frequency of VOC, also lowered cirDNA levels ²⁶ . In transgenic mice with SCD, administration of pro-inflammatory TNF-a increased cirDNA levels, induced severe lung injury and compromised survival ²⁷ . Together, the evidence suggests a possible causal link between cirDNA levels and VOC.

However, the handful of pioneering studies used various techniques and produced conflicting results regarding cirDNA levels in SCD patients at steady state versus healthy individuals. Yet, possible variations in cirDNA levels may also occur during the progression of VOC and remain to be investigated. CirDNA may be released by activated leukocytes, as neutrophil extracellular traps (NETs). NETs are genomic DNA-based net- like structures released by neutrophils after chromatin decondensation. NETs concentrate specific enzymatic activities, including neutrophil elastase (NE), myeloperoxidase (MPO) and a-defensins. NETs can participate in innate immunity and pathogen clearance by capturing and eliminating bacteria, fungi and viruses ^{29 0} . Recent reports suggest that NETs may also contribute to thrombosis ⁶ ' ^7,9 , by recruiting and activating platelets, or mixing with and strengthening fibrin meshwork ⁸ . It is the DNA strands of NETs that mediates their integration into forming thrombi. Thrombi formed during deep vein thrombosis are rich in cirDNA ⁶ , which makes them particularly resistant to plasmin ⁷ ' ³¹ , but sensitive to DNase ⁶ ' ^9,14 . In intensive care units, high cirDNA levels are thus considered a risk factor for thrombosis, systemic inflammation, organ failure and death. However, the pathophysiological sequelae and mechanism of action of cirDNA remain unknown in SCD.

The transit of RBC through small blood vessels, RBC aggregation and blood rheology are major contributors to vaso-occlusions, in a manner that is sensitive to the pro-oxidant and pro-inflammatory environment ³² . Impaired RBC circulation or RBC trapping favour acute and ischemic complications, and bear on clinical severity in SCD. Nevertheless, the effects of cirDNA and NETs on RBC remain unknown. They may not induce thrombosis and coagulation in a conventional manner in SCD. Indeed, SCD vaso-occlusions and VOC are not the product of disseminated coagulation, but rather of an extreme but reversible slowing down of RBC transit through capillaries and post-capillary venules. Some steps of the coagulation and thrombosis pathways may contribute to this phenomenon in an ill-understood manner specific to SCD.

In general, NETs and cirDNA have yet to be studied with respect to their natural antagonist, endogenous DNase activity. Moreover, previous studies in SCD often failed to discriminate VOC devoid of, or associated with severe infection and, most prominently, acute chest syndrome (ACS). In mouse models of SCD, NETs were studied after severe challenge with pro-inflammatory cytokines. As pathogens trigger NETosis, it remains unclear whether intravascular hemolysis can be responsible for increased cirDNA and NETs in SCD, independently from infection.

Some of the most severe consequence of SCD are thought to occur through repeated episodes of ischemia, leading to cumulative tissue injury and, eventually, organ failure. However, it is likely that not all vaso-occlusions and ischemic accidents lead to a recognized VOC. Some vaso-occlusion may remain too brief or silent. This is made even more complex by the highly heterogeneous reactions and interpretations of pain by SCD patients. Therefore, novel biological markers of vaso-occlusions, independent from the notion of pain or hospitalization, would be of great value to help clinicians identify SCD patients suffering from recurrent vaso-occlusions and ischemic episodes, and those most at risk of cardiovascular injury.

SUMMARY OF THE INVENTION:

The present invention relates to methods and kits for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Circulating cell-free DNA in plasma (cirDNA) has gained interest as an element of innate immunity, thrombosis and coagulation. The inventors hypothesized that cirDNA could favor and participate in vaso-occlusions (VOC) in sickle cell disease (SCD) by modulating RBC aggregation, adhesion and blood rheology. They investigated the impact of cirDNA on red blood cell (RBC) aggregation with respect to plasma DNase activity. CirDNA levels were increased by 300% in SCD patients at steady state versus controls, and a further 100% rise was observed during acute VOC. This latter rise disappeared within a week of hospitalization, as VOC terminated. In SCD plasma, increased cirDNA levels were coordinated with decreased endogenous plasma DNase activity, during steady state and acute phase. Therapeutic use of DNase- 1 diminished RBC aggregate robustness in SCD patient blood and released renal vaso- occlusions in transgenic mice with SCD. Hence, an imbalance between cirDNase activity and cirDNA levels controls vaso-occlusions in SCD and thus would represent a reliable biomarker for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions, as a part of a clinically obvious vaso-occlusive crisis, or as clinically silent events.

Accordingly, the first object of the present invention relates to a method of determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions comprising i) determining level of circulating cell-free DNA and the DNAse activity in a blood sample obtained from the patient, ii) calculating the ratio between DNAse activity and level of circulating cell- free DNA, iii) comparing the ratio calculated a step ii) with a predetermined reference value and iv) concluding that the patient has or is at risk of having vaso-occlusions when the ratio is lower than the predetermined reference value.

The inventors also showed that the DNase activity would represent a reliable biomarker for determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusions, as a part of a clinically obvious vaso-occlusive crisis, or as clinically silent events.

Accordingly, the second object of the present invention relates to a method of determining whether a patient suffering from sickle cell disease has or is at risk of having vaso- occlusions comprising i) determining level of the DNAse activity in a blood sample obtained from the patient, ii) comparing the level obtained a step ii) with a predetermined reference value and iii) concluding that the patient has or is at risk of having vaso-occlusions when the level is lower than the predetermined reference value.

As used herein, the term "sickle cell disease" or "SCD" has its general meaning in the art and refers to a hereditary blood disorder in which red blood cells assume an abnormal, rigid, sickle shape. Sickling of erythrocytes decreases the cells' flexibility and results in a risk of various life-threatening complications. The term includes includes sickle cell anemia, hemoglobin SC disease and Hemoglobin sickle beta-thalessemia.

As used herein, the term "vaso-occlusion" has its general meaning in the art and refers to a common complication of sickle cell disease which leads to the occlusion of capillaries and the restriction of blood flow to an organ, resulting in ischaemia, with vascular dysfunction, tissue necrosis, and often organ damage. Vaso-occlusions are usually a constituent of vaso- occlusive crises, but they may also be more limited, clinically silent, and not cause hospitalization for vaso-occlusive crisis. As used herein, the term "vaso-occlusive crisis" has its general meaning in the art and refers to a common painful complication of sickle cell disease which leads to hospitalization, in association with occlusion of capillaries and restrict blood flow to an organ resulting in ischaemia, severe pain, necrosis, and most often with transient vaso-occlusions, and organ damage.

As used herein, the term "risk" relates to the probability that an event will occur over a specific time period, as in the conversion to vaso-occlusion, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to vaso-occlusion conversion and therapeutic vaso-occlusion conversion risk reduction ratios. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to vaso-occlusion or to one at risk of developing vaso- occlusion. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of vaso-occlusion, such as alcohol consumption or cigarette smoking, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to vaso-occlusion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for vaso-occlusion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for vaso-occlusion. In some embodiments, the present invention may be used so as to discriminate those at risk for developing vaso-occlusion from those having vaso-occlusion, or those having vaso-occlusion from normal.

As used herein the term "blood sample" means a whole blood, serum, or plasma sample obtained from the patient. Preferably the blood sample according to the invention is a plasma sample. A plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the patient following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about l,500*g for about 15-20 minutes (room temperature), followed by pipeting of the plasma layer. Platelet- free plasma (PFP) will be obtained following centrifugation at about 13,000*g for 5 min. In order to collect or discard the microvesicles, the plasma sample may be centrifuged in a range of from about 15,000 to about 20,000*g. Preferably, the plasma sample is ultra-centrifuged at around 17,570*g at a temperature of about 4°C. Different buffers may be considered appropriate for resuspending the pelleted cellular debris, which contains the microvesicles. Such buffers include reagent grade (distilled or deionized) water and phosphate buffered saline (PBS) pH 7.4. Preferably, PBS buffer (Sheath fluid) is used. More preferably, the blood sample obtained from the patient is a platelet free platelet sample (PFP) sample. PFP may be separated from 10 ml citrated whole blood drawn from the fistula-free arm, 72 hours after the last dialysis. PFP may be obtained after citrate blood centrifugation at 1500*g (15 min), followed by 13000*g centrifugation (5 min, room temperature). As used herein, the term "circulating cell-free DNA" has its general meaning in the art and refers to the DNA released by the cell and present in the patient's blood stream. It is easy and routine for one of ordinary skill in the art to determining the level of circulating cell-free DNA in a blood sample obtained from the patient. In particular, the assay described in the EXAMPLE is particularly suitable for determining the level of circulating cell-free DNA. Briefly, circulating cell-free DNA may be quantified by colorimetric or fluorometric assays which are typically performed by adding reagents to the patient's blood sample, which produces a color change, the degree of which correlates with the level of circulating cell-free DNA. Other assays include hemagglutinin inhibition, complement fixation, and diffusion in agarose. Other assays involve RNA-DNA hybridization, RIA, and counter Immunoelectrophoresis assays that allow quantification of nanogram amounts of circulating DNA. With real-time PCR and PicoGreen double-stranded DNA quantification assays, picogram amounts of free DNA can be quantified. Those skilled in the art will readily appreciate various methods to determine the level of circulating cell-free DNA; the methods suggested are merely for purposes of example.

As used herein, term "DNase" has its general meaning in the art and refers to all enzymes having a phosphodiesterase activity and the ability to hydrolyse DNA. Accordingly, the term "DNAse activity" refers to the ability of the DNAses present in the blood sample to hydrolyse DNA in particular circulating cell-free DNA. Accordingly, the term "DNAse activity" has its general meaning in the art and refers to the capacity of DNAse to hydrolyse DNA. It is easy and routine for one of ordinary skill in the art to determining DNAse activity in a blood sample obtained from the patient. Conventional DNase activity assays can consist in monitoring the increase in UV absorbance that occurs when the base pairs unstack as the DNA is degraded. For instance, the assay mays consist in incubating the blood sample with an amount of DNA in a suitable buffer; separating the reaction products on an ethidium bromide agarose gel by electrophoresis and measuring the relative intensities of fluorescence of the DNA bands under UV light. Alternative relative changes in fluorescence of dye (e.g. SYBR green). Further methods are the unitz assay ( unitz, M; 1950, S. Gen Physiol, 33:363 and the modified Kunitz assay devised by Yamamoto (Yamamoto, M; 1971, Biochim Biophys Acta, 228:95).

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of ratios between DNAse activity and level of circulating cell-free DNA in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after calculating he ratio in a group of reference, one can use algorithmic analysis for the statistic treatment of the determined levels in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is Receiver Operator Characteristic Curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC .FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

The method of the present invention is thus particularly suitable for stratifying the risk in the patients and make the choice of the most appropriate therapeutic strategy. In particular, when it is concludes that the patient has or is at risk of having vaso-occlusions, then it can be decided to treat the patient with a therapeutically amount of an agent suitable for reperfusing the capillaries. In some embodiments, the agent is selected from the group consisting of DNase, antibodies (i.e. antibodies to histones or to a particular histone), and histone degrading enzymes (i.e. mast cell proteinase 1 (Gene ID: 1215)).

In some embodiments, the agent is a DNA-hydrolysing antibody such as described in ozyr AV, Gabibov AG. DNA-hydrolyzing Ab: is catalytic activity a clue for physiological significance? Autoimmunity. 2009 May;42(4):359-61.

In some embodiments, the agent is a DNase. Any suitable DNase may be used in the present invention. The DNase will most preferably be a DNase I (EC 3.1.21.1). It may, however, in some embodiments be a DNase II (EC 3.1.21.1). DNases occur in a number of species and any DNase capable of cleaving DNA may be used in the invention. The DNase may be from an animal source such as of bovine or porcine origin. It may be of plant, fungal, or microbial origin. However, typically and most preferably the DNase is of human origin and is preferably a recombinant human DNase. Commercially available DNase preparations such as Dornase™ and Pulmozyme™ may be used in embodiments of the invention. In some embodiments, the DNase has DNA hydrolytic activity, for example in the case of DNase I it may hydro lyse DNA to give '-phosphate nucleotides and in the case of DNase II it may hydro lyse DNA to give 3' phosphate nucleotides. A fluorescence-based assay using, for example, Hoechst Stain may be used such as that which was detected in Labarce & Paiden, 1980, Anal. Biochem., 102:344-352 to assay for DNA hydrolysis. Hydrolytic activity may be assessed in a variety of ways known in the art including analytical polyacrylamide and agarose gel electrophoresis, hyperchromicity assay (Kunitz, J. Gen. Physiol. 33:349-362 (1950); Kunitz, J. Gen. Physiol. 33:363-377 (1950)) or methyl green assay (Kurnick, Arch. Biochem. 29:41-53 (1950); Sinicropi, et al., Anal. Biochem. 222:351-358 (1994)). The breakdown of DNA molecules from high to lower molecular weight forms may be monitored preferably by techniques such as agarose gel electrophoresis. Control experiments in the absence of the enzyme and/or in the presence of a protein known to possess DNase activity may be performed. DNases are known to be prone to deamidation. Asparagine residues and in particular the asparagine at amino acid positions 7 and 74 of the mature human DNase I are prone to deamidation. This process converts the asparagine residues in question to aspartic acid or iso-aspartate residues. Deamidation reduces the activity of the enzyme and this is particularly the case for deamidation at the asparagine at amino acid position 74 of mature human DNase I. Techniques are available for removing deamidated forms of the enzymes to leave the amidated forms and these may be employed to prepare DNases for use in the invention. These techniques may include tentacle cation exchange (TCX) or affinity purification using DNA to purify the amidated forms of the enzyme which are still capable of binding DNA. A DNase preparation for use in the invention may typically comprise from 85 to 100%, preferably from 90 to 100%), more preferably from 95 to 100% and even more preferably from 99 to 100%) amidated, or partially amidated, enzyme by weight. In particular these figures refer to the amount of wholly amidated enzyme i.e. with all of the residues which are naturally amidated being amidated. They will typically have more than 95%>, preferably more than 99%> and even more preferably more than 99.9% of the DNase in an amidated form. In particular, these values refer to values at the time of production or to their values from one month to a year, preferably from two to six months and more preferably from three to four months after production. They may refer to the value during any point of the shelf-life of the product. Variants of naturally occurring or known DNases may be used in the invention. Thus the term DNase encompasses such variants. The term "variants" refers to polypeptides which have the same essential character or basic biological functionality as DNase. The essential character of a DNase is phosphodiesterase activity and the ability to hydrolyse DNA. Assays for measuring DNA cleavage are described herein and these may be used to determine whether a variant has hydro lytic activity. The sequence of the DNase may be modified so that it has extended half-life. For example the sequence of the enzyme may be changed to remove recognition sequences for certain proteases and in particular those derived from inflammatory cells. The DNase employed in the invention may also be chemically modified to alter its properties such as, for example, its biological half-life. To achieve this covalent modifications may be introduced by reacting targeted amino acid residues of the native or variant DNase with an organic derivatising agent that is capable of reacting with selected amino acid side-chains or N- or C-terminal residues. Suitable derivatising agents and methods are well known in the art. Residues which in particular may be derivatised include cysteinyl residues (most commonly by reaction with a-haloacetates), histidyl residues (by reaction with diethylpyrocarbonate at pH 5.5-7.0), lysinyl and amino terminal residues (by reaction with succinic or other carboxylic acid anhydrides), arginyl residues (by reaction with reagents such as phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin). Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides or may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. The covalent attachment of agents such as polyethylene glycol (PEG) or human serum albumin to the DNases may reduce their immunogenicity and/or toxicity of the variant and/or prolong its half-life and hence may be used in the invention. In some embodiments of the invention the DNase may be directly conjugated to the glycosaminoglycan or joined through an intermediate molecule. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

By a "therapeutically effective amount" of the agent is meant a sufficient amount of the agent to treat vaso-occlusion in the subject. It will be understood, however, that the total daily usage of the agent is decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific agent; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the agent may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the agent for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: CirDNA, NETs and cirDNase activity levels in SCD plasma. We studied our collection of platelet-depleted plasmas, with blood donors (o), matched non-SCD controls (o), SCD patients at steady state (o), early VOC (x) and late VOC (+). Early VOC combined with acute infection were shown (Δ). (a) We quantified total cirDNA using Sytox-Green™, a high affinity cell- impermeant cyanine nucleic acid-intercalating fluorescent dye, and subtracted background values of unlabeled plasma. Data was expressed as medians with interquartile ranges, (b) We measured NETs as DNA-neutrophil elastase (NE) complexes, using an anti-NE capture antibody for immunoadsorption onto gluteraldehyde polymer- covered culture plastic, and fluorescent Sytox-Green™ for DNA detection. Next, we measured cirDNase activity in plasma using a self-made radial diffusion and in-gel genomic DNA digestion assay, followed by Sybr-Green-1™ (Sigma- Aldrich, France) for DNA detection, (c) The test was validated using recombinant DNase-1 (positive control) and RNase-A (negative control), and performed with plasma.

Figure 2: Anti-DNA therapeutic intervention in a model of SCD vaso-occlusions We tested the potential therapeutic relevance of DNase-1 in our previously detailed mouse model of renal vaso-occlusions characterized by echo-Doppler. We used SAD transgenic mice with SCD injected with microvesicles purified from SAD RBC. (a) First, we quantified mouse plasma cirDNA and found increased levels in SAD versus control WT mice, (b) We injected RBC microvesicles and verified renal vaso-occlusions illustrated by reduced blood flow velocity in the renal artery, assessed by echo-Doppler (t=0 minutes, 'After MVP'). Spontaneous reperfusion started to increase significantly 2 hours post injection of vehicle (saline, lline). However, injection of DNase-1 (iv.) accelerated renal reperfusion. Velocity increased within 10 minutes and returned to normal within 30 minutes. (*) indicate significance, p<0.05 vs. velocity after microvesicle injection.

EXAMPLE:

Material & Methods

Human subjects and the HEMIR cohort.

87 adult SCD patients and 22 healthy volunteers were enrolled after informed consent and approval by the ethical committees of the French Scientific Research Ministry (collection DC-2011-1450-HEMIR) ³³ . We also recruited blood donors during self-appointed visits at Etablissement Francais du Sang of George Pompidou european hospital in Paris, according to running Inserm-EFS agreement I/DAJ/C2675 (renewed 2012). Blood was collected in citrated tubes, platelet-free plasma (PFP) was processed within 4 hours and stored at -80°C. We secured kidney biopsies from 3 SCD patients suffering from severe glomerulopathy, and non- malignant peritumoral renal tissue. Tissue sections were immunoreacted with phycoerythrin-coupled anti-human CD235a antibody and Sytox Green™ for DNA.

Quantification of cirDNA, cirRNA, NETs and plasma DNase activity

CirDNA and cirRNA were quantified by fluorometry using cyanine nucleic acid- intercalating fluorescent dyes Sytox-Green™ and SytoRNA-selec (ThermoFisher, France) 6,7,i5,3 i ci _rcu i _a u g NETs were quantified as DNA-NE complexes in plasma, using an in house- designed immunosorbant assay. Polyclonal anti-human NE antibody (Santa Cruz, SC-9521, Clinisciences, France) was immobilized in 96 well plates as published ⁴ . PFP (50% in buffer) were then incubated for 1 h at 37°C. Supernatants were eliminated. NE-bound DNA was revealed with Sytox Green™ (ThermoFisher, France) and expressed in relative fluorescence units (R.F.U.) to represent NETs. Circulating cell-free DNase (cirDNase) activity was measured using an in house-designed radial diffusion assay. Calf thymus DNA (Sigma- Aldrich, D-4522) was used as DNase substrate, diluted in 1% agarose gels. PFP samples (5 μΐ) were loaded in wells, and left to diffuse and digest DNA for 18 to 48 hours at 37°C. DNA was stained with fluorescent Sybr Green™ (Sigma-Aldrich). Images were collected under ultraviolet illumination. Digested DNA disks were measured using Image-J freeware and expressed in arbitrary units (A.U.).

Determination of RBC aggregation properties RBC aggregation properties were studied in whole blood within 4 hours of collection, by syllectometry (i.e., laser backscatter versus time) using Laser-assisted Optical Rotational Cell Analyzer (LORCA, RR Mechatronics, The Netherlands), after adjusting Hct to 40%, as previously described ^5~39 .

Neutrophil culture, induction of NETs and RBC adhesion assay.

Healthy or SCD human blood neutrophils depleted from platelets, monocytes, lymphocytes and RBC were cultured in Hanks-buffered saline and treated with heme or purified control and SCD RBC microvesicles (prepared as published ³ ' ⁴⁰ ), or synthetic multilayer vesicles (MLV). Extracellular DNA was stained with Sytox Green and NETs were expressed as a proportion of nuclei (%) with outward extracellular DNA filaments, distinguished from resting horseshoe- shaped nuclei, or nuclei full of decondensed chromatin. In some cases, purified RBC were cocultured with NETs. After washing, RBC bound to NETs, horseshoe-shaped nuclei, or decondensed nuclei were counted.

Murine model of sickle cell disease and kidney vaso-occlusions.

We used 10-18 week old SAD transgenic male mice ⁴¹ and induced VOC with purified heme-loaded microvesicles injected intravenously ⁴⁰ , generated from isolated SAD RBC, as previously described ³³ · ⁴⁰ · ⁴² . SAD mice are a validated model for inducible VOC, particularly suited to the study of RBC degradation products, as their spontaneous hemolysis remains moderate. SAD mice, like SCD patients, also present neutrophil hyperleukocytosis ^{4 ~45} , a relevant environment for NETs stimulation. Renal vaso-occlusions were characterized as before by Echo-Doppler ³³ ' ⁴⁰ ' ⁴⁶ .

Results

We studied cirDNA and cirDNase activity levels in our cohort 'HEMIR' of SCD patients (HbSS/HbSB°), recruited in steady state condition, or within 3 days of hospitalization for VOC (early VOC), or before hospital discharge (5-10 days after registration; late VOC). We also identified VOC with infection, either pulmonary or other systemic infections. Matched control non-SCD volunteers were recruited and a second control group of self-appointed blood donors was constituted.

CirDNA and NETs levels in SCD plasma

CirDNA levels were increased by 105% in SCD at steady state (n=40) versus matched controls (n=22, /?=0.0001 ; Figure la), and further increased by 34% over steady state during VOC (n=40, /?=0.001). CirDNA levels returned to steady state levels before hospital discharge (n=9, -22%, p=0.049). In patients with VOC complicated with infection cirDNA levels were increased versus simple VOC (n=7, +542%, p<0.003), with some extreme values, up to 8 fold. We also quantified circulating R A using highly R A-specific Syto- RNAselect™. CirR A levels dropped during VOC, validating the specific rise in cirDNA.

Next, we determined whether cirDNA potentially came from inflammatory neutrophils in SCD, in the form of NETs, using a self-designed plasma NE-capture assay coupled to cirDNA detection. NE -bound cirDNA levels, ie. NETs, were mostly absent from blood donors and matched healthy volunteer plasma (Figure lb). However, steady state SCD patients displayed a 2.5 fold increase in NETs versus controls (p<0.005), and a further rise (+2.6 fold) during early VOC (ie. +600% over controls; p<0.005). NETs tended to return to basal levels before hospital discharge (p=0A 1 vs. early VOC). NETs were similar in VOC with and without infection (p=0.16).

CirDNase activity levels in SCD plasma

We quantified plasma DNase activity using a self-made genomic DNA digestion assay based on radial diffusion (Figure lc). Significant cirDNase activity was detected in blood donors, matched healthy volunteers and SCD patients during steady state (Figure lc). In contrast, cirDNase activity fell by about 70% in early VOC (p=0.008) and sometimes down to undetectable levels. CirDNase activity dropped even further in VOC with infection (about - 95%, J O.001). CirDNase activity in all controls and steady state samples exceeded 400 activity units. The CirDNase activity can be considered as a significant biomarker for determining the risk of having vaso -occlusions in a patient suffering from sickle cell disease .

Alternatively, all cirDNA values in VOC exceeded 50 relative fluorescence units. A dot plot revealed a degree of negative correlation between cirDNase activity and cirDNA levels (data not shown; Spearman's coefficient p=-0.51 ; /?<0.0001). We calculated the ratio of cirDNase activity divided by cirDNA levels. This novel index of inflammation was closely associated to VOC. CirDNase activity increased again, but failed to return to basal values before hospital discharge (/?=0.025 vs. early VOC) (data not shown).

Extracellular DNA histology in SCD kidneys

We studied the occurrence of extracellular DNA in renal biopsies of SCD patients suffering from severe kidney damage (data not shown), and control tissue surrounding resected kidney tumors (data not shown). We used anti-CD235a antibody to stain RBC red (data not shown) and Sytox Green™ to stain DNA (data not shown). We readily observed RBC stacks in peritubular capillaries in most tissue sections (interrupted light blue lines). Most were negative for DNA. However, some RBC stacks within SCD capillaries displayed DNA staining. This green staining was faint, but CD235a and DNA colocalization appeared yellow and covered whole RBC, unlike nuclei. In the example, nucleated cells (CD235a-) stained vivid green (white circles) seemed to occupy the capillary immediately up- or down-stream of the RBC stack. Similar CD235a-DNA colocalization was not seen in non-SCD nephrons (data not shown), but could be observed in some SCD nephrons (data not shown).

Induction of NETs by circulating SCD microvesicles

We wondered whether DNA release in the vascular space could result from NETs formed in the course of intravascular hemolysis. We placed human neutrophils in culture and treated them with purified heme-loaded microvesicles, a known byproduct of hemolysis (data not shown), microvesicles were prepared from control and SCD RBC as described ³ . NETs were characterized as long extracellular DNA fibers released over 4 hours and stained with Sytox Green™. A fraction of cultured neutrophils treated with SCD RBC microvesicles , up to 10%, released NETs (/?<0.02 vs. control RBC microvesicles or vehicle). Control RBC microvesicles had little effects over vehicle (PBS). In other experiments, we stimulated neutrophils with near-physiological concentrations of heme (0.01 to 20 μΜ) (data not shown), synthetic MLV (20% phosphatidylserine:80% phosphatidylcholine), or MLV artificially loaded with heme ³³ (data not shown). Heme triggered NETs between 0.1 and 20 μΜ (p<0.02 vs. PBS). MLV had no effects, whereas heme-loaded MLV triggered up to 12% NETs (p<0.0\ vs. MLV). In comparison, known NETs stimuli, including LPS (1 Mg/ml) and PMA (500 nM) triggered NETs up to 10% (p<0.02 vs. PBS; data not shown).

Adhesion of SCD RBC to NETs in vitro

We asked whether extracellular DNA and NETs could contribute to RBC immobilization. We induced NETs with SCD RBC microvesicles, washed the cells gently and incubated them with control or SCD RBC for 20 minutes. Extracellular DNA fibers were stained with Sytox Green™ . Adherent RBC in direct contact with resting neutrophils, neutrophils with decondensed chromatin or stained extracellular DNA fibers (NETs) were counted (data not shown). We found that SCD RBC bound to NETs about 14 times more than neutrophils with intact nuclei (/?=0.002), and 5.4 times more than decondensed chromatin (p=0.006). Control RBC did not adhere to NETs significantly. In other experiments, we pre- incubated the stimulated neutrophils with recombinant DNase-1 for 20 minutes, which destroyed most extracellular DNA (data not shown). Subsequent RBC adhesion was reduced by half (p=0.049).

Contribution of cirDNA to SCD RBC aggregation ex vivo We investigated RBC aggregation properties in whole blood from SCD patients or matched controls. We used fresh blood collected during steady state or early VOC and added purified neutrophil DNA, or recombinant DNase- 1. Two main values were extracted from this analysis: γ (s ¹ ), ie. the lowest shear rate needed to dissociate RBC aggregates which reflects RBC aggregates robustness (data not shown), and the aggregation index (AI), which reflects the extent of RBC aggregation (data not shown). Adding purified DNA (5 μg/ml) increased γ by about 50% in blood from SCD patients at steady state (p<0.001), but had no effect during VOC, whose γ was already increased 2.5 fold over steady state (pO.001). In contrast, adding recombinant DNase-1 to VOC blood (10 g/ml, 20 minutes pre-incubation) strongly reduced Y (-50%; /?<0.001). In general, the AI was little modified in SCD. It was slightly reduced during VOC (-15%; / θ.001 vs. steady state), and adding DNase-1 corrected this variation back to control levels (p=0.005). Moreover, adding DNA or DNase-1 to control blood failed to modulate significantly γ or AI.

Contribution of cirDNA to vaso-occlusions in vivo

We assessed the therapeutic usefulness of DNase-1 to disperse vaso-occlusions in vivo.

We used our model of transgenic SAD mice with SCD, and triggered vaso-occlusions in kidneys by injecting SAD mice with SAD RBC microvesicles (100 10 ³ MV/mouse) ³ · ⁴⁰ · ⁴⁶ . After confirming reduced kidney perfusion by echo-Doppler (-30%; /?<0.01), which can be attributed to renal vascular occlusions, we administered either vehicle PBS, or recombinant DNase-1 (660μg/Kg) to the stressed mice and followed the evolving mBFV in renal arteries every 10 minutes (Figure 2 a-b). Spontaneous reperfusion is known to occur within 4 hours in this model ³³ . PBS infusion had little effect and mBFV remained below 10 cm/s after 40 minutes. Conversely, infusing DNase-1 accelerated reperfusion, which was near complete 20 minutes after administration (p<0.01 vs. PBS).

Discussion:

CirDNA gained intense attention since NETosis has been described, bringing the description of new and unexpected pathophysiological effects of genomic DNA during immune responses, thrombosis and coagulation. Our data reveal a novel pathological contribution of cirDNA to hemorheology, related to RBC aggregation and vaso-occlusions.

Increased cirDNA levels in SCD are observed during steady state and VOC

Our data brings a clearer insight into cirDNA levels in SCD. We excluded patients treated with hydoxyurea and analyzed separately those presenting overt signs of infection or ACS. We also opposed steady state to acute phase and resolving VOC, hoping to discriminate the contributions of chronic RBC injury versus immune reactions to pathogens in the release of cirDNA and NETs. CirDNA levels were increased in SCD versus healthy volunteers and blood donors, supporting previous reports ⁵ ' ^26-28 . However, unlike previous reports, cirDNA levels were significantly elevated (several fold) in steady state SCD versus controls. This may be explained by our quantification methods, using a fluorescent probe with linear representation of data, while signal amplification (PCR amplicons for instance), Log representations ⁵ , as well as mixing Hb genotypes ²⁸ and infections may have previously hidden some differences and favored the most extreme observations.

Here, cirDNA levels also increased transiently during acute phase VOC (within 48 hours), and returned to steady state levels before patient discharge. This was consistent with a putative contribution of cirDNA in the vascular manifestations of SCD. During acute phase of VOC, increased cirDNA levels might be explained by the induction of NETs in response to heme ^15,27 and heme-loaded microvesicles, as all are elevated. The drop in cirDNA levels before hospital discharge also implies that current handling of VOC helps limit the deleterious effects of cirDNA. Targeted anti-cirDNA therapy might however accelerate the process.

A subgroup of VOC patients with ACS displayed high and particularly variable levels of cirDNA. Infection is thus able, but not necessary, to upregulate cirDNA and NETs during vaso-occlusive events. Conversely, cirDNA levels might correlate with disease severity, as ACS is one of the most deleterious manifestations of SCD. Finally, the rise in cirDNA levels observed during VOC remained modest compared to the difference between steady state SCD and controls. We hypothesize that cirDNA may operate mostly as co-factor to RBC modifications thought to participate in vaso-occlusions, such as hemoglobin deoxygenation, sickle formation or other forms of RBC injury.

Markers of NETs are elevated in SCD

In general, the levels of NE bound to cirDNA increased in parallel with total cirDNA. Part of cirDNA may thus come from neutrophils, as NETs stimulated by hemolytic products. There has been controversy regarding the demonstration of NETs in vivo, but extracellular DNA has previously been imaged in lung vessels of transgenic mice with SCD challenged -quite severely- with a combination of heme and TNFa ²⁷ . To clarify the issue, we tried to identify NETs in human SCD kidney biopsies, based on DNA morphology. We failed to identify structures evoking bona fide cell-free DNA fibers. Instead, we could identify abnormal, faint DNA staining at the surface of some RBC stacks accumulating within capillaries of injured SCD kidneys, and in the vicinity of cell nuclei. We hypothesize that DNA can be digested and remodeled by plasma DNase activity. Shorter cirDNA fragments might then stick to and cover cell surfaces ^1,3 ' ¹⁸ ' ⁴⁷ ₅ modifying their appearance and providing new opportunities for cell-cell interactions.

In summary, we collected several lines of evidence that cirDNA accumulates in plasma and kidney biopsies in human SCD. We found possible markers of NETs (DNA- bound NE), while the stimulation of neutrophils in vitro with RBC degradation products triggered NETosis. CirDNA may thus, at least partly, relate to NETs in SCD. However, the proportion of cirDNA present as NETs, or in other forms, is challenging to establish. In our opinion, this remains a technical challenge, as classical release of NE, myeloperoxidase and other neutrophil-specific activities in plasma would provide multiple opportunities for their association with DNA released by other cell types, thereby confusing the issue.

The DNA/DNase ratio is off balance during VOC

Our exploration of DNase activity in plasma provided new information on SCD physiopathology. First, healthy controls and SCD patients during steady state displayed discrete levels of cirDNase activity. CirDNase activity was more variable, but largely similar in steady state SCD and controls. Nevertheless, cirDNA levels remained higher and the cirDNase/cirDNA index was strongly reduced in SCD steady state.

SCD patients displayed depressed plasma DNase activity during acute phase of VOC, concomitant with an amplified rise in cirDNA. In resolving VOC, DNase activity had begun to increase again, and excess cirDNA had mostly disappeared before discharge from hospital. An inverse correlation of average strength between cirDNA and cirDNase levels existed across 54 patients tested. Low cirDNase activity was thus generally linked to high cirDNA. A closer look suggests that patients with infections may not always verify this correlation.

These data lead us to hypothesize that 3 phases may exist in SCD. Chronic cell injury might lead to a continuous leak of DNA into plasma during steady state, in a form that resists endogenous DNase digestion. During acute phase VOC, intravascular hemolysis and RBC degradation products may participate in a further, sudden and transient boost in DNase- sensitive cirDNA, possibly in a low DNase-dependent fashion. Moreover, the presence of infection during VOC resulted in the worst profile, with the most extreme values of cirDNA and unsignificant cirDNase activity, coherent with disease severity and the unfavorable prognostic associated to ACS. Future studies will determine whether cirDNA levels increase due to a drop in DNase expression, the upregulation of DNase inhibitors, or by saturation of endogenous DNase activity when substrate accumulates. Extracellular DNA mediates SCD RBC aggregation

One reason for the lack of effective treatment of vaso-occlusion in SCD may relate to their unique mechanism, with slower blood flow resulting from RBC aggregation mixed with non- conventional aspects of coagulation and thrombosis. Here, we reveal a direct impact of cirDNA on RBC rheology. The aggregometry data suggest that VOC occur with a slightly lower aggregation index, but that RBC interactions are much tighter, as previously reported ^{37~ 39} . Conversely, DNase-1 invoked a few more RBC interactions, but of a much weaker nature, reverting RBC rheology back to control. In health and disease, cirDNA is virtually completely bound to cell surfaces ^1ι3 ' ¹⁸ ' ⁴⁷ _; as magnesium stimulates DNA-membrane interactions ^4S . CirDNA may thus contribute to RBC aggregation by providing binding material between stressed membranes, strengthening aggregates that would become more difficult to disperse with shear stress/rate. This is illustrated by the higher γ (i.e., increased aggregate robustness) in aggregometry and the DNA-covered stacks of RBC in renal biopsy capillaries. Stronger RBC aggregates may increase blood flow resistance, as aggregates need to be completely dispersed to negotiate the smaller capillaries at the appropriate velocity ⁴⁹ . Moreover, increased RBC aggregate robustness has been associated to a higher risk of ACS in SCD ³⁸ . Conversely, a direct participation of cirDNA in RBC aggregation, independent of platelet aggregation, might partly explain the limited effects of anti-platelet therapy against VOC ⁵⁰ ' ⁵¹ . DNase therapy might exert deeper or independent curative effects in SCD. Interestingly, cirDNA did not affect RBC aggregation in control blood. SCD RBC injury, exacerbated by hypoxia or other stimuli, may thus synergize with cirDNA to re-inforce RBC- RBC interactions.

DNase therapy against vascular occlusions in SCD

We used our model of acute vaso-occlusions in the kidneys of transgenic SAD mice challenged with RBC microvesicles ⁴⁰ . We tested DNase therapy in an attempt to disperse the occluding cellular aggregates. The intravenous administration of recombinant DNase-1 was indeed very potent and accelerated kidney reperfusion significantly. Hemodynamic parameters were restored within 40 minutes. The kinetics and efficacy of DNase in mice with SCD, as well as the fact that DNase administration was shown to be safe and well tolerated by patients with systemic lupus erythematosus or cystic fibrosis, form a promising basis for the investigation of curative strategies against VOC in SCD. The preliminary assessment of plasma DNase activity status might be a useful tool to identify patients who would benefit most from DNase therapy.

Conclusions We show for the first time that cirDNA release, including NETs, may relate to hemordynamic perturbations typical of SCD, and that cirDNA can impact SCD hemorheology by modifying RBC aggregation properties and adhesion. Moreover, significant plasma DNase activity might constitute a healthy protective barrier against deleterious effects of cirDNA. This mechanism is downregulated in SCD, allowing sudden increases in cirDNA to exert pathological effects, typically during VOC. Consequently, complementing plasma DNase activity by intravenous infusion can be viewed as a potential curative approach in SCD.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Tamkovich SN, Bryzgunova OE, Rykova EY, Permyakova VI, Vlassov VV, Laktionov PP. Circulating nucleic acids in blood of healthy male and female donors. Clin Chem. 2005;51(7):1317-1319.

2. van der Vaart M, Pretorius PJ. A method for characterization of total circulating DNA. Ann N Y Acad Sci. 2008; 1137:92-97.

3. Rykova EY, Morozkin ES, Ponomaryova AA, et al. Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content. Expert Opin Biol Ther. 2012;12 Suppl 1 :S141-153.

4. Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res. 2007;635(2-3): 105-117.

5. Vasavda N, Ulug P, ondaveeti S, et al. Circulating DNA: a potential marker of sickle cell crisis. Br J Haematol. 2007;139(2):331-336.

6. Brill A, Fuchs TA, Savchenko AS, et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012;10(1):136-144.

7. Fuchs TA, Kremer Hovinga JA, Schatzberg D, Wagner DD, Lammle B. Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies. Blood. 2012;120(6): 1157-1164.

8. Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vase Biol. 2012;32(8):1777-1783.

9. von Bruhl ML, Stark K, Steinhart A, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012;209(4):819-835. 10. Keshari RS, Jyoti A, Dubey M, et al. Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition. PLoS One. 2012;7(10):e4811 1.

1 1. Meng W, Paunel-Gorgulu A, Flohe S, et al. Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice. Crit Care.

2012; 16(4):R137.

12. Zeerleder S, Stephan F, Emonts M, et al. Circulating nucleosomes and severity of illness in children suffering from meningococcal sepsis treated with protein C. Crit Care Med. 2012;40(12):3224-3229.

13. Caudrillier A, Kessenbrock , Gilliss BM, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. Clin Invest. 2012;122(7):2661 - 2671.

14. Thomas GM, Carbo C, Curtis BR, et al. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood. 2012; 1 19(26):6335-6343.

15. ono M, Saigo K, Takagi Y, et al. Heme-related molecules induce rapid production of neutrophil extracellular traps. Transfusion. 2014;54(1 1):2811-2819.

16. Margraf S, Logters T, Reipen J, Altrichter J, Scholz M, Windolf J. Neutrophil- derived circulating free DNA (cf-DNA NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis. Shock. 2008;30(4):352-358.

17. Gupta A, Hasler P, Gebhardt S, Holzgreve W, Hahn S. Occurrence of neutrophil extracellular DNA traps (NETs) in pre-eclampsia: a link with elevated levels of cell-free

DNA? Ann N YAcad Sci. 2006; 1075: 1 18-122.

18. Kolesnikova EV, Tamkovich SN, Bryzgunova OE, et al. Circulating DNA in the blood of gastric cancer patients. Ann N Y Acad Sci. 2008; 1 137:226-231.

19. Tamkovich SN, Litviakov NV, Bryzgunova OE, et al. Cell-surface-bound circulating DNA as a prognostic factor in lung cancer. Ann N Y Acad Sci. 2008;1 137:214-

217.

20. Hebbel RP. Reconstructing sickle cell disease: a data-based analysis of the "hyperhemolysis paradigm" for pulmonary hypertension from the perspective of evidence- based medicine. Am J Hematol. 201 1 ;86(2): 123-154.

21. Muller-Eberhard U, Javid J, Liem HH, Hanstein A, Hanna M. Plasma concentrations of hemopexin, haptoglobin and heme in patients with various hemolytic diseases. Blood. 1968;32(5):811 -815. 22. Reiter CD, Wang X, Tanus-Santos JE, et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med. 2002;8(12): 1383-1389.

23. Westerman M, Pizzey A, Hirschman J, et al. Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy. Br J Haematol. 2008;142(1): 126-135.

24. Ergul S, Branson CY, Hutchinson J, et al. Vasoactive factors in sickle cell disease: in vitro evidence for endothelin- 1 -mediated vasoconstriction. Am J Hematol. 2004;76(3):245- 251.

25. Nouraie M, Lee JS, Zhang Y, et al. The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US andEurope. Haematologica. 2012.

26. Ulug P, Vasavda N, Kumar R, et al. Hydroxyurea therapy lowers circulating DNA levels in sickle cell anemia. Am J Hematol. 2008;83(9):714-716.

27. Chen G, Zhang D, Fuchs TA, Manwani D, Wagner DD, Frenette PS. Heme- induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease.

Blood. 2014;123(24):3818-3827.

28. Schimmel M, Nur E, Biemond BJ, et al. Nucleosomes and neutrophil activation in sickle cell disease painful crisis. Haematologica. 2013;98(11): 1797-1803.

29. Saitoh T, Komano J, Saitoh Y, et al. Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1. Cell Host Microbe. 2012; 12(1): 109-

116.

30. Jenne CN, Wong CH, Zemp FJ, et al. Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps. Cell Host Microbe. 2013; 13(2): 169-180.

31. Fuchs TA, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010;107(36): 15880-15885.

32. Connes P, Alexy T, Detterich J, Romana M, Hardy-Dessources MD, Ballas SK. The role of blood rheology in sickle cell disease. Blood Rev. 2016;30(2): 1 1 1 -118.

33. Camus SM, De Moraes JA, Bonnin P, et al. Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease. Blood. 2015; 125(24):3805-3814.

34. Angles-Cano E, Sultan Y. A solid-phase fibrin immunoassay for the specific detection of monoclonal antibodies against different epitopic determinants of tissue - plasminogen activators. J Immunol Methods. 1984;69(1): 1 15-127. 35. Hardeman MR, Dobbe JG, Ince C. The Laser-assisted Optical Rotational Cell Analyzer (LORCA) as red blood cell aggregometer. Clin Hemorheol Microcirc. 2001 ;25(1): 1-11.

36. Baskurt OK, Uyuklu M, Ulker P, et al. Comparison of three instruments for measuring red blood cell aggregation. Clin Hemorheol Microcirc. 2009;43(4):283-298.

37. Tripette J, Alexy T, Hardy-Dessources MD, et al. Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease. Haematologica. 2009;94(8): 1060-1065.

38. Lamarre Y, Romana M, Waltz X, et al. Hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease.

Haematologica. 2012;97( 1 1 ): 1641 - 1647.

39. Hierso R, Waltz X, Mora P, et al. Effects of oxidative stress on red blood cell rheology in sickle cell patients. Br J Haematol. 2014; 166(4):601 -606.

40. Camus SM, Gausseres B, Bonnin P, et al. Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease. Blood. 2012.

41. Trudel M, Saadane N, Garel MC, et al. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO J. 1991 ; 10(1 1):3157-3165.

42. Leroyer AS, Ebrahimian TG, Cochain C, et al. Microparticles from ischemic muscle promotes postnatal vasculogenesis. Circulation. 2009; 1 19(21):2808-2817.

43. De Franceschi L, Saadane N, Trudel M, Alper SL, Brugnara C, Beuzard Y. Treatment with oral clotrimazole blocks Ca(2+)-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease. J Clin Invest. 1994;93(4): 1670-1676.

44. Sabaa N, de Franceschi L, Bonnin P, et al. Endothelin receptor antagonism prevents hypoxia- induced mortality and morbidity in a mouse model of sickle-cell disease. J Clin Invest. 2008;1 18(5): 1924-1933.

45. de Franceschi L, Baron A, Scarpa A, et al. Inhaled nitric oxide protects transgenic SAD mice from sickle cell disease-specific lung injury induced by hypoxia/reoxygenation. Blood. 2003; 102(3): 1087- 1096.

46. Bonnin P, Sabaa N, Flamant M, Debbabi H, Tharaux PL. Ultrasound imaging of renal vaso- occlusive events in transgenic sickle mice exposed to hypoxic stress. Ultrasound Med Biol. 2008;34(7): 1076-1084. 47. Laktionov PP, Tamkovich SN, Rykova EY, et al. Cell-surface-bound nucleic acids: Free and cell-surface-bound nucleic acids in blood of healthy donors and breast cancer patients. Ann N Y Acad Sci. 2004; 1022:221-227.

48. Beliaev ND, Budker VG, Gorokhova OE, Sokolov AV. [Mg2+-dependent interaction of DNA with eukaryotic cells]. Mol Biol (Mosk). 1988;22(6): 1667-1672.

49. Baskurt OK, Meiselman HJ. RBC aggregation: more important than RBC adhesion to endothelial cells as a determinant of in vivo blood flow in health and disease. Microcirculation. 2008; 15(7):585-590.

50. Heeney MM, Hoppe CC, Abboud MR, et al. A Multinational Trial of Prasugrel for Sickle Cell Vaso-Occlusive Events. N Engl J Med. 2016;374(7):625-635.

51. Ataga KI, Kutlar A, Kanter J, et al. Crizanlizumab for the Prevention of Pain Crises in Sickle Cell Disease. N Engl J Med. 2017;376(5):429-439.