Methods for diagnosing fibrinolytic dysfunction associated with neutrophil extracellular trapping

Abstract

This invention relates to an in vitro process for predicting and / or detecting fibrinolytic insufficiency from a first biological sample, wherein the fibrinolytic insufficiency is preferably associated with symptoms and NETs with or without disseminated intravascular coagulation (DIC), the process comprising measuring the concentration of plasminogen and / or at least one fragment thereof. The invention also relates to a process for determining the efficacy of treatment for fibrinolytic insufficiency, as well as plasminogen and a pharmaceutical composition containing plasminogen, which is used as a medicine for treating fibrinolytic insufficiency, wherein the fibrinolytic insufficiency is preferably associated with septic shock and disseminated intravascular coagulation (DIC).

Description

Technical Field

[0001] This invention relates to an in vitro process for predicting and / or detecting fibrinolytic insufficiency from a first biological sample, wherein the fibrinolytic insufficiency is preferably associated with NETs with / without disseminated intravascular coagulation (DIC), the process comprising measuring the concentration of plasminogen and / or at least one fragment thereof. The invention also relates to a process for determining the treatment efficacy of fibrinolytic insufficiency.

[0002] This invention is particularly applicable to the medical and veterinary fields.

[0003] In the following description, the references enclosed in parentheses () refer to the reference list at the end of the text. Background Technology

[0004] Septic shock is a syndrome characterized by a dysregulated inflammatory response in the host to a pathogenic agent, inducing hemodynamic dysfunction and coagulation-fibrinolysis, leading to multiple organ failure syndrome.

[0005] Thrombolysis (a major line of defense against thrombus formation) is triggered by plasmin, which is produced after plasminogen bound to clot components (mainly fibrin or platelet membranes) is cleaved and activated. (1) Plasminogen is a single-chain molecule consisting of an N-terminal region, five kringle (K) domains, and a serine protease (SP) domain. (2) K1 and K4 each contain lysine binding sites that ensure the binding of plasminogen to the carboxyl-terminal lysine residue of fibrin. (3) Plasminogen thus bound is processed in Arg... 561 -Val 562 Plasminogen is cleaved at the fibrin site and converted into plasmin in a surface- and local-dependent manner via tissue-type or urokinase-type plasminogen activators tPA and uPA, respectively. Intentionally, tPA is fully active upon binding to fibrin, while uPA preferentially functions upon binding to its membrane-anchored receptor uPAR. Proteases such as human neutrophil elastase (HNE) degrade plasminogen without producing plasmin (4, 5). In circulation, HNE is cleaved at high concentrations (20 μM to 40 μM) by circulating α1-protease inhibitors (α1-PI, Ka = 6.5 x 10⁻⁶). 7 M -1 s -1(6) Rapid inhibition. The HNE-α1PI complex thus formed excludes the proteolytic degradation of circulating plasminogen. However, experimental evidence suggests that HNE bound to negatively charged membrane proteoglycans or other macromolecules escapes inhibition and remains active. (7, 8) Belorgey and Bieth (1995, 1998) (9, 10) demonstrated that HNE forms a tight complex with DNA that strongly attenuates the inhibition of α1-PI against it. In addition to this protective effect against HNE, Bieth’s group also documented the protective effect of DNA against other binding proteases. (11, 12) In 2004, it was discovered that neutrophils are squeezed into the extracellular space bound to a functional scaffold of intact DNA and attached HNE during the formation of neutrophil extracellular traps (NETs) or netosis. (13) PMA (phorbol 12-myristate 13-acetate) and bacterial infection use NET-induced pathways: activation of protein kinase C and production of reactive oxygen species. (14)

[0006] NETs are decondensed nuclear DNA fibers with binding nucleoproteins (histones) as well as cytoplasmic and granule proteases (including HNE, cathepsin G, and myeloperoxidase). (15) HNE is the most abundant (5.24 μmol / g NET-DNA), (16) a characteristic component of NETs that binds to DNA with nanomolar affinity. (17) NETs have proteolytic activity and were initially described as beneficial (16) extracellular killers of microorganisms. (18) Recent data have shown that NETs also have pathophysiological relevance in a variety of non-infectious inflammatory conditions, including autoimmune diseases, thrombosis (19, 20), and cancer (74). The combined effects of NETs and the coagulation system characterize immune thrombosis. (20) It has been shown that NETs are released during thrombosis and are integrated into the fibrin scaffold along with von Willebrand factor. (21, 22) The presence of NETs in human coronary artery and cerebral thrombosis has indeed been demonstrated (23-25). The insensitivity of the NET DNA backbone to plasmin and the formation of non-fibrinolytic plasmin-DNA-fibrin complexes are considered possible reasons for plasmin resistance that favors thrombosis (27, 29).

[0007] However, there are currently no processes that enable the safe and systematic detection of fibrinolytic dysfunction, particularly fibrinolytic dysfunction associated with netosis, especially in both infectious and non-infectious diseases. Nor are there processes and / or means that enable, for example, the prediction of the occurrence of fibrinolytic dysfunction from biological samples, particularly those associated with netosis and / or DIC.

[0008] Some potential biomarkers for DIC and secondary fibrinolysis have been disclosed.

[0009] However, results can be obtained under specific conditions and / or by using sophisticated devices and / or methods to detect / use known markers and / or allow for delays that are too long or potentially too long to be of any use to the patient and / or clinician and / or physician.

[0010] Therefore, there is indeed a need to find a process and / or means to overcome these defects, shortcomings and obstacles of the existing technology, especially a simple, rapid and effective process that enables the detection of fibrinolytic insufficiency and fibrinolytic insufficiency associated with netosis and DIC in order to improve individual life outcomes.

[0011] There is a real need to find a process and / or means to overcome these defects, shortcomings and obstacles of the existing technology, in particular a simple, rapid and effective process that enables the detection of fibrinolytic insufficiency and fibrinolytic insufficiency associated with netosis and DIC, in order to improve individual life outcomes and / or reduce treatment costs for said individuals. Detailed Implementation

[0012] The purpose of this invention is to overcome the shortcomings of the prior art by providing a process for predicting and / or detecting fibrinolytic dysfunction, preferably fibrinolytic dysfunction associated with netosis and / or disseminated intravascular coagulation (DIC), from biological samples, the process comprising the steps of detecting and / or measuring the concentration and / or content of at least one biomarker.

[0013] In this document, "biomarker" is intended to mean at least one biomarker selected from the group consisting of a protease component (preferably human neutrophil elastase (HNE)) comprising plasminogen or a fragment thereof and / or NET. Preferably, the biomarker is plasminogen or a fragment thereof, more preferably plasminogen and / or a protease-derived plasminogen fragment, and even more preferably an HNE-derived plasminogen fragment.

[0014] According to the present invention, the content of the at least one selected biomarker, particularly in a sample, can be measured by any suitable process known to those skilled in the art.

[0015] According to the present invention, the concentration of the at least one selected biomarker, particularly the concentration in a sample, can be measured by any suitable process known to those skilled in the art.

[0016] According to the present invention, the at least one selected biomarker can be detected by any suitable process known to those skilled in the art.

[0017] Because NETs can impair thrombolysis, the inventors investigated the effect of the HNE-DNA complex on fibrinolysis. Surprisingly, the inventors demonstrated that in the presence of NETs containing an active HNE-DNA complex, plasminogen is reduced to a fragment that inhibits plasminogen binding and plasmin formation on fibrin, thereby leading to impaired fibrinolysis. Based on the ability of the HNE-DNA complex to trigger novel and unconventional mechanisms of fibrinolytic dysfunction, the inventors surprisingly demonstrated new mechanistic insights into septic shock thrombosis.

[0018] The inventors also surprisingly demonstrated that the concentration of plasminogen was reduced in biological samples from patients with septic shock.

[0019] The inventors have also surprisingly demonstrated that plasminogen concentrations are reduced in biological samples from patients with fibrinolytic dysfunction, particularly those with fibrinolytic dysfunction associated with netosis and disseminated intravascular coagulation (DIC).

[0020] The inventors have also surprisingly demonstrated that in patients with septic shock-induced DIC and NETosis, plasminogen is degraded by DNA-bound human neutrophil elastase (HNE) and subsequently fails to form plasmin.

[0021] The inventors have also surprisingly demonstrated that NET induces a decrease in plasminogen concentration, particularly in plasma, especially in patients with disseminated intravascular coagulation (DIC) associated with septic shock.

[0022] One object of the present invention is an in vitro process for predicting and / or detecting fibrinolytic dysfunction (preferably fibrinolytic dysfunction associated with NET and disseminated intravascular coagulation (DIC)) from biological samples, comprising measuring the concentration of plasminogen and / or fragments thereof.

[0023] Another object of the present invention is an in vitro process for predicting and / or detecting fibrinolytic dysfunction with / without disseminated intravascular coagulation (DIC) in biological samples, comprising measuring the concentration of plasminogen and / or any fragment thereof.

[0024] Another object of the present invention is an in vitro process for predicting and / or detecting fibrinolytic dysfunction from a first biological sample, wherein the fibrinolytic dysfunction is preferably associated with symptoms of NET with / without disseminated intravascular coagulation (DIC), the process comprising measuring the concentration of plasminogen and / or at least one fragment thereof.

[0025] Another object of the present invention is an in vitro process for predicting and / or detecting fibrinolytic dysfunction and preferably fibrinolytic dysfunction associated with disseminated intravascular coagulation (DIC) from biological samples, comprising measuring the concentration of plasminogen and / or any fragment thereof.

[0026] Another object of the present invention is an in vitro process for predicting and / or detecting NET or Netosis-related symptoms (preferably sepsis, ischemic stroke, coronary thrombosis, cancer, and autoimmune diseases) from biological samples, comprising measuring the concentration of plasminogen and / or fragments thereof, preferably micro plasminogen (K5-SP).

[0027] Another object of the present invention is an in vitro process for predicting and / or detecting fibrinolytic dysfunction from biological samples, preferably comprising measuring the concentration of plasminogen and its fragments in patients with infectious and / or non-infectious diseases.

[0028] Another object of the present invention is an in vitro process for predicting and / or detecting symptoms associated with netosis from biological samples, comprising measuring the concentration of plasminogen and its fragments.

[0029] In this document, “netosis” refers to the process of neutrophil death, and NET specifically refers to DNA fibers with bound nucleoproteins and active enzymes, preferably DNA-HNE complexes.

[0030] In this document, “NET protease component” refers to the active enzyme that binds to the DNA fibers of NET.

[0031] In this document, "protease-derived plasminogen fragment" means any plasminogen fragment known to those skilled in the art.

[0032] In this document, "fibrinolytic insufficiency" refers to any disorder and / or defect in the fibrinolytic process. For example, it may be any deficiency that may involve fibrinolytic factors such as plasminogen, tissue plasminogen activator [tPA], urokinase [uPA], and inhibitors PAI-1 and α2-antiplasmin. Preferably, it refers to fibrinolytic insufficiency associated with the release of neutrophil extracellular traps (NETs) and / or with disseminated intravascular coagulation (DIC).

[0033] In this document, “disseminated intravascular coagulation” (DIC) refers to a condition characterized by systemic activation of coagulation, which may lead to thrombotic blockage of small and medium-sized blood vessels, resulting in organ dysfunction, as disclosed in Blood.

[0034] In this document, "symptoms associated with NET or Netosis" means any symptom involving NET or Netosis known to those skilled in the art. It can be, for example, sepsis, ischemic stroke, coronary thrombosis, cancer, and / or autoimmune diseases. Specifically, it can be NET associated with sepsis-DIC, NET associated with acute myocardial infarction or stent thrombosis, NET associated with ischemic stroke thrombosis, NET associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, or NET associated with breast, lung, or pancreatic cancer.

[0035] In this document, “plasminogen” refers to a 92-kDa protein that is a single-chain molecule consisting of an N-terminal region, five tricyclic (K) domains (K1 to K5), and a serine protease (SP) region. (2) K1 and K4 each contain lysine binding sites. In other words, “plasminogen” is intended to refer to a 92-kDa single-chain zymogen consisting of an N-terminal region, five tricyclic (K) domains, and a serine protease (SP) region. For example, it can be plasminogen as described by Xue Y et al. (2) and / or Law et al., Cell Reports | Vol. 1, No. 3, pp. 185-190, March 29, 2012 (75).It can be a protein with the amino acid sequence EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN (SEQ ID NO 1).

[0036] In this document, "plasminogen fragment" refers to any fragment of plasminogen known to those skilled in the art. It can be, for example, a fragment of plasminogen containing any domain of plasminogen, such as the tricyclic (K)1 to 5 domain, for example, the sequence CKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRT PENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWC FTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQC(SEQ Peptides containing the sequence VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR (SEQ ID NO 11) or any combination thereof, such as the EA fragment consisting of K1+K2+K3.For example, the sequence CKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNK NLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSC(SEQ ID NO 7), or comprising the sequence SEQ ID NO Fragments of peptides containing SEQ ID NO 7, fragments composed of K1+K2+K3+K4, such as the sequence CKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKC (SEQ ID NO 6) or peptides containing the sequence SEQ ID NO Fragments of the peptide with a K4 domain, or fragments consisting of a single K4 domain, such as the peptide with the sequence CYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKC (SEQ ID NO 12), or fragments containing a single K4 domain, such as VQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVV (SEQ ID NO 5). For example, it can be a fragment consisting of the K5 and SP domains, also known as miniature plasminogen (K5-SP).For example, the sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNL EPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR(SEQID The peptide of NO 8), or comprising the sequence SEQ ID Fragments of the NO8 peptide and / or any fragments of plasminogen obtained with an active protease-DNA complex (preferably the HNE-DNA complex of NET).

[0037] In this invention, "HNE-derived plasminogen fragment" refers to a fragment selected from those containing tricyclic 1 to 3 domains (K... 1+2+3 ), three-ring 1 to 4 structural domains (K 1+2+3+4 Fragments of plasminogen consisting of a group of segments containing a tricyclic 5-domain and a serine protease (SP) region are also known as miniature plasminogen (K5-SP). For example, fragments containing tricyclic 1-3 domains (K5-SP) and / or a tricyclic 5-domain and a serine protease (SP) region. 1+2+3The fragment of plasminogen can be, for example, a peptide containing the sequence SEQ ID NO 7, such as the sequence LFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPV (SEQ ID NO 7) 2) A peptide and / or sequence LFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPV (SEQ ID NO 9) containing tricyclic 1 to 4 domains (K 1+2+3+4The fragment of plasminogen can be, for example, a peptide containing the sequence SEQ ID NO 6, such as the sequence LFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVV (SEQ ID NO 6) A peptide of SEQ ID NO 3, containing a K5 domain and a serine protease (SP) region, is a fragment of plasminogen (also designated as miniature plasminogen (K5-SP)) that may, for example, contain the sequence SEQ ID NO 8 peptides such as the sequence APPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSY KVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN(SEQ The peptide of ID NO 4).

[0038] In this invention, "cytoplasmic and granule-active proteases" refers to cathepsin G, myeloperoxidase, or elastase.

[0039] In this invention, "human neutrophil elastase" (HNE) refers to neutrophil elastase (EC 3.4.21.37), i.e., leukocyte elastase. HNE is a trypsin / chymotrypsin-type serine protease in the same family as other leukocyte granule-associated proteases (e.g., protease 3 and cathepsin).

[0040] In this invention, "DNA-binding protein" refers to histones, preferably citrullinated histones, fibronectin, and von Willebrand factor.

[0041] In this document, "biological sample" is intended to mean any sample obtained from a mammal, such as mammals selected from the following groups: Monotremes, Didelphimorphia, Paucituberculata, Microbiotheria, Notoryctemorphia, Dasyuromorphia, Peramelemorphia, Diprotodontia, Tubulidentata, Sirenia, and Afrosoric. The order includes ida, macroscelidea, hyracoidea, proboscidea, and cingulata, such as rodents, lagomorphs, erinaceomorphs (e.g., hedgehogs), sorcerformes, chiroptera, phalidota, carnivora, perissodactyla, artiodactyla, and cetacea. For example, it can be a human or an animal. For example, it can be a domestic animal, a pet, a threatened animal species, or any other animal.

[0042] According to the present invention, the biological sample can be a blood sample, a plasma sample, or any biological fluid. For example, it can be a biological sample selected from the group consisting of blood, plasma, urine, tears, or tissue extracts. Preferably, the biological sample can be a blood sample or a plasma sample, more preferably a plasma sample, which can also be a culture supernatant obtained from cells cultured in vitro.

[0043] According to the present invention, biological samples may be derived from mammals at risk of septic shock or any other non-infectious inflammatory process.

[0044] According to the present invention, the concentration of plasminogen can be determined / measured by any method and / or process known to those skilled in the art. For example, it can be a concentration measured by antigenic assay or by functional assay using a plasminogen activator (e.g., urokinase) or streptokinase-plasminogen complex, as disclosed in “Criteria for specific measurement of plasminogen (enzymatic; procedure) in human plasma”, eJIFCC, Vol. 12, No. 2, 2000(77) or in Massberg, S et al. “Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases”, Nat Med 16, 887-896(45). The concentration of plasminogen can also be determined / measured by a process that includes measuring its potential fibrinolytic activity by immunoassay, cellular immunoassay, or flow cytometry.

[0045] For example, when measuring plasminogen concentration by immunoassay, this immunoassay allows for the detection of circulating plasminogen, and when measuring plasminogen concentration by cellular immunoassay, this cellular immunoassay can use flow cytometry to detect plasminogen on the cell surface. It can be, for example, an immunoassay as disclosed in Rife U, Milgrom F, Shulman S. "Antigenic analysis of plasminogen and plasmin". Blood 1963, 212, 322; Okamoto A et al. "Population-based distribution of plasminogen...". J Thromb Haemost 2003, 1, 2397(83).

[0046] For example, when measuring plasminogen concentration by flow, it may allow for the determination of plasminogen binding to identify cell surface-specific plasminogen binding.

[0047] For example, plasminogen concentration can be measured by colorimetric methods, such as those using streptokinase as an activator, or by using commercially available kits, such as the BIOPHEN plasminogen (LRT) Ref 221511 commercially available from Hyphen Biomed, or Chromogenix. Plasminogen Ref 82 2452 63; for example, STAGO's Stachrom plasminogen 00658. For example, when measuring plasminogen concentration using BIOPHEN plasminogen (LRT) Ref 221511, the reference or normal range is 70% to 130%. For example, when using... When measuring plasminogen concentration using Ref 82 2452 63, the reference or normal range is 80% to 120%. For example, when measuring plasminogen concentration using Stachrom plasminogen 00658, the reference or normal range is 80% to 120%.

[0048] For example, plasminogen concentration can be measured by immunoassay, such as using commercially available kits, like those from Technoclone. Glu plasminogen ELISA kit Ref TC12040 (normal Glu plasminogen values ​​are in the range of 60 μg / mL to 250 μg / mL). For example, Abcam's Human Plasminogen ELISA Kit (PLG) Ref ab108893. For example, when measuring plasminogen concentration using the Human Plasminogen ELISA Kit (PLG), the reference or normal range for plasma plasminogen concentration is 70 μg / mL to 135 μg / mL.

[0049] For example, plasminogen can be measured / detected by antigen assays, using, for example, polyclonal and / or monoclonal antibodies, such as commercially available plasminogen polyclonal and / or monoclonal antibodies, for example, for flow cytometry to detect plasminogen on the cell surface. For example, it can be a commercially available antibody such as Invitrogen's reference PA5-14196 and / or Abcam's ab154560.

[0050] According to the present invention, the concentration of plasminogen fragments can be measured by any method and / or process known to those skilled in the art. For example, it can be a concentration measured by proteomic analysis using mass spectrometry or by antigen assay using monoclonal antibodies or by any combination of methods. For example, the concentration of plasminogen fragments containing tricyclic 1-4 (also known as angiostatin-like) can be measured by an immunoassay, such as that disclosed in Lijnen et al. 2001 “Enzyme-linked Immunosorbent Assay for the Specific Detection of Angiostatin-Like Plasminogen Moieties in Biological Samples”, Thromb Research 2001 102(1), 53-9(76). Plasminogen fragments, including single K4, K1-K2-K3, K1-K2-K3-K4, or miniature plasminogen (K5-SP), can preferably be qualitatively detected by Western blotting or flow cytometry, for example, using specific antibodies.For example, it could be as in M ​​Sten-Linder et al., “Angiostatin Fragments in Urine From Patients With Malignant Disease,” *Anticancer Res*, 1999; 19; 3409-14(78), or Y Takada, “Potential Role of Kringle-Integrin Interaction in Plasmin and uPA,” *Actions Journal of Biomedicine and Biotechnology*, 2012, Vol. 136302, 8 pages, doi:10.1155 / 2012 / 136302, or Z Zhang, “Plasminogen Kringle5 Inhibits Alkali-Burn–Induced Corneal The process is disclosed in "Investigative" Ophthalmology & Visual Science, November 2005, Vol. 46, 4062-4071. doi:https: / / doi.org / 10.1167 / iovs.04-1330-(80).

[0051] For example, when measuring the concentration of a fragment of plasminogen containing tricyclic 1-4 (also known as angiostatin-like) by an immunoassay (e.g., in tumor fluid from a cancer patient), the process may include a step of removing other plasminogen fragments, for example, by immunoadsorption (e.g., in the sample, preferably in a diluted sample).

[0052] According to the present invention, the concentration of HNE-derived plasminogen fragments can be measured by any method and / or process known to those skilled in the art. For example, it can be a concentration measured by an antigen assay using a monoclonal antibody. For example, the concentration of HNE-derived plasminogen fragments can be measured by an immunoassay, as disclosed, for example, by Lijnen et al., 2001(76). HNE-derived plasminogen fragments comprising single K4, K1-K2-K3, K1-K2-K3-K4, or miniature plasminogen (K5-SP) can preferably be qualitatively detected by Western blotting or flow cytometry, for example, using specific antibodies. For example, it could be as in Rouy D et al., “Apolipoprotein(a) and Plasminogen Interactions With Fibrin: A Study With Recombinant Apolipoprotein(a) and Isolated Plasminogen Fragments”, Biochemistry, 1992; 31; 6333(56), or in Barbosa da Cruz D 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 J The process disclosed in 20189,33,14270(81). The concentration and quality of the HNE fragment can also be measured and evaluated, for example, by combining immunogenicity methods with any other methods for measuring the size of the fragment or determining the amino acid sequence of the fragment, such as by mass spectrometry, for example those combined for proteomics analysis.For example, the concentration of HNE-derived plasminogen fragments can be measured by flow cytometry to detect the binding of plasmin-generated protein fragments to cells, as disclosed in Constantinescu et al., “Amorphous protein aggregates stimulate plasminogen activation, leading to the release of cytotoxic fragments that are clients for extracellular chaperones”, Journal of Biochemistry (2017) 292(35) 14425–14437(82).

[0053] For example, when measuring the concentration of HNE-derived plasminogen fragments by immunoassay (e.g., in tumor fluid from a cancer patient), the process may include a step of removing other plasminogen fractions, for example, by immunoadsorption (e.g., in the sample, preferably in a diluted sample).

[0054] According to the present invention, a three-ring (K)1 to 3 structural domain (K) 1+2+3 The determination / measurement of the concentration of plasminogen fragments can be performed by any suitable method known to those skilled in the art. For example, it can be done by antigen assay. For example, for example, fragments containing tricyclic (K)1 to 3 domains (K 1+2+3 The concentration of plasminogen fragments can be measured by immunoassay, such as that published by Lijnen et al. 2001, “Enzyme-linked Immunosorbent Assay for the Specific Detection of Angiostatin-Like Plasminogen Moieties in Biological Samples”, Journal of Thrombosis Research 2001 102(1), 53-9(76).

[0055] According to the present invention, the determination / measurement of the concentration of mini-plasminogen (K5-SP) or fragments of plasminogen containing a tricyclic (K)5 domain and a serine protease (SP) region can be performed by any suitable method known to those skilled in the art. For example, it can be performed by an antigen assay. For example, a combination of protein separation by mass spectrometry and proteomic analysis can be used. For example, the concentration of fragments of plasminogen or mini-plasminogen (K5-SP) containing a tricyclic (K)5 domain and a serine protease (SP) region can be measured by an immunoassay, for example as disclosed in Moroz et al., 1986, “Mini-plasminogen-likfer agments of plasminogen in synovial fluid in acute inflammatory arthritis”, *Studies in Thrombosis*, 1986, 43, 417(5).

[0056] According to the invention, the detection process may further include a step of comparing the measured plasminogen concentration with a reference value. For example, this can be a reference value or a reference range, which typically represents 95% of the values ​​or ranges of a reference healthy population, preferably determined through statistical analysis, such as those disclosed by the Clinical & Laboratory Standards Institute (CLSI).

[0057] According to the present invention, the reference concentration value of plasminogen can be the concentration of plasminogen measured in a biological sample of a subject, or the average concentration measured in a group of reference healthy subjects. Preferably, the reference concentration value of plasminogen can be the concentration of plasminogen measured in a biological sample pool from healthy subjects. The reference concentration value of plasminogen can be defined as an internal standard for a biological sample of a subject titrated against a sample from a healthy subject. The reference concentration value of plasminogen can be, for example, a concentration of full-length plasminogen from 1 μmol / L to 2 μmol / L, preferably from 1.5 μmol / L to 2 μmol / L.

[0058] According to the present invention, the reference concentration value of the plasminogen fragment may be the concentration of the plasminogen fragment measured in a biological sample of a subject, or the average concentration measured in a group of reference healthy subjects. Preferably, the reference concentration value of the plasminogen fragment may be the concentration of the plasminogen fragment measured in a pool of biological samples from healthy subjects and / or in an internal standard defined as a sample titrated against a sample from a healthy subject.

[0059] According to the present invention, a three-ring (K)1 to 3 structural domain (K) 1+2+3 The reference concentration of the plasminogen fragment can be measured in the subject's biological sample and includes the tricyclic (K)1 to 3 domains (K). 1+2+3 The concentration of a fragment of plasminogen 1, or the average concentration measured in a group of reference healthy subjects. Preferably, it contains tricyclic (K)1 to 3 domains (K... 1+2+3 Reference concentrations of plasminogen fragments can be measured in a biosample pool from healthy subjects and / or in an internal standard containing tricyclic (K)1 to 3 domains of a subject's biosample, defined as being titrated against samples from healthy subjects. 1+2+3 The concentration of plasminogen fragments.

[0060] According to the present invention, the reference concentration value of the plasminogen fragment containing a tricyclic (K)5 domain and a serine protease (SP) region or a microplasminogen (K5-SP) can be the concentration of the plasminogen fragment containing a tricyclic (K)5 domain and a serine protease (SP) region or a microplasminogen (K5-SP) measured in a subject's biological sample, or the average concentration value measured in a group of reference healthy subjects. Preferably, the reference concentration value of the plasminogen fragment containing a tricyclic (K)5 domain and a serine protease (SP) region or a microplasminogen (K5-SP) can be the concentration of the plasminogen fragment (K5-SP) containing a tricyclic (K)5 domain and a serine protease (SP) region or a microplasminogen measured in a biological sample pool from healthy subjects or in an internal standard defined as a sample titrated against a sample from healthy subjects.

[0061] In this document, "reference healthy subject" or "healthy subject" is intended to mean a mammal, such as a human, who has not been subjected to infection, shock, hemorrhage, septic shock, and / or any other physical attack that could lead to fibrinolytic insufficiency and disseminated intravascular coagulation as defined above. For example, it can be a human who has not been subjected to infectious or non-infectious conditions associated with NET or Netosis, such as sepsis, ischemic stroke, coronary thrombosis, cancer, and autoimmune diseases as defined above.

[0062] According to the present invention, the term "reference subject group" or "reference healthy subject group" is intended to refer to a group in which reliable reference values ​​or reliable reference intervals can be defined. For example, it can be a group containing at least two reference subjects as defined above, such as at least 10, at least 40, at least 60, or at least 100 reference subjects or healthy subjects. For example, it can be a group containing 30 to 500 subjects, 40 to 200, or 45 to 110 reference subjects or healthy subjects.

[0063] Advantageously, the inventors have demonstrated that when the measured concentration of plasminogen is below a reference value or range, the process according to the invention enables the detection and / or prediction of disseminated intravascular coagulation with fibrinolytic insufficiency.

[0064] Advantageously, the inventors have also demonstrated that when the content / concentration of plasminogen fragments is greater than a reference value or range, the process according to the invention makes it possible to detect and / or predict the inhibition of fibrinolysis and / or fibrinolytic dysfunction, particularly fibrinolytic dysfunction in disseminated intravascular coagulation.

[0065] Advantageously, the inventors of the present invention have also demonstrated that a process for detecting fibrinolytic dysfunction comprising measuring the concentration of plasminogen and / or at least one fragment thereof can achieve the detection of fibrinolytic dysfunction.

[0066] Advantageously, the inventors have demonstrated that when detecting plasminogen, preferably fragments of micro plasminogen (K5-SP), in plasma, particularly the process according to the invention, it is possible to detect and / or predict conditions with NET, such as sepsis, ischemic stroke, coronary thrombosis, cancer, and autoimmune diseases, from a first biological sample or associated with netosis.

[0067] Advantageously, the inventors of the present invention have also demonstrated that a process for detecting fibrinolytic dysfunction comprising measuring the concentration of plasminogen and / or at least one fragment thereof can enable the detection of NET-related symptoms.

[0068] Another object of the present invention is an in vitro process for determining the efficacy of treatments for DIC-related fibrinolytic insufficiency and / or NET-related symptoms (preferably sepsis, ischemic stroke, coronary thrombosis, cancer, and autoimmune diseases) and / or NET-related symptoms, comprising:

[0069] a. Determine and / or measure the concentration C1 of plasminogen and / or fragments thereof from biological samples prior to treatment with the compound.

[0070] b. Determine and / or measure the concentration C2 of plasminogen and / or fragments thereof from biological samples treated with said compound, and optionally,

[0071] c. Compare concentrations and calculate the score (S) according to the following formula:

[0072] S = C2 / C1.

[0073] According to the present invention, a biological sample is a biological sample as defined above.

[0074] According to the present invention, the biological sample before treatment is the biological sample as defined above.

[0075] According to the present invention, the biological sample before treatment is the biological sample as defined above.

[0076] According to the present invention, the determination and / or measurement of the concentration of plasminogen and / or fragments can be performed by any method / process as defined / described above.

[0077] According to the present invention, the plasminogen fragment can be any plasminogen fragment defined / described above.

[0078] According to the present invention, when the process determines the efficacy of the treatment for fibrinolytic insufficiency and the biomarker is plasminogen, the treatment is effective when the S value obtained from the comparison step is greater than 1.

[0079] According to the present invention, when the process determines the efficacy of the treatment for fibrinolytic insufficiency and the biomarker is a plasminogen fragment, the treatment is effective when the S value obtained from the comparison step is less than 1.

[0080] According to the present invention, an in vitro process for determining the efficacy of the compound in treating DIC-related fibrinolytic insufficiency and / or NET-related symptoms (preferably sepsis, ischemic stroke, coronary thrombosis, cancer, and autoimmune diseases) and / or NET-related symptoms comprises:

[0081] a. Determine and / or measure the concentration C1 of plasminogen from a first biological sample prior to treatment with the compound.

[0082] b. Determine and / or measure the concentration of plasminogen fragment C1F from a second biological sample prior to treatment with the compound.

[0083] c. Determine and / or measure the concentration C2 of plasminogen from a third biological sample treated with the compound.

[0084] d. Determine and / or measure the concentration C2F of the plasminogen fragment from a fourth biological sample treated with the compound, and optionally,

[0085] e. Compare concentrations and calculate scores (S) and (SF) using the following formulas:

[0086] S = C2 / C1,

[0087] SF = C2F / C1F

[0088] According to the present invention, when the process determines the efficacy of the treatment for fibrinolytic insufficiency and the biomarker is plasminogen, the treatment is effective when the S value obtained from the comparison step is greater than 1.

[0089] According to the present invention, when the process determines the efficacy of the treatment for fibrinolytic insufficiency and the biomarker is a plasminogen fragment, the treatment is effective when the SF value obtained from the comparison step is less than 1.

[0090] According to the present invention, the first biological sample before treatment is the biological sample as defined above.

[0091] According to the present invention, the second biological sample before treatment is a biological sample as defined above.

[0092] According to the present invention, the second biological sample before treatment may be the same as or different from the first biological sample before treatment.

[0093] According to the present invention, the first biological sample and the second biological sample before treatment can be the same sample.

[0094] According to the present invention, the processed third biological sample is a biological sample as defined above.

[0095] According to the present invention, the treated fourth biological sample is a biological sample as defined above.

[0096] According to the present invention, the treated third biological sample may be the same as or different from the treated fourth biological sample.

[0097] According to the present invention, the treated third biological sample and the fourth biological sample can be the same sample.

[0098] In this invention, “treatment with compounds” refers to medical treatment, such as antagonistic therapy, which involves the use of molecules, such as chemical molecules, such as molecules obtained through organic synthesis, biologically derived molecules, such as proteins, molecules derived from and / or synthesized from living organisms (such as mammals, microorganisms, plants), or any other non-chemical treatment or any other device for delivering the aforementioned molecules, such as engineered or unengineered nanovesicles, for example, delivery of natural plasminogen.

[0099] The inventors have surprisingly demonstrated that supplying plasminogen to plasma samples from patients with septic shock-induced DIC allows for the restoration of plasmin formation and fibrinolysis.

[0100] One object of the present invention is a natural and / or recombinant plasminogen activator used as a medicament for treating fibrinolytic insufficiency, particularly fibrinolytic insufficiency caused by low plasminogen concentration in patients with netosis and / or disseminated intravascular coagulation (DIC).

[0101] Furthermore, one object of the present invention is a pharmaceutical composition comprising plasminogen, which is used as a medicine for treating fibrinolytic insufficiency, preferably associated with disseminated intravascular coagulation (DIC).

[0102] Another object of the present invention is a pharmaceutical composition comprising natural and / or recombinant plasminogen, which is used as a medicine for treating fibrinolytic insufficiency (preferably, fibrinolytic insufficiency caused by low plasminogen concentration in patients with netosis and / or disseminated intravascular coagulation (DIC)).

[0103] Another object of the present invention is natural and / or recombinant plasminogen, which is used as a medicine for treating disseminated intravascular coagulation (DIC) (e.g., DIC associated with moderate / severe plasminogen depletion / deficiency), particularly in patients with septic shock-induced DIC.

[0104] One object of the present invention is a pharmaceutical composition comprising natural and / or recombinant plasminogen, which is used as a medicine for treating disseminated intravascular coagulation (DIC) (e.g., DIC associated with moderate / severe plasminogen depletion / deficiency), particularly in patients with septic shock and DIC.

[0105] In this application, the term "treatment" means prevention and / or therapy, particularly where the aim is to prevent or mitigate (alleviate) undesirable physiological changes or conditions, such as the development and / or progression of disseminated intravascular coagulation (DIC). Beneficial or desired clinical outcomes include, but are not limited to, improvement in organ failure, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and healing (whether partial or complete). "Treatment" may also mean prolonged survival and / or improved quality of life compared to expected survival and / or quality of life without treatment.

[0106] "Subject" or "patient" includes mammals, such as humans, including mammals that require treatment for a disease or condition, such as mammals that have been diagnosed with a disease or condition or identified as being at risk of developing a disease or condition.

[0107] The pharmaceutical composition may be in any form that can be administered to humans or animals.

[0108] The pharmaceutical composition may contain plasminogen, such as natural and / or recombinant plasminogen. For example, it may be Glu-plasminogen at a concentration of 6.6 mg / kg of the subject.

[0109] Administration can be performed directly, i.e., pure or substantially pure, or after plasminogen and / or fragments thereof have been mixed with a pharmaceutically acceptable carrier and / or medium. According to the invention, the pharmaceutical composition can be a syrup or an injectable solution. For example, when the pharmaceutical composition is an injectable solution, it can be injected and / or administered as an intravenous infusion over 10 to 30 minutes.

[0110] According to the present invention, the pharmaceutical composition containing plasminogen can be administered daily, every two days, every three days, etc. Those skilled in the art will adjust the administration frequency of the pharmaceutical composition containing plasminogen, taking into account their technical knowledge.

[0111] According to the present invention, when fibrinolytic insufficiency is associated with netosis, the pharmaceutical composition may be eye drops or any formulation suitable for external application.

[0112] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition for oral administration, selected from the group comprising liquid formulations, oral effervescent formulations, oral powders, multi-particle systems, and oral dispersible formulations. For example, when the pharmaceutical composition is for oral administration, it can be in the form of a liquid formulation selected from the group comprising solutions, syrups, suspensions, emulsions, and oral drops. When the pharmaceutical composition is in the form of an oral effervescent formulation, it can be in the form selected from the group comprising tablets, granules, and powders. When the pharmaceutical composition is in the form of an oral powder or multi-particle system, it can be in the form selected from the group comprising beads, granules, small tablets, and microparticles. When the pharmaceutical composition is in the form of an oral dispersible formulation, it can be in the form selected from the group comprising oral dispersible tablets, lyophilized tablets, films, chewable tablets, tablets and capsules, and medical chewing gum. According to the present invention, the pharmaceutical composition can be used via the buccal and sublingual routes, and is, for example, selected from the group comprising buccal or sublingual tablets, mucosal adhesive formulations, lozenges, oral mucosal drops, and sprays.

[0113] According to the present invention, when fibrinolytic insufficiency is associated with netosis, the pharmaceutical composition can be used for topical transdermal application, such as being selected from the group comprising ointments, creams, gels, lotions, patches and foams.

[0114] According to the present invention, pharmaceutical compositions can be formulated for nasal administration, such as those selected from the group comprising nasal drops, nasal sprays, and nasal powders. According to the present invention, pharmaceutical compositions can be formulated for rectal administration, such as suppositories or hard gelatin capsules. According to the present invention, pharmaceutical compositions can be used for parenteral administration, such as subcutaneous, intramuscular, or intravenous administration. It will be readily understood by those skilled in the art that the term "form," as used herein, refers to the pharmaceutical preparation intended for its practical use.

[0115] Pharmaceutically acceptable carriers can be any known pharmaceutical carrier for administering natural or recombinant plasminogen, depending on the subject. For example, pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / coloring agents, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers.

[0116] The form of the pharmaceutical composition can be selected based on whether the person or animal is to be treated.

[0117] In another aspect, the present invention provides a method for treating a subject suffering from disseminated intravascular coagulation (DIC), particularly DIC during septic shock. The method may include the step of administering natural and / or recombinant plasminogen to the subject.

[0118] Natural or recombinant plasminogen and available formulations are as defined above. Administration may be carried out by any pharmaceutical method known to those skilled in the art and suitable for administering natural and / or recombinant plasminogen, preferably parenteral, and preferably intravenous. Examples of administerable forms of the medicament / drug are provided above.

[0119] Other advantages will become apparent to those skilled in the art from the following examples illustrated in the accompanying drawings, which are given by way of example. Attached Figure Description

[0120] - Figure 1 Neutrophil extracellular traps (NETs) generated from neutrophils seeded onto a fibrin matrix: DNA and HNE activities of NETs are shown. Neutrophils isolated from human blood and seeded onto the wells of 96-well microtiter plates with or without a fibrin matrix were activated with 50 nM PMA in HBSS. After incubation at 37°C in a humidified 5% CO2 incubator for 4 hours, the plates were centrifuged at 3000 g for 5 min, the supernatant was collected, and the cells and NETs in the wells were stained with a solution of 10 g / ml Hoechst 33342 (a minor groove DNA binder). Elastase activity bound to NETs was detected using the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline. Figure 1A shows a fluorescence microscopy image (X20) of cell nuclei and extracellular DNA fibers stained with Hoechst. Scale bar: 20 μm. Neutrophils: Unstimulated neutrophils incubated without PMA show a typical polymorphonuclear appearance. NET: Extracellular traps of neutrophils generated in the absence of fibrin. NET-fibrin: NETs generated on a fibrin matrix have a denser and more reticular appearance than NETs extruded without fibrin. Figure 1 B indicates the HNE activity detected on the HNE-ADN complex (black bar) and in the supernatant (gray bar) in the absence of inhibitors (No Inh) or in the presence of α1-protease inhibitors (α1-PI, 10 μM), aprotinin (10 μM), or the synthesis inhibitor N-methoxysuccinyl-Ala-Pro-Val-chloromethyl ketone (AAPV-cmk, 100 μM). C. Detection of soluble HNE activity in buffer (·) and its inhibitory effect when added to normal human plasma (■). Purified HNE was incubated in buffer or plasma at different concentrations (15 min) before activity measurement with 1.5 mM of the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline.

[0121] - Figure 2 This indicates that NET-related elastases digest plasminogen into fragments. For example... Figure 1 As shown, plasminogen (1 μM) was incubated with NETs generated from neutrophils treated with 50 nM PMA for 4 hours. Samples were analyzed by SDS-PAGE and Western blotting using a sheep-specific antibody against human plasminogen. Figure 2 A. Fragmentation of plasminogen via the NETs HNE·DNA complex after incubation for 30 to 240 minutes (A) and 2 to 10 hours (B). C. Western blots of purified plasminogen (Pg), plasmin (Pn) (left panel), and HNE-DNA-derived plasminogen fragments (middle panel). No plasminogen fragments produced by PMA-stimulated neutrophils were observed (middle panel) in the presence of the elastase-specific inhibitor (EI) N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (right panel).

[0122] - Figure 3 This indicates a decrease in tPA-mediated plasmin formation caused by NETs formed on fibrin. Neutrophils isolated from human blood were seeded onto fibrin in 96-well microtiter plates and activated by 50 nMPMA in HBSS. NETs released by the activated neutrophils... Figure 1The identification is shown. After tPA binds to fibrin, different concentrations of plasminogen are added to the interlaced NET-fibrin structure. Figure 3 AB. Plasmin formation from plasminogen (Pg) was monitored at 50 nM (A) and 500 nM (B) by measuring the release of the plasmin-selective chromogenic substrate pNA at 0.75 mM. □ Plasmin formation on fibrin. ● Plasmin formation on fibrin-NET. ■ Plasmin formation on fibrin-NET treated with the elastase inhibitor MeO-Suc-AAPV-CMK. Figure 3 C. After plasminogen activation, the wells were carefully washed, and the amount of fibrin-bound plasmin was detected by adding 0.75 mM of a plasmin-selective chromogenic substrate. Gray bars: Plasmin binds to the fibrin-NET surface. White bars: Plasmin binds to the fibrin-NET surface treated with the elastase inhibitor MeO-Suc-AAPV-CMK. Figure 3 D. Analysis of plasminogen fragmentation in protein extracts recovered from the fibrin-NET surface after 2 hours of incubation. Western blotting of non-reduced samples was performed using a plasminogen-specific monoclonal antibody. 1: In the absence of NET, plasminogen is activated by fibrin-bound tPA (in non-reduced gels, plasminogen and plasmin migrate in similar locations). 2 and 4: Plasminogen incubated with the fibrin-NET lattice; production of plasminogen fragments and trace amounts of plasmin. 3, 5. In the presence of the elastase inhibitor MeO-Suc-AAPV-CMK, plasmin production was restored, and plasminogen fragment formation was inhibited.

[0123] - Figure 4 This indicates the circulating plasminogen fragment in patients with septic shock-induced disseminated intravascular coagulation (DIC). Plasma samples from patients and controls were diluted 1:5 in 125 mM Tris-HCl buffer (pH 6.8) containing 20% ​​glycerol and 4% SDS. Proteins were separated by SDS-PAGE (10%) and then blotted using a sheep-specific antibody against human plasminogen or a horseradish peroxidase-labeled monoclonal antibody CPL15-PO against tricyclic 1. C1-C4: Plasma from healthy donors. S1-S7: Samples from patients with septic shock-induced DIC. Pg: Plasminogen in samples from patients and healthy donors. The bands between 49 kDa and 38 kDa in S1-S7 represent plasminogen fragments (Pg frg). Plasma samples treated with euglobulin precipitation or lysine affinity fractionation to remove high-abundance proteins were used for proteomic analysis of plasminogen fragments.

[0124] - Figure 5This represents the fibrinolytic activity of plasma isolated from septic shock. The fibrinolytic activity of the plasma is tested by incubating the corresponding euglobulin fraction with a fibrin surface previously bound with tPA (50 iu / ml). The euglobulin fraction contains plasminogen and plasminogen fragments with K1, K4, and K5, as identified by mass spectrometry (see text and supplementary documents). Figure 3 As shown, the amount of plasmin formed was quantified by measuring the change in absorbance at A405 nm using a chromogenic substrate selective for plasmin. C (hollow bar): Control plasma pool (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). Gray bars: Activity of plasma samples S1 to S6 at their native plasminogen concentration. Solid bars: Activity of plasma samples S1 to S6 after plasminogen concentration normalized to 1.5 μM.

[0125] Figure 6 illustrates how the NET DNA-HNE complex digests plasminogen into fragments with known structures identified by proteomic analysis. As described above, plasminogen and its fragments were identified by mass spectrometry in purified samples and plasma protein fractions. A schematic diagram of plasminogen and its major fragments, modified from the plasminogen structural sequence, is shown (http: / / www.chem.cmu.edu / groups / Llinas / res / structure / kringle-big.html). Figure 6A shows that the full-length plasminogen consists of an N-terminal region, five tricyclic (K) domains, and a serine protease (SP) region. Arrows indicate the major HNE cleavage sites in plasminogen. (68), Figure 6B. 27kDa K1-K3 Leu74-Val338, Figure 6C. 34kDa K1-K3 Leu74-Val354, Figure 6D. 39kDa K1-K3 Leu74-Val354, Figure 6E. 14kDa K4 Val355-Val443 and Figure 6F. 38kDa K5-SP Ala444-Asn791. K 1+2+3 The difference in molecular weight is related to the sites of cleavage and glycosylation.

[0126] - Figure 7A Western blot image representing a fragment of human neutrophil elastase-derived Glu-plasminogen. The fragment was prepared by incubating 10 μM purified protein with 250 nM of purified HNE sufficient to completely convert plasminogen into the fragment at 37 °C for 30 min. The corresponding fragments were separated by affinity chromatography on lysine-agarose and identified by proteomics analysis using mass spectrometry. M. Reference molecular markers. 1. K5-SP. 2. First band K1+2+3, second and third bands K1+2+3. 3. K1+2+3+4, 4. K4.

[0127] - Figure 8 This diagram represents plasminogen reduced to fragments by the active HNE-DNA complex of NET. Plasminogen is reduced to fragments by the active HNE-DNA complex of NET. The HNE-DNA complex is central to a mechanism that leads to severe fibrinolytic failure by consuming plasminogen via proteolysis, thereby producing antifibrinolytic plasminogen fragments. This fibrinolytic function is partly responsible for the impaired dissolution of microthrombi, thus promoting their stability in the microcirculation and contributing to organ failure during septic shock.

[0128] - Figure 9 This represents the functional plasminogen concentration in septic shock patients with or without disseminated intravascular coagulation (DIC). Plasminogen is assessed using a functional assay that measures plasmin formation from available natural plasminogen. D1, D3, D7: Days following admission to the intensive care unit. The dashed line represents the mean plasminogen concentration in healthy subjects (n=31).

[0129] Example

[0130] Example 1: Determination of plasminogen concentration and detection of DIC.

[0131] In septic shock, platelet and neutrophil activation leads to the formation of microvascular clots containing an intricate fibrin scaffold and neutrophil extracellular traps (NETs). NETs contain various components that can influence endogenous fibrinolysis, resulting in the inability to dissolve clots in the microcirculation and residual systemic microthrombus formation. The fibrin-NET matrix was prepared by seeding fibrin surfaces and activating neutrophils, and the construction was used to monitor plasminogen activation or degradation. We demonstrated that the elastase activity of the HNE-DNA complex was protected from inhibition by plasma antiproteases and maintained its ability to degrade plasminogen. Using mass spectrometry proteomics analysis, the activity of the HNE-DNA complex was identified by the tricyclic (K) domain (K... 1+2+3 k 1+2+3+4The inventors further demonstrated that septic shock patients with disseminated intravascular coagulation (DIC) possess circulating HNE-DNA complexes, HNE-derived plasminogen fragments, low plasminogen concentrations, and reduced ability to produce plasmin on fibrin. The inventors demonstrated that NETs carrying active HNE-DNA complexes reduce plasminogen to fragments, thereby weakening fibrinolysis by reducing local plasminogen concentration, plasminogen binding to fibrin, and local plasmin formation. Therefore, the reservoir of human neutrophil elastase (HNE) on NETs directly interferes with the fibrinolytic mechanism via the plasminogen hydrolysis pathway.

[0132] Materials and methods

[0133] abbreviation:

[0134] ABTS, 2,2'-Zylated-bis(3-ethylbenzothiazole-6-sulfonic acid)

[0135] AEBSF, aminoethylbenzenesulfonyl fluoride

[0136] α1-PI, α1-protease inhibitor

[0137] DIC, disseminated intravascular coagulation

[0138] HBSS, Hank's equilibrium salt solution (HBSS)

[0139] HNE, human neutrophil elastase

[0140] K, the tricyclic domain of plasminogen

[0141] NET, neutrophil extracellular trap

[0142] MeO-Suc-AAPV-CMK, N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethyl ketone

[0143] MeO-Suc-AAPV-pNA, N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine p-nitroaniline

[0144] MM-H-Pro-Arg-pNA, Methyl(malonyl)hydroxyproline-arginine-p-nitroaniline, CBS0065

[0145] NET, neutrophil extracellular trap

[0146] PMA, Pheromonyl 12-Myristate 13-Acetate

[0147] PMN, polymorphonuclear leukocytes

[0148] PMSF, Benzylsulfonyl fluoride

[0149] tPA, tissue plasminogen activator

[0150] uPA, urokinase plasminogen activator

[0151] uPAR, urokinase plasminogen activator receptor

[0152] Plasma from septic shock patients and healthy subjects

[0153] Seven patients (mean age 74.5 ± 10 years) diagnosed with septic shock and multiple organ failure (mean SOFA score of 13 ± 2) and presenting with disseminated intravascular coagulation within the first 24 hours after admission, according to the Japanese Association for Acute Medicine JAAM 2016 score (31), and ten healthy individuals were included (trial registration: Clinicaltrial.gov identifier NCT#02391792). This study was approved by the Ethics Committee of Strasbourg University Hospital. Informed consent was obtained from the patients or their relatives at admission and confirmed by the patients as soon as possible. Samples were collected from 0.13 M sodium citrate (Vacutainer) by a double centrifugation procedure (2500 g for 15 minutes). TM Platelet-free plasma was prepared from blood samples from Becton Dickinson (BD) in the United States, aliquoted, and immediately frozen at -80°C. Periodic hemostasis studies and the detection of HNE-α1PI and plasmin-α2-antiplasmin complex were evaluated in human plasma.

[0154] Human proteins and antibodies

[0155] Human Glu-plasminogen was purified as described and had a purity exceeding 99%, as assessed by SDS / PAGE and by N-terminal sequence analysis (3, 32). Plasminogen-elastase-derived plasminogen fragments were prepared by incubating 10 μM of purified protein with 250 nM of purified HNE at 37 °C for 30 min. The reaction was terminated by adding 5 mM PMSF. The fragments were then separated by affinity chromatography on lysine-agarose. 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 obtained from Calbiochem (Merck KGaA, Darmstadt, Germany). Sheep polyclonal antibodies against plasminogen and monoclonal IgG1 antibodies against plasminogen tricyclic 1 (CPL15) have been previously produced and characterized. (34, 35) Horseradish peroxidase (HRP) conjugated CPL15 mAbs were obtained using a peroxidase labeling kit according to the manufacturer's instructions (Roche, Mannheim, Germany). Polyclonal rabbit anti-sheep (HRP) conjugated IgG was obtained from DakoCytomation (Glostrup, Denmark).

[0156] Neutrophil segregation and the formation of extracellular traps in neutrophils

[0157] Venous blood from healthy volunteers is collected by the Blood Donor Center (Etablissement). (du Sang) was collected using acid-citric acid-glucose. Neutrophils were isolated from anticoagulated blood as described. (36) Briefly, leukocytes were separated from erythrocytes on separation medium containing 9% Dextran T-500 in Radioselectan. After erythrocyte sedimentation, the leukocyte suspension was centrifuged on a Pancoll 1077. The cell pellet was washed with phosphate-buffered saline and contaminated erythrocytes were removed by hypotonic lysis. Polymorphonuclear neutrophils (PMNs) were then resuspended in RPMI-1640 or Hank balanced salt solution (HBSS) (see below) and used immediately after separation. Flow cytometry showed the absence of CD14+, CD3+, and CD19+ cells, confirming the recovery of highly purified PMNs (CD16+) identified by nuclear morphology on May-Grünwald-Giemsa staining (not shown). Typically, the purity and cell viability (trypan blue) of the neutrophil preparation were ≥98%. The isolated neutrophils were resuspended in RPMI-1640 medium or HBSS at a concentration allowing for a distribution of 200,000 cells per well. Experiments were performed on 96-well flat plates with or without a well-defined fibrin surface prepared and characterized as described below.

[0158] Because PMA and bacteria utilize related pathways (protein kinase C activation and reactive oxygen species generation for inducing proteolytic activity of NETs), we used a well-standardized technique for PMA stimulation. (14) Neutrophils plated with 50 nM PMA were treated at 37°C in a humidified 5% CO2 atmosphere for 1 hour and then for 4 hours. NETs were identified by (1) DNA staining with 10 μg / ml Hoechst 33342 solution and (2) measurement of DNA-binding elastase activity.

[0159] Using the X20 objective of a Zeiss AxioObserver D1 fluorescence microscope equipped with a CCD imaging camera, and Histolab software from Microvision Instruments (Evry, France), stained nuclei of unstimulated neutrophils and PMA-induced extracellular traps were detected in three well fields of view for each condition.

[0160] To detect NET-bound elastase, the substrate MeO-Suc-AAPV-pNA (100 μl per well, final concentration 1.5 mM in HBSS) was incubated with NET 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 A405 nm. Elastase inhibitors MeO-Suc-AAPV-CMK (final 100 μM) or α1-PI (final 10 μM) and AEBSF (1 mM) were used to verify the preservation of DNA-bound elastase activity.

[0161] Preparation and characterization of fibrin matrix

[0162] The fibrin matrix was prepared as previously described (WO / 1985 / 004425). (34, 37, 38) Briefly, a monolayer of fibrinogen was immobilized onto a poly(glutaraldehyde) activated flat-bottomed 96-well plate. The immobilized fibrinogen (410 ± 4 fmol / cm³) was stabilized with a thrombin solution containing 2 mM CaCl₂ at a concentration of 10 N·L Hu / ml. 2 ) is converted into fibrin. The release of fibrin peptide A was tracked using the monoclonal antibody Y18. (39) The resulting fibrin interacts specifically with the finger domain of tPA, while plasminogen binds to newly exposed carboxyl-terminal lysine residues revealed during ongoing fibrin degradation. (3, 33, 37) Fibrin degradation was tracked using mAb DD3B6, which is specific for D-dimer structures revealed by plasmin on immobilized fibrin. (40) Advantageously, the fibrin-NET matrix can be prepared alternatively using various supports (e.g., beads or slides).

[0163] reagents

[0164] The chromogenic substrate selective for plasmin (methylmalonyl)hydroxypropionylarginine-p-nitroaniline (CBS0065) was courtesy of G. Contant (Diagnostica Stago, Asnières, France). Phorbolol 12-myristate 13-acetate (PMA), serine protease inhibitors aminoethylbenzenesulfonyl fluoride (AEBSF) and phenylmethylsulfonyl fluoride (PMSF), elastase-specific substrates N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (MeO-Suc-AAPV-pNA), and elastase-specific inhibitors 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 was 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 binding agent for dsDNA) was from BioProbes (distributed by Thermo Fisher Scientific, France).

[0165] Detection of HNE-α1-PI and plasmin-α2-anti-plasmin complex

[0166] Human neutrophil elastase (HNE) as a complex with an α1-protease inhibitor (α1-PI) was detected using the Human PMN Elastase Platinum ELISA kit from Affymetrix (Bender MedSystems GmbH, Vienna, Austria). The plasmin-α2-antiplasmin complex was detected by ELISA (Technozym, Technoclone GmnH, Vienna, Austria).

[0167] Identification of plasminogen and its fragments through mass spectrometry database search and interpretation of MS datasets.

[0168] The samples used were purified proteins (human plasminogen, plasmin, and HNE-derived plasminogen fragments) or plasma protein fractions of interest depleted of high-abundance proteins (euglobulin and lysine affinity fractions). Euglobulin fractions from 100 μl plasma (patient or control) were precipitated in the presence of protease inhibitors at low ionic strength and pH 5.8 and resuspended in 2.5% SDS, 62.5 mM Tris-HCl pH 6.8 sample buffer; this fraction was free of albumin and α-globulin but contained all β-globulins and γ-globulins, including plasminogen and its fragments. As previously described, proteins bound to lysine-agarose gels from 3 ml plasma (from a plasma pool of 6 patients) were eluted with 30 mM ε-aminocaproic acid. (66) Lysine affinity fractions specifically contained proteins with lysine binding sites.

[0169] Proteins in the sample were separated on an 8% or 15% SDS-PAGE gel. Strips were manually cut from the gel and diced. Then, following a published procedure, intragel digestion was performed using trypsin, with slight modifications (67): the sample was decolorized twice for 30 minutes at room temperature with a mixture of 100 mM ammonium bicarbonate (ABC) and 50% (v / v) acetonitrile (ACN), followed by dehydration with 100% ACN for 20 minutes, reduction with 25 mM ABC containing 10 mM DTT at 57 °C for 1 hour, and alkylated in the dark at room temperature with 55 mM iodoacetamide in 25 mM ABC for 30 minutes. The gel sheets were washed twice with 25 mM ABC and dehydrated twice with 100% ACN (20 minutes each time). Gel cubes were incubated overnight at 37°C with sequencing-grade modified trypsin (Promega, USA; 12.5 ng / μl in 40 mM ABC containing 10% ACN, pH 8.0). After digestion, peptides were extracted twice from the gel sheets using a mixture of 50% ACN and 5% formic acid (FA) and then with 100% ACN. The extracts were dried using a vacuum centrifuge plus (Eppendorf).

[0170] Mass spectrometry analysis was performed online using an Ultimate 3000 rapid separation liquid chromatography (RSLC) system with a Q Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific). Briefly, the peptide was dissolved in 3.5 μL of 10% ACN-0.1% trifluoroacetic acid (TFA). Then, it was separated into a C18 reversed-phase pre-column (3 μm particle size). Peptides were loaded onto C18 reverse-phase resin (75 μm inner diameter, 2 cm length) and washed. The loading buffer contained 98% H2O, 2% CAN, and 0.1% TFA. Peptides were separated using a 30-minute gradient from 99% A (0.1% FA and 100% H2O) to 40% B (80% ACN, 0.085% FA and 20% H2O) on a pore size of 75 μm inner diameter and 25 cm length.

[0171] The mass spectrometer acquired data throughout the elution process and operated with a data-dependent scheme, using Orbitrap to acquire a full MS scan, followed by up to 10 MS / MS HCD spectra acquired in Orbitrap. The mass spectrometer was configured for: full MS (AGC: 3 × 10e6, resolution: 7 × 10e4, m / z range 400–2000, maximum ion implantation time: 100 ms) and MS / MS (AGC: 1 × 10e5, maximum implantation time: 100 ms, isolation window: 4 m / z, dynamic exclusion time setting: 15 s). Fragmentation was permitted for precursors with charge states of +2, 3, and 4. For spectral processing, Proteome Discoverer 1.4 (Thermo Fisher Scientific) was used to generate the .mgf files. A database search was performed using Mascot version 2.5.1 (MatrixScience, London, UK) on “human” proteins (20,273 sequences) from the SwissProt database, which contains 550,552 sequences (196,472,675 residues) (February 2016). Search parameters were as follows: enzyme specificity as a trypsin, carbamoyl methylation as a fixed modification of cysteine, and oxidation as a variable modification of methionine. A maximum of one deletion cleavage was allowed, and a precursor quality accuracy tolerance of 4 ppm and a fragment quality accuracy tolerance of 20 mmu were used for all trypsin quality searches. Positive identification was based on a Mascot score above the significance level (i.e., 5%). Reported proteins were always those with the highest number of peptide-matched proteins.

[0172] Study on plasminogen activation / fragmentation on fibrin-NET matrix

[0173] The fibrin-NET matrix was prepared by activating neutrophils seeded onto a fibrin surface prepared as described above using PMA. Neutrophils were seeded at 200,000 cells per well onto the fibrin matrix and stimulated with PMA 1 hour after plating, as described above. After 4 hours of stimulation, the fibrin-NET matrix was obtained, as observed by Hoechst staining and elastase activity assay. Figure 1 After NET formation, remove the supernatant solution, thoroughly clean the wells once with HBSS, and perform the following experiments:

[0174] 1. Plasminogen activation on the fibrin-NET matrix. After washing with HBSS, 50 i.u. / ml tPA was incubated with the surface at 37°C for 1 h to allow tPA binding to fibrin. Unbound tPA was carefully discarded, and solutions of plasminogen at different concentrations were added to trigger plasmin formation via tPA bound to the fibrin-NET lattice. In parallel experiments, the effect of elastase on plasminogen fragmentation was blocked by the elastase inhibitor MeO-Suc-AAPV-CMK. The kinetics of plasminogen to plasmin conversion were tracked over 1 h by measuring pNA release at A405 nm in a microplate counter. The initial reaction rate was calculated using a dedicated computer program.

[0175] 2. Plasminogen fragmentation via NET-bound elastase. After NE formation and washing with HBSS, 1 μM plasminogen was incubated with NET at 37°C for 15 min to 4 h. When prompted, NET was pretreated with the elastase inhibitor MeO-Suc-AAPV-CMK or α1-PI before adding plasminogen. After incubation, the plate was centrifuged at 3000 g for 5 min, and the supernatant from each plate was collected and stored at -20°C for further analysis. The material remaining on the fibrin surface was recovered by scraping with 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 HNE activity detection, respectively. HNE activity in samples collected in HBSS was measured as described above using the chromogenic substrate MeO-Suc-AAPV-pNA (100 μl per well, final concentration in HBSS 1.5 mM). In samples collected in Tris-SDS, plasminogen hydrolysis was investigated by Western blotting using SDS-10% PAGE followed by either a mAb against plasminogen K1 (CPL15-HRP, 100 ng / ml) (35) or polyclonal sheep IgG (125 μg / ml) followed by secondary anti-sheep IgG-HRP (1:20,000). Signal detection was performed using an ECL kit from Amersham (Arlington Heights, UK) and the Curix-60-AGFA developed for testing.

[0176] Detection of plasminogen, plasminogen fragments and plasmin formation in plasma from patients with septic shock

[0177] Plasminogen was quantified using the modified functional activity assay described above. (41) Briefly, plasma was diluted 1:50 in assay buffer (80 mM phosphate, 50 mM NaCl, 2 mg / ml BSA) supplemented with 75 i.u. / ml urokinase, 1 mM tranexamic acid, and 0.75 mM plasmin-selective chromogenic substrate, to a final volume of 50 μl. The kinetics of the reaction were tracked by measuring the release of pNA at A405 nm in a microplate spectrophotometer in kinetic mode. Results were given in nM concentrations using a standard curve prepared with plasminogen-depleted plasma supplemented with known concentrations of purified plasminogen (0.2 μM to 1 μM). As described above, the presence of plasminogen fragments in plasma was initially detected by Western blotting. Further identification was obtained by mass spectrometry (see above). As described above, euglobulin fractions from plasma of healthy controls and patients were used as the source of plasminogen to measure plasmin formation on the fibrin matrix. Plasminogen activation occurred on the fibrin-NET matrix. The kinetics of plasminogen conversion to plasmin were tracked over 1 hour by measuring pNA release at A405 nm, and the initial rate of the reaction was calculated as shown above.

[0178] Detection of extracellular traps of neutrophils in human plasma

[0179] The plasma HNE-DNA complex of NET was identified using the ELISA capture assay described in (19), with the following modifications. Since HNE is an enzyme involved in plasminogen fragmentation and is the most abundant particulate component of NET, we chose to use an anti-HNE antibody to capture the HNE-DNA complex. As described above for fibrinogen, the antibody (10 μg / ml) was immobilized on a poly(glutaraldehyde) activated U 96-well polyvinyl chloride plate. Following the manufacturer's instructions (Roche Diagnostic, Mannheim, Germany), 50 μl of diluted human plasma and peroxidase-labeled anti-DNA monoclonal antibody (component 2 of the commercial cell death detection ELISA kit) were added to each well. After incubation at 37°C for 1 hour, the samples were washed three times with 100 μl of PBS per well and 100 μl / well of 1 mg / ml peroxidase substrate ABTS was added. As shown above, the absorbance at 405 nm was measured in the dark at 37°C using a multi-well plate spectrophotometer.

[0180] result:

[0181] NETs released onto the fibrin matrix form a dense lattice.

[0182] NETs were identified as extracellular fibrous networks primarily composed of Hoechst-stained DNA. Further identification was achieved by detecting DNA-bound HNEs using an activity assay. Figure 1 Unstimulated neutrophils exhibit typical intracellular segmented nuclei and are easily identifiable on plates containing or without fibrin matrix before or after incubation at 37°C for 4 hours. Figure 1 A, neutrophils). Extracellular DNA was not observed in unstimulated neutrophils, indicating that fibrin alone cannot promote NET formation and release. In the absence of fibrin, NETs released under similar conditions appear to be isolated and unwoven ( Figure 1 A, NET). Conversely, extracellular DNA released by stimulated neutrophils seeded on fibrin forms a dense fibrous weave, clearly visible after Hoechst staining ( Figure 1 A, NET-fibrin), indicating that the regular distribution of positive charge along the fibrin filaments favors DNA diffusion on the fibrin. (42) Notably, fibrin alone did not exhibit autofluorescence (not shown) that could potentially affect the dense appearance of NET. The presence of HNE coupled with NET was detected using the elastase-selective chromogenic substrate MeO-Suc-AAPV-pNA ( Figure 1 B). The proteolytic activity of the HNE-DNA complex was completely blocked by the peptidyl chloromethyl ketone elastase inhibitor MeO-Suc-AAPV-CMK ( Figure 1 B). In contrast, it effectively inhibits soluble HNE in the supernatant or when added to plasma ( Figure 1 α1-PI of C) only reduced the activity of DNA-associated HNE by about 20% ( Figure 1 B). Aprotinin (an unrelated inhibitor) had no effect on HNE activity when added to NET or cell supernatant at a concentration similar to α1-PI (40 μM). Figure 1 B). These data indicate that the HNE-DNA lattice diffused onto the fibrin matrix is ​​fully active.

[0183] The NET HNE-DNA complex cleaves plasminogen into well-defined fragments.

[0184] It is well known that HNE in solution (purified or released from neutrophils) produces plasminogen fragments with known structures (4, 44). However, it remains unknown whether HNE bound to NET can cleave plasminogen into fragments. To investigate this hypothesis, we incubated plasminogen with a NET lattice dispersed on a fibrin matrix. Figure 2A clearly demonstrates that plasminogen fragments were present after 30 minutes and 1 hour of incubation with NET. Three major bands with relative molecular masses of 92 kDa, 47 kDa, and 32 kDa were observed, along with a smaller band of approximately 50 kDa above the 47 kDa band. Longer incubation times did not alter this pattern. Figure 2 B). Proteomics analysis identified the 92 kDa band as human plasminogen, the 47 kDa and 50 kDa bands as fragments K1+2+3, and the 32 kDa band as K5-SP (Figure 6). Plasminogen treated with NET-HNE (Figure 6) and purified plasminogen treated with HNE (Figure 6) were also analyzed. Figure 7 Proteomic analysis in the study clearly identified K4. The HNE-DNA complex was validated to specifically cleave plasminogen by pre-incubating NET with a selective protease inhibitor. Pre-incubation with MeO-Suc-AAPV-CMK (an elastase-specific inhibitor) completely inhibited HNE and eliminated plasminogen from fragments, confirming HNE's activity on NET. Figure 2 C). Conversely, aprotinin (a plasmin and kallikrein inhibitor) cannot alter the activity of elastase or the appearance of plasminogen fragments. Figure 1 B). Because the specific inhibitor of elastase, α1-PI, only slightly reduces the activity of the HNE-DNA complex. Figure 1 B) This indicates that the observed plasminogen fragment was generated by the proteolytic activity of the HNE-DNA complex.

[0185] NET HNE-derived plasminogen fragments interfere with fibrinolysis

[0186] It has been demonstrated that the HNE·DNA complex of NET cleaves plasminogen into fragments K1+2+3, K4, and K5-SP, seeking answers to key questions about their roles in fibrinolysis. To this end, neutrophils seeded on a fibrin matrix were shown to release NETs with the aforementioned PMA. A plasmin-selective chromogenic substrate was then used to assess the conversion of plasminogen to plasmin via tPA binding to the fibrin-NET lattice. Compared to fibrin alone, the time- and concentration-dependent conversion of plasminogen to plasmin at the fibrin-NET lattice was reduced. Figure 3 (AB). When MeO-Suc-AAPV-CMK (a selective inhibitor of elastase) was pre-incubated with fibrin-NET lattices, plasmin formation was restored to control levels, indicating that HNE proteolytic activity prevented plasminogen conversion to plasmin. Compared to similar conditions with MeO-Suc-AAPV-CMK, the amount of plasmin formed and retained bound to the fibrin-NET lattice was reduced by approximately 50%. Figure 3 C). This reduction in the amount of plasmin formed on fibrin is associated with plasminogen fragmentation via the HNE-DNA complex, as indicated by Western blot analysis of the molecular species eluted from the fibrin surface. Figure 3 D). In summary Figure 3 The data indicate that plasminogen fragments were identified in fibrin matrices containing HNE·DNA complexes and exhibiting low fibrinolytic activity. Conversely, when fibrin-NET matrices were pretreated with MeO-Suc-AAPV-CMK, plasminogen fragments were absent and fibrinolytic activity was similar to that of control fibrin.

[0187] NET, plasminogen fragment, HNE·α1-PI and fibrinolytic activity in septic shock plasma.

[0188] The results of the test parameters are shown in Table 1 below.

[0189] Table 1: Fibrinolytic parameters and net plasma density in plasma of septic shock and healthy controls

[0190]

[0191] Pool 91: A pool consisting of plasma isolated from the blood of 18 healthy controls. ND: Not completed.

[0192] NET was 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, A 405 nm). Plasminogen concentration, as measured by 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 compared to healthy controls, at 437 ± 274 ng / ml versus 43 ± 19 ng / ml. Patients had elevated D-dimer (12 ± 8 mg / L) and plasmin-α2-antiplasmin (range 4814–159 ng / ml, median 1207 ng / ml) without signs of hepatic dysfunction, despite moderate cytolysis. The presence of HNE-derived plasminogen fragments was confirmed by SDS-PAGE and Western blotting. Figure 4The patterns of plasminogen fragments detected in seven selected patients are shown compared to healthy controls. In addition to plasminogen, three major plasminogen fragments were identified by mass spectrometry analysis of the amino acid sequences of protein bands from samples depleted of high-abundance proteins (euglobulin fraction or lysine affinity fraction): plasminogen fragments containing K1+2+3, K1+2+3+4, and plasminogen fragments consisting of K5-SP (Figure 6). K4 was not detected in plasma samples from patients with septic shock despite the use of acrylamide gels with small pores or short migration times. In contrast, K4 was clearly identified in purified plasminogen treated individually by NET or HNE (Figures 6–7).

[0193] To investigate the effects of NET and plasminogen fragments on fibrinolysis, euglobulin fractions from septic shock plasma samples were incubated with tPA bound to the fibrin surface, and the amount of plasmin formed was quantified using a chromogenic substrate. When different septic shock plasma samples were compared to normal plasma pools, plasmin production was reduced (>50%). Figure 5 To compensate for the relatively low plasminogen concentration in these plasmas, purified plasminogen was added to a concentration similar to that of the normal plasma pool. As a result, only in the three plasmas supplemented with plasminogen (S1, S3, S5) did the amount of plasmin formed show a normal trend of increase. Inadequate or insufficient responses were observed in the remaining plasma samples (S2, S4, S6), indicating competition for the added plasminogen.

[0194] discuss

[0195] Netosis (a major antimicrobial mechanism) plays a substitute role in both infectious and non-infectious diseases. For example, in septic shock, activated neutrophils release extracellular traps that both trap pathogens and activate coagulation, leading to fibrin formation. The ensuing microvascular clots (a mechanism known as immune thrombosis) facilitate the restriction and killing of pathogens by NET enzymes. (20, 45, 46) Several factors in this interrelationship between coagulation and innate immunity may also contribute to uncontrolled thrombosis, thus hindering the initial benefits of the response. (47) Therefore, NET may contribute significantly to intravascular coagulation associated with septic shock. Indeed, we have detected circulating NET in septic shock patients with intravascular coagulation, (48) a finding confirmed in recent studies. (49–52) This example focuses on the role of NET on the other side of the hemostatic balance: fibrinolytic response.

[0196] Using a fluorescent DNA adhesive, the inventors noted that NETs formed in vitro on the surface of fibrin exhibited a denser and more reticular appearance than NETs released in the absence of fibrin. The positively charged DNA, regularly distributed along the fibrin fibers, interacted with the negatively charged DNA, producing a denser appearance of DNA fibers within the tangled fibrin-NET matrix. These findings are consistent with previously reported data suggesting that cell-free DNA incorporated into clots alters the structure of fibrin, making it resistant to plasmin-mediated degradation. (26, 28) The assembly of DNA and fibrin tangles within the thrombus may be associated with NET-mediated in vitro resistance to tPA-induced fibrinolysis (21, 29) and thrombolysis in patients with acute ischemic stroke. (54) This type of fibrinolytic resistance may lead to persistent thromboembolic occlusion due to sustained platelet activation via histones (21) and inactivation of tissue factor pathway inhibitors (TFPIs) via HNE. (45) In fact, the inventors demonstrated that fibrin-entangled NETs contain active HNEs in their DNA complexes, which are insensitive to inhibition by α1-PI. Similarly, NET-associated mouse NEs were found to have proteolytic activity in the hepatic vascular system. (55) Conversely, elastases bound to the plasma membrane of neutrophils are readily released to form soluble, irreversible complexes with circulating α1-PIs. (7) The protective effect of DNA on HNE activity is consistent with previous findings, indicating that leukocyte granule serine proteases bind to DNA with nanomolar affinity and confer resistance to plasma inhibitors to HNEs, thereby ensuring local degradation of the protein substrate. (9, 11, 12, 17)

[0197] This example clearly demonstrates that in the presence of NET containing an active HNE-DNA complex, plasminogen is reduced to well-defined fragments: K1+2+3, K1+2+3-4, K4, and K5-SP, which have lost their ability to generate plasmin via fibrin-bound tPA. These plasminogen fragments were detected when plasminogen was added to NET released from in vitro activated neutrophils. The nature of the fragments was identified by mass spectrometry analysis of the amino acid sequences. Since plasminogen fragments containing K1 have previously been shown to be strong inhibitors of plasminogen binding to fibrin, plasmin production, and fibrinolysis (56-58), the inventors investigated the role of NET in these mechanisms. The experimental fibrin-NET lattice used by the inventors is an excellent setup for detecting the competitive effects of NET-HNE-derived plasminogen fragments and K1-containing fragments on plasminogen binding and activation. The inventors have demonstrated that when plasminogen is activated by fibrin-bound tPA on the NET-fibrin lattice, a decrease in the amount of plasmin produced by the tPA occurs alongside the appearance of such fragments. These data indicate that these plasminogen fragments compete with plasminogen for fibrin binding, thereby reducing its ability to produce plasmin.

[0198] Unlike the antifibrinolytic activity described herein, there are other hemostatic protein components that may be substrates of HNE, some of which may have direct or indirect effects on thrombus formation and dissolution. In particular, HNE can exert procoagulant activity by inactivating TFPI (45), or by directly cleaving fibrin (59) by degrading α2-antiplasmin or PAI-1 (60, 61) or by generating micro-plasminogen (K5-SP). (5) Although micro-plasminogen (K5-SP) can be activated by urokinase in solution, it does not bind to fibrin and is therefore not activated by tPA on the fibrin surface; therefore, the effect of K5-SP on tPA-fibrin-dependent clot dissolution is negligible. (5, 62)

[0199] In summary, the results clearly demonstrate that the HNE-DNA complex exerts its antifibrinolytic effect by generating plasminogen fragments and reducing plasminogen concentration.

[0200] Indicators of NET-induced in vitro fibrinolysis failure were investigated in plasma samples from septic shock patients with DIC and circulating NET. The inventors clearly demonstrated that this group of patients had low plasminogen concentrations, circulating plasminogen fragments, and an inability to produce plasmin on fibrin (less than 50% compared to normal plasma pools), which significantly contributed to and / or caused the observed intravascular coagulation and multiple organ dysfunction. Protein bands detected by Western blot and identified by mass spectrometry amino acid analysis in plasma fractions from selected patients corresponded to intact plasminogen and plasminogen fragments: K1+2+3, K1+2+3+4, and K5-SP. These fragments were identical to those derived from plasminogen cleaved in vitro by purified HNE. (44)

[0201] Since α1-PI circulates at a concentration of μM and inhibits all free HNE with an extremely high binding constant (Ka = 6.5 × 10⁷ M⁻¹ s⁻¹), (6) elastase released by neutrophils is readily found as an HNE-α1PI complex in the plasma of patients with septic shock. Therefore, the HNE-α1PI complex is a marker of neutrophil activation in sepsis. The inventors have demonstrated that plasminogen fragments found in the plasma of these patients, in the absence of active elastase, are generated by active HNE-DNA complexes. These data strongly support a mechanism in which plasminogen fragments found in patients with septic shock are locally generated on the fibrin-NET matrix of clots. However, in addition to fibrin, histone H₂B with a C-terminal lysine residue may capture plasminogen near HNE bound to DNA fibrils. (64) Circulating NET containing active HNE-DNA complexes that are insensitive to α1-PI may also be involved in plasminogen fragmentation.

[0202] As demonstrated, the proteolysis of plasminogen by the HNE-DNA complex is partly responsible for the low concentrations (below 1 μM) of full-length plasminogen detected in the plasma of patients with septic shock. Low concentrations of plasminogen, along with the antifibrinolytic activity of plasminogen fragments, lead to inadequate fibrinolysis in septic shock, resulting in disseminated intravascular coagulation (DIC). These parameters, plasminogen concentration, plasminogen fragments, and the inability to form plasmin are relevant novel prognostic biomarkers for assessing the role of NET in sepsis and septic shock-induced DIC, and as predictors of mortality and organ failure in patients with septic shock.

[0203] Since the low plasminogen concentrations in these plasmas could partially explain the low fibrinolytic activity, the plasmas were supplemented with plasminogen prior to testing. Despite achieving plasminogen concentrations similar to those in normal plasma pools, three of the six plasma samples tested failed to restore their fibrinolytic activity to normal levels. These results suggest that the competitive effect on plasminogen is likely related to the presence of a K1-containing circulating HNE-derived plasminogen fragment.

[0204] The results clearly demonstrate that the significant reduction in fibrinolysis induced by the NET HNE-DNA complex contributes to the persistence and stability of thrombi in the microcirculation during septic shock.

[0205] The inventors have also demonstrated that septic shock thrombosis in the microcirculation is related to the ability of the HNE-DNA complex to trigger fibrinolytic dysfunction. For example... Figure 5 As shown, identifying this reduction in fibrinolytic potential in the plasma of patients with septic shock has practical implications for finding feasible alternatives to improve the fibrinolytic response in these patients. For example, active enzymes (i.e., HNE-DNA complexes) and substrates (i.e., plasminogen) represent novel targets and / or compounds that can be used for therapies of sepsis-DIC.

[0206] The inventors demonstrated that NET or NET fragments contain active elastase (a competitor of plasminogen in binding to fibrin) responsible for producing plasminogen fragments with inhibitory activity, which reduces plasminogen concentration and plasmin formation. In summary, low plasminogen and the inhibitory activity of elastase-derived plasminogen fragments contribute to the elimination of the fibrinolytic system, thereby promoting microthrombus formation and organ failure.

[0207] This example clearly demonstrates the need for fragmentation of plasminogen via an active HNE-DNA complex, along with a concurrent decrease in plasminogen concentration and plasmin formation, to inhibit fibrinolysis. Figure 4 It also showed that the low ability to generate plasmin was associated with a reduction in the amount of plasminogen bound to fibrin, resulting from low plasminogen concentrations and a competitive effect caused by plasminogen fragments derived from elastase. The data clearly indicate that the HNE-DNA complex is at the center of a mechanism that leads to severe fibrinolytic failure via the depletion of plasminogen through proteolysis, resulting in the production of antifibrinolytic plasminogen fragments. This incomplete fibrinolytic function is the cause of impaired dissolution of microthrombi, thereby promoting their stability in the microcirculation and contributing to DIC and organ failure during septic shock.

[0208] This example clearly demonstrates that plasminogen levels are a biomarker for fibrinolytic responses associated with DIC, especially during septic shock.

[0209] This example also clearly demonstrates that plasminogen restores fibrinolytic activity in patients with netosis and septic shock-related DIC, and constitutes a novel treatment option for these patients through supplementation.

[0210] Example 2: Plasminogen, an indicator of fibrinolytic response in sepsis and septic shock DIC

[0211] Plasminogen is a precursor to plasmin, the enzyme required to dissolve imprinted clots. Plasminogen is converted to plasmin by endothelial plasminogen activator (tPA). Plasminogen circulates in the blood at concentrations ranging from 1.5 μmol / L to 2 μmol / L (138 μg / ml to 184 μg / ml), exceeding the amount required to produce full plasmin activity from fibrin via tPA. However, concentrations below 1 μM of plasminogen are the rate-limiting factor for plasmin production, potentially leading to inadequate fibrinolysis (Biochem J, 1995).

[0212] In fact, previous studies have shown that the rate of thrombolysis is limited by the amount of plasminogen available to control its accumulation on fibrin (Biochemistry 1991, Thrombosis and Hemostasis 1991; J Lab Clin Med 1992, Circulation 1995). Therefore, a continuous supply of plasminogen from the circulation is necessary to ensure its accumulation on the thrombus and maintain its optimal dissolution potential. However, under pathological conditions combining thrombus formation in the microcirculation with a significant reduction in plasminogen, this supply is limited; impaired blood flow due to inadequate thrombolysis can thus worsen clinical outcomes.

[0213] Example 1 above clearly demonstrates that in patients with septic shock presenting with disseminated intravascular coagulation (DIC), functional plasminogen concentrations below 1 μM, or even below 0.5 μM in severe cases, are found. Figure 9 ).

[0214] The decreased plasminogen concentration observed in patients with septic shock may be partly due to consumption via fibrinolysis secondary to DIC (as indicated by circulating plasmin-α2-anti-plasmin complex and D-dimer plasma concentrations). However, the persistent presence of microthrombi in DIC suggests a failure of the fibrinolytic response.

[0215] In septic shock with disseminated intravascular coagulation (DIC), neutrophils and platelets work synergistically in an intercoupled process known as immunothrombosis to promote coagulation and the formation of neutrophil extracellular traps (NETs) (20, 45). NET formation is a mediating mechanism in septic shock. Figure 4 ).

[0216] NETs are DNA fibers containing elastase, myeloperoxidase, and other particulate proteins. Unlike free elastase, NET-associated elastase is resistant to α1 protease inhibitors, and therefore can cleave plasminogen into well-known fragments that lose their ability to produce plasmin.

[0217] The results clearly demonstrate that the significant decrease in the concentration of functional plasminogen detected in septic shock patients with DIC is primarily associated with proteolysis of the elastase-DNA complex via NET. Proteolysis of the full-length plasminogen and the N-ter region -K1-K2-K3-K4-K5-SP (K: tricyclic domain, SP: serine-protease domain) by elastase produces plasminogen fragments K1+2+3, K1+2+3+4, K4, and K5-SP (also known as miniature plasminogen).

[0218] Data obtained in a cohort of patients (n=100) showed a significant reduction in plasminogen activator production in patients with septic shock and low plasminogen activator levels compared to healthy volunteers. This reduced capacity to form plasmin leads to inadequate fibrinolysis, which contributes to the presence of thrombi in the microcirculation (DIC) and associated multi-organ dysfunction in patients with septic shock. Extensive ischemic tissue damage associated with DIC results in failure of multiple organs, particularly the lungs, kidneys, liver, and brain.

[0219] Data from the generation of plasmin on the fibrin surface using these plasmas indicate that, in addition to being a marker of plasminogen hydrolyzed by elastase-DNA, miniature plasminogen (K5-SP) is most importantly a source of non-fibrinolytic miniature plasminogen, thus contributing to fibrinolytic dysfunction. Miniature plasminogen generated from plasma containing miniature plasminogen lacks the ability to bind to fibrin, meaning it is easily inhibited by plasma inhibitors and therefore lacks specificity for fibrinolysis. Therefore, K5-SP in plasma interferes with the functional determination of full-length plasminogen and the assessment of fibrinolysis.

[0220] Administration of plasminogen to patients with septic shock exhibiting a significant reduction in endogenous circulating plasminogen is beneficial for improving effective fibrinolysis, thrombolysis, and patient recovery. In this context, plasminogen monitoring (full-length protein and / or fragments thereof) is a key determinant for assessing the fibrinolytic response to DIC in patients with septic shock and its evolution into organ dysfunction, as well as for monitoring plasminogen recovery during ongoing human Glu-plasminogen replacement therapy.

[0221] Plasminogen (and / or fragments thereof) is a key analytical measure for the diagnosis and / or follow-up of patients (e.g., those with netosis-related septic shock), for the diagnosis of DIC and / or plasminogen-related fibrinolytic dysfunction and the treatment of plasminogen replacement therapy.

[0222] fibrinolysis and NET components / parameters will be measured in patients with septic shock (n=100) compared to healthy individuals (n=31).

[0223] Furthermore, this example clearly supports the identification of microplasminogen (K5-SP) as a direct biomarker of elastase-DNA activity and a marker of NET-related symptoms such as sepsis, ischemic stroke, coronary thrombosis, cancer, and autoimmune diseases. Assessing microplasminogen through functional assays can also provide information about its role in fibrinolytic dysfunction.

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Journal of Thrombosis and Hemostasis, 2003, 1, 2397. sequence list <110> Strasbourg University Hospital National Institute of Health and Medical Sciences <120> Methods for diagnosing fibrinolytic dysfunction associated with neutrophil extracellular trapping <130> BNT228237PC00 <150> EP19306378.1 <151> 2019-10-24 <150> EP20174840.7 <151> 2020-05-14 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 791 <212> PRT <213> Homo sapiens <400> 1 Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser 1 5 10 15 Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala 20 25 30 Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His 35 40 45 Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser 50 55 60 Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr 65 70 75 80 Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met 85 90 95 Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser 100 105 110 Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu 115 120 125 Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp 130 135 140 Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu 145 150 155 160 Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly 165 170 175 Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser 180 185 190 Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys 195 200 205 Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro 210 215 220 Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile 225 230 235 240 Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys 245 250 255 Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val 260 265 270 Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His 275 280 285 Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr 290 295 300 Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn 305 310 315 320 Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser 325 330 335 Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr 340 345 350 Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly 355 360 365 Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser 370 375 380 Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala 385 390 395 400 Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro 405 410 415 Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu 420 425 430 Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val 435 440 445 Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe 450 455 460 Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly 465 470 475 480 Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile 485 490 495 Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys 500 505 510 Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn 515 520 525 Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro 530 535 540 Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly 545 550 555 560 Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln 565 570 575 Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu 580 585 590 Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser 595 600 605 Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val 610 615 620 Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu 625 630 635 640 Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala 645 650 655 Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr 660 665 670 Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr 675 680 685 Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val 690 695 700 Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val 705 710 715 720 Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser 725 730 735 Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys 740 745 750 Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro 755 760 765 Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile 770 775 780 Glu Gly Val Met Arg Asn Asn 785 790 <210> 2 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> Plasminogen fragment Leu74-val338 <400> 2 Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly 1 5 10 15 Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys 20 25 30 Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala 35 40 45 Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp 50 55 60 Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg 65 70 75 80 Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys 85 90 95 Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu 100 105 110 Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile 115 120 125 Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn 130 135 140 Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys 145 150 155 160 Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser 165 170 175 Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg 180 185 190 Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser 195 200 205 Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys 210 215 220 Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala 225 230 235 240 Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys 245 250 255 Ile Pro Ser Cys Asp Ser Ser Pro Val 260 265 <210> 3 <211> 370 <212> PRT <213> Artificial Sequence <220> <223> Plasminogen Fragment Leu 74 - Val443 <400> 3 Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly 1 5 10 15 Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys 20 25 30 Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala 35 40 45 Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp 50 55 60 Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg 65 70 75 80 Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys 85 90 95 Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu 100 105 110 Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile 115 120 125 Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn 130 135 140 Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys 145 150 155 160 Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser 165 170 175 Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg 180 185 190 Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser 195 200 205 Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys 210 215 220 Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala 225 230 235 240 Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys 245 250 255 Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro 260 265 270 Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly 275 280 285 Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys 290 295 300 Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr 305 310 315 320 Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn 325 330 335 Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val 340 345 350 Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser 355 360 365 Val Val 370 <210> 4 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> Plasminogen Fragment Ala 444 - Asn 791 <400> 4 Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu 1 5 10 15 Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala 20 25 30 Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro 35 40 45 His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu 50 55 60 Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp 65 70 75 80 Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro 85 90 95 Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro 100 105 110 Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His 115 120 125 Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe 130 135 140 Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His 145 150 155 160 Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly 165 170 175 Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val 180 185 190 Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys 195 200 205 Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu 210 215 220 Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr 225 230 235 240 Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu 245 250 255 Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe 260 265 270 Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala 275 280 285 Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys 290 295 300 Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu 305 310 315 320 Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg 325 330 335 Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn 340 345 <210> 5 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Plasminogen Fragment Val355-Val433 <400> 5 Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser 1 5 10 15 Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr 20 25 30 Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu 35 40 45 Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys 50 55 60 Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys 65 70 75 80 Cys Ser Gly Thr Glu Ala Ser Val Val 85 <210> 6 <211> 352 <212> PRT <213> Synthetic Sequence <220> <223> Plasminogen Fragment Cys84 - Cys435 <400> 6 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu 65 70 75 80 Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser 85 90 95 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 145 150 155 160 Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly 165 170 175 Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His 180 185 190 Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn 210 215 220 Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val 225 230 235 240 Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser 245 250 255 Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val 260 265 270 Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser 275 280 285 Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro 290 295 300 His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr 305 310 315 320 Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe 325 330 335 Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys 340 345 350 <210> 7 <211> 250 <212> PRT <213> Artificial sequence <220> <223> Plasminogen fragment Cys84 - Cys333 <400> 7 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu 65 70 75 80 Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser 85 90 95 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 145 150 155 160 Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly 165 170 175 Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His 180 185 190 Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn 210 215 220 Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val 225 230 235 240 Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 245 250 <210> 8 <211> 328 <212> PRT <213> Synthetic Sequence <220> <223> Plasminogen Fragment Cys462 - Arg789 <400> 8 Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr 1 5 10 15 Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg 20 25 30 His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys 35 40 45 Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr 50 55 60 Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys 65 70 75 80 Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys 85 90 95 Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp 100 105 110 Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly 115 120 125 Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu 130 135 140 Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His 145 150 155 160 Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg 165 170 175 Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser 180 185 190 Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser 195 200 205 Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp 210 215 220 Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln 225 230 235 240 Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn 245 250 255 Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly 260 265 270 Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu 275 280 285 Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys 290 295 300 Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val 305 310 315 320 Thr Trp Ile Glu Gly Val Met Arg 325 <210> 9 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> Plasminogen Fragment Leu 74 - Val 354 <400> 9 Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly 1 5 10 15 Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys 20 25 30 Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala 35 40 45 Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp 50 55 60 Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg 65 70 75 80 Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys 85 90 95 Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu 100 105 110 Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile 115 120 125 Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn 130 135 140 Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys 145 150 155 160 Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser 165 170 175 Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg 180 185 190 Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser 195 200 205 Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys 210 215 220 Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala 225 230 235 240 Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys 245 250 255 Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro 260 265 270 Thr Ala Pro Pro Glu Leu Thr Pro Val 275 280 <210> 10 <211> 458 <212> PRT <213> Synthetic Sequence <220> <223> Plasminogen Fragment Cys84-Cys541 [[ID=,44]]<400> 10 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu 65 70 75 80 Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser 85 90 95 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 145 150 155 160 Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly 165 170 175 Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His 180 185 190 Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn 210 215 220 Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val 225 230 235 240 Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser 245 250 255 Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val 260 265 270 Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser 275 280 285 Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro 290 295 300 His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr 305 310 315 320 Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe 325 330 335 Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys 340 345 350 Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu 355 360 365 Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly 370 375 380 Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys 385 390 395 400 Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro 405 410 415 Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro 420 425 430 Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys 435 440 445 Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys 450 455 <210> 11 <211> 228 <212> PRT <213> Artificial sequence <220> <223> Plasminogen: Serine protease domain (SP) Val562-Arg789 <400> 11 Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile 20 25 30 Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro 35 40 45 Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn 50 55 60 Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu 65 70 75 80 Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val 85 90 95 Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val 100 105 110 Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln 115 120 125 Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile 130 135 140 Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln 145 150 155 160 Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys 165 170 175 Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr 180 185 190 Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn 195 200 205 Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu 210 215 220 Gly Val Met Arg 225 <210> 12 <211> 78 <212> PRT <213> Artificial Sequence <220> <223> Plasminogen Fragment Single K4 Cys358 - Cys435 <400> 12 Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr 1 5 10 15 Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg 20 25 30 His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys 65 70 75

Claims

1. Use of plasminogen and at least one fragment thereof in the preparation of a kit for predicting and / or detecting fibrinolytic insufficiency, comprising measuring the concentration of plasminogen and at least one fragment thereof in a first biological sample; wherein the first biological sample is derived from a patient suffering from septic shock.

2. The use according to claim 1, wherein the fibrinolytic insufficiency is associated with NET symptoms with / without disseminated intravascular coagulation (DIC).

3. The use according to claim 2, wherein the symptoms are infectious or non-infectious.

4. The use according to claim 2, wherein the NET-related symptoms are selected from the group comprising sepsis, septic shock, ischemic stroke, coronary thrombosis, cancer, trauma, and autoimmune diseases.

5. The use according to any one of claims 1 to 4, wherein the first biological sample is selected from the group comprising blood samples and plasma samples.

6. The use according to any one of claims 1 to 4, wherein the fragment of said plasminogen is selected from those containing tricyclic (K)1 to 3 domains (K... 1+2+3 ), three rings (K) 1 to 4 structural domains (K) 1+2+3+4 Groups of fragments containing the tricyclic (K)5 domain and / or the serine protease (SP) region (miniature plasminogen) (K5-SP).

7. The use according to any one of claims 1 to 4, comprising measuring the concentration of micro plasminogen (K5-SP).

8. Use of plasminogen and its fragments in the preparation of kits for determining the efficacy of treatment for fibrinolytic insufficiency associated with disseminated intravascular coagulation (DIC), comprising: a. Determine and / or measure the concentrations C1 of plasminogen and its fragments from a first biological sample prior to compound treatment. b. Determine and / or measure the concentration C2 of plasminogen and its fragments from a second biological sample treated with said compound, and optionally... c. Compare concentrations and calculate the score (S) according to the following formula: S = C2 / C1, An S-value greater than 1 indicates that the treatment is effective; The first biological sample and the second biological sample were derived from patients suffering from septic shock.

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