Anticoagulant proteins and their use for treating diseases associated with neutrophil activation - Patents.com

Abstract

To provide pharmaceutical compositions for inhibiting the extrinsic coagulation pathway and for treating and / or preventing diseases and conditions associated with the recruitment and activation of neutrophils following activation of the extrinsic coagulation pathway.SOLUTION: The present invention relates to a protein or polypeptide comprising an Ixodes ricinus salivary gland polypeptide, a fragment or a variant thereof, and its use.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to proteins and polypeptides, including tick salivary gland polypeptides, and their use for treating and / or preventing diseases and conditions associated with neutrophil activation. [Background technology]

[0002] Homeostasis is the biological function that stops bleeding and protects the integrity of the blood circulation at both the molecular and macroscopic levels. Homeostasis traditionally involves the coagulation cascade, which is divided into two converting enzyme cascades: one induced by blood-borne components of the vasculature (intrinsic pathway) or one induced by exposure of blood to damaged vessel walls (extrinsic pathway).

[0003] The intrinsic pathway and contact phase (also known as the plasma kallikrein-kinin system) are initiated by contact phase proteins, including the zymogens factor XII (FXII), factor XI (FXI), and prekallikrein (pKK or PK), as well as the cofactor high molecular weight kininogen (HK). Factor XII undergoes autoactivation upon binding to negatively charged surfaces, resulting in a conformational change to produce activated factor XII (FXIIa or αFXIIa). αFXIIa then converts PK to activated plasma kallikrein (KK or PKa). When small amounts of PKa are formed, they catalyze the conversion of surface-bound factor XII to αFXIIa, providing a powerful positive feedback loop for the system. During this process, factor XII activation leads to a series of proteolytic steps, resulting in the production of a series of different active enzymes (XIa, IXa, VIIIa, and Xa), ultimately leading to the activation of prothrombin to thrombin and the formation of fibrin.

[0004] The extrinsic coagulation pathway, or tissue factor (TF) pathway, consists of many serine proteases, cofactors, calcium, and cell membrane components. TF, also known as platelet tissue factor, factor III, thromboplastin, and CD142, is expressed in the subendothelium and activates the extrinsic coagulation pathway. TF is then delivered by exposed subendothelial cells and binds to plasma factor VII, triggering the extrinsic coagulation pathway. The resulting complex initiates the coagulation cascade, converting factor X to factor Xa and ultimately leading to the production of thrombin. Thrombin activates platelets and converts fibrinogen to fibrin. Both platelets and fibrin are important components of the hemostatic plug, contributing to the sealing of vascular breaches. The extrinsic pathway has been proposed to be the primary activator of the coagulation protease cascade in vivo. Subsequent clot propagation involves the recruitment of additional platelets and the amplification of the coagulation cascade.

[0005] Furthermore, under pathological conditions such as inflammatory stimuli, TF is expressed by monocytes, neutrophils, endothelial cells, and platelets, resulting in elevated levels of circulating TF-positive microparticles. Thus, the extrinsic coagulation pathway also involves cells in addition to activating coagulation factors. Importantly, platelets play a key role in amplifying the coagulation cascade by providing a surface for clot formation.

[0006] Over the past decade, research has supported the growing belief that neutrophils significantly contribute to the thrombotic process. Attenuated thrombus formation in neutrophil-deficient animal models indicates the contribution of these cells to thrombosis. Neutrophils can contribute to pathogenic venous and arterial thrombosis through the release of neutrophil extracellular traps (NETs). NET release has emerged as a major trigger for thrombus formation in pathologies such as sepsis, deep vein thrombosis, and malignancy. Furthermore, it has been suggested that NETs provide a scaffold for fibrin deposition and platelet entrapment, as well as subsequent activation. Furthermore, blood-cell-derived microparticles, including those derived from neutrophils, are involved in thrombus formation. Several studies have also supported the production of TFs by neutrophils in vivo and ex vivo (Kambas et al., 2012).

[0007] The active role of neutrophils in experimental thrombosis and inflammation-driven thrombotic diseases in vivo has been demonstrated. Their contribution to the activation of the extrinsic coagulation cascade is the degradation of TFPI via the release of elastase (TFPI is the primary inhibitor of TF) (Massberg et al., 2010). Furthermore, neutrophil binding to damaged endothelium was demonstrated to be the first step in the chain of events leading to thrombus formation in a model of laser-induced endothelial injury. This crucial role of neutrophils was reinforced by the finding that these cells are the primary source of TF, which is required for thrombus formation. Inhibition of neutrophil binding to the vessel wall was shown to reduce the presence of TF, leading to decreased fibrin production and platelet accumulation (Darbousset et al., 2012; Darbousset et al., 2014). Furthermore, in the same model, factor XII deficiency did not attenuate thrombus formation (Darbousset et al., 2012). Thus, neutrophils play a key role in the activation of the extrinsic coagulation system, and NETs may provide a scaffold for fibrin deposition and platelet capture and subsequent activation.

[0008] For the past 50 years, anticoagulant treatment has relied primarily on two classes of agents: heparin and antivitamin K. Heparin accelerates the inhibitory action of antithrombin by indirectly inhibiting several activated coagulation factors (particularly thrombin and factors IXa and Xa) and is only active when administered parenterally. Antivitamin K prevents the final synthesis of four coagulation factors (prothrombin, and factors VII, IX, and X). Both classes of agents inhibit the extrinsic pathway of coagulation. However, the therapeutic window for these agents is narrow, requiring careful patient monitoring. Indeed, these agents require careful clinical trials to ensure sufficient antithrombotic efficacy while avoiding the risk of bleeding.

[0009] Therefore, there is a strong need for new anticoagulants that do not have bleeding side effects.

[0010] Previously, the present applicant demonstrated that Ir-CPI (Ixodes ricinus) polypeptides isolated from the salivary glands of the tick, I. ricinus (Ixodes ricinus), specifically target the coagulation factors Factor XIa and Factor XIIa (EP Patent Nos. 1892297 and 2123670; Decrem et al., 2009). This finding indicated that tick salivary gland polypeptides are specific inhibitors of the intrinsic coagulation pathway. Indeed, in vitro studies have shown that Ir-CPI interferes with the intrinsic pathway by prolonging the activated partial thromboplastin time (aPTT), while having no effect on the prothrombin time (PT), thrombin time (TT), or diluted Russell's viper venom time (dRVVT)—three tests that test the induction of blood coagulation by activators of the extrinsic or common pathway. Furthermore, in contrast to the strong dose-dependent reduction in thrombin generation induced by ellagic acid (an intrinsic pathway activator), Ir-CPI was reported to be highly inactive in a thrombin generation assay in which thrombin generation was induced by low concentrations of TF (5 pM) (an extrinsic pathway activator). The small inhibition of thrombin generation at 5 pM TF has been explained by the fact that at low concentrations of TF, thrombin can activate FXI in the intrinsic pathway, creating a feedback loop involving thrombin, FXI(α), FIX(α), FX(α), and (pro)thrombin, maintaining the extrinsic pathway (Keularts, Zivelin et al. 2001).

[0011] As a possible mechanism of action, this molecule was shown to inhibit the activation of factor XI and PK by factor XIIa and the activation of factor XII by factor XIa, but was totally inactive against other targets of the coagulation pathway, particularly thrombin and factor Xa, which are targets of currently available parenteral and / or oral anticoagulant and antithrombotic drugs. Previously disclosed results were obtained in in vitro assays that included only factors of the coagulation pathway and therefore could not be indicative of activity of cells involved in the extrinsic coagulation pathway.

[0012] Due to its specific action on the intrinsic coagulation pathway, Ir-CPI is expected to have a larger therapeutic window with respect to the risk of bleeding, which constitutes the main side effect of current parenteral anticoagulants. Indeed, the applicant has shown that this molecule, at pharmacologically therapeutically effective doses, does not increase bleeding in experimental rodent models (Decrem, Rath et al., 2009).

[0013] Previously, we have found that tick salivary gland polypeptides unexpectedly inhibit the recruitment of polymorphonuclear neutrophils (PMNs) and platelets at the lesion site in an experimental mouse arteriolar laser injury model, which has been reported to be highly dependent on TF and independent of FXII (Darbousset et al., 2012). Furthermore, we have found that tick salivary gland polypeptides inhibit PMN activation and neutrophil extracellular trap formation (also known as NETosis) in vitro. Without being bound by theory, we have concluded that Ir-CPI inhibits the recruitment and activation of neutrophils at the lesion site, thereby playing a role in the recruitment and activation of cells involved in extrinsic coagulation pathway activation.

[0014] Tick ​​salivary gland polypeptides are therefore useful for the treatment and / or prevention of diseases and conditions associated with activation of the extrinsic coagulation pathway, such as thrombosis, and for the treatment and / or prevention of inflammatory diseases and thromboinflammation associated with a tendency to thrombosis.

[0015] The discovery of these novel features in the mechanism of action of Ir-CPI polypeptides allows these molecular parent compounds to not only inhibit the intrinsic pathway but also act on the coagulation and / or thrombosis processes involving TF via a mechanism entirely different from that of thrombin and / or factor Xa inhibitors.

[0016] Thus, the present invention relates to anticoagulant proteins comprising tick salivary gland polypeptides and their uses for use in the treatment and / or prevention of diseases and conditions associated with the activation of neutrophils and / or NETosis. Summary of the Invention

[0017] The present invention relates to proteins or polypeptides, including polypeptides having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1, for use in inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or neutrophil extracellular trap formation (NETosis).

[0018] In one embodiment, inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis treats and / or prevents a disease or condition associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0019] In one embodiment, the disease or condition associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis is selected from the group consisting of venous thrombosis, arterial thrombosis, thromboinflammation, and cardiovascular disease. In one embodiment, the disease or condition associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis is thromboinflammation.

[0020] In one embodiment, the thromboinflammation is selected from the group comprising atherosclerosis, plaque rupture, device-induced thromboinflammation, thrombosis induced by catheterization and / or stent placement procedures, thrombosis induced by extracorporeal circulation, thrombus formation after brain injury, coronary artery disease, acute myocardial infarction, cancer-related thrombosis, metastasis-related thrombosis, stroke-related thrombosis, Behcet's disease (BD), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Takayasu's arteritis, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, familial Mediterranean fever, thromboangiitis obliterans (TAO), sepsis, inflammatory bowel disease, heparin-induced thrombocytopenia, immunological thrombosis, thrombosis associated with preeclampsia, thrombotic complications in cell and cell cluster transplantation and whole organ transplantation or grafts, venous thromboembolism, aneurysms, and ischemia-reperfusion syndrome in skeletal muscle.

[0021] In one embodiment, the thromboinflammation is thrombosis induced by catheterization and / or stent placement procedures at the site of localized vascular stenosis. In another embodiment, the thromboinflammation is thrombosis induced by a medical device in contact with blood. In another embodiment, the thromboinflammation is thrombosis and / or coagulation associated with extracorporeal circulation.

[0022] The present invention also relates to pharmaceutical compositions comprising a protein or polypeptide, including a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1, and at least one pharmaceutically acceptable excipient, for use in inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and / or for treating and / or preventing a disease or condition associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0023] The present invention also relates to a medicament comprising a protein or polypeptide, comprising a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1, for use in inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and / or for treating and / or preventing a disease or condition associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0024] Another object of the present invention is a medical device coated with an isolated polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 for use in inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and / or for treating and / or preventing diseases or conditions associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0025] Yet another object of the present invention is a kit comprising a protein or polypeptide, including an isolated polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1, for use in inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and / or for treating and / or preventing a disease or condition associated with platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0026] The present invention further relates to a protein or polypeptide comprising a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 for use in inhibiting the extrinsic coagulation pathway in a subject in need thereof, wherein inhibiting the extrinsic coagulation pathway comprises inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or neutrophil extracellular trap formation (NETosis).

[0027] The present invention also relates to a method for inhibiting the extrinsic coagulation pathway in a subject in need thereof, comprising administering to the subject a protein or polypeptide comprising an isolated polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0028] In one embodiment, the inhibition of the extrinsic coagulation pathway treats and / or prevents a disease or condition associated with activation of the extrinsic coagulation pathway. In one embodiment, the inhibition of the extrinsic coagulation pathway treats and / or prevents thrombosis associated with activation of the extrinsic coagulation pathway. In one embodiment, the inhibition of the extrinsic coagulation pathway treats and / or prevents venous thrombosis. In one embodiment, the inhibition of the extrinsic coagulation pathway treats and / or prevents arterial thrombosis. In one embodiment, the inhibition of the extrinsic coagulation pathway treats and / or prevents cancer-related thrombosis. In one embodiment, the inhibition of the extrinsic coagulation pathway treats and / or prevents stroke-related thrombosis. In one embodiment, the inhibition of the extrinsic pathway treats and / or prevents an inflammatory disease associated with thrombotic propensity. In one embodiment, the inhibition of the extrinsic pathway treats and / or prevents thromboinflammation.

[0029] The present invention also relates to a pharmaceutical composition comprising a protein or polypeptide comprising a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 and at least one pharmaceutically acceptable excipient, for use in inhibiting the extrinsic coagulation pathway in a subject in need thereof as described herein above.

[0030] The present invention also relates to a pharmaceutical comprising a protein or polypeptide comprising a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 for use in inhibiting the extrinsic coagulation pathway in a subject in need thereof as described herein above.

[0031] The present invention also relates to a kit comprising a protein or polypeptide comprising a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 for use in inhibiting the extrinsic coagulation pathway in a subject in need thereof as described herein above.

[0032] The present invention also relates to a medical device coated with an isolated polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 for use in inhibiting the extrinsic coagulation pathway in a subject in need thereof as described herein above.

[0033] definition In the present invention, the following terms have the following meanings:

[0034] The term "about" preceding a value means ±10% of said value.

[0035] The term "amino acid substitution" refers to the replacement of one amino acid in a polypeptide with another amino acid. In one embodiment, one amino acid is replaced with another amino acid having similar structural and / or chemical properties (e.g., a conservative amino acid substitution). "Conservative amino acid substitutions" can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions involve exchanging a member of one of these classes for another class. For example, amino acid substitutions can also result in the replacement of one amino acid with another amino acid having different structural and / or chemical properties, for example, replacing an amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., basic). Amino acid substitutions can be effected by genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods of altering the side chain groups of amino acids by methods other than genetic engineering, such as chemical modification, can also be useful.

[0036] The term "identity" refers to the measurement of the identity of nucleotide or amino acid sequences. Generally, these sequences are aligned to obtain the highest degree of correspondence. "Identity" itself has a recognized meaning in the art and can be calculated using published techniques. See, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds, Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, von Heijne, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds, M Stockton Press, New York, 1991. There are many methods for measuring the identity between two polynucleotide or polypeptide sequences, and the term "identity" is well known to those skilled in the art (Carillo and Lipton, SIAM J Applied Math, 1998, 48:1073). Commonly used methods for determining identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994; and Carillo and Lipton, SIAM J Applied Math, 1998, 48:1073. Methods for determining identity and similarity are codified in computer programs.Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al., J Molec Biol, 1990, 215:403). Most preferably, the program used to determine the level of identity was the GAP program, which is used in the following examples.

[0037] For example, a polynucleotide having a nucleotide sequence with at least, for example, 95% "identity" with a reference nucleotide sequence is intended to be identical to the reference sequence, except that the nucleotide sequence of this polynucleotide may contain an average of up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' end of the reference nucleotide sequence, or anywhere between these end positions, or may be individually dispersed among the nucleotide sequences of the reference sequence, or may be dispersed in one or more contiguous groups within the reference sequence.

[0038] The term "peptide linker," also known as a "spacer peptide," refers to a peptide used to link two peptides or polypeptides together. In one embodiment, a peptide linker of the invention comprises 3 to 50 amino acids. Peptide linkers are known in the art or described herein.

[0039] The term "pharmaceutically acceptable excipient" refers to an excipient that does not produce adverse, allergic, or other undesirable reactions when administered to animals, preferably humans. This includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation aid. For human administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biologics standard.

[0040] The term "polynucleotide" refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. Furthermore, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. A variety of modifications have been made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also encompasses shorter polynucleotides often called oligonucleotides.

[0041] The term "polypeptide" refers to any peptide or protein containing two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., a peptide isostere. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids.

[0042] The term "protein" refers to a sequence of more than 100 amino acids and / or a multimeric entity. Proteins of the present invention are not limited to products of a particular length. The terms "polypeptide" or "protein" do not refer to or exclude naturally occurring and non-naturally occurring post-expression modifications of proteins, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide or protein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. It is understood that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide or protein. A given polypeptide or protein can also contain many types of modifications. Polypeptides or proteins can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides or proteins can result from post-translation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, formation of a GPI anchor, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, e.g., arginylation, and ubiquitination.See, e.g., "Proteins - structure and molecular properties," 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York, 1993; Wolt, F., "Posttranslational Protein Modifications: Perspectives and Prospects," Posttranslational covalent modification of proteins, B.C. Johnson, Ed., Academic Press, New York, 1983, pp. 1-12; Seifter et al., "Analysis for protein modifications and nonprotein cofactors," Meth Enzymol, 1990, 182:626-646; Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging," Ann NY Acad Sci, 1992, 663:48-62. The protein may be an entire protein or a subsequence thereof.

[0043] An "isolated protein or polypeptide" refers to a protein or polypeptide that has been identified and separated and / or recovered from a component of its natural environment. In a preferred embodiment, an isolated protein or polypeptide is (1) to greater than 80, 85, 90, or 95% by weight of protein or polypeptide as determined by the Lowry method, most preferably to greater than 96, 97, 98, or 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) SDS-PAGE under reducing or non-reducing conditions using Coomassie blue staining, preferably silver staining, until homogeneous. It is refined.

[0044] Isolated protein or polypeptide includes protein in situ within recombinant cells since at least one component of the protein's or polypeptide's natural environment will not be present. Ordinarily, however, isolated protein or polypeptide will be prepared by at least one purification step.

[0045] The term "fusion protein" refers to a molecule comprising two or more proteins or fragments thereof covalently linked via their respective peptide backbones, most preferably resulting from genetic expression of polynucleotide molecules encoding these proteins. The polypeptides forming the fusion protein are usually linked C-terminally to N-terminally, but they can also be linked C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally. The polypeptides of the fusion protein can be fused in any order.

[0046] The term "fused" refers to components that are joined by a peptide bond, either directly or via one or more peptide linkers.

[0047] The term "native Ir-CPI" refers to a naturally occurring Ir-CPI, as opposed to a "modified Ir-CPI" that has been modified from a naturally occurring Ir-CPI to alter one or more of its properties, such as stability. A modified Ir-CPI polypeptide can include, for example, an amino acid sequence modification, such as an amino acid substitution, deletion, or insertion.

[0048] The term "subject" refers to a mammal, preferably a human. In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting or seeking medical attention, or who has been / is / may be the subject of medical attention in the past / present / future, or who is being monitored for the development of a disease or condition. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age).

[0049] The term "therapeutically effective amount" means a level or amount of an agent intended to (1) slow or prevent a disease or condition associated with neutrophil recruitment, neutrophil activation, or activation of the extrinsic coagulation pathway; (2) slow or stop the progression, exacerbation, or worsening of one or more symptoms of a disease or condition associated with neutrophil recruitment, neutrophil activation, or activation of the extrinsic coagulation pathway; (3) cause an improvement in the symptoms of a disease or condition associated with neutrophil recruitment, neutrophil activation, or activation of the extrinsic coagulation pathway; (4) reduce the severity or frequency of a disease or condition associated with neutrophil recruitment, neutrophil activation, or activation of the extrinsic coagulation pathway; or (5) prevent a disease or condition associated with neutrophil recruitment, neutrophil activation, or activation of the extrinsic coagulation pathway, without causing significant negative or harmful side effects to the target. In one embodiment, the therapeutically effective amount is administered prior to the onset of a disease or condition associated with neutrophil recruitment, neutrophil activation, or activation of the extrinsic coagulation pathway for a preventative or prophylactic effect.

[0050] The terms "treating" or "treatment" or "alleviation" refer to both therapeutic treatment and preventative or prophylactic measures, where the purpose is to prevent or slow (attenuate) a disease or condition associated with neutrophil recruitment, neutrophil activation, or the extrinsic coagulation pathway. Those in need of treatment include those already suffering from a disease or condition associated with neutrophil recruitment, neutrophil activation, or the extrinsic coagulation pathway, as well as those prone to have a disease or condition associated with neutrophil recruitment, neutrophil activation, or the extrinsic coagulation pathway, or those in whom a disease or condition associated with neutrophil recruitment, neutrophil activation, or the extrinsic coagulation pathway is to be prevented. A subject or mammal is successfully "treated" for infection if, after administering a therapeutic amount of a protein or polypeptide according to the methods of the invention, the patient exhibits an observable and / or measurable reduction or absence of one or more of the following: a reduction in the number of pathogenic cells; a reduction in the percentage of the total number of cells that are pathogenic; and / or alleviation to some extent of one or more of the symptoms associated with a disease or condition associated with neutrophil recruitment, neutrophil activation, or the extrinsic coagulation pathway; a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement of a disease or condition associated with neutrophil recruitment, neutrophil activation, or the extrinsic coagulation pathway can be readily measured by routine techniques familiar to physicians.

[0051] The term "variant" refers to a polynucleotide or polypeptide that differs from the respective reference polynucleotide or polypeptide but retains essential properties. A typical polynucleotide variant differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations of the polypeptide encoded by the reference sequence, as described below. A typical polypeptide variant differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. A variant and a reference polypeptide can differ in amino acid sequence by one or more substitutions (preferably conservative substitutions), additions, or deletions in any combination. A substituted or inserted amino acid residue may or may not be an amino acid encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring variant, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be produced by mutagenesis techniques or by direct synthesis. A variant must retain one or more of the biological activities of the reference polypeptide.

[0052] Detailed Description Thus, the present invention relates to proteins or polypeptides comprising tick salivary gland polypeptides or fragments or variants thereof, and their uses for inhibiting the extrinsic coagulation pathway and thereby treating and / or preventing diseases and conditions associated with the extrinsic coagulation pathway, in particular treating and / or preventing thrombi associated with activation of the extrinsic coagulation pathway.

[0053] In one embodiment, the present invention relates to a protein or polypeptide comprising a tick salivary gland polypeptide or a fragment or variant thereof, and its use for inhibiting a cellular portion of the extrinsic coagulation pathway. In one embodiment, the term "cellular portion of the extrinsic coagulation pathway" refers to platelet recruitment, neutrophil activation, and / or neutrophil recruitment, and / or NET formation. Thus, in one embodiment, inhibiting a cellular portion of the extrinsic coagulation pathway comprises inhibiting platelet recruitment, neutrophil activation, and / or neutrophil recruitment, and / or NET formation.

[0054] In one embodiment, inhibiting the extrinsic coagulation pathway comprises inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis. Thus, in one embodiment, the present invention relates to the use of a protein or polypeptide comprising a tick salivary gland polypeptide or a fragment or variant thereof for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and the use thereof. The present invention also relates to a protein or polypeptide of the present invention for treating and / or preventing diseases and conditions associated with platelet recruitment, neutrophil activation, neutrophil recruitment, and / or NETosis.

[0055] In one embodiment, inhibiting the extrinsic coagulation pathway, in particular inhibiting the cellular portion of the extrinsic coagulation pathway, comprises inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0056] In one embodiment, the present invention relates to the use of a protein or polypeptide comprising a tick salivary gland polypeptide or a fragment or variant thereof for inhibiting the extrinsic coagulation pathway, preferably the cellular portion of the extrinsic coagulation pathway, or for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0057] In one embodiment, inhibiting the extrinsic coagulation pathway, and in particular inhibiting the cellular portion of the extrinsic coagulation pathway, does not include inhibiting tissue factors specific to the extrinsic coagulation pathway, such as tissue factor. In one embodiment, inhibiting the extrinsic coagulation pathway, and in particular inhibiting the cellular portion of the extrinsic coagulation pathway, does not include inhibiting coagulation factors specific to the extrinsic coagulation pathway.

[0058] In one embodiment, a protein or polypeptide of the invention is an isolated protein or polypeptide.

[0059] In one embodiment, the tick salivary gland polypeptide of the present invention is an Ir-CPI (tick contact phase inhibitor). As used herein, Ir-CPI may also be referred to as IrCPI.

[0060] In one embodiment, an Ir-CPI polypeptide of the invention has an amino acid sequence that comprises or consists of SEQ ID NO: 1. In one embodiment, an Ir-CPI polypeptide of the invention is encoded by a cDNA that corresponds to the amino acid sequence of SEQ ID NO: 1.

[0061] In one embodiment, the amino acid sequence of an Ir-CPI polypeptide of the invention is at least 75% identical, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1.

[0062] In one embodiment, fragments of Ir-CPI polypeptides are also included in the present invention. As used herein, the term "fragment" refers to a polypeptide having the same amino acid sequence as part, but not all, of the amino acid sequence of an Ir-CPI polypeptide described above. As with Ir-CPI polypeptides, fragments may be "free-standing" or may be included within a larger polypeptide that forms a portion or region, most preferably as a single, contiguous region. Representative examples of polypeptide fragments of the present invention include, for example, fragments of about 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of the polypeptide. In this context, "about" includes the specifically stated range, which may be several, 5, 4, 3, 2, or 1 amino acid greater or less at either or both ends.

[0063] Preferred fragments include, but are not limited to, truncated polypeptides having the amino acid sequence of an Ir-CPI polypeptide, except for the deletion of a contiguous series of residues including the amino terminus, or a contiguous series of residues including the carboxyl terminus and / or transmembrane region, or a deletion of two contiguous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional properties, such as fragments containing α-helix and α-helix-forming regions, β-sheet and β-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, amphipathic α regions, amphipathic β regions, flexible regions, surface-forming regions, substrate-binding regions, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate the activity of an Ir-CPI polypeptide, including fragments with similar activity, improved activity, or decreased undesirable activity.

[0064] In one embodiment, all of these polypeptide fragments retain some of the biological activity of the Ir-CPI polypeptide.

[0065] In one embodiment, variants of Ir-CPI polypeptides are also included in the present invention. Preferred variants differ from Ir-CPI polypeptides by conservative amino acid substitutions, i.e., variants in which a residue is replaced with another residue of similar characteristics. Typical such substitutions are among Ala, Val, Leu, and Ile; among Ser and Thr; among acidic residues Asp and Glu; among Asn and Gln; and among basic residues Lys and Arg; or among aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5 to 10, 1 to 5, or 1 to 2 amino acids are substituted, deleted, or added in any combination. Most preferred variants are naturally occurring allelic variants of Ir-CPI polypeptides present in the salivary glands of ticks.

[0066] In one embodiment, an Ir-CPI polypeptide of the present invention has an amino acid sequence that includes a mutation resulting in the replacement of aspartic acid with glutamine at the position corresponding to position 54 of the amino acid sequence of SEQ ID NO: 1 to prevent N-glycosylation. In one embodiment, an Ir-CPI polypeptide of the present invention has an amino acid sequence that includes or consists of the sequence of SEQ ID NO: 2.

[0067] In one embodiment, the amino acid sequence of an Ir-CPI polypeptide of the invention is at least 75%, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2.

[0068] In one embodiment, the Ir-CPI polypeptides of the present invention can be prepared by any suitable method. Examples of peptides prepared by suitable methods include, but are not limited to, isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

[0069] In one embodiment, the protein of the invention is a fusion protein comprising Ir-CPI.

[0070] As used herein, the term "fusion protein" refers to a fusion of an Ir-CPI polypeptide, as described hereinabove, with at least one other polypeptide.

[0071] In one embodiment, the Ir-CPI polypeptide is linked to at least one other polypeptide in such a way as to produce a single protein that retains the biological activity of Ir-CPI.

[0072] In one embodiment, the polypeptides of the fusion protein are linked C-terminally to N-terminally. In another embodiment, the components of the fusion protein are linked C-terminally to C-terminally. In another embodiment, the components of the fusion protein are linked N-terminally to N-terminally. In another embodiment, the components of the fusion protein are linked N-terminally to C-terminally.

[0073] In one embodiment, the polypeptides of the fusion protein may be fused in any order.

[0074] In one embodiment, the polypeptides are arranged in a single continuous polypeptide chain.

[0075] In one embodiment, the fusion protein of the invention further comprises at least one peptide linker. In one embodiment, the polypeptides of the fusion protein of the invention are linked to each other via one or more peptide linkers.

[0076] In one embodiment, the term "Ir-CPI fusion protein" or "fusion protein" refers indiscriminately to a fusion protein without a linker sequence or to a protein with a linker sequence.

[0077] In one embodiment, the linker peptide provides greater physical separation between the two moieties, thus maximizing the availability of the tick salivary gland polypeptide. The linker peptide may be composed of amino acids that are flexible or more rigid.

[0078] In one embodiment, the peptide linker of the present invention comprises 1 to 60 amino acids, preferably 2 to 50 amino acids, and more preferably 4 to 40 amino acids. In one embodiment, the peptide linker of the present invention comprises 5 to 50 amino acids, 10 to 50 amino acids, 15 to 50 amino acids, or 20 to 50 amino acids. In another embodiment, the peptide linker of the present invention comprises 6 to 20 amino acids, 8 to 20 amino acids, or 10 to 20 amino acids. In another embodiment, the peptide linker of the present invention comprises 2 to 10 amino acids, 5 to 20 amino acids, 10 to 30 amino acids, or 15 to 40 amino acids.

[0079] In one embodiment, the peptide linker of the invention is not an in vivo cleavable linker. In one embodiment, the peptide linker of the invention is an in vivo cleavable linker.

[0080] The fusion proteins of the present invention can be obtained, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or by recombinant production. In recombinant production, one or more polynucleotides encoding the fusion protein (fragments), e.g., as described herein above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional techniques.

[0081] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the fusion protein (fragment) along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., "MOLECULAR CLONING: A LABORATORY MANUAL," Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY," Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector can be part of a plasmid, a virus, or a nucleic acid fragment. The expression vector contains an expression cassette into which a polynucleotide encoding the fusion protein (fragment) (i.e., a coding region) is cloned in operative association with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, and when present may be considered part of the coding region, all adjacent sequences, e.g., promoters, ribosome binding sites, transcription terminators, introns, 5'- and 3'-untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present on a single polynucleotide construct, e.g., a single vector, or on separate polynucleotide constructs, e.g., separate (different) vectors. Furthermore, any vector may contain a single coding region or may contain two or more coding regions; for example, a vector of the invention may encode one or more polypeptides that are post- or co-translationally separated into the final protein via proteolytic cleavage.Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions, or variants or derivatives thereof, fused or unfused to the polynucleotide encoding the fusion protein (fragment) of the present invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable linkage occurs when a coding region for a gene product, e.g., a polypeptide, is linked to one or more regulatory sequences in such a way that expression of the gene product is placed under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the nature of the expression control sequences directing expression of the gene product or the nature of the DNA template being transcribed. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of this nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to a promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (e.g., the immediate early promoter associated with intron A), promoters and enhancer segments from simian virus 40 (e.g., the early promoter), and promoters and enhancer segments from retroviruses (e.g., Rous sarcoma virus).Other transcription control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit α-globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., promoters inducible tetracyclines). Similarly, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). Expression cassettes may also include other features, such as origins of replication and / or chromosomal integration elements, such as the long terminal repeats (LTRs) of retroviruses or the inverted terminal repeats (ITRs) of adeno-associated viruses (AAV).

[0082] Fusion proteins prepared as described herein can be purified by techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and molecular sieve chromatography. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those of skill in the art. Affinity chromatography purification can use an antibody, ligand, receptor, or antigen to which the fusion protein binds. The purity of the fusion protein can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-pressure liquid chromatography, and the like.

[0083] The present invention also relates to polynucleotides or nucleic acids encoding proteins or polypeptides comprising the Ir-CPI polypeptides, fragments or variants thereof described hereinabove, and uses thereof for inhibiting the extrinsic coagulation pathway, preferably for inhibiting the cellular portion of the extrinsic coagulation pathway, or for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and thereby treating and / or preventing diseases and conditions associated with the extrinsic coagulation pathway, or platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0084] In one embodiment, a polynucleotide or nucleic acid of the invention has a nucleotide sequence that comprises or consists of the sequence of SEQ ID NO:3, or that consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3.

[0085] In one embodiment, a polynucleotide encoding a polypeptide of the invention has the nucleotide sequence set forth in SEQ ID NO:3.

[0086] In one embodiment, polynucleotides encoding the fusion proteins of the invention can be expressed as a single polynucleotide encoding the entire fusion protein, or as multiple (e.g., two or more) co-expressed polynucleotides. The polypeptides encoded by the co-expressed polynucleotides can be linked, for example, via disulfide bonds or other means, to form a functional fusion protein.

[0087] In one embodiment, the polynucleotide or nucleic acid is DNA. In another embodiment, the polynucleotide or nucleic acid is RNA, for example, RNA in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0088] Another object of the present invention is a vector comprising one or more polynucleotides encoding the proteins or polypeptides of the present invention, and its use, for inhibiting the extrinsic coagulation pathway, preferably the cellular portion of the extrinsic coagulation pathway, or for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, and thereby treating and / or preventing diseases and conditions associated with the extrinsic coagulation pathway, or platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis. In a preferred embodiment, the vector of the present invention is an expression vector.

[0089] The present invention also relates to a composition comprising a protein or polypeptide, a polynucleotide or nucleic acid, or a vector as described herein above.

[0090] The present invention further relates to a pharmaceutical composition comprising a protein or polypeptide, a polynucleotide or nucleic acid, or a vector of the present invention and at least one pharmaceutically acceptable excipient.

[0091] Pharmaceutically acceptable excipients that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0092] Another object of the present invention is a medicament comprising a protein or polypeptide, a polynucleotide or nucleic acid, a vector, a composition, or a pharmaceutical composition as described herein above.

[0093] In one embodiment, the pharmaceutical composition or medicament of the present invention comprises a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, or vector as described herein above.

[0094] A further object of the present invention is a medical device coated with a protein or polypeptide as described herein above.

[0095] Furthermore, another object of the present invention is the use of a protein or polypeptide as described herein above for coating a medical device. Yet another object of the present invention is a protein or polypeptide as described herein above for coating a medical device.

[0096] Thus, the present invention also relates to medical devices coated with the proteins or polypeptides described herein above.

[0097] Examples of medical devices that may be coated with a protein or polypeptide of the present invention include, but are not limited to, coronary stents, artificial hearts, artificial heart valves, central venous lines, cardioplegia delivery systems, dilators, tunneling devices, stent graft cannulae, catheters, extracorporeal circulation systems including, but not limited to, extracorporeal membrane oxygenation systems, (auto)transfusion systems, arterial filters, hemodialysis systems, plasmapheresis systems; medical devices used for blood collection outside the body; and / or accessories of any one of the above devices including, but not limited to, tubing, cannulae, centrifugal pumps, valves, ports, and / or diverters, arterial stents for stenosis and aneurysms, and left ventricular assist devices.

[0098] The present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention to inhibit the extrinsic coagulation pathway, preferably to inhibit the cellular portion of the extrinsic coagulation pathway, more preferably to treat a thrombotic disorder associated with the extrinsic coagulation pathway.

[0099] As described hereinabove, in one embodiment, inhibiting the extrinsic coagulation pathway, and in particular inhibiting the cellular portion of the extrinsic coagulation pathway, includes inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis.

[0100] Unexpectedly, the applicant has discovered that the Ir-CPI polypeptides of the present invention inhibit thrombosis associated with the extrinsic coagulation pathway by inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and NETosis.

[0101] Indeed, we have demonstrated that Ir-CPI polypeptides have a striking protective effect in an experimental animal model in which cremaster arterioles are laser-injured, a condition in which the extrinsic coagulation pathway is known to be a key factor in thrombotic events (Darbousset et al., 2012). In this model, Ir-CPI inhibits not only fibrin production but also platelet recruitment at the lesion site. Furthermore, Ir-CPI polypeptides were active when applied to tumor-bearing mice in a model in which laser injury is primarily dependent on tissue factor expressed by cancer cell-derived microparticles (Thomas et al., 2009). Because neutrophils play a key role in activating tissue factor-dependent pathways in the laser injury model (Darbousset et al., 2012), the presence and activation of neutrophils were compared in the presence or absence of Ir-CPI. The present applicants observed in vivo that Ir-CPI significantly inhibited the amount of neutrophils accumulating and the activity of neutrophil elastase at injury sites. The present applicants also demonstrated in vitro that Ir-CPI significantly inhibited the activation of neutrophils primed with TNFα (tumor necrosis factor α) or incubated with adenosine triphosphate (ATP), as observed through the detection of CD11b, CD11b-activated, or Ly6G expression on the neutrophil surface. Following activation, neutrophils can produce "neutrophil extracellular traps" (NETs), which are structures of histone-coated chromatin filaments, proteases, and granule and cytoplasmic proteins. Research suggests that NETs contribute to thrombosis by promoting fibrin deposition and platelet aggregation. Applicants have also observed in vitro that Ir-CPI significantly reduced the formation of NETs (i.e., NETosis) by TNFα-primed and PAF-activated neutrophils. The contribution of neutrophils in the development of thrombotic events has been demonstrated in various experimental paradigms.The influence of autophagy in NET release and tissue factor delivery to NETs, ​​as well as the association between NETs and thrombosis, suggest a critical role for neutrophils in the interplay between inflammatory and thrombotic pathways (Demers et al., 2012; Leal et al., 2017; Mauracher et al., 2018). Neutrophil depletion results in fewer thrombi in animal models of thrombosis, demonstrating the contribution of these cells to thrombosis (von Bruhl et al., 2012). Expression of TFs produced and / or acquired by neutrophils suggests their involvement in the pathogenesis of the thrombotic events that characterize several inflammatory disorders. Thus, Ir-CPI inhibits thrombus formation following activation of the extrinsic coagulation pathway through inhibition of platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NET formation, rather than through inhibition of coagulation factors specific to this pathway, such as tissue factor.

[0102] Thus, unexpectedly, Applicants have discovered that Ir-CPI polypeptides may be used for therapeutic indications aimed at preventing and / or treating thrombotic events in diseases and conditions in which thrombosis and / or coagulation depend on platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NET formation. Applicants have also shown that Ir-CPI may be used for therapeutic indications aimed at preventing and / or treating thrombotic events in diseases and conditions associated with activation of the extrinsic coagulation pathway.

[0103] The present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention to treat and / or prevent diseases and conditions associated with platelet recruitment, neutrophil activation, neutrophil recruitment, and / or NET formation in a subject in need thereof.

[0104] In one embodiment, treating and / or preventing diseases and conditions associated with platelet recruitment, neutrophil activation, neutrophil recruitment, and / or NET formation in a subject in need thereof comprises treating and / or preventing diseases and conditions associated with activation of the extrinsic coagulation pathway.

[0105] The present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing diseases and conditions associated with activation of the extrinsic coagulation pathway, preferably thrombosis associated with the extrinsic coagulation pathway, in a subject in need thereof.

[0106] In one embodiment, diseases and conditions associated with activation of the extrinsic coagulation pathway are selected from the group comprising or consisting of thrombosis (including venous thrombosis and arterial thrombosis), inflammatory diseases associated with thrombotic tendency, thromboinflammation (including device-induced thromboinflammation), diseases and conditions associated with cardiac surgical intervention, cardiovascular disease, cancer, and metastasis.

[0107] Diseases and conditions associated with activation of the extrinsic coagulation pathway include inflammatory diseases with thrombotic tendencies. As used herein, the term "inflammatory diseases with thrombotic tendencies" may be interchanged with "thrombotic complications in inflammatory diseases."

[0108] In one embodiment, the invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing thrombosis associated with activation of the extrinsic coagulation pathway in a subject in need thereof.

[0109] In one embodiment, the thrombosis associated with activation of the extrinsic coagulation pathway is selected from the group consisting of venous thrombosis, cancer-related thrombosis, and stroke-related thrombosis. In one embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the present invention to treat and / or prevent venous thrombosis. In another embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the present invention to treat and / or prevent cancer-related thrombosis. In another embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the present invention to treat and / or prevent stroke-related thrombosis.

[0110] Thus, in one embodiment, the present invention relates to the use of a protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the present invention for treating and / or preventing an inflammatory disease associated with thrombotic tendencies in a subject in need thereof.

[0111] In one embodiment, the present invention relates to the use of a protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing thrombosis or thromboinflammation associated with an inflammatory disease in a subject in need thereof.

[0112] In one embodiment, the present invention provides a method for treating cancer-associated thrombosis, metastasis-associated thrombosis, stroke-associated thrombosis, Behcet's disease (BD), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Takayasu's arteritis, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, familial Mediterranean fever, thromboangiitis obliterans (TAO), sepsis, inflammatory bowel disease, atherosclerosis and plaque rupture, coronary artery disease, acute myocardial infarction, thrombus formation after brain injury, medical device-induced thromboinflammation in contact with blood, thrombosis induced by catheterization and / or stent positioning procedures at the site of a localized vascular stenosis, The present invention relates to the use of the protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the present invention for treating and / or preventing inflammatory diseases or thromboinflammation accompanied by a tendency to thrombosis, selected from the group consisting of: thrombosis induced by extracorporeal circulation, heparin-induced thrombocytopenia (HIT), immunological thrombosis formation, thrombosis associated with preeclampsia, thrombotic complications in cell and cell cluster transplantation and whole organ transplantation or grafts, venous thromboembolism, aneurysm, and ischemia-reperfusion syndrome in skeletal muscle.

[0113] In one embodiment, the thromboinflammation is selected from the group consisting of thrombosis induced by catheterization and / or stent placement procedures at the site of a localized vascular stenosis, atherosclerosis, plaque rupture, coronary artery disease, and device-induced thromboinflammation. In one embodiment, device-induced thromboinflammation includes thromboinflammation induced by a medical device in contact with blood. In another embodiment, the thromboinflammation is selected from the group consisting of cancer-related thrombosis, metastasis-related thrombosis, and stroke-related thrombosis.

[0114] Diseases and conditions associated with activation of the extrinsic coagulation pathway include blood-contacting medical device-induced thromboinflammation, which in one embodiment includes, but is not limited to, inflammation induced by catheterization (e.g., percutaneous transluminal angioplasty) and / or stent placement procedures at the site of localized vascular stenosis, given the risk of atherosclerotic plaque rupture and the risk of subsequent re-thrombosis and catheter thrombosis after such intervention; and inflammation induced by extracorporeal circulation.

[0115] In one embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing device-induced thromboinflammation in a subject in need thereof.

[0116] Thus, in one embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention to treat and / or prevent catheterization (e.g., percutaneous transluminal angioplasty) and / or stent positioning procedures; and / or extracorporeal circulation-induced thrombosis at the site of localized vascular stenosis in a subject in need thereof.

[0117] Diseases and conditions associated with activation of the extrinsic coagulation pathway include thrombosis and / or coagulation associated with extracorporeal circulation performed in the context of cardiac surgical intervention (e.g., coronary artery bypass graft surgery (CABG), open-heart surgery).

[0118] Thus, in one embodiment, the present invention relates to the use of a protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing thrombosis and / or coagulation associated with extracorporeal circulation performed in the context of cardiac surgical intervention in a subject in need thereof.

[0119] Diseases and conditions associated with activation of the extrinsic coagulation pathway include cancer, particularly cancer-associated thrombosis.

[0120] Indeed, microparticles containing tissue factor and NETosis have also been implicated in cancer-associated thrombus formation ( Thomas et al., 2009 ; Demers et al., 2012 ; Mauracher et al., 2018 ).

[0121] Thus, in one embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing cancer and / or metastasis in a subject in need thereof.

[0122] In one embodiment, the cancer and / or metastasis is associated with the extrinsic coagulation pathway. In one embodiment, the cancer and / or metastasis is associated with thrombosis. In a particular embodiment, the cancer and / or metastasis is associated with thrombosis associated with the extrinsic coagulation pathway.

[0123] In one embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing thrombosis associated with cancer and / or metastasis in a subject in need thereof.

[0124] In one embodiment, the present invention relates to the use of a protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device of the invention for treating and / or preventing stroke-related thrombosis in a subject in need thereof.

[0125] The present invention also relates to a method for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition, or medicament, or coated medical device of the present invention.

[0126] The present invention further relates to a method for treating and / or preventing a disease or condition associated with platelet mobilization, neutrophil recruitment, neutrophil activation, and / or NETosis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition, or medicament, or coated medical device of the present invention.

[0127] In one embodiment, the method of the present invention is a method for treating and / or preventing diseases and conditions associated with activation of the extrinsic coagulation pathway selected from the group including or consisting of venous thrombosis, arterial thrombosis, thromboinflammation, cardiovascular disease, cancer, and metastasis.

[0128] The present invention also relates to a method for inhibiting the extrinsic coagulation pathway, preferably inhibiting the cellular portion of the extrinsic pathway, or for inhibiting platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition, or medicament, or coated medical device of the present invention.

[0129] The present invention further relates to a method for treating and / or preventing a disease or condition associated with platelet mobilization, neutrophil recruitment, neutrophil activation, and / or NETosis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition, or medicament, or coated medical device of the present invention.

[0130] The present invention further relates to a method for treating and / or preventing a disease or condition associated with the extrinsic coagulation pathway in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition or medicament, or coated medical device of the present invention.

[0131] In one embodiment, the method of the invention is a method for treating and / or preventing diseases and conditions associated with activation of the extrinsic coagulation pathway, or platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis, selected from the group including or consisting of thrombosis, inflammatory diseases with thrombosis tendency, device-induced thromboinflammation, diseases and conditions associated with cardiac surgical intervention, and cancer and metastasis.

[0132] Another object of the present invention is a method for treating and / or preventing inflammatory diseases associated with thrombotic tendency or thromboinflammation, in particular inflammatory diseases associated with thrombosis or thrombosis associated with inflammatory diseases, in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition or medicament, or coated medical device of the present invention.

[0133] In one embodiment, the method of the present invention is directed to treating inflammatory diseases such as thrombosis-associated inflammatory diseases, thrombosis associated with inflammatory diseases, cancer-associated thrombosis, metastasis-associated thrombosis, stroke-associated thrombosis, Behcet's disease (BD), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Takayasu's arteritis, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, familial Mediterranean fever, thromboangiitis obliterans (TAO), sepsis, inflammatory bowel disease, atherosclerosis and plaque rupture, coronary artery disease, acute myocardial infarction, thrombus formation after brain injury, blood-contacting medical device-induced thrombosis, and the like. A method for treating and / or preventing a disease or condition selected from the group including or consisting of thromboinflammation, thrombosis induced by catheterization and / or stent positioning procedures at sites of localized vascular stenosis, thrombosis induced by extracorporeal circulation, heparin-induced thrombocytopenia (HIT), immunological thrombosis formation, thrombosis associated with preeclampsia, thrombotic complications in cell and cell cluster transplantation and whole organ transplants or grafts, venous thromboembolism, aneurysms, and ischemia-reperfusion syndrome in skeletal muscle.

[0134] Another object of the present invention is a method for treating and / or preventing thrombosis in all catheterization (such as percutaneous transluminal angioplasty) and / or stent placement procedures at the site of localized vascular stenosis in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition or medicament, or coated medical device of the present invention.

[0135] Another object of the present invention is a method for treating and / or preventing thrombosis and / or coagulation associated with extracorporeal circulation performed in the context of cardiac surgical intervention in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition or medicament, or coated medical device of the invention.

[0136] Another object of the present invention is a method for treating cancer and / or metastasis, in particular cancer-associated thrombosis, in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of a protein or polypeptide, polynucleotide or nucleic acid, vector, pharmaceutical composition or medicament, or coated medical device of the present invention.

[0137] In one embodiment, the subject is susceptible to forming a thrombus, particularly a thrombus associated with the extrinsic coagulation pathway. In one embodiment, the subject is at risk of developing a thrombus, particularly a thrombus associated with the extrinsic coagulation pathway. In another embodiment, the subject has or has had a thrombus, particularly a thrombus associated with the extrinsic coagulation pathway.

[0138] Examples of the risk of developing thrombi, particularly thrombi associated with the extrinsic coagulation pathway, include, but are not limited to, the presence of inflammatory diseases associated with thrombosis tendency, the presence of atherosclerotic lesions requiring intervention aimed at enhancing local blood flow at the level of vascular stenosis, the performance of extracorporeal circulation, coronary artery bypass graft surgery (CABG) associated with cardiopulmonary bypass, and the presence of cancer.

[0139] Extracorporeal circulation may indeed mediate the generation of thrombin by activation of the extrinsic pathway through exposure of blood to air and wound TF and subsequent recirculation of this blood through open heart surgery suction used to reduce blood loss.

[0140] In one embodiment, the subject is susceptible to developing cancer and / or metastasis. In one embodiment, the subject is at risk of developing cancer and / or metastasis. In one embodiment, the subject has or has had cancer and / or metastasis.

[0141] In one embodiment, the subject has recently undergone surgery. In one embodiment, "recently" means within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or more. In another embodiment, the term "recently" means within 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, or 24 hours.

[0142] In another embodiment, the subject is scheduled to undergo surgery. "Scheduled to" in one embodiment means within 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, or 24 hours. In another embodiment, the term "planned to" means within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or more.

[0143] In one embodiment, the subject has not been previously treated for the thrombus, hi another embodiment, the subject has previously received one, two, or more other treatments for the thrombus.

[0144] As used herein, the term "treatment" includes preventative and therapeutic treatment.

[0145] In one embodiment, a subject of the present invention is an elderly person. As used herein, the term "elderly person" means that the subject is at least 50 years old, at least 55, 60, 65, 70, 75, 80, 85, or 90 years old.

[0146] In one embodiment, the subject is male. In another embodiment, the subject is female.

[0147] It should be understood that the total daily usage of the proteins, polynucleotides, vectors, compositions, pharmaceutical compositions, and medicaments of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular patient will vary depending on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific agent used; the particular composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific agent used; the duration of treatment; drugs used in combination or concomitantly with the specific agent used; and similar factors well known in the medical field. For example, it is well within the skill of one in the art to start administering an agent at a level lower than that required to achieve the desired therapeutic effect and gradually increase this dosage until the desired effect is achieved. However, the daily dosage of the product can vary over a wide range, from about 10 to about 10,000 mg per adult per day, preferably from 100 to about 5,000 mg per adult per day, and more preferably from about 200 to about 2,000 mg per adult per day. Preferably, the compositions contain 10, 50, 100, 250, 500, 1000, and 2000 mg of the active ingredient, with dosage adjustment depending on the symptoms of the patient being treated. Pharmaceuticals typically contain about 10 to about 10,000 mg of the active ingredient, preferably 5 to about 5,000 mg, and more preferably about 10 to about 2,000 mg. An effective amount of the drug is typically supplied at a dosage level of 0.01 mg / kg to about 100 mg / kg of body weight per day, preferably about 0.05 mg / kg to 40 mg / kg of body weight per day, more preferably about 0.1 mg / kg to 20 mg / kg of body weight per day, and more preferably about 0.2 mg / kg to 1 mg / kg of body weight per day.

[0148] For use in administering to a subject, the compositions, pharmaceutical compositions, and medicaments of the present invention are formulated for administration to a subject. The compositions, pharmaceutical compositions, and medicaments of the present invention can be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, bucally, intravaginally, or via an implanted reservoir. As used herein, the term administration includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0149] In one embodiment, the composition, pharmaceutical composition or medicament of the present invention is in a form suitable for topical administration.

[0150] Examples of forms suitable for topical administration include, but are not limited to, liquid, paste, or solid compositions, more particularly in the form of aqueous solutions, drops, eye drops, ophthalmic solutions, dispersions, sprays, microcapsules, microparticles or nanoparticles, polymer patches, or sustained-release patches.

[0151] In one embodiment, the composition, pharmaceutical composition, or medicament of the present invention comprises one or more pharmaceutically acceptable carriers for formulation suitable for topical administration.

[0152] In one embodiment, the composition, pharmaceutical composition, or medicament of the present invention is in a form suitable for injection, such as intraocular, intramuscular, subcutaneous, intradermal, transdermal, or intravenous injection or infusion.

[0153] Examples of forms suitable for injection include, but are not limited to, liquid solutions, such as sterile aqueous solutions, dispersions, emulsions, suspensions, and solid forms suitable for use in preparing solutions or suspensions by adding a liquid prior to use, such as powders, liposomal forms, and the like.

[0154] The sterile injectable forms of the compositions, pharmaceutical compositions, and medicaments of the present invention may be aqueous or oily suspensions. These suspensions may be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Alternatively, the sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil, including synthetic mono- or diglycerides, may be used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersing agents commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for the formulation.

[0155] In certain embodiments, the composition, pharmaceutical composition, or medicament of the invention is in a form suitable for intraocular administration.

[0156] In one embodiment, the composition, pharmaceutical composition, or medicament of the present invention is administered to a subject in need thereof at least once a day. For example, the composition, pharmaceutical composition, or medicament of the present invention can be administered once a day, twice a day, or three times a day. In a preferred embodiment, the composition, pharmaceutical composition, or medicament of the present invention is administered to a subject in need thereof once a day.

[0157] In another embodiment, the composition, pharmaceutical composition, or medicament of the present invention is administered to a subject in need thereof at least once a week. For example, the composition, pharmaceutical composition, or medicament of the present invention can be administered once a week, twice a week, three times a week, four times a week, or up to seven times a week.

[0158] In another embodiment, the composition, pharmaceutical composition, or medicament of the present invention is administered to a subject in need thereof once a month, twice a month, every two months, every two or three months, twice a year, or once a year.

[0159] In one embodiment, the composition, pharmaceutical composition, or medicament of the present invention is administered to a subject in need thereof before exposure to a risk of developing a blood clot. In one embodiment, the term "before" refers to at least one week before exposure. In another embodiment, the term "before" refers to 5, 4, 3, 2, or 1 day before exposure. In another embodiment, the term "before" refers to 24, 18, 15, 12, 6, 4, 2, or 1 hour before exposure. In another embodiment, the term "before" refers to less than one hour before exposure, such as 45, 30, 15, 10, or 5 minutes before exposure, or at the moment of exposure. For example, the composition, pharmaceutical composition, or medicament of the present invention can be administered to a subject 24 hours, 12 hours, 6 hours, or 1 hour before long-term travel, or at the start of long-term travel.

[0160] In another embodiment, the composition, pharmaceutical composition, or medicament of the present invention is administered to a subject in need thereof after exposure to a risk of developing a thrombus. In one embodiment, the term "after" refers to 5, 10, 15, 30, or 45 minutes after exposure. In another embodiment, the term "after" refers to 1, 2, 4, 6, 12, 15, 18, or 14 hours after exposure. In another embodiment, the term "after" refers to 1, 2, 3, 4, or 5 days after exposure. In another embodiment, the term "after" refers to 1 week or later after exposure. For example, the composition, pharmaceutical composition, or medicament of the present invention can be administered to a subject immediately after a long trip, or 1, 2, 6, or 12 hours after a long trip.

[0161] In one embodiment, a medical device coated with a protein of the present invention is assembled for administration to a subject. The medical device coated with a protein of the present invention may be administered parenterally or topically, or may be implanted.

[0162] In one embodiment, a medical device coated with a protein of the present invention is implanted in the cardiac or circulatory system.

[0163] The present invention also relates to a kit comprising the protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition or medicament, or coated medical device according to the present invention.

[0164] In one embodiment, the kit of the invention further comprises a means for administering the protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device to a subject in need thereof.

[0165] In one embodiment, the kit of the present invention further comprises instructions for administering the protein or polypeptide, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or coated medical device to said subject.

[0166] In one embodiment, the kit of the present invention is used to inhibit the extrinsic coagulation pathway, particularly the cellular portion of the extrinsic coagulation pathway, or to inhibit platelet recruitment, neutrophil recruitment, neutrophil activation, and / or NETosis. In one embodiment, the kit of the present invention is used to treat and / or prevent diseases and conditions associated with activation of the extrinsic coagulation pathway. In one embodiment, the kit of the present invention is used to treat and / or prevent thrombosis associated with the extrinsic coagulation pathway, preferably cancer-related thrombosis. In one embodiment, the kit of the present invention is used to prevent thrombosis formation and / or thrombus growth associated with the extrinsic coagulation pathway. In one embodiment, the kit of the present invention is used to treat and / or prevent cancer and / or metastasis. [Brief explanation of the drawings]

[0167] [Figure 1] Figure 1 is a composite of graphs showing in vivo detection of platelets (A) and fibrin (B) after thrombus formation was induced by vessel wall injury in mice. Fibrin and platelets were detected using an anti-fibrin antibody conjugated with Alexa Fluor 488 and an anti-GPIb antibody labeled with DyLight649, respectively. Median integrated fluorescence over time (short-term) is presented for 53 (control) and 54 (Ir-CPI) thrombi produced in five mice per group. AU: arbitrary units. [Figure 2]Figure 2 is a composite of representative fluorescence microscopy images taken in vivo 30, 60, 120, and 180 seconds after laser injury of the vessel wall to induce thrombus formation in mice treated with NaCl (control) or Ir-CPI. Fibrin and platelets were detected using an anti-fibrin antibody conjugated with Alexa Fluor 488 and an anti-GPIb antibody labeled with DyLight649, respectively. The area enclosed by the dashed line represents platelet accumulation, and the area enclosed by the dotted line represents fibrin accumulation. Bar: 10 μm. [Figure 3] Figure 3 is a graph showing in vivo detection of fibrin over a 90-minute time course following thrombus formation induction in mice via vessel wall injury. Fibrin was detected using an anti-fibrin antibody conjugated with Alexa Fluor 488. Median integrated fluorescence over time (long-term) is presented for 53 (control) and 50 (Ir-CPI) thrombi produced in 9 mice (n = 9 mice per group). AU: arbitrary units. [Figure 4] Figure 4 is a graph showing in vivo detection of fibrin over a 90-minute time course after thrombus formation was induced by vessel wall injury in mice. Fibrin was detected using an anti-fibrin antibody conjugated with Alexa Fluor 488. Median values ​​within interquartile ranges are presented for control-treated mice (n=53 thrombi) and Ir-CPI-treated mice (n=50 thrombi) generated in 9 mice (n=9 mice per group). AU: arbitrary units. [Figure 5] Figure 5 is a composite of representative fluorescence microscopy images taken in vivo 30, 60, 120, and 180 seconds after laser injury of the vessel wall to induce thrombus formation in mice treated with NaCl (control) or Ir-CPI. PMNs were detected using a PE-labeled anti-Ly6G antibody. The area enclosed by the dashed line represents PMN accumulation. Bar: 10 μm. [Figure 6]Figure 6 is a composite of graphs showing in vivo detection of PMNs after thrombus formation was induced by vessel wall injury in mice. PMN accumulation was assessed using a PE-labeled anti-Ly6G antibody. The median integrated fluorescence over time (A) and the corresponding area under the curve (B) are presented for 26 (control) or 27 (Ir-CPI) thrombi generated in eight mice (n = 4 mice per group). AU: arbitrary units. Statistical analysis was performed using the Mann-Whitney test (***p<0.001). [Figure 7] Figure 7 is a graph showing in vivo detection of neutrophil elastase activity after thrombus formation was induced by vessel wall injury in mice. Neutrophil elastase activity was assessed by using a BODIPY-FL-labeled DQ-elastin conjugate fragment as a fluorescently labeled substrate for elastase. Median integrated fluorescence over time is presented for 31 (control) or 23 (Ir-CPI) thrombi produced in 7 mice (n=3 control mice and n=4 Ir-CPI-treated mice). AU: arbitrary units. [Figure 8] Figure 8 is a graph showing tumor volume after transplantation of murine Panc02 cells in mice. Before the laser injury experiment, tumor volume was evaluated in mice bearing ectopic pancreatic tumors induced by Panc02 cells. In black, mice received control treatment, i.e., NaCl (n=5), and in gray, mice received Ir-CPI treatment (n=4). The mean tumor volume ± SEM is shown. [Figure 9]Figure 9 is a composite of graphs showing in vivo platelet detection and fibrin production after thrombus formation was induced in mice by vessel wall injury. Fibrin and platelets were detected using an anti-fibrin antibody conjugated with Alexa Fluor 488 and an anti-GPIb antibody labeled with DyLight649. The area under the curve for platelets (A) or the integrated fluorescence intensity of fibrin (B) is shown for 41 (NaCl WT, i.e., cancer-free; n = 3 mice) or 40 (NaCl cancer; n = 6 mice) thrombi produced. Statistical analysis was performed using the Mann-Whitney test (*p < 0.05). [Figure 10] Figure 10 is a composite of graphs showing in vivo detection of fibrin (A) and platelets (B) after thrombus formation was induced in tumor-bearing mice by vessel wall injury in mice treated with 20.5 mg / kg (bolus) + 3.7 mg / kg / h (infusion) (i.e., high dose). Fibrin and platelets were detected using an Alexa Fluor 488-conjugated anti-fibrin antibody and a DyLight649-labeled anti-GPIb antibody, respectively. Median integrated fluorescence over time is presented for 44 (corresponding to administration of NaCl to tumor-free mice, NaCl cancer-free), 34 (corresponding to administration of NaCl to tumor-bearing mice, NaCl cancer), and 39 (corresponding to administration of Ir-CPI to tumor-bearing mice, Ir-CPI cancer) thrombi produced in 14 mice (n = 5 for NaCl cancer-free mice, n = 5 for NaCl cancer mice, and n = 4 for Ir-CPI cancer mice). AU: arbitrary units. [Figure 11]Figure 11 shows the integrated fluorescence intensity (area under the curve) of platelets present in 44 (NaCl tumor-free mice), 34 (NaCl tumor-bearing mice), and 39 (Ir-CPI tumor-bearing mice) thrombi produced in 14 mice treated with high doses of Ir-CPI (n = 5 NaCl tumor-free mice, n = 5 NaCl tumor-bearing mice, and n = 4 Ir-CPI tumor-bearing mice). Platelets were detected using an anti-GPIb antibody labeled with DyLight649. Statistical analysis was performed using the Mann-Whitney test (*p < 0.05; ***p < 0.001). [Figure 12] Figure 12 is a composite of graphs showing in vivo detection of fibrin (A) and platelets (B) after thrombus formation was induced in tumor-bearing mice by vessel wall injury in mice treated with 10.3 mg / kg (bolus) + 1.9 mg / kg / h (infusion) (i.e., low dose). Fibrin and platelets were detected using an Alexa Fluor 488-conjugated anti-fibrin antibody and a DyLight649-labeled anti-GPIb antibody, respectively. Median integrated fluorescence over time is presented for 41 (corresponding to administration of NaCl to tumor-free mice, NaCl cancer-free), 40 (corresponding to administration of NaCl to tumor-bearing mice, NaCl cancer), and 40 (corresponding to administration of Ir-CPI to tumor-bearing mice, Ir-CPI cancer) thrombi produced in 14 mice (n = 3 for NaCl cancer-free mice, n = 6 for NaCl cancer mice, and n = 5 for Ir-CPI cancer mice). AU: arbitrary units. [Figure 13]Figure 13 shows the integrated fluorescence intensity (area under the curve) of platelets present in thrombi of 41 (NaCl tumor-free mice), 40 (NaCl tumor-bearing mice), or 40 (Ir-CPI tumor-bearing mice) generated in 14 mice (n = 3 mice without NaCl tumor, n = 6 mice with NaCl tumor, and n = 5 mice with Ir-CPI tumor). Mice with Ir-CPI tumor were treated with 10.3 mg / kg (bolus) + 1.9 mg / kg / h (infusion) of Ir-CPI (i.e., low dose). Platelets were detected using an anti-GPIb antibody labeled with DyLight649. Statistical analysis was performed using the Mann-Whitney test (*p < 0.05; ***p < 0.001). [Figure 14] 14 is a graph showing the bleeding time (seconds) of NaCl wild-type (i.e., cancer-free) mice, wild-type (i.e., cancer-free) mice treated with Ir-CPI (low and high doses), and NaCl cancer-bearing mice or cancer-bearing mice treated with Ir-CPI (low and high doses). One-way ANOVA was used for statistical analysis (***p<0.001). All groups tested contained 7 mice, except for the NaCl wild-type mice group, which contained 8 mice. [Figure 15] Figure 15 shows a graph comparing NETosis under different in vitro experimental conditions (untreated control, tumor necrosis factor alpha (TNFα) priming with or without platelet-activating factor (PAF) activation, and / or incubation with Ir-CPI, as indicated). The median percentage of NETosis was calculated after immunofluorescence staining and analysis of NET length (n = 5 independent experiments; 5 random fields per condition were analyzed). Statistical analysis was performed using the Wilcoxon test (ns: not statistically significant; *p < 0.05; **p < 0.01; ***p < 0.001). [Figure 16]Figure 16 is a graph comparing the expression of CD11b on the surface of neutrophils under different experimental conditions (untreated control, priming with TNFα, and / or incubation with Ir-CPI, as indicated). Data are presented as mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test (ns: not statistically significant; *p < 0.05). [Figure 17] Figure 17 is a graph comparing the expression of Ly6G (A), CD11b (B), and activated CD11b (C) on the surface of neutrophils under different experimental conditions (ATP-activated control, ATP-activated neutrophils incubated with Ir-CPI [0.65 or 2 μM]). Data are presented as mean ± SEM (n = 2 independent experiments). DETAILED DESCRIPTION OF THE INVENTION

[0168] Example The invention is further illustrated by the following examples.

[0169] Example 1: Model involving activation of the extrinsic coagulation pathway material and method material Test substance Ir-CPI (MW = 7660 Da) was obtained from Bachem (powder, batch 1060411, purity: 98.4%) and stored at −80°C until use.

[0170] Ir-CPI was freshly prepared on the day of testing by dissolution in NaCl (0.9%) until a concentration of 50 mg / mL was reached (corresponding to the maximum concentration reached to avoid solubility problems).

[0171] NaCl (0.9%) was used as a control.

[0172] animal Male mice (C57BL / 6JRj) purchased from Janvier LABS were used in this study (5-8 weeks old).

[0173] method Administration Ir-CPI was administered intravenously as a bolus (20.5 mg / kg) followed immediately by a continuous infusion at a rate of 3.7 mg / kg / h (see Table 1 below). Due to the rapid distribution and elimination of Ir-CPI in mice (t1 / 2α = 14 min and t1 / 2β = 236 min), a combination of bolus and infusion was chosen to maintain a constant Ir-CPI concentration in the blood. [Table 1] n = number of mice used

[0174] Intravital microscopy of the cremaster muscle microcirculation Intravital videomicroscopy of the cremaster muscle microcirculation was performed as previously described ( Falati et al., 2002 ).

[0175] Mice were anesthetized using an intraperitoneal injection of ketamine (125 mg / kg), xylazine (12.5 mg / kg), and atropine (0.25 mg / kg) and maintained at 37°C with a thermo-controlled rodent blanket.

[0176] A tracheal tube was inserted into the trachea to guide respiration. A cannula was inserted into the jugular vein for the administration of pentobarbital. Pentobarbital (12 mg / kg) was administered into the jugular vein every 20–30 min to maintain the mouse under anesthesia throughout the procedure. The same jugular vein cannula was used for the administration of fluorescent dye-conjugated antibodies and enzyme substrates required to track thrombus development (detection of fibrin and / or platelets and / or neutrophil and / or neutrophil elastase activity). A cannula was also inserted into a second, contralateral jugular vein for the administration of vehicle or test items (Ir-CPI or NaCl).

[0177] After incision of the scrotum, the testes and surrounding cremaster muscle were exposed on a biomicroscope tray and continuously perfused with aerated (95% N, 5% CO) bicarbonate-buffered saline under thermoregulation (37°C) for the duration of the experiment.

[0178] Microvascular data were obtained using an Olympus AX61WI microscope with a 60x 0.9-numerical aperture water immersion objective. The digital wide-field fluorescence microscope system was previously described (Falati et al., 2002). Digital images were captured in 640x480 format using a Cooke Sensicam CCD camera.

[0179] Laser-induced damage Vessel wall injury was induced using a Micropoint Laser System (Photonics Instruments), focused through the microscope objective, parfocal with the focal plane, and aimed at the vessel wall, as previously described (Dubois et al., 2006). Typically, one to two pulses were required to induce vessel wall injury.

[0180] Multiple thrombi were tested in a single mouse, with new thrombi formed upstream of earlier thrombi to avoid any contribution from earlier thrombi in the animal under test. Laser beam injury was performed at various times after the start of administration of the test item (+5 min up to approximately 150 min after bolus administration of Ir-CPI or NaCl (control)).

[0181] Image analysis was performed using Slidebook 4.1 (Intelligent Imaging Innovations). Fluorescence data were digitally captured at up to 50 frames per second and analyzed as previously described (Falati et al., 2002). The kinetics of thrombus formation were analyzed by determining the median fluorescence value over time in a minimum of 20 thrombi.

[0182] After this procedure, mice were euthanized by an overdose of pentobarbital (120 mg / kg).

[0183] Detection of fibrin, platelet, PMN, and neutrophil elastase activity in vivo In vivo detection of fibrin was performed using an anti-fibrin antibody conjugated with Alexa Fluor 488 from Darbousset et al., 2014, which was administered intravenously at 0.5 μg per gram of mouse.

[0184] In vivo detection of platelets was performed using anti-GPIb antibody labeled with DyLight649 from Emfret, which was administered intravenously at 0.5 μg per gram of mouse.

[0185] In vivo detection of PMNs was performed using PE-labeled anti-Ly6G antibody.

[0186] In vivo detection of neutrophil elastase activity was performed using a BODIPY-FL labeled DQ-elastin conjugate fragment from Life Technologies, which was administered intravenously at 0.3 μg per gram of mouse.

[0187] Blood sampling and plasma preparation At the end of the experiment (approximately 150 min after the bolus), blood was collected from the inferior vena cava and placed in 0.109 M 3.2% sodium citrate tubes (citrate / blood, v / v 1 / 9). Plasma (approximately 250 μL) was centrifuged at 2500 g for 15 min at 18–22°C. Plasma was centrifuged again (2500 g for 15 min at 18–22°C) and stored at −80°C in polypropylene tubes. Plasma samples were then frozen on dry ice for determination of circulating concentrations of Ir-CPI by ELISA.

[0188] Quantification of Ir-CPI in plasma samples Quantification of circulating Ir-CPI in plasma samples was assessed by ELISA at Eurofins, ADME Bioanalyses (Vergeze, France). A sandwich immunoassay was performed using two anti-Ir-CPI antibodies. The capture monoclonal antibody (Ir-CPI3) was coated onto microtiter plates (Nunc, ThermoFischer Scientific, USA) at 1 μg / ml in PBS (Hyclone, USA) overnight at 4°C. The wells were blocked with PBS BSA (2%) (Sigma-Aldrich, USA) for 1 hour at room temperature (RT) and then washed. Samples were added to the wells and incubated for 2 hours at RT. A biotinylated detection antibody (L39-biotin), pre-diluted in PBS BSA (1%) (Sigma-Aldrich, USA), was added to the wells and complemented with streptavidin-HRP (REF) diluted in PBS BSA (1%). TMB SureBlue (KPL, REF) and H2SO4 (1N) (VWR, USA) were used for spectrophotometric detection (450 nm).

[0189] Data processing and statistical analysis Data are expressed as median and interquartile range relative to the sample size (n). Statistical comparison of data from the Ir-CPI-treated group versus the control group was performed using the Mann-Whitney test (***p<0.001).

[0190] result The aim of this study was to evaluate the antithrombotic efficacy of Ir-CPI in an experimental model of thrombosis in mice, in which thrombosis was induced by localized injury of cremaster arterioles with a laser beam. This experimental model of thrombosis is thought to involve activation of the extrinsic coagulation pathway, resulting in tissue factor-mediated thrombin generation (Dubois et al., 2007; Darbousset et al., 2012). Indeed, in such models, the kinetics of thrombin formation and fibrin production have been reported to be significantly reduced in tissue factor (TF)-low mice, as opposed to those in the absence of factor XII (FXII). - / - The laser-induced vessel wall injury model (mice) was ineffective (Darbousset et al., 2012). Therefore, the laser-induced vessel wall injury model allows the evaluation of the inhibitory effect of Ir-CPI on thrombosis involving the extrinsic coagulation pathway.

[0191] Monitoring platelet and fibrin accumulation after laser injury in control and Ir-CPI-treated mice (short-term measurements) Platelet accumulation and fibrin production in the injured vessel wall were monitored using the following fluorescently labeled antibodies: DyLight649-labeled anti-GPIb antibody and Alexa Fluor 488-conjugated anti-fibrin antibody, respectively.

[0192] In control mice, platelets rapidly accumulated at the injury site, peaking at approximately T114 seconds (median platelet integrated fluorescence: 508,541 AU), then decreased until the end of the measurement period at T228 seconds (median platelet integrated fluorescence: 209,893 AU). Fibrin production at the injury site continued to increase until the end of the measurement period, with a median fibrin integrated fluorescence of 16,686 AU at T228 seconds (Figures 1 and 2).

[0193] In mice treated with Ir-CPI, the kinetics of platelet accumulation differed from those in control mice. Platelet accumulation was significantly reduced compared to control mice. The maximum median value of measured platelet integrated fluorescence was 121,783 AU at T214 s. At T228 s (i.e., the end of the measurement period), the median value of platelet integrated fluorescence was 101,887 AU. Furthermore, fibrin production appeared to be less significant in Ir-CPI-treated mice than in control mice during the first 3.8 min after laser injury. At T228 s, the maximum median value of fibrin integrated fluorescence was 7,539.7 AU (Figures 1 and 2).

[0194] The mean plasma concentration measured in mice treated with Ir-CPI after approximately 150 minutes of the procedure was 6.48±1.02 μg / mL (mean±SEM; n=5).

[0195] Kinetics of fibrin production after laser injury in control and Ir-CPI-treated mice (long-term measurements) The kinetics of fibrin production was measured in control and Ir-CPI-treated mice over a 90-minute period after laser injury of the vessel wall. At all measured times (0, 15, 30, 45, 75, and 90 minutes), the median integrated fibrin fluorescence intensity was lower in Ir-CPI-treated mice than in control mice (Figures 3 and 4).

[0196] Effect of Ir-CPI on neutrophil accumulation Polymorphonuclear neutrophils (PMNs) have been demonstrated to be the first cells to accumulate in the vessel wall after laser-induced injury, and their interaction with activated endothelial cells is required for the initiation of thrombus formation ( Darbousset et al., 2012 ).

[0197] To determine whether Ir-CPI has an effect on PMN accumulation at the injury site, PMN accumulation in the injured vessel wall was monitored using a PE-fluorescently labeled antibody directed against Ly6G (Figure 5).

[0198] In control mice, PMNs rapidly accumulated at the injury site, peaking at T2 seconds (median platelet integrated fluorescence: 1.60 × 10 7 AU). The median integrated fluorescence of PMNs was 1.10 × 10 at T of 70 seconds. 7 AU (Figure 6).

[0199] In mice treated with Ir-CPI, the kinetics of neutrophil accumulation differed from that in control mice. Indeed, during the first 4.5 minutes after laser injury, PMN accumulation was significantly reduced compared to control mice. The maximum median integrated platelet fluorescence measured was 3.02 × 10 at T261 seconds. 6 At T270 s (i.e., the end of the measurement period), the median integrated fluorescence of PMNs was 2.78 × 10 6 AU (Figure 6). Similar results were obtained when using a fluorescently labeled substrate of elastase as a marker of PMN activity. The signal corresponding to neutrophil elastase activity was significantly reduced in mice treated with Ir-CPI compared with control mice (Figure 7).

[0200] conclusion Taken together, these results unexpectedly demonstrate that Ir-CPI is active in an experimental model known to involve activation of the extrinsic coagulation pathway. These results indicate that Ir-CPI exerts antithrombotic effects not necessarily related to its known targets (i.e., factors XIa and XIIa). Surprisingly, Ir-CPI inhibits not only fibrin formation at the lesion site but also platelet accumulation and the early emergence of PMNs, which are known to play a key role in activation of the extrinsic coagulation system because they are the main source of TF required for clot formation at the site of laser injury (Darbousset, Thomas et al. 2012).

[0201] Example 2: In vitro platelet aggregation assay material and method Blood from healthy human donors who had not taken antiplatelet medications within 15 days prior to collection was collected in citrate tubes and centrifuged at 200 g for 13 minutes. Platelet-rich plasma was collected and centrifuged at 900 g for 13 minutes in Tyrode's buffer (138 mM NaCl, 2.9 mM KCl, 12 mM NaHCO , 5.5 mM glucose, 1.8 mM CaCl , and 0.4 mM MgCl , pH 7.4) containing 0.2% BSA, apyrase (0.02 U / mL), and PGI (500 nM). Washed platelets were resuspended in Tyrode's / BSA (0.2%) / apyrase / PGI solution and centrifuged at 900 g for 13 minutes. Platelets were collected at 3.10 8 Platelets were resuspended in Tyrode's / BSA (0.2%) solution at a concentration of 1000 platelets / mL. Platelets were stored in a solution containing 4 μM calcium and 6 μM magnesium at 37°C for 30 minutes. Platelets were then incubated with ADP (adenosine diphosphate), TRAP (thrombin receptor-activating peptide), and / or Ir-CPI for 5–10 minutes at 37°C with agitation in an APACT 4004 aggregometer.

[0202] result The effect of Ir-CPI on platelet aggregation was evaluated in vitro by using isolated human washed platelets (n=1).

[0203] Washed platelets were first incubated with ADP (adenosine diphosphate), a platelet activator that does not induce platelet aggregation, or TRAP (thrombin receptor-activating peptide), an activator of platelet aggregation. As expected, and as shown in Table 2 below, ADP did not induce platelet aggregation (maximum aggregation: 6.9%), while TRAP activated platelet aggregation (maximum aggregation: 58.6-59.5%). [Table 2]

[0204] The addition of Ir-CPI to washed platelets did not activate platelet aggregation (maximum aggregation: 4.1%). The addition of both Ir-CPI and TRAP to washed platelets did not significantly alter platelet aggregation (maximum aggregation: 71.7%) compared to the positive control condition (TRAP alone: ​​maximum aggregation: 58.6-59.5%). When TRAP was added to Ir-CPI, this effect produced similar results (maximum aggregation: 73.5%) but with a longer latency period (231.2 seconds) (Table 2).

[0205] The results of Example 1 showed that Ir-CPI inhibits platelet accumulation in vivo. However, Ir-CPI does not inhibit platelet aggregation in an in vitro platelet aggregation assay. Taken together, these results suggest that Ir-CPI may act on targets other than platelets that are involved in thrombus formation.

[0206] In laser-induced vessel wall injury models, polymorphonuclear neutrophils (PMNs) are known to adhere to injured vessels within seconds of injury, preceding platelets by binding to activated endothelium. By binding to the vessel wall, PMNs are the primary source of blood-borne TFs for early thrombus formation (Darbousset et al., 2012). The interaction of PMNs with endothelial cells is a critical step preceding platelet accumulation for thrombosis initiation.

[0207] The results of Example 1 show that Ir-CPI reduced the recruitment of PMNs to the injured vascular wall. Similarly, a decrease in neutrophil elastase activity was detected under Ir-CPI treatment. However, this decrease in elastase activity may result from a decrease in the number of neutrophils recruited to the lesion site.

[0208] Example 3: Model of cancer-associated thrombosis material and method material Test substance Ir-CPI (MW = 7660 Da) was obtained from Bachem (powder, batch 1060411, purity: 98.4%) and stored at −80°C until use.

[0209] Ir-CPI was freshly prepared on the day of testing by dissolution in NaCl (0.9%) to reach a concentration of 50 mg / mL (corresponding to the maximum concentration reached to avoid solubility problems).

[0210] NaCl (0.9%) was used as a control.

[0211] animal Male mice (C57BL / 6JRj) purchased from Janvier LABS were used in this study (5 weeks old).

[0212] method Administration Ir-CPI was administered as described herein above (see Example 1) and summarized in Table 3 below. [Table 3] n = number of mice used; WT (wild type) corresponds to cancer-free mice.

[0213] [Table 4] n = number of mice used; WT (wild type) corresponds to cancer-free mice.

[0214] Induction of ectopic tumors Mouse Panc02 cells (a mouse pancreatic ductal adenocarcinoma cell line) were cultured to 80% confluence in RPMI-1640 medium (Life Technologies) supplemented with 10% FCS (PAA), 1000 U / mL penicillin (Life Technologies), 100 μg / mL streptomycin (Life Technologies), and 0.1% Fungizone (Life Technologies) at 37°C in a humidified atmosphere of 5% CO2. After reaching logarithmic growth phase, cells were washed three times with PBS and briefly exposed to non-enzymatic cell dissociation buffer (Life Technologies) to detach the cells. The cells were carefully washed three times, resuspended in PBS, and diluted to the desired concentration.

[0215] Five-week-old C57BL / 6 mice were inoculated with a tumor cell suspension (10 in 100 μL of PBS) into the right flank. 6 When tumors became palpable (0.2 cm), they were measured in two dimensions with calipers and the volume of each tumor was calculated using the formula for the volume of an ellipsoid: π / 6 × a(b). 2 (where a is the largest diameter of the tumor and b is the smallest diameter of the tumor).

[0216] Intravital microscopy of the cremaster muscle microcirculation Intravital videomicroscopy of the cremaster muscle microcirculation was performed as previously described (Falati et al., 2002) and as described herein above (see Example 1).

[0217] Laser-induced damage Vessel wall injury was induced with a Micropoint Laser System (Photonics Instruments) as described herein above (see Example 1).

[0218] Platelet and fibrin accumulation after injury was monitored and detected as described herein above (see Example 1).

[0219] Assessment of tail bleeding time Assessment of tail bleeding time under treatment with NaCl or Ir-CPI (low or high doses) was performed in anesthetized wild-type (i.e., cancer-free) or cancer-bearing mice as previously described (Mezouar et al., 2015). Briefly, a distal 1-3 mm portion of the tail was removed from the mice; the tail was immersed in isotonic saline (37°C), and the time until complete cessation of blood flow was recorded. Bleeding time was monitored for up to 10 min.

[0220] Data processing and statistical analysis For assessment of tumor volume, data were expressed as mean ± SEM along with the relevant sample size (n). For assessment of the effect of Ir-CPI on platelet accumulation and fibrin production in tumor-bearing mice, data were expressed as median integrated fluorescence over time. Statistical analysis used the Mann-Whitney test, and for tail bleeding time, one-way ANOVA with Tukey's multiple comparison test was used.

[0221] result Using the same thrombosis-induced model, the antithrombotic effects of Ir-CPI were evaluated in mice bearing pancreatic cancer (ectopic model). Indeed, pancreatic cancer cells have been shown to produce microparticles (MPs) carrying tissue factor (TF), which plays a key role in thrombus formation in vivo (Thomas et al., 2009; Mezouar et al., 2015). Two doses (high and low) of Ir-CPI were tested in this model.

[0222] In another experiment, the time to cessation of tail bleeding in wild-type (cancer-free) and cancer-bearing mice was assessed under NaCl (control) or Ir-CPI treatment (low and high doses).

[0223] Assessment of tumor volume before laser injury experiments In the mouse model of this study, cancer was induced by subcutaneous injection of murine pancreatic (Panc02) cancer cells. Tumor-bearing mice were randomly divided into two groups: control and Ir-CPI. Twenty-two days after implantation of Panc02 cells, mice from both groups were injected with vehicle (NaCl (control)) or test item (Ir-CPI) and subjected to laser injury of the cremaster muscle microcirculation for thrombus induction. As shown in Figure 8, the mean tumor volumes of the two groups were not statistically different at day 22 (control: 372.6 ± 45.3 mm). 3 ;n=5 and Ir-CPI: 365.0±52.1mm 3 (n=4). The latter mice were used for experiments using high-dose Ir-CPI treatment. Similarly, mean tumor volumes were not statistically different in mice treated with low-dose Ir-CPI (data not shown).

[0224] Monitoring platelet and fibrin accumulation after laser injury Tumor-bearing mice were from the Ir-CPI-treated group (Ir-CPI cancer) or the control group (NaCl cancer). Furthermore, cancer-free mice treated with NaCl (NaCl cancer-free) were used as a negative control. All mice were subjected to laser injury to the cremaster arteriole to induce thrombosis. Mice treated with Ir-CPI were administered low or high doses of the test product.

[0225] Platelet accumulation and fibrin production in the injured vessel wall were monitored using the following fluorescently labeled antibodies: DyLight649-labeled anti-GPIb antibody and Alexa Fluor 488-conjugated anti-fibrin antibody, respectively.

[0226] Mice with NaCl tumors exhibited a prothrombotic state at the injury site, as assessed by significant platelet accumulation and abundant fibrin production, compared with mice without NaCl tumors. Indeed, the integrated fluorescence intensity (area under the curve) of platelet and fibrin labels was significantly higher in control tumor-bearing mice compared with control WT mice (*p<0.05). In control tumor-bearing and control WT mice, the median integrated fibrin fluorescence was 59,172 AU and 23,005 AU at T278 s, respectively (Figures 9 and 10).

[0227] A significant decrease in platelet accumulation and fibrin production was observed in both high- and low-dose Ir-CPI-treated cancer mice compared with NaCl cancer-bearing mice. As shown in Figure 11, the integrated fluorescence intensity (area under the curve) of the platelet label was significantly lower in high-dose Ir-CPI-treated cancer mice than in NaCl cancer-free mice (*p<0.05) and NaCl cancer-bearing mice (***p<0.001). Similar results were observed in mice treated with low-dose Ir-CPI (Figures 12 and 13).

[0228] Assessment of tail bleeding time Tail bleeding time was significantly reduced in mice that developed tumors (e.g., median bleeding time of NaCl-treated mice administered Panc02 cancer cells was 75 seconds) compared to mice that did not develop tumors (e.g., median bleeding time of wild-type (i.e., cancer-free) NaCl-treated mice was 147 seconds) (***p<0.001) (Figure 14). No significant effect of Ir-CPI treatment (low and high doses) on tail bleeding time was observed in cancer-free and cancer-bearing mice when compared to NaCl-treated mice (Figure 14).

[0229] conclusion The pathogenesis of the prothrombotic state in cancer is associated with the production of local and systemic hypercoagulable / thrombotic conditions that provide tumor cells with a growth advantage. Pancreatic cancer cells have been shown to produce microparticles (MPs) bearing tissue factor (TF), which plays a key role in thrombus formation in vivo (Thomas et al., 2009; Mezouar et al., 2015). PMNs from cancer-bearing mice exhibit an increased tendency to form neutrophil extracellular traps (NETs)—spider-web-like structures formed by exogenous chromatin and secreted proteases, a process termed NETosis (Leal et al., 2017). Furthermore, extracellular chromatin released through NET formation has been shown to contribute to cancer-associated thrombosis (Demers et al., 2012; Mauracher et al., 2018).

[0230] The results of Example 3 demonstrate that Ir-CPI inhibits the prothrombotic state associated with cancer in a murine laser injury model. Indeed, in mice bearing ectopic pancreatic tumors, Ir-CPI significantly reduced platelet accumulation and fibrin formation at the laser injury site. Importantly, in both non- and cancer-bearing mice, Ir-CPI had no effect on tail bleeding time, indicating that it does not compromise homeostasis.

[0231] Example 4: NETosis assay material and method material Neutrophils were isolated from the femoral bone marrow of mice (C57BL / 6) using FR-1-conjugated magnetic beads (anti-Ly6G Microbead Kit, Miltenyi Biotec) as previously described (Hu 2012).

[0232] method In vitro NETosis assay Cells were harvested, centrifuged, and incubated in Hank's solution (Gibco) with or without 2 ng / mL TNFα for 30 minutes. Neutrophils were seeded onto poly-L-lysine-coated slides and incubated in Hank's solution at 37°C for 1 hour. Neutrophils were then activated with or without 25 μM platelet-activating factor (PAF) ± Ir-CPI (0.65 or 2 μM) in Hank's solution (37°C, 3 hours). NET formation was assessed by immunofluorescence (Leica fluorescence microscope) after fixation of neutrophils with 4% PAF (paraformaldehyde) and fluorescent labeling of citrullinated histones (H3 antibody) and nuclei (Hoechst). Image processing and analysis were performed using FIJI software.

[0233] Data processing and statistical analysis Median NETosis percentages are presented for five independent experiments. Statistical analysis was performed using the Wilcoxon test (ns: not significant; *p<0.05; **p<0.01; ***p<0.001).

[0234] result Following the results of Example 3, the effect of Ir-CPI on the activation state of PMNs was evaluated in vitro by assessing the formation of NETs. Indeed, it has been reported that PMNs from cancer-bearing mice have an increased tendency to form neutrophil extracellular traps (NETs), which have been shown to be responsible for cancer-associated thrombosis (Demers, Krause et al. 2012, Mauracher, Posch et al. 2018).

[0235] Effect of Ir-CPI on NETosis in vitro It has been reported that PMNs from cancer-bearing mice have an increased tendency to form neutrophil extracellular traps (NETs), spider web-like structures formed by exogenous chromatin and secreted proteases; a process called NETosis (Leal et al., 2017). Furthermore, extracellular chromatin released through NET formation has been shown to contribute to cancer-associated thrombosis (Demers et al., 2012; Mauracher et al., 2018).

[0236] To assess the effect of Ir-CPI on NETosis, immunofluorescence staining (Hoechst for DNA and H3Cit for citrullinated histone marker) was performed on PMNs isolated from femoral bone marrow of mice and subjected to different in vitro experimental conditions (untreated control, priming with TNFα with or without activation with PAF, and / or incubation with Ir-CPI), as shown in Figure 15.

[0237] As a positive control for NET formation, we used TNFα-primed and PAF-activated PMNs, which increased the percentage of NETosis compared to untreated PMNs (***p<0.001), thereby validating this model (Figure 15).

[0238] As shown in Figure 15, when PMNs were primed with TNFα, activated with PAF, and incubated with Ir-CPI (0.65 and 2 μM), a significant decrease in the percentage of NET formation was observed compared to PMNs incubated with TNFα and PAF (*p<0.05 for 0.65 μM Ir-CPI and ***p<0.001 for 2 μM Ir-CPI).

[0239] These results demonstrate that Ir-CPI reduces NETosis when incubated with activated neutrophils in vitro.

[0240] Example 5: Effect of Ir-CPI on neutrophil activation material and method Neutrophils were isolated from the femoral bone marrow of mice (C57BL / 6) using FR-1-conjugated magnetic beads (anti-Ly6G Microbead Kit, Miltenyi Biotec) as previously described (Hu 2012). For TNFα activation, cells were harvested, centrifuged, and incubated with or without 2 ng / mL TNFα in Hank's solution (Gibco) for 30 min. Next, neutrophils were incubated with Ir-CPI (0.65 or 2 μM) in Hank's solution for 1 h at 37°C. For ATP activation, cells were harvested, centrifuged, and incubated with ATP (10 μM) and Ir-CPI (0.65 or 2 μM) in Hank's solution for 1 h at 37°C. After washing, the expression of CD11b, activated CD11b, or Ly6G was assessed by flow cytometry (Beckman Coulter Gallios). Antibodies used for neutrophil identification and activation analysis were IRR IgG1-FITC (irrelevant antibody), anti-CD45-APC, anti-Ly6G-PE, and anti-CD11b-FITC, or Alexa Fluor 647 anti-activated CD11b. Results were analyzed using Kaluza software (Beckman Coulter).

[0241] Data processing and statistical analysis Percentages of activation marker expression are presented as mean ± SEM (n = 3 independent experiments for TNFα activation and n = 2 independent experiments for ATP activation). For experiments with TNFα activation, statistical analysis used one-way ANOVA followed by Tukey's multiple comparison test (ns: not significant; *p < 0.05).

[0242] result Following the results of Example 4, the effect of Ir-CPI on PMN activation status was evaluated in vitro by assessing the expression of neutrophil activation markers after incubation with molecules known to activate neutrophils, namely adenosine triphosphate (ATP) and tumor necrosis factor alpha (TNFα). ATP has been shown to contribute to neutrophil activation, resulting in neutrophil adhesion at the site of laser-induced endothelial injury, a necessary step leading to fibrin production and subsequent platelet-dependent thrombus formation (Darbousset, Delierneux et al. 2014). Additionally, another neutrophil activator, TNFα (Futosi, Fodor et al. 2013), was tested in this example.

[0243] Effect of Ir-CPI on neutrophil activation after incubation with TNFα Neutrophils isolated from the femoral bone marrow of mice were subjected to different in vitro experimental conditions (untreated control, priming with TNFα, and / or incubation with Ir-CPI) to evaluate the effect of Ir-CPI on their activation state, which was assessed by analyzing the percentage of CD11b expression on the surface of neutrophils (Figure 16).

[0244] Incubation of untreated neutrophils with Ir-CPI (0.65 and 2 μM) had no effect on CD11b expression. TNFα-primed neutrophils were used as a positive control for neutrophil activation. As shown in Figure 16, TNFα-primed neutrophils had increased CD11b expression compared with untreated neutrophils. Remarkably, TNFα-primed neutrophils incubated with Ir-CPI (2 μM) showed a significant decrease in CD11b expressed on the cell surface (*p<0.05).

[0245] These results indicate that Ir-CPI alters TNFα-induced neutrophil activation through a reduction in CD11b expression in vitro.

[0246] Effect of Ir-CPI on neutrophil activation after incubation with ATP Neutrophils isolated from the femoral bone marrow of mice were subjected to different in vitro experimental conditions (untreated control, incubation with Ir-CPI and ATP, or incubation with Ir-CPI but without ATP) to evaluate the effect of Ir-CPI on their activation state, which was assessed by analyzing the percentage of CD11b, activated CD11b, and Ly6G expression on the surface of neutrophils (Fig. 17).

[0247] Incubation of naive neutrophils with Ir-CPI (0.65 and 2 μM) had no effect on CD11b expression (Figure 17B). Surprisingly, however, Ir-CPI reduced the expression of activated CD11b on the surface of neutrophils (Figure 17C). Ly6G expression was also reduced on the surface of neutrophils incubated with Ir-CPI (Figure 17A).

[0248] These results indicate that Ir-CPI alters ATP-induced neutrophil activation in vitro via a decrease in the expression of activated CD11b.

[0249] Thus, Ir-CPI inhibits thrombus formation following activation of the extrinsic coagulation pathway, not through inhibition of tissue factors specific to this pathway, such as tissue factor (TF), but through inhibition of neutrophil recruitment, activation, and NET formation. These events are the initial steps leading to activation of the extrinsic coagulation system and subsequent thrombus formation after laser endothelial injury.

[0250] References Darbousset, R., et al. (2014). ”P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice.” Blood. 124(16):2575-2585. Darbousset, R., et al. (2012). ”Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation.” Blood. 120(10):2133-2143. Decrem, Y., et al. (2009). ”Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis.” J Exp Med. 206(11):2381-2395. Demers, M., et al. (2012). ”Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.” Proc Natl Acad Sci U S A. 109(32):13076-13081. Dubois, C., et al. (2007). ”Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model.” J Clin Invest; 117(4):953-960. Dubois, C., et al. (2006). ”Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo.” Blood. 107(10):3902-3906. Falati, S., et al. (2002). ”Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.” Nat Med. 8(10):1175-1181. Futosi, K., et al. (2013). ”Neutrophil cell surface receptors and their intracellular signal transduction pathways.” Int Immunopharmacol. 17(3):638-650. Hu, Y. (2012). ”Isolation of human and mouse neutrophils ex vivo and in vitro.” Methods Mol Biol. 844:101-113. Kambas, K., et al. (2012). ”The emerging role of neutrophils in thrombosis-the journey of TF through NETs.” Front Immunol. 3:385. Keularts, I. M., et al. (2001). ”The role of factor XI in thrombin generation induced by low concentrations of tissue factor.” Thromb Haemost. 85(6):1060-1065. Leal, A. C., et al. (2017). ”Tumor-Derived Exosomes Induce the Formation of Neutrophil Extracellular Traps: Implications For The Establishment of Cancer-Associated Thrombosis.” Sci Rep. 7(1):6438. Massberg, S., et al. (2010). ”Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.” Nat Med. 16(8):887-896. Mauracher, L. M., et al. (2018). ”Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients.” J Thromb Haemost. Mezouar, S., et al. (2015). ”Inhibition of platelet activation prevents the P-selectin and integrin-dependent accumulation of cancer cell microparticles and reduces tumor growth and metastasis in vivo.” Int J Cancer. 136(2):462-475. Thomas, G. M., et al. (2009). ”Cancer cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo.” J Exp Med. 206(9):1913-1927. von Bruhl, M. L., et al. (2012). ”Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.” J Exp Med. 209(4):819-835.

Claims

1. A pharmaceutical composition for treating and / or preventing thromboinflammation, comprising a protein or polypeptide comprising a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 and at least one pharmaceutically acceptable excipient.

2. 2. The pharmaceutical composition of claim 1, wherein the protein or polypeptide comprises a polypeptide having the amino acid sequence of SEQ ID NO:

2.

3. 3. The pharmaceutical composition of claim 1, wherein the thromboinflammation is selected from the group comprising atherosclerosis, plaque rupture, device-induced thromboinflammation, thrombosis induced by catheterization and / or stent positioning procedures, thrombosis induced by extracorporeal circulation, thrombus formation after brain injury, coronary artery disease, acute myocardial infarction, cancer-related thrombosis, metastasis-related thrombosis, stroke-related thrombosis, Behcet's disease (BD), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Takayasu's arteritis, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, familial Mediterranean fever, thromboangiitis obliterans (TAO), sepsis, inflammatory bowel disease, heparin-induced thrombocytopenia, immunological thrombosis, thrombosis associated with preeclampsia, thrombotic complications in cell and cell cluster transplantation and whole organ transplantation or grafts, venous thromboembolism, aneurysm, and ischemia-reperfusion syndrome in skeletal muscle.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the thromboinflammation is thrombus formation after brain injury.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the thromboinflammation is stroke-related thrombosis.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the thromboinflammation is plaque rupture.

7. A medicament comprising the pharmaceutical composition according to any one of claims 1 to 6.

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