New generation of antithrombotics targeting LRP5

Abstract

The invention relates to the field of therapeutics and, more in particular, it concerns the use of a low-density lipoprotein receptor-related protein 5 (LRP5) inhibitor for the treatment and / or prevention of thrombosis in a patient as well as genetically modified megakaryocyte or progenitor thereof in which LRP5 gene expression has been silenced and the uses thereof for the treatment and / or prevention of thrombosis in a patient.

Description

[0001] NEW GENERATION OF ANTITHROMBOTICS TARGETING LRP5

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of therapeutics and, more in particular, it concerns the use of a low-density lipoprotein receptor-related protein 5 (LRP5) inhibitor for the treatment and / or prevention of thrombosis in a patient as well as genetically modified megakaryocyte or progenitor thereof in which LRP5 gene expression has been silenced and the uses thereof for the treatment and / or prevention of thrombosis in a patient.

[0004] BACKGROUND ART

[0005] Platelets are key players in haemostasis and thrombosis and have also important roles in angiogenesis and inflammation. They are found in circulating blood in a nonadhesive, inactive state. In the presence of vascular damage, platelets adhere to matrix proteins, become activated and change their characteristic round shape into widened cytoplasm structures with extended pseudopodia. Activated platelets turn on receptors including the ADP activated P2Y12 and the GPIIb / llla receptor involved in platelet aggregation or the GPIb receptor involved in platelet adhesion, and secrete chemical messengers, vasoactive substances and inflammatory mediators. Platelets aggregate and form a haemostatic plug that by activation of the coagulation cascade result in fibrin deposition. Myocardial infarction and stroke are major consequences of anomalous platelet activation leading to pathological thrombus formation, causing vessel occlusion and tissue ischemia.

[0006] Preventive treatments for thrombosis (blood-thinners, blood pressure-lowering medications and cholesterol-lowering medications), acute medications (blood-thinners and clot-busting drugs), surgery and catheter procedures, and other treatments are rapidly advancing.

[0007] The inhibition of the platelet P2Y12 receptor is one of the most widely studied strategies. For example, in Sibbing D, et al., Updated Expert Consensus Statement on Platelet Function and Genetic Testing for Guiding P2Y12 Receptor Inhibitor Treatment in Percutaneous Coronary Intervention, JACC Cardiovasc Interv. 2019; 12(16):1521- 1537, the beneficial effects and limitations of current therapeutic strategies and future perspectives related to platelet P2Y12 receptor inhibitors, including clopidogrel are described. Variability in response to clopidogrel is a well-known phenomenon, the significance of which is highlighted by the fact that a multitude of studies have observed an association between low response and adverse cardiovascular events. To overcome the limitations of clopidogrel, new P2Y12 receptor antagonists have been studied, including: prasugrel, ticagrelor, cangrelor and elinogrel. Prasugrel irreversibly inhibits P2Y12 and is more effectively converted to its active metabolite than clopidogrel. Ticagrelor directly and reversibly inhibits the P2Y12 receptor and this is a superior inhibition to that obtained with clopidogrel. Cangrelor is an ATP analogue, which inhibits P2Y12 reversibly and directly without biotransformation. Elinogrel has a direct and reversible action, with a higher degree of platelet inhibition than clopidogrel.

[0008] Successful strategies to inhibit thrombosis are presently clinically available; however they can cause significant bleeding. Therefore, the deep understanding of the regulation of platelet reactivity is essential to develop new therapeutic strategies with reduced bleeding risk.

[0009] SUMMARY OF THE INVENTION

[0010] The authors of the present invention have found that LRP5 regulates platelet reactivity. The inventors have analyzed the role of LRP5 in platelet reactivity. The inventors have shown that blocking LRP5 abolishes flow-dependent platelet deposition in wild-type (Wt) mice and human blood.

[0011] In addition, the inventors have shown that LRP5 interacts directly with the ADP receptor P2Y12 and that treatment of human platelets with a compound which is capable of specifically blocking the interaction of LRP5 with P2Y12 prevents or reduces P2Y12 translocation to the plasma membrane and thus, reduces flow-dependent platelet deposition.

[0012] Moreover, the inventors have demonstrated that human blood treated with an LRP5 mimetic or with an agent that is capable of specifically blocking the interaction of LRP5 with P2Y12 results in decreased platelet adhesion and aggregation, indicating that interference with LRP5 inhibits platelet deposition. In view of the aforementioned, the present invention relates to a low-density lipoprotein receptor-related protein 5 (LRP5) inhibitor or a genetically modified megakaryocyte or progenitor thereof with LRP5 gene expression inhibited for use in the treatment and / or prevention of thrombosis in a subject.

[0013] In addition, the invention relates to a genetically modified megakaryocyte or progenitor thereof with LRP5 gene expression inhibited and to uses thereof in the treatment and / or prevention of thrombosis in a subject.

[0014] Lastly, the present invention relates to a blood contact surface wherein the surface is coated with a LRP5 inhibitor.

[0015] DESCRIPTION OF THE FIGURES

[0016] Figure 1. LRP5 in platelet adhesion and aggregation. A LRP5 expression in mice and human platelets. B Representative images of thrombus formation in perfused blood of

[0017] 5 Wt +vehicle, 5 Lrp5- / - +vehicle and 6 Wt mice+25pM LRP5 inhibitor GTX89521 at high shear rate (975 s-1). Quantification of the area covered by thrombi. C Representative images of the aggregation analyses of mice Platelet Rich Plasma (PRPN=14 / genotype). D Representative images of human PRP treated with ADP, collagen and LRP5 inhibitor (N=4). E Carotid artery occlusion times after FeCh treatment in Wt (N=8), Lrp5- / + mice (N=8) and Lrp5- / - mice (N=7). *p<0.05.

[0018] Figure 2. Thromboelastographic analyses in mice. Thromboelastografic analyses performed in blood of 8 Wt and 9 Lrp5- / - mice A Intrinsically Activated Thromboelastometric Test (InTEM) and B Externally Activated Thromboelastometric Test (ExTEM). C Tissue Factor-Procoagulant activity (TF-PCA) performed in blood from

[0019] 6 Wt and 6 Lrp5- / - mice. D Platelet count in blood of 8 Wt and 8 Lrp5- / - mice. *p<0.05, **p<0.01 ***p<0.005.

[0020] Figure 3. Tail vein and liver bleeding in mice. A Tail vein bleeding time (TVBT) (%) in 23 Wt and 18 Lrp5- / - mice. B TVBT (%) in 5 Wt mice after 4h administration of acetylsalicylic acid (ASA) or Clopidogrel. C TVBT (in seconds) in Wt and Lrp5- / - mice and D after ASA or Clopidogrel administration in Wt mice (in seconds). E Liver Bleeding (%) in 5 Wt and 5 Lrp5- / - mice. F Liver Bleeding (%) in 5 Wt mice after 4h administration of ASA or Clopidogrel. G Liver bleeding (in mg) in Wt and Lrp5- / - mice and H after 4h administration of ASA or Clopidogrel in Wt mice. *p<0.05, **p<0.01 ***p<0.005.

[0021] Figure 4. Mice platelet adhesion. A Quantification of images of static binding of Wt and Lrp5- / - mice platelets to 2mg / ml fibrinogen (FG) and 0.1 mg / ml collagen (Col) coated surfaces (N=6). B Quantification of expression of Glycoprotein VI (GPVI) and GPIIb in Wt and Lrp5- / - mice platelets attached to fibrinogen and collagen coated surfaces *p<0.05, **p<0.01 ***p<0.005.

[0022] Figure 5. GPVI expression in Wt and Lrp5- / - mice platelets. A Representative Western Blot (WB) and quantification of GPVI expression in washed platelets of Wt and Lrp5- / - mice (N=7 mice / genotype). B Cellular fractionation analyses of Wt and Lrp5- / - mice PRP treated or not with 30pg / ml collagen. C: cytoplasm; M: membranes (N= 6 mice / genotype). pPLCy2 / PLCy2 ratio quantification in collagen treated M sections of Wt and Lrp5- / - mice. C Schematic of GPVI (grey) and GPIIb (black) expression in Wt and Lrp5- / - mice platelets attached to collagen coated surfaces showing colocalization of GPVI receptor and GPIIb in Wt mice platelets. However, platelets from Lrp5- / - mice showed less deposition density and there was no colocalization between GPVI and GPIIb. D Platelets plasma membrane expression of GPVI, FcRy and a2pi before and after collagen stimulate on in 6 Wt and 6 Lrp5- / - mice Bar: 2pm. **p<0.01 , ***p<0.005, ns=non significant.

[0023] Figure 6. Mice platelets granules release. A ELISA analyses of PF4 release in washed platelets from 5 Wt, 5 Lrp5- / - and 5 Lrp5- / + mice after ADP treatment. B WB analysis of TSP-1 expression in washed platelets of 5 Wt and 5 Lrp5- / - mice after ADP treatment and quantification of TSP-1 cellular content. C ELISA analyses of Serotonin release in washed platelets from 6 Wt, 6 Lrp5- / - and 6 Lrp5- / + mice after ADP treatment. D ADP receptors expression in washed platelets from Wt (N=5) and Lrp5- / - (N=5) mice. Quantification of WB analyses. E Flow cytometry analysis of VASP phosphorylation in platelets of Wt (N=7) and Lrp5- / - (N=7) mice. *p<0.05, **p<0.01 , ***p<0.005.

[0024] Figure 7. Canonical Wnt signaling protein expression. A Representative WB of the expression of different canonical Wnt proteins in Wt (N=5), Lrp5- / - (N=5) mice platelets and B in human platelets (N=6). C Cellular fractionation analyses and quantification of washed platelets from 4 healthy individuals after ADP treatment. *p<0.05. Figure 8. Interaction of LRP5 with purinergic receptors. A WB analyses of cellular subfractionation experiments of human platelets immunoprecipitated with LRP5 (N=5) or with P2Y12 (N=5) B Cellular fractionation analyses and quantification of human platelets treated with ADP, LRP5 inhibitor or both (N=3). C Cellular fractionation analyses and quantification (M / (C+M)) of washed platelets from 5 healthy individuals before (Basal) and after 150 mg Clopidogrel treatment in the presence and absence of 10pm ADP for 5 minutes. D Thrombus formation in perfused human blood treated or not with 25pM of the LRP5 inhibitor GTX89521 at high shear rate (975 s-1 ; N=7). Quantification of the area covered by thrombi. TP: Total Protein; C: cytoplasm; M: membranes *p<0.05, **p<0.01 , ***p<0.005.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The authors of the present invention have discovered that LRP5 regulates platelet reactivity. The inventors have shown for the first time that LRP5 is involved in platelet deposition, participates in human flow-mediated platelet deposition and directly interacts with P2Y12. Hence, the present invention opens up a new therapeutic window to thrombosis patients.

[0027] As shown in the examples of the present invention, the inventors have measured the bleeding time after a tail incision in Wt and Lrp5- / - mice and have observed that the bleeding time is longer in Lrp5- / - mice indicating that these mice take longer to heal their wounds. They have also observed that the bleeding time after treatment with clopidogrel or aspirin in Wt mice is much longer than in Lrp5- / - mice. LRP5 inhibition therefore induces less bleeding than treatment with clopidogrel or aspirin. These results indicate that LRP5 antagonists unexpectedly induce less bleeding than aspirin or clopidogrel treatments.

[0028] Thus, in a first aspect, the present invention relates to a low-density lipoprotein receptor- related protein 5 (LRP5) inhibitor for use in the treatment and / or prevention of thrombosis in a subject.

[0029] The low-density lipoprotein receptor-related protein 5 (LRP5, UniProtKB identifier 075197) is a protein that in humans is encoded by the LRP5 gene (Ensembl accession number ENSG00000162337). In the present invention, the term “LRP5 inhibitor” refers to any molecule capable of completely or partially inhibiting LRP5 gene expression in platelets, both by preventing the expression product of said gene from being produced (interrupting LRP5 gene transcription, blocking the translation of the mRNA coming from LRP5 gene expression and / or promoting the premature degradation of the mRNA originating from the LRP5 gene), by directly inhibiting LRP5 protein activity and by promoting / targeting LRP5 protein degradation by targeting it to the ubiquitin-proteasome pathway or to lysosomal proteolysis. Methods for decreasing / abrogating the expression of the gene encoding the LRP5 protein include, without being limited to, editing technologies such as CRISPR / cas9 or Cas9 nickase technology.

[0030] The expression of a protein or nucleic acid is considered reduced when its levels decrease with respect to the reference value by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100% (i.e., absent).

[0031] Suitable methods for determining whether an inhibitor specific for LRP5 is capable of decreasing LRP5 mRNA levels include, without limitation, standard assays for determining mRNA expression levels such as qPCR, RT-PCR, RNA protection analysis, Northern blot, RNA dot blot, in situ hybridization, microarray technology, tag based methods such as serial analysis of gene expression (SAGE), including variants such as LongSAGE and SuperSAGE, microarrays, fluorescence in situ hybridization (FISH), including variants such as Flow-FISH, qFiSH and double fusion FISH (D-FISH), and the like.

[0032] Suitable methods for determining whether an inhibitor acts by decreasing LRP5 protein levels include the quantification by means of conventional methods, for example, using antibodies with a capacity to specifically bind to the protein encoded by LRP5 gene (or to fragments thereof containing antigenic determinants) and subsequent quantification of the resulting antibody-antigen complexes. There are a wide variety of well-known assays that can be used in the present invention, which use non-labeled antibodies (primary antibody) and labeled antibodies (secondary antibodies); among these techniques are included Western blot or Western transfer, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzymatic immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. Other ways of detecting and quantifying the levels of the protein of interest include techniques of affinity chromatography, bindingligand assays, etc.

[0033] Suitable methods for determining whether an inhibitor acts by decreasing LRP5 activity include, for instance, the determination of the translocation of P2Y12 purinergic receptor to the cytoplasm in platelets after ADP activation, the determination of the translocation of LRP5 to the platelet cytoplasm after ADP activation or the determination of the amount of platelets which are deposited on collagen-coated slides.

[0034] Any LRP5 inhibitor may be used in the use of the present invention. However, in a particular embodiment of the use of the invention, the LRP5 inhibitor may be selected from the group consisting of:

[0035] (i) a LRP5-specific aptamer;

[0036] (ii) a LRP5 specific antibody;

[0037] (iii) a protein inhibiting the LRP5 expression;

[0038] (iv) a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant or a peptide mimetic thereof;

[0039] (v) a scaffold presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof.

[0040] LRP5 specific aptamer

[0041] In a particular embodiment, the specific LRP5 inhibitor for use according to the invention is a LRP5 specific aptamer.

[0042] In the context of the present invention, “LRP5 specific aptamer” is understood as any aptamer capable of binding specifically to the LRP5 protein and inhibiting one or more of the functions of said protein. Aptamers as used herein refer to short sequences of artificial DNA, RNA, XNA, or peptide that bind the LRP5. The term “XNA” refers to xeno nucleic acids (XNA), which are synthetic nucleic acid analogues that have a different sugar backbone than the natural nucleic acids DNA and RNA. In XNA nucleotides, the deoxyribose and ribose sugar groups of DNA and RNA have been replaced with other chemical structures. XNA-based aptamers can introduce additional chemical diversity to increase binding affinity or greater durability in serum or in vivo.

[0043] In one embodiment, the LRP5 specific aptamer is capable of preventing deposition of platelets over collagen when blood is contacted with a collagen-containing surface. A suitable assay for measuring the capacity of the aptamer to prevent or to block deposition of platelets over collagen is explained in the examples of the present document.

[0044] In one embodiment, the LRP5 specific aptamer is capable of preventing platelet aggregation after platelet stimulation with collagen and ADP. A suitable assay for measuring the capacity of the aptamer to prevent or to block collagen and ADP- dependent platelet aggregation is explained in the examples of the present document.

[0045] In one embodiment, the LRP5 specific aptamer is capable of preventing association of LRP5 with P2Y12 and / or translocation of P2Y12 to the plasma membrane in platelets activated with ADP. Suitable assays for measuring the association of LRP5 and P2Y12 and for detecting translocation of P2Y12 to the plasma membrane in platelets are provided in the examples of the present document.

[0046] LRP5 Inhibitory antibodies

[0047] In a particular embodiment, the specific LRP5 inhibitor for use according to the invention is an inhibitory antibody for a LRP5-specific sequence.

[0048] In the context of the present invention, "inhibitory antibody" is understood as an antibody which specifically binds to LRP5 and is capable of inhibiting at least partially the biological activity of LRP5. In one embodiment, the LRP5 specific antibody is capable of preventing deposition of platelets over collagen when blood is contacted with a collagen-containing surface. A suitable assay for measuring the capacity of the antibody to prevent or to block deposition of platelets over collagen is explained in the examples of the present document.

[0049] In one embodiment, the LRP5 specific antibody is capable of preventing platelet aggregation after platelet stimulation with collagen and ADP. A suitable assay for measuring the capacity of the antibody to prevent or to block collagen and ADP- dependent platelet aggregation is explained in the examples of the present document.

[0050] In one embodiment, the LRP5 specific antibody is capable of preventing association of LRP5 with P2Y12 and / or translocation of P2Y12 to the plasma membrane in platelets activated with ADP. Suitable assays for measuring the association of LRP5 and P2Y12 and for detecting translocation of P2Y12 to the plasma membrane in platelets are provided in the examples of the present document.

[0051] Methods for obtaining antibodies are known by the skilled in the art.

[0052] The term "antibody" is understood as a polypeptide including at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as LRP5, or a fragment thereof. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Antibodies of the present disclosure include those that are specific for LRP5 and reduce or inhibit the biological activity of a LRP5 protein.

[0053] The term "antibody" includes intact immunoglobulins, as well the variants and portions thereof, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, diabodies or bispecific antibodies).

[0054] The antibodies can be prepared using any of the methods which are known by the person skilled in the art, some of which have been mentioned above. Thus, the polyclonal antibodies are prepared by means of immunizing an animal with the protein, or a fragment of said protein, to be inhibited. In the context of the present invention, suitable antibodies include intact antibodies comprising a variable antigen binding region and a constant region, "Fab", "F(ab')2" and "Fab"', Fv, scFv fragments, disulfide stabilized Fv proteins ("dsFv"), diabodies and bispecific antibodies. Once antibodies with LRP5 protein binding capacity are identified, those capable of inhibiting the activity of this protein will be selected using an inhibitory agent identification assay.

[0055] Examples of LRP5 specific antibodies which may be used in the context of the present invention include, without limiting to, an antibody that binds specifically to the extracellular region of LRP5. The “term extracellular region” refers to the space external to the outermost structure of a cell. For cells without external protective or external encapsulating structures, this refers to space outside of the plasma membrane. In LRP5 protein, about 85% of its 1615-amino-acid length is extracellular. In a particular embodiment, the antibody anti-LRP5 binds to the extracellular region of the LRP5 protein that comprises amino acids 32 to 1384 of the sequence SEQ ID NO: 2. In a more particular embodiment, the LRP5 specific antibody binds to the, to a region within said extracellular region that comprises the sequence ERVEKTTGDKRT (SEQ ID NO: 1). or to an epitope that comprises the sequence ERVEKTTGDKRT (SEQ ID NO: 1). As used herein, the term “epitope” refers to the part of an antigen that is recognized by the immune system, specifically by the LRP5 specific antibody.

[0056] Inhibitory proteins

[0057] In a particular embodiment, the specific LRP5 inhibitor for use according to the invention is an inhibitory protein or peptide.

[0058] As used herein, the term "inhibitory protein" or “protein inhibiting the LRP5 expression” refers to those proteins capable of binding to the LRP5 protein and inhibiting its activity. In one embodiment, the LRP5 inhibitory protein or peptide is capable of preventing deposition of platelets over collagen when blood is contacted with a collagen-containing surface. A suitable assay for measuring the capacity of the protein or peptide to prevent or to block deposition of platelets over collagen is explained in the examples of the present document.

[0059] In one embodiment, the LRP5 inhibitory protein or peptide is capable of preventing platelet aggregation after platelet stimulation with collagen and ADP. A suitable assay for measuring the capacity of the protein or peptide to prevent or to block collagen and ADP-dependent platelet aggregation is explained in the examples of the present document.

[0060] In one embodiment, the LRP5 inhibitory protein or peptide is capable of preventing association of LRP5 with P2Y12 and / or translocation of P2Y12 to the plasma membrane in platelets activated with ADP. Suitable assays for measuring the association of LRP5 and P2Y12 and for detecting translocation of P2Y12 to the plasma membrane in platelets are provided in the examples of the present document.

[0061] In addition, the term “inhibitory proteins” also includes fragments of proteins wherein said fragments have the LRP5 inhibitory effect. Proteins capable of inhibiting LRP5 are known in the state of the art, such as mesoderm development LRP chaperone (Mesd), the sclerotin (SOST) and Dickkopf homolog-1 (DKK1).

[0062] Polypeptides and peptides

[0063] Another suitable LRP5 inhibitor for use in the present invention is a polypeptide or peptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant or a peptide mimetic thereof. SEQ ID NO: 1 corresponds to amino acids 1178-1189 of the LRP5 protein (UniProtKB identifier 075197).

[0064] In some embodiments, the LRP5 inhibitor for use in the present invention is a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT). The polypeptide may comprise, comprise essentially or consist of 100,150, 200, 250, 300, 350, 400, 450, 500 amino acids. In some embodiments, the LRP5 inhibitor for use in the present invention is a peptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT). The peptide may comprise, comprise essentially or consist of 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90 or 95 amino acids.

[0065] The term “functionally equivalent variant” refers to a protein (or polypeptide or peptide) comprising one or more alterations, such as substitutions, insertions, deletions and / or truncations of one or more specific amino acid residues at one or more specific positions in the protein, and having the same function that the original polypeptide, particularly maintain the LRP5 inhibitory activity. In one embodiment, the inhibitory peptide for use according to the present invention has the sequence

[0066] EDQK / RQHK / VI LM / EDQK / KRNQE / TANS / TANS / GPA / DNE / KRNQE / RQ / TANS

[0067] The “functionally equivalent variant” preferably has a sequence identity with this peptide of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0068] In some embodiments, variants of the peptide of SEQ ID NO:1 include peptides which show conservative substitutions of amino acid residues with respect to SEQ ID NO:1 and which do substantially not impair the activity or properties of the peptide of variants described herein. Examples of such conservative amino acid substitutions include, but are not limited to, Ala to Gly, Ser or Thr; Arg to Gin, His or Lys; Asn to Asp, Gin, His, Lys, Ser, or Thr; Asp to Asn or Glu; Cys to Ser; Gin to Arg, Asn, Glu, His, Lys or Met; Glu to Asp, Gin, or Lys; Gly to Pro or Ala; His to Arg, Asn, Gin, or Tyr; lie to Leu, Met or Vai; Leu to lie, Met, Phe, or Vai; Lys to Arg, Asn, Gin, or Glu; Met to Gin, lie, Leu, or Vai; Phe to Leu, Met, Tip, or Tyr; Ser To Ala, Asn, Met, or Thr; Thr to Ala, Asn, Ser, or Met; Trp to Phe or Tyr; Tyr to His, Phe or Trp; and Vai to lie, Leu, or Met.

[0069] In the context of the present invention, it is considered that peptides or polypeptides or proteins with an identity of 70% with SEQ ID NO: 1 are functionally equivalent variants of the SEQ ID NO: 1 and maintain the same properties as the sequence to which they refer, i.e., they show LRP5 inhibitory activity when measured using one of the assays shown in the examples. The term "identity" or "sequence identity" as used herein refers to the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences, i.e. the proportion of identical nucleotide or amino acids between two compared nucleotide sequences or peptides / polypeptides / proteins along their full- length sequence. Depending on the number of common residues between the aligned sequences, a different degree of identity, expressed as a percentage, will be obtained. The degree of identity between two amino acids or nucleotides sequences may be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST [Altschul S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3): 403-10], The BLAST programs, for example, BLASTN, BLASTX, and T BLASTX, BLASTP and TBLASTN, are in the public domain at The National Center for Biotechonology Information (NCBI) website. The skilled person in the art understands that mutations in the nucleotide sequence of genes that lead to conservative amino acid substitutions at non-critical positions for the functionality of the protein are evolutionarily neutral mutations which do not affect its global structure or its functionality.

[0070] The term peptide also encompasses derivatized peptide molecules. Briefly, in the context of the present invention, a derivative is a peptide in which one or more of the amino acid residues of the peptide have been chemically modified (for instance by alkylation, acylation, ester formation, or amide formation) or associated with one or more non-amino acid organic and / or inorganic atomic or molecular substituents (for instance a polyethylene glycol (PEG) group, a lipophilic substituent (which optionally may be linked to the amino acid sequence of the peptide by a spacer residue or group such as P-alanine, y-aminobutyric acid (GABA), L / D-glutamic acid, succinic acid, and the like), a fluorophore, biotin, a radionuclide, etc.). Additionally or alternatively, the peptide may comprise non-essential, non-naturally occurring, and / or non-L amino acid residues. Non-limiting examples of such amino acid residues include for instance 2-aminoadipic acid, 3-amino- adipic acid, p-alanine, p-aminopropionic acid, 2-aminobutyric acid, 4- aminobutyric acid, 6- aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-di- aminopropionic acid, N-ethylglycine, N- ethylasparagine, hydroxylysine, allohydroxylysine, 3- hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methyl- isoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine, and statine halogenated amino acids.

[0071] As used herein, the term “peptide-mimetic” refers to a protein-like structure designed to mimic a peptide, capable of replacing it in its interactions with receptors and enzymes.

[0072] In the present invention, the peptide-mimetic of the polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or of the functionally equivalent variant is capable of preventing deposition of platelets over collagen when blood is contacted with a collagen-containing surface. A suitable assay for measuring the capacity of the peptide-mimetic to prevent or to block deposition of platelets over collagen is explained in the examples of the present document.

[0073] In one embodiment, the peptide-mimetic of the polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or of the functionally equivalent variant is capable of preventing platelet aggregation after platelet stimulation with collagen and ADP. A suitable assay for measuring the capacity of the peptide-mimetic to prevent or to block collagen and ADP-dependent platelet aggregation is explained in the examples of the present document.

[0074] In one embodiment, the peptide-mimetic of the polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or of the functionally equivalent variant is capable of preventing association of LRP5 with P2Y12 and / or translocation of P2Y12 to the plasma membrane in platelets activated with ADP. Suitable assays for measuring the association of LRP5 and P2Y12 and for detecting translocation of P2Y12 to the plasma membrane in platelets are provided in the examples of the present document.

[0075] Scaffolds

[0076] Another suitable LRP5 inhibitor is a scaffold presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof.

[0077] In a particular embodiment, the scaffold is a protein scaffold presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof. As used herein, the term “protein scaffold” refers to peptide-presenting structures such as designed ankyrin repeat proteins (DARPins), repebodies, anticalins, fibronectins, affibodies and engineered Kunitz domains. As noted above, in certain embodiments, the inhibitory peptides of the present disclosure (e.g., peptides comprising the SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant or a peptide mimetic thereof) can be linked, conjugated, complexed, bound, coupled, or otherwise attached to a carrier molecule, such as a protein or polymeric material such as polyethylene glycol (PEG) via PEGylation. For example, the variant peptides may be conjugated or otherwise attached to a suitable carrier molecule such as, but not limited to, keyhole limpet haemocyanin (KLH), ovalbumin, bovine serum albumin (BSA), or human serum albumin (HSA).

[0078] In another particular embodiment, the scaffold is a non-protein scaffold. In a more particular embodiment, the non-protein scaffold is a polymeric nanoparticle presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof. Polymeric nanoparticles (NPs) are particles within the size range from 1 to 1000 nm.

[0079] In another more particular embodiment, the non-protein scaffold is a dendrimer presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof. The term dendrimer refers to highly ordered, branched polymeric molecules. Dendrimers are nano-sized, radially symmetric molecules with well-defined, homogeneous, and monodisperse structure that has a typically symmetric core, an inner shell, and an outer shell.

[0080] As mentioned above, the present invention relates to the LRP5 for use in the treatment and / or prevention of thrombosis in a subject.

[0081] As used herein, the term "treating" (or "treat" or "treatment" or “therapy”) refers to processes involving a slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders. Furthermore, the “treatment” also refers to a process involving the avoiding of the appearance of symptoms which have not manifested themselves yet, but which will manifest themselves due to an untreated progression of the disorder, condition or disease. The treatment of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The "treatment" of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject / patient suffering from the disorder or disease. Accordingly, the "treatment" of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject / patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above).

[0082] The term “preventing” or (“prevent” or “prevention”) refers to the capacity of the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof to minimize or hinder the progression of a disease, condition or disorder in a subject.

[0083] In a particular embodiment, the prevention of thrombosis is a secondary prevention of thrombosis. A substantial proportion of patients with thrombosis will develop recurrent events. The term “secondary prevention” refers to the use of antithrombotic agent to prevent thrombotic events in patients with previous thrombosis. Some of the antithrombotic drugs used in secondary prevention of thrombosis include, but are not limited to, aspirin, rivaroxaban or sulodexid.

[0084] In the present invention, the condition or disorder to be treated and / or prevented is thrombosis.

[0085] The term “thrombosis” as used herein refers to the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel (a vein or an artery) is injured, the body uses platelets (thrombocytes) and fibrin to form a blood clot to prevent blood loss. Even when a blood vessel is not injured, blood clots may form in the body under certain conditions. Thrombosis may occur in veins (venous thrombosis) or in arteries (arterial thrombosis). Venous thrombosis leads to a blood clot in the venous system while arterial thrombosis affects the blood supply by arteries and leads to damage of the irrigated producing organ ischemia and necrosis. A piece of either an arterial or a venous thrombus can break off as an embolus, which could then travel through the circulation and lodge somewhere else as an embolism. This type of embolism is known as a thromboembolism. Complications can arise when a venous thromboembolism (commonly called a VTE) lodges in the lung as a pulmonary embolism.

[0086] In a particular embodiment of the present invention, the thrombosis is arterial and / or venous thrombosis.

[0087] In another particular embodiment, the LRP5 inhibitor is for use in the treatment and / or prevention of myocardial infarction, brain infarction, pulmonary infarction, arteriosclerosis obliterans, Buerger disease, thrombophlebitis, venous thromboembolism (economy class syndrome), arterial thromboembolism, and disseminated intravascular coagulation (DIC), atherosclerosis, angina, transient ischemic attacks and strokes, peripheral vascular diseases, preeclampsia, embolism and carotid endarterectomy, deep vein thrombosis (DVT) or a pulmonary embolism (PE).

[0088] In a more particular embodiment, the LRP5 inhibitor is for use in the treatment and / or prevention of deep vein thrombosis (DVT) or a pulmonary embolism (PE).

[0089] As used herein, the term "subject" or “patient” refers to human and non-human animals, such as veterinary subjects. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dog, cat, horse, cow, chickens, amphibians, and reptiles. In a particular embodiment, the subject is a human of any age or ethnical background.

[0090] In another particular embodiment, the treatment and / or prevention of thrombosis using the LRP5 inhibitor results in a reduction in the platelet deposition at the site of vascular damage.

[0091] Platelets or thrombocytes are a component of blood whose function (along with the coagulation factors) is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot.

[0092] Assays to determine platelet deposition are well known by an expert in the field. For example, immunofluorescence assays can be used. Perfusion experiments in a flat chamber can also be used. In this type of experiment, glass slides are coated with collagen type I and placed in flat perfusion chambers. Then, blood is perfused and platelet deposition can be measured using immunofluorescence.

[0093] In another particular embodiment, the treatment and / or prevention of thrombosis using the LRP5 inhibitor does not lead to an increased bleeding in the subject.

[0094] The term “bleeding” refers to blood escaping from the circulatory system from damaged blood vessels. Assays that can be used to determine the bleeding are well known by an expert in the field. For example, tail bleeding assays or liver bleeding assays can be used. According to the instant invention, the term “bleeding” designates any clinically relevant bleeding (major or clinically relevant non-major hemorrhage).

[0095] The term “major bleedings” designates the following clinical situations: bleeding that is associated with a fall in hemoglobin of 2 g per deciliter or more, bleeding resulting in transfusion of 2 or more units of packed red cells or whole blood (a red cell unit being defined as the quantity of red cells obtained from or corresponding to approximately 500 ml of whole blood), bleeding involving a critical organ (intracranial, intraocular, intraspinal, retroperitoneal or pericardial), and bleeding contributing to death;

[0096] The term “clinically relevant non-major bleeding” designates the following clinical situations: any bleeding compromising hemodynamics, any bleeding leading to hospitalization, subcutaneous hematoma larger than 25 cm2, or 100 cm2if there is a traumatic cause, intramuscular hematoma documented by ultrasonography, epistaxis that lasts for more than 5 minutes, is repetitive (i.e., two or more episodes of bleeding more extensive than spots on a handkerchief within 24 hours), or results in an intervention (e.g., packing or electrocoagulation), gingival bleeding occurring spontaneously (i.e., unrelated to eating or tooth brushing) or lasting for more than 5 minutes, hematuria that is macroscopic and is spontaneous or lasts for more than 24 hours after instrumentation (e.g., catheter placement or surgery) of the urogenital tract, macroscopic gastrointestinal hemorrhage, including at least one episode of melena or hematemesis, if clinically apparent with positive results on a fecal occult-blood test, rectal blood loss, hemoptysis, if more than a few speckles in the sputum and not occurring within the context of pulmonary embolism.

[0097] As mentioned above, successful strategies to inhibit thrombosis are presently clinically available, but they can cause significant bleeding. In the present invention, the use of LRP5 inhibitor in the treatment and / or prevention of thrombosis does not lead to an increased bleeding. According to the invention, the decrease in the incidence of bleedings, or the effect of minimizing the risk of bleedings, are meant in comparison with the incidence rate and risk, respectively, of either a treatment with a standard antithrombotic treatment. In particular, the incidence of bleedings is decreased compared with a treatment by either acetylsalicylic acid or clopidogrel.

[0098] The LRP5 inhibitors may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), inhalation spray, nasal, vaginal, rectal, sublingual, buccal or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.

[0099] The LRP5 inhibitors thereof can be incorporated into pharmaceutical compositions for its administration to the subject, wherein the pharmaceutical composition further comprises at least one inert pharmaceutically acceptable excipient.

[0100] The term “pharmaceutical composition” is such a form that allows the biological activity of the active ingredient contained therein to be effective and has unacceptable toxicity for the subject to which the composition is administered. The term "excipient" refers to a substance that aids the absorption of any of the components or compounds of the pharmaceutical composition, or stabilises the components or compounds and / or aids the preparation of the pharmaceutical composition in the sense of giving it consistency or flavours to make it more palatable. Thus, excipients may have the function, by way of example, but not limited to, binding the components (e.g. starches, sugars or cellulose), sweetening, colouring, protecting the active substance (e.g. to insulate it from air and / or moisture), filling a pill, capsule or any other presentation or a disintegrating function to facilitate the dissolution of the components, not excluding other excipients not listed in this paragraph. The term 'excipient' is therefore defined as a material which, included in the dosage forms, is added to the active substances or their associations to enable their preparation and stability, to modify their organoleptic properties or to determine the physical and chemical properties of the pharmaceutical composition and their bioavailability.

[0101] The expression “pharmaceutically acceptable excipient”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof.

[0102] The “dosage form” is the configuration to which the active ingredients and excipients are adapted to provide a pharmaceutical composition or medicinal product. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

[0103] The pharmaceutical composition comprises the LRP5 inhibitor in a therapeutically effective amount. The “therapeutically effective amount” is any amount of the component or compound of the composition which, when administered to a subject, is sufficient to produce the desired effect. Said component or compound of the composition refers to the LRP5 inhibitors. The therapeutically effective amount may vary depending on, for example, the age, body weight, general state of health, sex and diet of the subject, as well as the mode and timing of administration, the rate of excretion or any possible cotreatment with other drugs. In a particular embodiment, the LRP5 inhibitor or the pharmaceutical composition comprising the LRP5 inhibitor is administered intradermally, subcutaneously, via dermal infusion, via subcutaneous infusion, intravenously, or orally.

[0104] The LRP5 inhibitor may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

[0105] Formulations for oral use may also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0106] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0107] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0108] Furthermore, various systems are known which can be used for sustained release administration of the pharmaceutical composition, including, without limitation, encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. Suitable sustained release forms, as well as materials and methods for their preparation, are well known in the prior art. Thus, the orally administrable form of the pharmaceutical composition is in a sustained release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, without limitation, semisynthetic or synthetic, water insoluble or modified natural polymers, waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticisers or a combination of two or more thereof. Enteric coatings can be applied by conventional processes known to the person skilled in the art.

[0109] For topical use, creams, ointments, jellies, solutions or suspensions and the like, containing the LRP5 inhibitors or the genetically modified megakaryocytes or progenitors thereof are employed. Similarly, transdermal patches may also be used for topical administration. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0110] The LRP5 inhibitor may be used in combination with one or more other drugs in the treatment and / or prevention of thrombosis. Such other compound(s) / drug(s) may be administered by a route and in an amount commonly used therefore, contemporaneously or sequentially with the LRP5 inhibitor. When the LRP5 inhibitor is used contemporaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such other drugs and the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof is preferred. However, the combination therapy may also include therapies in which the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof and one or more other drugs are administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions include those that contain one or more other active ingredients, in addition to the LRP5 inhibitor.

[0111] Thus, in a particular embodiment, the LRP5 inhibitor is administered in combination with at least another suitable drug for the treatment of disorders associated with undesirable coagulation, wherein said at least another suitable drug is administered simultaneously, sequentially or separately.

[0112] Examples of other compounds useful for treating thrombosis which may be used in conjunction with the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof include, without being limited to, antithrombotic agents.

[0113] An antithrombotic agent is a substance which inhibits the formation or development of a thrombus (or thrombosis). For present purposes, the term "thrombus" or its equivalent includes the term "embolus", unless otherwise specifically indicated. In general, an antithrombotic agent, in the presence of mammalian blood or appropriately-prepared plasma, may display anticoagulant activity and / or platelet anti-aggregatory activity.

[0114] In a particular embodiment, the suitable drug for the treatment of disorders associated with undesirable coagulation is an antithrombotic agent. In a more particular embodiment, the antithrombotic agent has anticoagulant activity and / or platelet antiaggregation activity.

[0115] Anticoagulants are agents which limit or prevent the conversion of fibrinogen to fibrin and the consequent formation of a clot. A platelet anti-aggregating agent is a substance which inhibits platelet aggregation.

[0116] Examples of anticoagulants which can be used in conjunction with LRP5 inhibitors include, without being limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin and fondaparinux. Examples of antiplatelet agents which can be used in conjunction with LRP5 inhibitors or genetically modified megakaryocytes or progenitors thereof include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole / aspirin, ticlopidine and eptifibatide.

[0117] Genetically modified megakaryocyte or progenitor thereof

[0118] In another aspect, the present invention relates to a genetically modified megakaryocyte or progenitor thereof, in which the expression of the LRP5 gene is inhibited.

[0119] The term “megakaryocyte progenitor” as used herein refers to an hematopoietic stem cell committed to at least the megakaryocyte lineage cell with the propensity to generate proplatelets and / or platelets, and includes, but is not limited to, cells in the umbilical cord blood, bone marrow, and peripheral blood as well as hematopoietic stem cells, human embryonic stem cells, and induced pluripotent stem cells. In a particular embodiment, the megakaryocyte or progenitor thereof is chosen from the group consisting of a hemocytoblast, a megakaryoblast and a promegakaryocyte. In a more particular embodiment, the megakaryocyte or progenitor thereof is obtained from bone marrow or from circulating blood. The term "genetically modified”, when applied to the megakaryocyte or the progenitor thereof as used in the present invention herein refers to a megakaryocyte or the progenitor thereof which has been genetically modified by disrupting or deleting the LRP5 gene, or inhibiting its transcription or translation.

[0120] The term “disrupting the gene” (or “disrupt”) as used herein refers to interrupt the normal function of the gene. The disruption of the gene could be caused by the modification of any number of nucleotides.

[0121] The term “deleting the gene” (or “delete”) as used herein refers to a mutation in which from a nucleotide to a gen is removed. Any number of nucleotides can be deleted, from a single base to a complete gene. The gene deletion can be done through a variety of methods, including but not limited to homologous recombination, site-specific nucleases, Zinc-finger nucleases, CRISPR-Cas9, TALENs, gene knock-in and gene knock-out.

[0122] In another embodiment, the genetically modified megakaryocyte or progenitor thereof is modified so that the LRP5 gene is silenced.

[0123] The terms "silence" and "inhibit the expression of", "down-regulate the expression of," "suppress the expression of", and the like, in as far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a target gene, as manifested by a reduction of the amount of target mRNA, which may be isolated from a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

[0124] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene or the number of cells displaying a certain phenotype. In principle, target genome silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given nucleic acid inhibits the expression of a target gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below and those known in the art shall serve as such reference. For example, in certain instances, expression of a target gene is suppressed by at least about 5 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, or 50 percent by administration of the double-stranded oligonucleotide. In some embodiments, a target gene is suppressed by at least about 60 percent, 70 percent, or 80 percent by administration of the double- stranded oligonucleotide. In some embodiments, the target gene is suppressed by at least about 85 percent, 90 percent, or 95 percent by administration of the double-stranded oligonucleotide.

[0125] For instance, the nucleic acid sequence can be introduced into a cell that expresses the LRP5 target gene. The mRNA level of the LRP5 target gene in the cell can be detected by using RT-PCR, Northern blot or any other standard methods. Alternatively, the level of the polypeptide encoded by the target mRNA can be measured using Western blot, ELISA or any other immunological or non-immunological method. A substantial change in the expression level of mRNA or of the protein encoded by the target gene after the introduction of the RNAi sequence is indicative of the effectiveness of the RNAi sequence in suppressing the expression of the target gene. A RNAi sequence which has inhibitory effect on target gene expression but does not significantly affect the expression of other genes can be selected. In another specific example, multiple RNAi sequences can be introduced into the same target cell. These RNAi sequences specifically inhibit target gene expression but not the expression of other genes. In yet another specific example, RNAi sequences that inhibit the expression of the target gene and other gene or genes can be used.

[0126] The skilled person will appreciate that the specific choice of nucleic acid molecule which is incorporated into the conjugates of the invention will depend on the type of selectivity agent present in the conjugate.

[0127] The term “inhibiting” when referred to the transcription or translation of the LPRP5 gene, as used herein, refers to the stoppage or decrease of the transcription of a certain gene and / or the translation of the corresponding mRNA. In a more particular embodiment, the LRP5 gene expression is inhibited by using: a LRP5-specific RNA interference construct (RNAi), a microRNA (miRNA), an LRP5-specific antisense oligonucleotide or a LRP5- specific ribozyme or DNAzyme. In a particular embodiment, the LRP5 inhibitor for use according to the invention is a LRP5 specific RNA interference (RNAi), i.e. RNAi which are capable of knocking down the expression of LRP5 or any component gene necessary for LRP5 function. The double stranded oligonucleotides used to effect RNAi are preferably less than 30 base pairs in length and, more preferably, comprise about 25, 24, 23, 22, 21 , 20, 19, 18 or 17 base pairs of ribonucleic acid. Optionally the dsRNA oligonucleotides of the invention may include 3' overhang ends. The specific sequence utilized in design of the oligonucleotides may be any contiguous sequence of nucleotides contained within the expressed gene message of the target. Several different types of molecules have been used effectively in the RNAi technology including small interfering RNA (siRNA), microRNA (miRNA) and short hairpin RNA (shRNA).

[0128] The term small interfering RNA ("siRNA") refers to small inhibitory RNA duplexes that induce the RNA interference pathway. These molecules may vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3' end of the sense strand and / or the antisense strand. The term "siRNA" includes duplexes of two separate strands. As used herein, siRNA molecules are not limited to RNA molecules but further encompass nucleic acids with one or more chemically modified nucleotides, such as morpholinos. Exemplary, non-limiting, siRNAs for LRP5 include commercially available LRP5 siRNAs from Santa Cruz Biotechnology (Catalog Nos. sc-43900, sc-43900-PR) and from ThermoFisher (Catalog Nos. 1299001)

[0129] The term "shRNA" or "short hairpin RNA" as used herein refers to a dsRNA where the two strands are connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5' end of the respective other strand to form a duplex structure. Exemplary, non-limiting, shRNAs for LRP5 include commercially available LRP5 shRNAs from Santa Cruz Biotechnology (Catalog Nos. sc-43900-SH, sc-43900-V) and from Abbexa (Catalogue No.:abx952732).

[0130] The term "micro RNA" or "miRNA" refers to short single-stranded RNA molecules, typically of about 21-23 nucleotides in length capable of regulating gene expression. miRNAs may be synthetic (i.e., recombinant) or natural. Natural miRNAs are encoded by genes that are transcribed from DNA and processed from primary transcripts ("pri- miRNA") to short stem-loop structures ("pre-miRNA"), and finally to mature miRNA. Mature miRNA molecules are partially complementary to one or more mRNA molecules, and downregulate gene expression via a process similar to RNA interference, or by inhibiting translation of mRNA. In a preferred embodiment, the LRP5-specific miRNA is, but not limited, to microRNA-23a, miR-9-5p, miR-675-5p, miR-138-5p and miR-545-3p. In a more preferred embodiment, the LRP5-specific miRNA is miR-545-3p.

[0131] An "antisense sequence," as used herein includes antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).

[0132] A LRP5-specific RNA interference construct (RNAi), a microRNA (miRNA) for use in the genetically modified megakaryocyte or progenitor thereof according to the invention contain one or more sequence stretches which are complementary to the nucleic acid sequence of the LRP5 target mRNA. Different criteria are available to the skilled person for selecting the most adequate nucleic acid. By way of example, when the nucleic acid is a RNAi, this can be selected by scanning the mRNA sequence of the target for AA dinucleotides and recording the 19 nucleotides immediately downstream of the AA. Other methods can also be used to select the nucleic acid targets. In one example, the selection of the RNAi target sequence is purely empirically determined (see, e.g., Sui G et al., Proc. Natl. Acad. Sci. USA 99:5515-20 (2002)), as long as the target sequence starts with GG and does not share significant sequence homology with other genes as analyzed by BLAST search. In another example, a more elaborate method is employed to select the RNAi target sequences. This procedure exploits an observation that any accessible site in endogenous mRNA can be targeted for degradation by synthetic oligodeoxyribonucleotide / RNase H method (see, e.g., Lee NS et al., Nature Biotechnol. 20:500-05 (2002)).

[0133] Alternatively, the hairpin RNAi expression cassette is constructed to contain the sense strand of the target, followed by a short spacer, the antisense strand of the target, and 5-6 Ts as transcription terminator. The order of the sense and antisense strands within the RNAi expression constructs can be altered without affecting the gene silencing activities of the hairpin RNAi. In certain instances, the reversal of the order may cause partial reduction in gene silencing activities.

[0134] The length of nucleotide sequence being used as the stem of RNAi expression cassette can range, for instance, from 19 to 29. The loop size can range from 3 to 23 nucleotides. Other lengths and / or loop sizes can also be used.

[0135] Additional criteria can also be used for selecting RNAi target sequences. For instance, the GC content of the remaining 19-mer sequence can be limited to between 45% and 55%. Moreover, any 19-mer sequence having three consecutive identical bases (i.e., GGG, CCC, TTT, or AAA) or a palindrome sequence with 5 or more bases is excluded. Furthermore, the remaining 19-mer sequence can be selected to have low sequence homology to other genes. In one specific example, potential target sequences are searched by BLASTN against NCBI's human UniGene cluster sequence database. The human UniGene database contains non- redundant sets of gene-oriented clusters. Each UniGene cluster includes sequences that represent a unique gene. 19-mer sequences producing no hit to other human genes under the BLASTN search can be selected. During the search, the e-value may be set at a stringent value (such as "1").

[0136] The effectiveness of any RNAi sequence derived according to the present invention in silencing expression of the target gene can be evaluated using various methods known in the art.

[0137] As used herein, the term "ribozyme" or "RNA enzyme" or "catalytic RNA" refers to an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome, the ligase activity of a DNA ligase, and a number of other chemical reactions performed by conventional protein enzymes.

[0138] “DNA enzyme”, as used herein, relates to enzymes that incorporate some of the mechanistic characteristics of both antisense and ribozymes technologies. The DNA enzymes are designed such that they recognize a particular target nucleic acid sequence, similar to the antisense oligonucleotide, however like ribozymes, they are catalytic and specifically cleave the target nucleic acid. In a preferred embodiment, the DNA enzyme is specific to LRP5 sequence.

[0139] Therapeutic uses of the genetically modified megakaryocyte or progenitor thereof

[0140] The present invention relates to a genetically modified megakaryocyte or progenitor thereof as defined above for use in medicine.

[0141] As mentioned above, the present invention relates to the genetically modified megakaryocyte or progenitor thereof in which the LRP5 gene expression is inhibited as defined above for use in the treatment and / or prevention of thrombosis in a subject.

[0142] The terms “treating”, “preventing”, “secondary prevention”, “thrombosis”, “subject”, “Platelets”, “bleeding”, including “major bleedings” and “clinically relevant non-major bleeding”, ““pharmaceutical composition”, "excipient", “therapeutically effective amount", “pharmaceutically acceptable excipient”, “dosage form” “anticoagulants”, “platelet anti-aggregating agent” have been described in the context of the use of LRP5 inhibitors and are equally applicable to the present method.

[0143] In a particular embodiment, the prevention of thrombosis is a secondary prevention of thrombosis. Some of the antithrombotic drugs used in secondary prevention of thrombosis include, but are not limited to, aspirin, rivaroxaban or sulodexid.

[0144] In the present invention, the condition or disorder to be treated and / or prevented is thrombosis. In yet another embodiment, the thrombosis is arterial and / or venous thrombosis.

[0145] In another particular embodiment, the genetically modified megakaryocyte or progenitor thereof is for use in the treatment and / or prevention of myocardial infarction, brain infarction, pulmonary infarction, arteriosclerosis obliterans, Buerger disease, thrombophlebitis, venous thromboembolism (economy class syndrome), arterial thromboembolism, and disseminated intravascular coagulation (DIC), atherosclerosis, angina, transient ischemic attacks and strokes, peripheral vascular diseases, preeclampsia, embolism and carotid endarterectomy, deep vein thrombosis (DVT) or a pulmonary embolism (PE). In a more particular embodiment, the genetically modified megakaryocyte or progenitor thereof is for use in the treatment and / or prevention of deep vein thrombosis (DVT) or a pulmonary embolism (PE).

[0146] In another particular embodiment, the treatment and / or prevention of thrombosis using the genetically modified megakaryocyte or progenitor thereof in which the expression of the LRP5 gene expression has been inhibited results in a reduction in the platelet deposition at the site of vascular damage.

[0147] In another particular embodiment, the treatment and / or prevention of thrombosis using the genetically modified megakaryocyte or progenitor thereof in which the expression of the LRP5 gene has been inhibited does not lead to an increased bleeding in the subject. According to the invention, the decrease in the incidence of bleedings, or the effect of minimizing the risk of bleedings, are meant in comparison with the incidence rate and risk, respectively, of either a treatment with a standard antithrombotic treatment. In particular, the incidence of bleedings is decreased compared with a treatment by either acetylsalicylic acid or clopidogrel.

[0148] The genetically modified megakaryocytes or progenitors thereof may be administered by any route which is suitable for the administration of a cell-therapy composition.

[0149] In one embodiment, the genetically modified megakaryocytes or progenitors thereof are administered parenterally, which includes, among others, intramuscular, intraperitoneal, intravenous, ICV or subcutaneous injection.

[0150] The genetically modified megakaryocytes or progenitors thereof can be incorporated into pharmaceutical compositions for its administration to the subject, wherein the pharmaceutical composition further comprises at least one inert pharmaceutically acceptable excipient. The pharmaceutical composition comprises the genetically modified megakaryocyte or progenitor thereof in a therapeutically effective amount.

[0151] In a particular embodiment, the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof or the pharmaceutical composition comprising the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof is administered intradermally, subcutaneously, via dermal infusion, via subcutaneous infusion, intravenously, or orally.

[0152] It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0153] The genetically modified megakaryocyte or progenitor thereof may be used in combination with one or more other drugs in the treatment and / or prevention of thrombosis. Such other compound(s) / drug(s) may be administered by a route and in an amount commonly used therefore, contemporaneously or sequentially with the genetically modified megakaryocyte or progenitor thereof. When the genetically modified megakaryocyte or progenitor thereof is used contemporaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such other drugs and the genetically modified megakaryocyte or progenitor thereof is preferred. However, the combination therapy may also include therapies in which the genetically modified megakaryocyte or progenitor thereof and one or more other drugs are administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the genetically modified megakaryocyte or progenitor thereof and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions include those that contain one or more other active ingredients, in addition to the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof with the LRP5 gene expression inhibited.

[0154] Thus, in a particular embodiment, the genetically modified megakaryocyte or progenitor thereof is administered in combination with at least another suitable drug for the treatment of disorders associated with undesirable coagulation, wherein said at least another suitable drug is administered simultaneously, sequentially or separately. Examples of other compounds useful for treating thrombosis which may be used in conjunction with the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof include, without being limited to, antithrombotic agents.

[0155] An antithrombotic agent is a substance which inhibits the formation or development of a thrombus (or thrombosis). In a particular embodiment, the suitable drug for the treatment of disorders associated with undesirable coagulation is an antithrombotic agent. In a more particular embodiment, the antithrombotic agent has anticoagulant activity and / or platelet anti-aggregation activity. Examples of anticoagulants which can be used in conjunction with the genetically modified megakaryocytes or progenitors thereof include, without being limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin and fondaparinux. Examples of antiplatelet agents which can be used in conjunction with LRP5 inhibitors or genetically modified megakaryocytes or progenitors thereof include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole / aspirin, ticlopidine and eptifibatide.

[0156] Derivatized blood contact surfaces and uses thereof

[0157] In another aspect, the present invention relates to a blood contact surface wherein the surface is coated with a LRP5 inhibitor, wherein the LRP5 inhibitor can be any of the inhibitors defined above.

[0158] The term “blood contact surface” as used herein refers to surfaces that come into contact with a patient's blood. These surfaces can be comprised in medical devices. Medical devices that come into contact with a patient's blood can be treated with or manufactured with blood compatible surfaces to reduce adverse biological reactions associated with the use of such medical devices. These surfaces can act as a barrier between the medical device and blood, thus allowing a wider range of materials to be used for the medical device itself. The surfaces that are in contact with blood must not interact adversely with any blood component or activate or destroy blood components.

[0159] Catheters, guidewires, dialyzers, oxygenators (artificial lungs), heart-supporting systems, cardiac pacemakers, vascular grafts, stents, heart valves, micro-, and nanoparticles are widely used medical devices and materials coming in direct contact with blood. In a particular embodiment, the blood contact surface is a medical device selected from the group consisting of: catheters, guidewires, dialyzer, oxygenators (artificial lungs), heart-supporting systems, cardiac pacemaker, vascular grafts, stents, heart valves, micro-, and nanoparticles.

[0160] In a more particular embodiment, the blood contact surface is from a catheter. The term “catheter” refers to a medical device in the form of a narrow, elongated tube that is inserted into a tissue or vein.

[0161] In the present invention the blood contact surface is coated with a LRP5 inhibitor. In a particular embodiment, the LRP5 inhibitor that is coating the blood contact surface is selected from the group consisting of:

[0162] (i) a LRP5-specific aptamer;

[0163] (ii) a LRP5 specific antibody;

[0164] (iii) a protein inhibiting the LRP5 expression;

[0165] (iv) a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant or a peptide mimetic thereof;

[0166] (v) a scaffold presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof.

[0167] In a more particular embodiment, the LRP5 inhibitor that is coating the blood contact surface is a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant thereof.

[0168] EXAMPLES

[0169] The following examples illustrate the invention and must not be considered as limiting the scope thereof.

[0170] Material and Methods

[0171] Human Platelet Isolation Human blood was collected into acid citrate dextrose (29.9 mM sodium citrate, 113.8 mM glucose, 72.6 mM NaCI, 2.9 mM citric acid, pH 6.4) and was centrifuged (10 min, 280 x g, 20 °C w / o brake) to obtain PRP. PRP was removed and centrifuged in the presence of 5mg / mL of PGE1 . Platelet pellets were suspended in Hepes-Tyrode buffer. Washed platelets were then incubated 5 min, 37 °C with 10 pM ADP or 10 min, 37°C with 30pg / ml collagen. After the incubation period platelets were centrifuged 10 min at 4°C to stop the reaction, supernatants were removed and platelet pellets stored deep- frozen (-80 °C). The pellet was lysed with 1 mL of cold lysis buffer (1 % Triton X-100, 10 mM Tris, pH 7.5, 150 mM KCI, and protease inhibitors) and then centrifuged at 10 000 g for 10 min at 4°C to obtain platelet lysates. The procedure for human platelets withdrawing was approved by the Sant Pau Hospital Ethics Committee (ICCC025 / 4356).

[0172] Wt and Lrp5- / - mice Platelet Isolation

[0173] Blood was drawn by cardiac puncture under terminal anesthesia into acid citrate dextrose, 1 :7 ratio, v / v. Blood was diluted with 250 pl of modified Tyrode’s-HEPES buffer (134 mM NaCI, 0.34 mM Na2HPO4, 2.9 mM KCI, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, and 1 mM MgCh, pH 7.3) and centrifuged at 180 g for 6 minutes at room temperature. Platelet-rich plasma was removed, and platelets were isolated by centrifugation at 550 g for 6 minutes in the presence of the platelet adenylyl cyclase prostaglandin PGE1 (5mg / ml). Pelleted platelets were suspended to the required density in standard Tyrode’s-HEPES buffer (119 mM NaCI, 5 mM KCI, 25 mM HEPES, 2mM CaCh,5 mM glucose, and 2 mM MgCh) and rested for 30 minutes at 37°C prior to stimulation.

[0174] Platelet aggregation

[0175] Blood from Wt and Lrp5- / - mice was obtained by cardiac puncture into a syringe containing acid citrate dextrose anticoagulant (9:1). Blood was centrifuged 100 g for 10 min and PRP was used for aggregation experiments. Aggregation was measured by a turbidimetric method in a dual-channel Payton aggregometer. A 300 pl aliquot of platelet suspension was stirred at 1100 rpm and activated by the addition of 10 pM ADP. PRP from healthy donors was obtained as for mice but before aggregation with 1 pM ADP or 1 pg collagen, PRP was incubated for 30 min with or without 25 pM of LRP5 inhibitor treatment (GTX89521-PEP, Gene Tex). The extent of aggregation was estimated by measuring the maximum curve height above baseline level. Carotid artery occlusion in mice

[0176] Mice (N=8 per genotype) were anesthetized and their right carotid was exposed by cervical incision, separated from the adherent tissue and vagus nerve, and carotid flow was registered using a perivascular flowmeter (Transonic System INC, Ts 420). Thrombus formation by ferric chloride was induced by applying one 1x2 mm filter paper saturated with 7.5% FeCh for 90 seconds. A small piece of parafilm was kept below the artery prior to FeCh application so that it did not contact the adjacent tissue. The end points of the experiment were when blood flow had ceased for more than 30 seconds; if occlusion was not seen after 30 min, the time was recorded as 30min for statistical comparisons.

[0177] Perfusion experiments in the flat chamber

[0178] Glass slides were coated with collagen type I (0.1 mg / mL) and placed in flat perfusion chambers. Human blood or blood of Wt mice with or without 25 pM of LRP5 inhibitor or Lrp5- / - mice blood was perfused for 2 min at a constant shear rate of 975 s-1. Platelets were rendered fluorescent by mepacrine 10 mol L / 1 (Sigma).

[0179] Pharmacological LRP5 Inhibitor

[0180] LRP5 inhibitor (GTX89521-PEP, Gene Tex) is a peptide that binds to and blocks LRP5 activity. It was reconstituted in 200 pl distilled water to obtain a 0.5 mg / ml peptide solution.

[0181] Thrombelastographic coagulation analysis

[0182] Dynamic whole blood clot formation in Wt and Lrp5- / - mice was performed with the rotation thromboelastometry (ROTEM) coagulation analyzer (Pentapharm, Germany), which is based on the thrombelastograph system using the tissue factor-activated extrinsic pathway assay (ExTEM, n=6 mice / genotype) and the ellagic acid activated intrinsic pathway assay (InTEM n=7 mice / genotype). The following variables were determined: clotting time (CT, s), clot formation time (CFT, s), clot firmness (A10, mm) and maximum clot firmness (MCF, mm).

[0183] Tissue Factor activity TF-PCA was measured from citrated blood by using a factor (F) Xa generation test previously described. TF-PCA values were obtained from a standard curve performed with FXa.

[0184] Tail Vein Bleeding Time and Liver Bleeding measurements

[0185] Wt (n=23) and Lrp5- / - (n=18) mice were anesthetized with isofluorane and placed on a prewarmed pad. Because Lrp5- / - mice are smaller in size, their tails and tail veins are also about 10% to 20% smaller. To monitor bleeding times under conditions similar for Wt and Lrp5- / - mice they were anesthetized with ketamine / metodomidine by intraperitoneal injection and tails were cut with a scalpel at the position were the diameter of tails was 2.5 mm. Each tail was immersed in normal saline (37°C). The time from the incision to cessation of bleeding was recorded as the TVBT. Any continued bleeding was stopped by cauterization at 5 minutes to prevent hypovolemic shock.

[0186] Acetylsalicylic acid (1 ,5mg / kg) or clopidogrel (5mg / kg) was given to 5 Wt mice diluted in water through oral gavages and after 4h tail bleeding assays were performed. The same procedure was used in Wt mice that underwent Liver Bleeding assays. After 4h of oral gavages, mice were chest opened and the liver was exposed. A Whatmann paper (previously weighed) was located underneath the same lobe of the liver in each mouse, a 4mm incision was made and blood was collected in the Whatmann paper during the following 5 minutes. The weight: Whatmann paperwith blood -Whatmann paper without blood was recorded as liver bleeding.

[0187] Platelet spreading and adhesion assay

[0188] Coverslips were coated with either 0.1 mg / ml collagen type I or 2mg / ml fibrinogen and left overnight, followed by nonspecific blocking step with 2% BSA for 1 hour. Platelets were suspended to 2.5 x io6 / ml in Tyrodes-HEPES buffer, dispensed onto the coverslip and left for 90 minutes when cells were washed and fixed with 4% paraformaldehyde. Adhesion of platelets was observed by immunofluorescence as explained below. Fifteen images in different random parts of the coverslip area were taken and processed with a Leica microscope. The surface area and number of adherent platelets was estimated using Imaged software.

[0189] Western blot Platelet extracts from mice or human were resolved by SDS-PAGE and electrotransferred to nitrocellulose membranes. LRP5 (Abeam), p-catenin (Millipore), c- jun (Santa Cruz Biotechnology), BMP2 (R+D Systems), LEF1 (Sigma), OPN (Sigma), GPVI (LifeSpan Biosciences) pPLCy2 (Cell Signalling), PLCy2 (Cell Signalling), p-actin (Abeam), P2X1 (Novusbio), P2Y1 (Santa Cruz Biotechnology), and P2Y12 (Abeam) primary antibodies were used. Secondary anti-rabbit or anti-mouse antibodies were from Dako. Band densities were determined with the ChemiDoc™ XRS system (Bio-Rad) in chemiluminescence detection modus and Quantity-One software (Bio-Rad). Normalization was performed against p-actin (Abeam).

[0190] Determination of GPVI, FCRy and a2P1 by flow cytometry

[0191] Blood was drawn from Wt and Lrp5- / - mice and anticoagulated with 1 / 10 volume of 150 mM trisodium citrate. To obtain platelet-rich plasma (PRP), blood was centrifuged at 125 g for 10 minutes. To avoid contaminations with other cell types only the upper two thirds of the PRP fraction were used. Each PRP sample was treated or not with 30 pg / ml collagen an analyzed with GPVI (Biotechne Ltd), FCRyll and III (Abeam) and a2pi (Abeam) antibodies.

[0192] P2Y12-mediated VASP phosphorylation assay

[0193] VASP phosphorylation was measured by flow cytometry using a kit (BioCytex, Marseilles, France) essentially according to the manufacturer’s recommendations. In brief, citrated whole blood (10 pL) and prostaglandin E1 (9 pL) or the same volume of PGE1 plus ADP were incubated for 10 minutes at room temperature. The samples were then fixed, permeabilized, and labeled with a fluorescently conjugated monoclonal antibody (clone 16C2) directed against the serine 239 phosphorylated form of VASP and, as a platelet marker anti-CD61 antibody. Analysis was performed in a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).

[0194] Immunofluorescence

[0195] Mice platelets were fixed with 4% PFA and permeabilized with 0.5% Tween in PBS at room temperature. After incubation in blocking buffer (3% bovine serum albumin in PBS), primary GPIIb (CD41-GeneTex) or GPVI (LifeSpan Biosciences) antibodies were added for 1 h at RT in a moist chamber. Secondary antibodies and Hoechst 33342 were added for 1 h and cells were washed and covered with Prolong Gold antifade reagent (Molecular Probes). Images were recorded on a Leica inverted fluorescence confocal microscope (Leica TCS SP2-AOBS). Fluorescent images were acquired in a scan format of 1024 x 1024 pixels in a spatial data set (xyz) and were processed with the Leica Standard Software TCS-AOBS. Controls without primary antibodies showed no fluorescence labeling.

[0196] Platelet subfractionation in mice and human

[0197] Human blood was collected from 5 healthy individuals at day 0 (basal) and immediately after blood extraction individuals were given 75 mg clopidogrel. 23h later, another 75 mg clopidogrel pill was administered. 1h after this last intake, blood was extracted (Clopidogrel). Platelet subfractionation from mice or human experiments was performed with the ProteoExtract Subcellular Proteome Extraction Kit following the manufacturer instructions.

[0198] Immunoprecipitation of LRP5 or P2Y12 in human platelets

[0199] Total protein content in cell lysates of human platelets was estimated using the BCA protein assay (Pierce). IP was carried out on 500 pg of total protein. Total proteins, cytoplasmic and membrane fractions were incubated with 5 pl of unspecific IgG, LRP5 or P2Y12 specific antibodies at room temperature for 1 h. Immunoprecipitated proteins were subjected to Western blotting as described.

[0200] Serotonin and PF4 release assays

[0201] Washed mice platelets were stimulated with 10pm ADP for 5 minutes at 37°C and immediately pelleted by centrifugation for 30 seconds. The serotonin and PF4 levels in the supernatant were determined using the Serotonin ELISA (LDN, Germany) and PF4 ELISA (Abeam) Kits following the manufacturer’s instructions.

[0202] Statistical analysis

[0203] Results are expressed as mean ± S.E.M. StatView statistical package was used for all the analysis. Comparisons among groups were performed by ANOVA analysis. Statistical significance was considered when p<0.05. All the experiments were performed at least three times.

[0204] Results

[0205] 1. LRP5 is expressed in platelets and is needed for flow dependent platelet deposition and platelet aggregation The authors demonstrated that LRP5 is found in Wt mice and human platelets (Fig 1A). Lrp5- / - mice were then used to examine the role of LRP5 in platelet adhesion to collagen. The authors perfused blood from Wt and Lrp5- / - mice over collagen-coated slides at high-shear-rate (975 s-1). Platelet deposition was analyzed by immunofluorescence showing that there was a highly significant inhibition in Lrp5- / - platelet deposition in comparison to Wt platelet deposition (Fig 1B). Similarly, when blood from Wt mice was treated with 25 pM of the LRP5 inhibitor, GTX89521-PEP for 30 min and perfused over collagen, abrogation of platelet deposition was observed. These results showed that LRP5 has a significant role in flow dependent platelet deposition. For light transmission platelet aggregation (LT A) PRP from Wt, Lrp5- / + and Lrp5- / - mice was stimulated with 10pM ADP to induce aggregation (Fig 1 C, Table 1). While Wt and Lrp5- / + mice platelets responded to ADP in a similar manner, Lrp5- / - mice platelets showed a different aggregation pattern without the first wave of aggregation. Maximum aggregation at 1, 2 and 4 minutes is shown in Table 1. Differences between Wt and Lrp5- / - mice aggregation patterns were significant as soon as 1 minute post incubation with 10pM ADP (Fig 10, Table 1).

[0206] Blood from healthy human volunteers was treated for 30 min with 25 pM of LRP5 inhibitor or vehicle and stimulated with 1 pM ADP and 1 pg collagen. Again, differences in maximum aggregation between LRP5 inhibitor treated and untreated platelets could be observed after 1 minute of ADP stimulation (Fig 1 D, Table 2). When platelets were treated with LRP5 inhibitor and stimulated with 1 pg collagen, a significant reduction in maximum aggregation after 2 minutes of stimulation and up to 4 minutes was observed indicating that LRP5 contributes to platelet aggregation in humans (Fig 1D; Table 2).

[0207] Table 1. Aggregation analyses in mice platelets. Aggregation analyses of mice PRP (N=14 / genotype). *p<0.05, ***p<0.005. Table 2. Aggregation analyses in human platelets. Aggregation analyses of human PRP

[0208] (N=4). *p<0.05, ***p<0.005.

[0209] 2. Lrp5- / - mice show delayed carotid artery occlusion time

[0210] Carotid artery thrombosis was induced in vivo in Wt, Lrp5- / + and Lrp5- / - mice and time to total occlusion (TTO) determined. Wt animals had a TTO of 21±2 min while Lrp5 null mice did not reach total occlusion at the end of the study period (30 min) (p<0.05; Fig 1 E). Interestingly, heterozygous Lrp5- / + mice showed full occlusion at 23±3 min similar to Wt animals. This experimental procedure did not induce bleeding in any of the intervened animals independent of genotype.

[0211] 3. Coagulation in Wt and Lrp5- / - mice.

[0212] Thrombelastographic analyses performed in blood from Wt and Lrp5- / - mice showed that LRP5 expression did not affect the activated intrinsic coagulation pathway (InTEM) since all ROTEM parameters representing the initiation (CT), propagation (CFT) and clot firmness (A10, MCF) were similar for Wt and Lrp5- / - mice (Fig 2A). Absence of LRP5 expression did affect tissue factor-triggered extrinsic pathway (ExTEM). Indeed, whole blood from Lrp5- / - mice showed a 36.34 s increase in CT and a 53.7 s increase in CFT indicating that platelets from Lrp5- / - mice were less reactive and took more time to activate after a challenge. However, there was no difference between Wt and Lrp5- / - mice regarding clot firmness (A10, MCF) indicating that clotting is not affected by LRP5 (Fig 2B).

[0213] The authors further investigated tissue factor procoagulant activity (TF-PCA) in Wt and Lrp5- / - mice and found a 40.2% reduction in TF-PCA in platelets of Lrp5- / - mice (Fig 2C). Platelet count was similar in Wt and Lrp5- / - mice (Fig 2D). Tail bleeding time experiments showed a mild prolongation in Lrp5- / - mice (Fig 3A; 42±9 s Fig 3C). For comparative purposes Wt mice were given oral acetylsalicylic acid (ASA, 1.5mg / kg) or clopidogrel (clopi, 5mg / kg) and after 4h, tail vein bleeding assays were performed showing 155±12 s and 297±9 s tail bleeding time respectively (Fig 3B, Fig 3D). Liver bleeding measurements were also performed in Wt mice showing similar results: Lrp5- / - mice showed mild longer bleeding to haemostasis than Wt mice (Fig 3E, 3G) while Wt mice treated with ASA or Clopidogrel showed significant increased loss of blood from incision to cessation of bleeding (Fig 3F, 3H).

[0214] 4. LRP5-mediated surface attachment in platelets from Wt and Lrp5- / - mice

[0215] The authors next studied LRP5 involvement in platelet surface attachment as an indication of function inhibition. They isolated platelets from Wt and Lrp5- / - mice and analyzed their binding to fibrinogen and collagen coated surfaces in static conditions. There was a significant reduction in the attachment of platelets from Lrp5- / -m'\ce to fibrinogen and collagen coated surfaces (Fig 4A). Collagen-adhered platelets (GPVI++) and fibrinogen-adhered platelets (GPI I b++) were observed in Wt mice (Fig 4A). Platelets from Lrp5- / - mice showed less adhesion to both fibrinogen and collagen although they have similar GPVI expression levels than Wt mice platelets (Fig 4B).

[0216] 5. Collagen activation in Lrp5- / - mice platelets

[0217] Glycoprotein GPVI expression levels in Wt and Lrp5- / - mice washed platelets were similar (Fig 5A). Platelet rich plasma (PRP) from Wt and Lrp5- / - mice was treated with 30pg / ml collagen at 37°C and membranes and cytoplasms were separated. Collagen induced GPVI translocation to the cell membranes and phosphorylation of PLCy2 with no change in PLCy2 total protein (Fig 5B). There were no differences in pPLCy / PLCy ratio (Fig 5B) or in GPVI signaling between Wt and Lrp5- / - mice platelets after collagen stimulation.

[0218] Schematic of GPVI (grey) and GPI I b (black) expression in Wt and Lrp5- / - mice platelets attached to collagen coated surfaces showing colocalization of GPVI receptor and GPI lb in Wt mice platelets. However, platelets from Lrp5- / - mice showed less deposition density and there was no colocalization between GPVI and GPI I b (Fig 5C).

[0219] Similarly activated (30pg / ml collagen) PRP of Wt and Lrp5- / - mice was analyzed by flow cytometry for GPVI, FcRy and a2pi integrin levels. Results showed that GPVI levels were similarly increased in Wt and Lrp5- / - mice platelets after collagen stimulation (Fig 5D). Interestingly, a2 i levels were highly induced in Lrp5- / - platelets by collagen. FcRy levels were not upregulated in the membrane of Lrp5- / - mice platelets after collagen activation.

[0220] 6. ADP induced activation of Lrp5- / - mice platelets

[0221] Washed platelets of Wt and Lrp5- / - mice were treated with 10pM ADP and secreted proteins collected. The authors found an increased release of PF4 from a-granules in Wt but not in Lrp5- / - or in Lrp5- / + mice activated platelets (Fig 6A). Consistently, after stimulation with ADP, TSP-1 was released from a-granules in the cell lysates in Wt mice washed platelets but in Lrp5- / - mice platelets there was no effect on TSP-1 release by ADP stimulation (Fig 6B). ADP activation promoted serotonin release from dense granules of Wt mice platelets. However both Lrp5- / + and Lrp5- / - mice platelets showed a reduced release of serotonin upon ADP activation (Fig 6C). All together these results indicated a defect in the granules secretory machinery in Lrp5- / - mice platelets.

[0222] Authors then analyzed the purinergic ADP receptor levels in these platelets. Wt mice predominantly expressed P2Y12 while Lrp5- / - mice showed reduced expression of P2Y12 and higher P2Y1 and P2X1 expression levels than Wt mice (Fig 6D). Activation of P2Y12 by ADP induced the downregulation of cytosolic cAMP and the consequent reduction in VASP phosphorylation. They analyzed VASP-P in platelets of Wt and Lrp5- / - mice after 3pM ADP treatment. While in Wt mice platelets ADP induced a significantly decreased VASP phosphorylation, in Lrp5- / - mice platelets there was no response to ADP and no modulation in VASP phosphorylation. In summary in Lrp5- / - mice the downstream signaling pathway of purinergic receptors was impaired (Fig 6E).

[0223] 7. Wnt signaling proteins in mice and human platelets

[0224] The different components of the canonical Wnt signaling pathway (including LRP5, - CAT, LEF1 , C-JLIN, BMP2 and OPN) were investigated in platelets of Wt and Lrp5- / - mice. Western blot results showed that the expression of canonical Wnt proteins was similar in Wt and Lrp5- / - mice platelets (Fig 7A). Therefore the observed effects were related to LRP5 and no other protein in the canonical WNT pathway. The analyses of canonical WNT pathway components in human by western blotting in washed platelets showed that the canonical WNT pathway is also operational in human platelets (Fig 7B). Stimulation of human platelets with 10pM ADP for 5 minutes at 37°C showed activation of canonical WNT signaling (Fig 7C). The cytoplasmic and membrane fractions of unstimulated and ADP stimulated human platelets were separated and blotted against LRP5 and p-catenin that showed translocation from the cytoplasm into the membrane fraction after ADP activation.

[0225] The WNT pathway downstream protein BMP2 was released to the extracellular compartment, causing a decrease in its cytoplasmic levels (Fig 7C). Therefore, WNT pathway activation was fully active in human platelets and responded to ADP activation.

[0226] 8. Interaction of LRP5 with P2Y12 in human platelets

[0227] The authors next investigated the interaction between LRP5 and purinergic receptor by immunoprecipitation experiments in human washed platelets. Results showed that LRP5 coimmunoprecipitates with P2Y12 (Fig 8A). This result was equally evidenced when platelets were immunoprecipitated with P2Y12 and blotted against LRP5 (Fig 8A).

[0228] They then activated human platelets with 10pM ADP for 5 minutes, fractionated cytoplasms (C) and plasma membranes (M) and blotted the fractions against P2Y12 in order to observe whether there is translocation of P2Y12 (Fig 8B). Quantification of P2Y12 was expressed as a ratio between membrane fraction M / total protein (M+C). ADP induced a translocation of P2Y12 to the platelet membrane. Contrarily, when they pre-treated platelets with 25 pM of LRP5 inhibitor and then proceeded to platelet fractionation, a reduced translocation of P2Y12 to the plasma membrane was observed (Fig 8B). However, when platelets were pretreated with the same concentration of LRP5 inhibitor and afterwards activated with ADP for 5 minutes, P2Y12 was able to translocate to the membrane indicating that the inhibition was reversible.

[0229] Human blood was collected from 5 healthy individuals at day 0 (Basal) and 24 h after clopidogrel treatment (Clopidogrel). Blood was withdrawn and platelets isolated and activated with 10pM ADP. The cytoplasm and membrane fractions were separated and LRP5 expression analyzed by western blotting. They found that in basal conditions LRP5 was primarily in the cytoplasm. After ADP activation, LRP5 was translocated to the membrane fraction; however, in clopidogrel treated platelets, LRP5 was not translocated to the membrane but remained in the cytoplasm. Blockade of P2Y12 receptor by clopidogrel reduced the translocation of LRP5 to the membrane. As proof of mechanism, von Willebrand Factor (vWF; an a-granule component) and p-selectin (present in membranes of a and dense granules) were also measured and their translocation to the membrane were also found inhibited by the P2Y12 receptor blockade (Fig 8C). These results indicated that LRP5 mobilization is directly involved in platelet response to activation and inhibition.

[0230] Finally, they perfused human blood treated or not with the LRP5 inhibitor (25 pM) over collagen coated slides at high shear rate (975 s-1) and platelet deposition was analyzed by mepacrine staining. Inhibition of LRP5 induced a significant reduction in platelet deposition over collagen (Fig 8D).

Claims

CLAIMS1 . A low-density lipoprotein receptor-related protein 5 (LRP5) inhibitor for use in the treatment and / or prevention of thrombosis in a subject.

2. The LRP5 inhibitor for use according to claim 1 , wherein the LRP5 inhibitor is selected from the group consisting of:(i) a LRP5 specific aptamer;(ii) a LRP5 specific antibody;(iii) a protein inhibiting the LRP5 expression;(iv) a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant or a peptide mimetic thereof;(v) a scaffold presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof.

3. The LRP5 inhibitor for use according to any of claims 1 or 2, wherein the LRP5 inhibitor is a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant thereof.

4. A genetically modified megakaryocyte or progenitor thereof, in which the expression of the LRP5 gene is inhibited.

5. The genetically modified megakaryocyte or progenitor thereof according to claim4, wherein the expression of the LRP5 gene is inhibited by disrupting or deleting the gene, by inhibiting the transcription of the gene or by inhibiting the translation of the corresponding mRNA.

6. The genetically modified megakaryocyte or progenitor thereof according to claim5, wherein the LRP5 gene expression is inhibited by using: a LRP5-specific RNA interference construct (RNAi), a microRNA (miRNA), an LRP5-specific antisense oligonucleotide or a LRP5-specific ribozyme or DNAzyme.

7. The genetically modified megakaryocyte or progenitor thereof according to any of claims 4 to 6, wherein the megakaryocyte progenitor thereof is chosen from the group consisting of a hemocytoblast, a megakaryoblast and a promegakaryocyte.

8. The genetically modified megakaryocyte or progenitor thereof according to any of claims 4 to 7, wherein the megakaryocyte or progenitor thereof is obtained from bone marrow or from circulating blood9. The genetically modified megakaryocyte or progenitor thereof according to any of claims 4 to 8 for use in medicine.

10. The genetically modified megakaryocyte or progenitor thereof according to any of claims 4 to 8 for use in the treatment and / or prevention of thrombosis in a subject.

11. The LRP5 inhibitor for use according to any of claims 1 to 3 or the genetically modified megakaryocyte or progenitor thereof for use according to claim 10, wherein the thrombosis is in arterial and / or venous thrombosis: myocardial infarction, brain infarction, pulmonary infarction, arteriosclerosis obliterans, Buerger disease, thrombophlebitis, venous thromboembolism (economy class syndrome), arterial thromboembolism, and disseminated intravascular coagulation (DIC), atherosclerosis, angina, transient ischemic attacks and strokes, peripheral vascular diseases, preeclampsia, embolism and carotid endarterectomy, deep vein thrombosis (DVT) or a pulmonary embolism (PE).

12. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to claim 11 , wherein the prevention is secondary prevention of thrombosis.

13. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to claim 12, wherein the subject is a human.

14. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to any of claims 11 to 13 wherein the treatment and / or prevention results in a reduction in the platelet deposition at the site of vascular damage.

15. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to any of claims 11 to 14, wherein the treatment and / or prevention does not lead to an increased bleeding in the subject.

16. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to any of claims 11 to 15, wherein the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof is administered intradermally, subcutaneously, via dermal infusion, via subcutaneous infusion, intravenously, or orally.

17. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to any of claims 11 to 16, wherein the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof is administered in combination with at least another suitable drug for the treatment of disorders associated with undesirable coagulation, wherein said at least another suitable drug is administered simultaneously, sequentially or separately.

18. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to claim 17, wherein the at least another suitable drug for the treatment of disorders associated with undesirable coagulation is an antithrombotic agent.

19. The LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof for use according to any of claims 11 to 18, wherein the LRP5 inhibitor or the genetically modified megakaryocyte or progenitor thereof is comprised within a pharmaceutical composition wherein the pharmaceutical composition further comprises at least one inert pharmaceutically acceptable excipient.

20. A blood contact surface wherein the surface is coated with a LRP5 inhibitor.

21. The blood contact surface according to claim20, wherein the LRP5 inhibitor is selected from the group consisting of:(i) a LRP5 specific aptamer;(ii) a LRP5 specific antibody;(iii) a protein inhibiting the LRP5 expression;(iv) a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant or a peptide mimetic thereof;(v) a scaffold presenting the peptide of SEQ ID NO: 1 or the functionally equivalent variant thereof.

22. The blood contact surface according to any of claims 20 or 21 , wherein the LRP5 inhibitor is a polypeptide comprising the sequence SEQ ID NO: 1 (ERVEKTTGDKRT) or a functionally equivalent variant thereof.

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