Peptide derived from endoglin for treating bleeding disorders

Abstract

Inventors show that both iKO and Eng+ / - experienced increased bleeding compared to WT mice (longer bleeding times, more rebleeding events, greater hemoglobin loss, p<0.05). Treatment with pEng effectively reduced bleeding in all genotypes (p<0.05), reverting to the control WT condition. Furthermore, pEng induced strong platelet aggregation in both murine and human platelets compared to controls (p<0.01), demonstrating its efficacy in promoting hemostasis. No differences were found in term of co-culture, wound healing and sprouting considering controls and endoglin siRNA ECs, while a beneficial effect on ENG-free endothelial cells was found in capillary flow conditions, restoring the control condition. Accordingly, pEng appears to be a promising treatment for epistaxis in HHT, without deleterious effect on EC tested functions. The present invention relates to a peptide derived from endoglin (pEng) comprising at least the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (SEQ ID NO: 1) wherein X1 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X2 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X3 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X4 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X5 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X6 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X7 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X8 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X9 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), X10 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Val (V), Wherein peptide derived from endoglin (pEng) comprises at least one of each Phe(F), Leu(L), Tyr(Y), His(H), Thr(T), Ser(S), and Val(V).

Description

[0001] PEPTIDE DERIVED FROM ENDOGLIN FOR TREATING

[0002] BLEEDING DISORDERS

[0003] FIELD OF THE INVENTION:

[0004] The invention is in the field of medicine, more particularly relates to bleeding disorders.

[0005] BACKGROUND OF THE INVENTION:

[0006] Hereditary Hemorrhagic Telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is an autosomal dominant disorder characterized by vascular malformations that result in chronic bleeding and acute hemorrhage in patients (Faughnan ME et al. Ann Intern Med. 2020). HHT typically follows an autosomal dominant inheritance pattern; however, it can also manifest de novo, occasionally in mosaic form, where the genetic mutation is present in only some of the individual's cells (Gedge, F. et al. 2007; McDonald, J. et al. 2018). The prevalence of HHT is estimated to be between 1 :5000 and 1 :8000 (Shovlin CL 2018). All known genes whose mutations cause HHT are found within the transforming growth factor-B (TGF-P)-signaling pathway. Mutations in the membrane TGF-P receptors endoglin (ENG) and activin A receptor type Il-like 1 (ACVRL1, also known as ALK1) cause HHT1 (MIM 131195) and HHT2 (MIM 601284) variants, respectively. Interestingly, these two genes, ENG or ACVRL1, are mutated in over 80% of patients with HHT (McDonald J, et al Front Gen 2015). In addition, a combined syndrome of juvenile polyposis (JP) and HHT was reported to be caused by mutations in MADH4 (also known as SMAD4) that encodes a transcription factor (Smad4) of the TGF-P signaling pathway (Gallione C, et al. Am J Med Genet. 2010). This combined syndrome (JP-HHT; MIM600993) occurs only in approximately 2% of HHT patients. Recently, mutations in BMP9 (GDF2), a member of the TGF-P family able to interact with endoglin and ALK1, have also been shown to cause an HHT -like phenotype (Wooderchak- Donahue WL, et al. Am J Hum Genet. 2013), which has been named as the HHT5 variant of the disease (MIM 615506). Overall, all HHT variants share common symptoms, but they differ in the frequency of specific vascular lesions. The decrease in BMP / TGF-beta signaling leads to abnormal vascular development and remodeling, resulting in multiple fragile mucocutaneous telangiectases and arterial malformations (MAV) in the gastrointestinal tract, brain, liver, and lungs (Garcia J, et al. 2020). Although visceral MAVs are generally silent, the most frequent and earliest clinical manifestation of HHT is spontaneous and recurrent epistaxis resulting from the rupture of fragile telangiectasias of the nasal mucosa. Furthermore, abnormal levels of angiogenesis factors such as vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-P), along with other factors such as trauma, inflammation, and fluctuations in blood flow, can trigger epistaxis in patients with HHT. More than 90% of patients affected by HHT suffer from epistaxis, with this symptom occurring more frequently and more severely as individuals age. The treatments for HHT patients often require continuous analysis of symptoms: blood analyses, blood transfusions, iron supplements, outpatient procedures, hospitalization, surgery, and radiographs (CT scans and MRI). (Garcia J, et al. 2020). According to the Second International HHT Guidelines, recommendations vary among various interventions, including hydrating topical therapies, tranexamic acid (TA), ablative therapies of nasal telangiectasias, systemic anti angiogenic agents, septodermoplasty, and nasal retainers to prevent symptoms (Faughnan ME et al. 2020, McDonald et al. Gene Rev 2000). However, to date, no treatment seems to be entirely effective, and unfortunately, the majority of them lead to long-term resistance. There is no consensus treatment available to improve epistaxis in these patients, as HHT is an orphan disease. Endoglin (Eng) is a membrane coreceptor for the transforming growth factor-beta (TGF-P) family that is overexpressed on proliferating endothelial cells and involved in different cardiovascular conditions. Mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia (HHT) type. The inventors previously demonstrated that upon the proteolytic processing of membrane-bound Eng, catalyzed by the metalloprotease matrix metalloproteinase- 14 (MMP14) or -12 (MMP12), a circulating form (soluble endoglin, sEng), encompassing the extracellular region of Eng, can be released. They show that sEng bind the allbp3 integrin via the RGD motifs and can inhibit platelet aggregation and prevents thrombus formation (WO / 2023 / 152291).

[0007] Recently, the inventors has discovered that endoglin (Eng) can also be cleaved by thrombin, resulting in the release of different fragments, one of which resembles the terminal sequence of PARI . Protease-activated receptors (PAR) are a subfamily of related G protein- coupled receptors that are activated by cleavage of part of their extracellular domain and are highly expressed in platelets. Their hypothesis is that the peptide derived from Eng (pEng) has platelet pro-aggregating properties and that it can be used for the treatment of epistaxis in HHT patients without generating vascular secondary effects. In this work, the inventors provide evidence for the first time that the pEng reduces the bleeding time and loss of hemoglobin after tail clip in HHT mouse models, Eng+ / - (Bourdeau A. et al. Trends Cardiovasc Med. 2000) and iKO (Kim YH et al Circ Res 2020) mice, the latter presenting a more prominent hemorrhagic phenotype. They also demonstrated its pro-aggregant effect on human and murine blood. Finally, they provided evidence that pEng has no negative effects on the endothelial cell and that it seems to improve the condition of endoglin haploinsufficiency under flow conditions. pEng appears to be a promising therapeutic strategy for the treatment of epistaxis in HHT.

[0008] SUMMARY OF THE INVENTION:

[0009] The invention relates to a peptide derived from endoglin (pEng) comprising at least the amino acid sequence X1-X2-X3- X4-X5- X6- X7- X8- X9- X10 (SEQ ID NO: 1) wherein

[0010] XI is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0011] X2 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0012] X3 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0013] X4 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0014] X5 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0015] X6 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0016] X7 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0017] X8 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0018] X9 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0019] XI 0 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V), wherein peptide derived from endoglin (pEng) comprises at least 1 each of Phe(F), Leu(L), Tyr(Y), His(H), Thr(T), Ser(S), and Val(V).

[0020] In particular, the invention is defined by the claims.

[0021] DETAILED DESCRIPTION OF THE INVENTION:

[0022] Amino acid sequence analysis of endoglin identified the presence of a conserved sequence in mammalian genome RESELL (SEQ ID NO : 15) highly similar to the a-thrombin cleavage sequence of the protease-activated receptor (PAR)-1.

[0023] According to N-t sequence analysis, the primary structure of recombinant soluble Endoglin of 70 kDa starts at Glu 26 ETVHC (SEQ ID NO : 4) and ends in Gly 586 CTSKG (SEQ ID NO : 6), from the commercial datasheet provided by the manufacturer and similar to the described cleavage produced by MMP147,37. N-t analysis demonstrated that both the 60 kDa and 40 kDa fragments start with ETVHC (SEQ ID NO : 4) but C-t analysis was not able to identify with precision the corresponding C-terminal. Interestingly, this cleavage site GGRLQT (SEQ ID NO : 7) maps within the 18-residue linker between the OR and the ZP module and it can be hypothesized that this precise location, right in the boundary between those two different functional regions, has a potentially relevant biological role. Considering the fragment of 20 kDa, N-t starts with SAYSS (Ser 407) (SEQ ID NO : 11) whereas the C-t ends with the fragment corresponding to CTSKG (Gly 586) (SEQ ID NO : 6). Of note, the N-t cleavage site for the 20kDa fragment, between residues 406 and 407, is exactly the last residue previously mapped in sENG purified from PE patients’ plasma (Venkatesha, S. et al. Soluble endoglin contributes to the pathogenesis of preeclampsia. 698 Nat Med 12, 642-649 (2006). The 10 kDa fragment presents the N-t starting with AAKGN (Ala 511) (SEQ ID NO : 10) and the C-t ends with the peptide corresponding to CTSKG (Gly 586) (SEQ ID NO : 6). The N-t starting sequence of 20kDa and 10 kDa fragments confirmed the prediction of Thr cleavage suggested by modelling. The fragment of 8 kDa was also analyzed by N-t analysis and starts with SFLLH (Ser 531) (SEQ ID NO : 52).

[0024] Inventor synthesized two peptides, namely pEng and pSCR, comprising a sequence of 10 amino acids: H2N-FSFLLHFYTV-COOH (pEng, (SEQ ID NO : 17) and H2N- VTFSLHFLFY-COOH (pSCR, SEQ ID NO : 18), respectively. Inventor tested these peptides on platelets aggregation.

[0025] While pEng was able to induce a strong platelets aggregation on human platelets pSCR wasn’t able to induce platelets aggregation. Then both peptides were tested on mouse model of HHT1 (Eng+ / - and iKO) and WT.

[0026] They performed tail clip assays on wild type (WT) n=55, heterozygous (Eng+ / -) n=18, and induced knockout (iKO) mice n=24. Additionally, murine and human platelets were isolated and treated with pEng to assess platelet aggregation (n=4 respectively). Inventors then studied the effect of pEng on endothelial cells (ECs) by conducting angiogenic tests on both control and endoglin siRNA knockdown ECs to assess its safety (minimum of n=3).

[0027] Their results show that both iKO and Eng+ / - experienced increased bleeding compared to WT mice (longer bleeding times, more rebleeding events, greater hemoglobin loss, p<0.05). Treatment with pEng effectively reduced bleeding and / or reduced Hemoglobin loss in all genotypes (p<0.05), reverting to the control WT condition. Furthermore, pEng induced strong platelet aggregation in both murine and human platelets compared to controls (p<0.05), demonstrating its efficacy in promoting hemostasis. No differences were found in term of pEng treatment of co-culture, wound healing and sprouting considering controls and endoglin siRNA ECs, while a beneficial effect on endoglin-free endothelial cells was found in capillary flow conditions, restoring the control condition.

[0028] Accordingly, pEng appears to be a promising treatment for epistaxis in HHT, without deleterious effect on EC tested functions. Accordingly, the invention relates to peptide derived from endoglin for use in the method for treating, ensuring long-term benefits, stopping or preventing bleedings disorders such as HHT epistaxis.

[0029] Definitions

[0030] As used herein, the term "bleeding disorders" refers to any disorders associated with excessive bleeding, such as a congenital coagulation disorder, an acquired coagulation disorder, administration of an anticoagulant, or a trauma induced hemorrhagic condition. Bleeding disorders may include, but are not limited to hereditary hemorrhagic telangiectasia (HHT), hemophilia A, hemophilia B, von Willebrand disease, idiopathic thrombocytopenia, a deficiency of one or more contact factors, such as Factor XI, Factor XII, prekallikrein, and high molecular weight kininogen (HMWK), a deficiency of one or more factors associated with clinically significant bleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia), and von Willebrand factor (vWD Type 1, vWD Type 2 A, vWD Type 2B, vWD Type 2N, vWD Type 2M, vWD Type 3, and acquired vWD), a vitamin K deficiency, a disorder of fibrinogen, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia, an alpha2-antiplasmin deficiency, and excessive bleeding such as caused by liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, bleeding is associated with surgery, e.g. in a subject with a type of hemophilia, bleeding is associated with a medical procedure, e.g., a dental procedure, trauma (caused during battlefield), hemorrhagic wound, injury, traumatic injury, traumatic brain injury, intracerebral hemorrhage, intracranial hemorrhage, traumatic intracranial hemorrhage, spontaneous intracranial hemorrhage without traumatic injury), hypothermia, menstruation, pregnancy and delivery bleeding such as preeclampsia Bernard- Soulier syndrome, Glanzmann’s thrombasthenia, platelet storage pool deficiencies, Gastrointestinal (GI) Bleeding disorder (caused by Crohn's disease, Esophageal cancer, Esophageal varices, Esophagitis, Gastritis, Gastrointestinal stromal tumor (GIST), GERD (Chronic Acid Reflux), Liver cancer, Pancreatic cancer, Peptic ulcers., Stomach cancer, Anal cancer, Anal fissures, Colon polyps, Colorectal cancer, Crohn's disease, Diverticulitis and diverticulosis, Gastrointestinal stromal tumor (GIST), Hemorrhoids, Rectal ulcers, Ulcerative colitis), arteriovenous malformation (AVM) or wound-healing disorder

[0031] Typically, in the context of the invention, the peptide derived from endoglin according to the invention is suitable to reduce or stop the excessive bleeding. In a particular embodiment the peptide derived from endoglin according to the invention is suitable to prevent the excessive bleeding.

[0032] In a particular embodiment, the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT).

[0033] As used herein, the term “hereditary hemorrhagic telangiectasia” (HHT), also known as 01 ser-Web er-Rendu syndrome, has its general meaning in the art and refers to an inherited disorders causing abnormal blood vessel formation, known as arteriovenous malformations (AVMs), between arteries and vein. More than 80% of all cases of HHT are due to mutations in either ENG or ACVRL1, which cause HHT1 and HHT2, respectively. Hereditary Hemorrhagic Telangiectasia (HHT) is characterized by overwhelming bleeding such as nosebleeds, acute and chronic digestive tract bleeding. However, as explained in Gaetani et al, Journal of Clinical Medicine, 2020, patients afflicted with HTT require also antithrombotic therapy (AT). For instance, they may have coronary artery disease (CAD), venous thromboembolism (VTE), or atrial fibrillation (AF). AT was generally well tolerated, with no fatal bleedings and no significant changes in hemoglobin levels.

[0034] As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human. Preferably, the subject according to the invention is a human afflicted with or susceptible to be afflicted with bleeding disorder.

[0035] In some embodiments, the subject is a human afflicted with or susceptible to be afflicted with hereditary hemorrhagic telangiectasia (HHT).

[0036] As used herein, the term “endoglin” refers to a type I membrane glycoprotein located on cell surfaces and is part of the TGF beta receptor complex. It is a membrane co-receptor for the transforming growth factor-beta (TGF-P) family that is overexpressed on proliferating endothelial cells and involved in different cardiovascular conditions. Mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia (HHT) type.

[0037] Human endoglin has the following amino acid sequence (SEQ ID NO: 3): MDRGTLPLAV ALLLASCSLS PTSLAETVHC DLQPVGPERG EVTYTTSQVS

[0038] KGCVAQAPNA ILEVHVLFLE FPTGPSQLEL TLQASKQNGT WPREVLLVLS VNSSVFLHLQ ALGIPLHLAY NSSLVTFQEP PGVNTTELPS FPKTQILEWA

[0039] AERGPITSAA ELNDPQSILL RLGQAQGSLS FCMLEASQDM GRTLEWRPRT PALVRGCHLE GVAGHKEAHI LRVLPGHSAG PRTVTVKVEL SCAPGDLDAV

[0040] LILQGPPYVS WLIDANHNMQ IWTTGEYSFK IFPEKNIRGF KLPDTPQGLL GEARMLNASI VASFVELPLA SIVSLHASSC GGRLQTSPAP IQTTPPKDTC

[0041] SPELLMSLIQ TKCADDAMTL VLKKELVAHL KCTITGLTFW DPSCEAEDRG DKFVLRSAYS SCGMQVSASM ISNEAVVNIL SSSSPQRKKV HCLNMDSLSF

[0042] QLGLYLSPHF LQASNTIEPG QQSFVQVRVS PSVSEFLLQL DSCHLDLGPE

[0043] GGTVELIQGR AAKGNCVSLL SPSPEGDPRF SFLLHFYTVP IPKTGTLSCT

[0044] VALRPKTGSQ DQEVHRTVFM RLNIISPDLS GCTSKGLVLP AVLGITFGAF

[0045] LIGALLTAAL WYIYSHTRSP SKREPVVAVA APASSESSST NHSIGSTQST

[0046] PCSTSSMA.

[0047] Peptide of the invention

[0048] In a first aspect, the invention relates to a peptide derived from endoglin (pEng) comprising the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (SEQ ID NO: 1), wherein

[0049] XI is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X2 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X3 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X4 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X5 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X6 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X7 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X8 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X9 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X10 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V), wherein peptide derived from endoglin (pEng) comprises at least 1 each of Phe(F), Leu(L), Tyr(Y), His(H), Thr(T), Ser(S), and Val(V).

[0050] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention comprising the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (SEQ ID NO: 2), wherein

[0051] XI is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X2 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X3 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X4 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X5 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V) X6 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0052] X7 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0053] X8 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0054] X9 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0055] X10 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V), wherein peptide derived from endoglin (pEng) comprises 3 Phe(F), 2Leu(L), ITyr(Y), lHis(H), IThr(T), ISer(S), and IVal(V).

[0056] Typically, the peptide derived from endoglin comprises H2N-X1-X2-X3-X4-X5-X6-

[0057] X7-X8-X9-X10-COOH (SEQ ID NO: 56), wherein

[0058] XI is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0059] X2 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0060] X3 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0061] X4 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0062] X5 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0063] X6 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0064] X7 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0065] X8 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0066] X9 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V)

[0067] X10 is Phe (F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), or Vai (V), wherein peptide derived from endoglin (pEng) comprises at least 1 each of Phe(F), Leu(L), Tyr(Y), His(H), Thr(T), Ser(S), and Val(V).

[0068] In some embodiments, the peptide derived from endoglin of the invention comprises 10, 11, 12, 13, 14, 15 or 16 amino acids.

[0069] In some embodiments, the peptide derived from endoglin of the invention comprises 10 amino acids.

[0070] In a second aspect, the invention relates to a peptide derived from endoglin (pEng) comprising or consisting of the amino sequence in table 1.

[0071] Table 1 : Peptide derived from endoglin (pEng)

[0072] In other words, in some embodiment, the peptide derived from endoglin comprises or consists of the amino sequence SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 SEQ ID NO 35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56.

[0073] In some embodiments, the peptide derived from endoglin of the invention comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.

[0074] In some embodiments, the peptide derived from endoglin of the invention comprises 10 amino acids.

[0075] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: ETVHC (SEQ ID NO: 4).

[0076] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: CGGRLQTS (SEQ ID NO: 5).

[0077] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: CTSKG (SEQ ID NO: 6).

[0078] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: GGRLQT (SEQ ID NO: 7).

[0079] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: CGGRLQT (SEQ ID NO: 8).

[0080] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: SFFLH (SEQ ID NO: 9).

[0081] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: AAKGN (SEQ ID NO: 10).

[0082] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: SAYSS (SEQ ID NO: 11).

[0083] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: GDPRFSFLLH (SEQ ID NO: 12).

[0084] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: PRSFLL (SEQ ID NO: 13).

[0085] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: FSFLLH (SEQ ID NO: 14).

[0086] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: RFSFLL (SEQ ID NO: 15). In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises or consists of the amino acid sequence: FSFLLHFYTV (“pEng”, SEQ ID NO: 16)

[0087] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: RFSFLL (SEQ ID NO: 15) and wherein the derived peptide from endoglin (pEng) comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.

[0088] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention, comprises at least the amino acid sequence: FSFLLHFYTV (SEQ ID NO: 16) and wherein the derived peptide from endoglin (pEng) comprises 10, 11, 12, 13, 14, 15 or 16 amino acids.

[0089] In some embodiment, the peptide derived from endoglin comprises or consists of the comprising or consisting of the amino acid sequence LV-X3-H-X5-LSTFF (SEQ ID NO:57), wherein X3 is F or Y, and X5 is F or Y.

[0090] In some embodiment, the peptide derived from endoglin comprises or consists of the amino sequence LVFHYLSTFF (SEQ ID NO: 21, “pVl”) or the amino sequence LVYHFLSTFF (SEQ ID NO:23, “pV2”).

[0091] In some embodiment, the peptide derived from endoglin comprises or consists of the amino sequence LVFHYLSTFF (SEQ ID NO: 21, “pVl”) or the amino sequence LVYHFLSTFF (SEQ ID NO:23, “pV2”), and wherein the derived peptide from endoglin (pEng) comprises 10, 11, 12, 13, 14, 15 or 16 amino acids.

[0092] As used herein, the term “peptide” corresponds to the chemical agents belonging to the protein family. A peptide is composed of a mixture of several amino acids. Depending on the number of amino acids involved, peptides are categorized as dipeptides, composed of 2 amino acids, tripeptides, made up of 3 amino acids, and so on. Peptides composed of more than 10 amino acids are called polypeptides. Thus, the peptide of the invention can be considered as a polypeptide. The innovative peptide according to the invention has the following biological and clinical properties:

[0093] Binds to human PARI (hPARl) and murin PAR4 (mPAR4): therapeutic peptide acts on the PARI and PAR4. This duality allows for a reliable animal model for preclinical data; Induces strong platelet aggregation in both murine and human platelets compared to controls, suggesting its efficacy in promoting hemostasis : Reduces bleeding in all genotypes reverting to the control WT condition ; modifies calcium mobilization^ has no negative effects on the endothelial cell (EC) and that it seems to improve the condition of endoglin haploinsufficiency under flow conditions, restoring control condition.

[0094] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention is able to bind to human PARI (hPARl).

[0095] In a particular embodiment, the peptide derived from endoglin (pEng) according to the invention is able to bind to murin PAR4 (mPAR4).

[0096] As used herein, the term “PAR” refers to protease-activated receptors. PARs are a unique family of G-protein coupled receptors, which play important roles in vascular physiology, neural tube closure, hemostasis, and inflammation. There are four members of PARs in humans:PARl, PAR2, PAR3 and PAR4. PARI and PAR4 are both thrombin receptors and share the same ligands and the mechanism of activation.

[0097] The peptides according to the invention, may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art.

[0098] Bioinformatic analysis in search of possible cleavage sites on the Eng sequence by Thr through a Profile Specific Scoring Matrix (PSSM) analysis was performed. In ref26 53 peptide sequences were reported to be substrates for Thr cleavage and aligned around a central arginine from position P3 to position P4’. This alignment constitutes the dataset to extract a profile characterizing the cleavage motif. In a PSSM a score is associated with the presence of a given amino acid at a specific position of the motif, generating a scoring matrix. In our case the matrix is of dimension 20 x 8, i.e. 20 amino acids times 8 positions in the motif. The score is calculated computing the probability of occurrence of each amino acid at a given position from the known sequence alignments, normalized by its relative abundance in nature. Based on this scoring matrix it is then possible to analyze a new sequence in search for subsequences of the size of the motif. For the new sequence a score is computed adding the values of the PSSM corresponding to the amino acids of the subsequence at the position in which they appear in the motif. In our case, we scanned the whole endoglin sequence and for each arginine we computed the score of motifs P3 to P4’. Incubation of purified endoglin with thrombin resulted in the generation of several fragments, whose C- and N-terminal sequencing confirmed the predicted thrombin cleavage sites. Thrombin treatment of endoglin-expressing cells, including endothelial and mesenchymal stem cells, led to the release of sEng concomitantly with a decrease of cell surface endoglin. These findings suggest the existence of multiple cleavage sites sequentially targeted by various proteases, leading to sEng composed by different fragments.

[0099] When searching for potential new cleavage sites, amino acid sequence analysis of endoglin highlighted the presence of a GDPRFSFLLH (SEQ ID NO: 12) sequence (residues 526-534). Of note, a clear similarity was found to the protease activated receptor PAR-1 site PRSFLL (SEQ ID NO: 13). Alignment of the human endoglin amino acid sequence with those of 10 other mammal species revealed that this sequence (amino acids 528 to 534) is highly conserved among different species, potentially suggesting that this may be an important recognition site for a putative protease. Given the high sequence similarity to the Thr cleavage site of PARI, inventors hypothesized that Thr might also be able to cleave endoglin. The crystal structure of the ZP module of ENG29 shows that the GDPRFSFLLH (SEQ ID NO: 12) sequence is not highly exposed within the context of the ZP-C module and is thus unlikely to be readily accessible to Thr or other proteases. However, it could become accessible upon structural rearrangements of ENG, possibly triggered by cleavage at other sites occurring prior. Indeed, a Profile Specific Scoring Matrix analysis identified several other potential cleavage sites with relatively high scores and accessibility. As a proof of principle, computational docking of Thr to the site 329-CGGRLQTS-338 (SEQ ID NO: 5) suggests that the latter may be cleaved by the protease. Looking at the crystal structure of ENG, this site appears to be the most exposed to the solvent, and it could be a plausible first target for the protease, since it is easily accessible.

[0100] Peptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. Peptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides. A variety of expression vector / host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below. U.S. Pat. No. 6,569,645; U.S. Pat. No. 6,043,344; U.S. Pat. No. 6,074,849; and U.S. Pat. No. 6,579,520 provide specific examples for the recombinant production of peptides and these patents are expressly incorporated herein by reference for those teachings. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

[0101] In a particular embodiment, to identify optimized peptide derived from Endoglin, the methodology comprised several sequential steps. Initially, sequence selection involved obtaining the relevant amino acid sequences from UniProt (The UniProt Consortium, 2023) (id:P25116), with the signal peptide (1-21) omitted for PARI, as it is cleaved in the mature receptor (Zampatis et al., 2012) , which we will refer to as matPARl from now on. All scripts for deep learning models are obtained via ColabFold (Mirdita et al., 2022), which is a package that offers a user-friendly interface to run deep learning models for protein’s 3D predictions such as ESFMFOLD, Alphafold 2 and Alphafold 2 multimer, and then executed on Google Colab (Bisong, 2019), providing access to GPU-accelerated computing. Refinement of predicted structures via steepest descent optimization was performed in UCSF Chimera software (Pettersen et al., 2004). All intermediate and final results were visualised using UCSF ChimeraX software (Goddard et al., 2018).

[0102] In a particular embodiment, the ESMFOLD (Rives et al., 2021) was used to make the first structure prediction, which was subsequently minimised. ESMFOLD’ s prediction was used as a template for the Alphafold2 (AF2) (Jumper et al., 2021) with MMseqs2 (Steinegger and Sbding, 2017). The third step used the prediction of AF2 as template for AF2 multimer (Evans et al., 2022), where both the sequence of PARI and peptide of interest were given as input to make a prediction of the PARl / peptide complex‘s 3D structure. For the last step, the peptide poses were refined using HADDOCK (van Zundert et al., 2016), a method computing physical interactions to perform peptide / protein docking. All residues of the peptide of interest and PARl’s residues within a 5-angstrom of the peptide in the predicted complex were defined as active residues. Active residues are used to define the Ambiguous Interaction restraint (AIR) of HADDOCK. Based on an energy score, HADDOCK predicted several optimal structures for the complex that were grouped by structural similarity (clustered). All clusters were then analysed using Protein Ligand Profiler (PLiP) (Salentin et al., 2015) to detect and quantify the contacts between PARI and peptide of interest.

[0103] Typically, in the context of the invention, A DNA library has been created for Phage Display that contains at least 88% of the theoretically possible scrambled peptide sequences. From this DNA library, a Phage Display library has been created by restriction-ligation of the plasmid DNA library. To facilitate setting up an appropriate screening (panning) protocol, four sequence-specific phages have also been created, three as positive controls of binding and a fourth one as negative control of binding. The in vivo biopanning process has been validated by flow cytometry in terms of the number of cells that yield strong GFP fluorescence and the capacity to bind to phages expressing specific peptides. 4 cycles of biopanning has been running with the PDLIB library against cells expressing hPARl-EGFP or mPAR4-EGFP, eluting and amplifying the phage population after each cycle of cell sorting and elution. For mPAR4-EGFP it was necessary to re-make the Phage Display library and re-run the complete protocol. After the fourth cycle of biopanning, the amplified phage population was transduced into A. coli cells to isolate individual phages (e.g. phage genomes). 3 X 96 (288) phage genomes for hPARl- EGFP and 2X 96 (192) phage genomes for mPAR4-EGFP have thus far been sequenced, obtaining 23 hits for hPARl-EGFP (including the V1PC sequence) and 14 hits for mPAR4- EGFP.

[0104] As used herein, the term “amino acid” refers to natural or unnatural amino acids in their D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptidyl structure. Natural and unnatural amino acids are well known in the art. Common natural amino acids include, without limitation, alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V). Uncommon and unnatural amino acids include, without limitation, a-aminoisobutyric acid (Aib, U), allyl glycine (AllylGly), norleucine (Nle), norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn), Cyclohexylalanine (Cha, Fx), and pentafluorophenylalanine.

[0105] In the context of the invention, the amino acid sequence comprises 7 distinct amino acids: Phe(F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), and Val(V) with the same frequency of occurrence as the target sequences: at least 1 Phe, at least 1 Leu, at least ITyr, at least IHis, at least IThr, at least ISer, and at least IVal.

[0106] In a particular embodiment, the amino acid sequence comprises 7 distinct amino acids: Phe(F), Tyr(Y), His(H), Thr(T), Ser(S), Leu(L), and Val(V) with the same frequency of occurrence as the target sequences: 3 Phe, 2 Leu, and 1 each of Tyr, His, Thr, Ser, and Vai.

[0107] In a further embodiment, a penetrating enhancer is coupled to the peptide derived from endoglin according to the invention.

[0108] As used herein, the term “penetration enhancers” also called chemical penetration enhancers, absorption enhancers or sorption promotors refers to chemical compounds that can facilitate the penetration of active pharmaceutical ingredients (API) into or through the poorly permeable biological membranes. These compounds are used in some pharmaceutical formulations to enhance the penetration of APIs in transdermal drug delivery and transmucosal drug delivery (for example, ocular, nasal, oral and buccal). They typically penetrate into the biological membranes and reversibly decrease their barrier properties.

[0109] In some embodiments, a cell penetrating sequence is coupled to the peptide derived from endoglin.

[0110] In some embodiment, the cell penetrating sequence is coupled in N-terminal or C- terminal of the peptide derived from endoglin.

[0111] As used herein, the term “cell penetrating sequence” has its general meaning in the art and refers to short sequence that facilitate cellular intake and uptake of the peptide of the invention. Based on the origin of peptides, CPPs are divided into chimeric, protein-derived and synthetic. Cell penetrating sequence include but are not limited to Penetratin, octaarginine (R8), tat, Transportan and Xentry. Penetratin is a cell penetrating peptide from the first generation, which is derived from Drosophila Antennapedia Homeodomain. Penetratin overcomes the plasma membrane barrier of mammalian cells through the macropinocytotic pathway and efficiently delivers molecular cargoes in a biologically active form. The tat peptide is derived from the transactivator of transcription (tat) of human immunodeficiency virus. TAT is an arginine-rich peptide which directly penetrates plasma membrane and stabilized DNA. Transportan is a chimeric CPP, which derived from galanin and mastoparan. Xentry is a short- peptide derived from an N-terminal region of the X-protein of the hepatitis B virus. Xentry permeates adherent cells using syndecan-4 as a portal for entry. Horton peptide is a synthetic cell-permeable peptide that are able to enter mitochondria. The sequences of the MPPs were designed to display two properties known to be important for passage across both the plasma and mitochondrial membranes: positive charge and lipophilic character as explained in Horton et al, Chem Biol. 200858.

[0112] In a particular embodiment, the penetration enhancer is selected from the group consisting of but not limited to: Alkylsaccharides such as alkyl maltosides, including dodecyl maltoside (DDM); Bile salts and derivatives such as Cholates (e.g., sodium glycocholate, sodium taurocholate); Cyclodextrins such as methyl Beta-cyclodextrin, capryl ocaproyl macrogol-8 glycerides, 2-(2-ethoxyethoxy)ethanol ; Transcutol P (Diethylene glycol monoethyl ether) ; Labrasol (Caprylocaproyl macrogol-8 glycerides) ; Cyclopentadecanolide ; Diethylene glycol monoethyl ether ; Fatty acids ; Phospholipids ; Surfactants such as Pol oxamer 188, Polysorbate 20 and Polysorbate 80, Thiomers ; Mucoadhesives such as Cellulose and derivatives ; Chitosan and derivatives.

[0113] In a particular embodiment, the peptide derived from endoglin of the present invention can be cyclized with cyclisation method. Such method is well known in the art for exerting biological functions, maintaining stability under harsh conditions and conferring proteolytic resistance, as demonstrated both in nature and in the laboratory (Heather C Hayes et al 2021 ; doi: 10.1039 / dlob00411e).

[0114] In a third aspect, the invention relates to a vector comprising the peptide derived from endoglin of the present invention.

[0115] Typically, the peptide may be delivered in association with a vector. The peptide derived from endoglin of the present invention is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. So, a further object of the invention relates to a vector comprising the peptide of the invention. Typically, the vector is a viral vector, which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus.

[0116] In some embodiments, the vector is an AAV vector.

[0117] As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and / or env genes but without the LTR and / or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein that allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

[0118] In some embodiments, the vector is an adeno-associated virus (AAV).

[0119] Diagnostic methods

[0120] Inventors have performed Western blot analysis of plasma and serum from preeclamptic women. They demonstrated that the presence of endoglin fragments consistent with a proteolysis by thrombin. Incubation of purified endoglin with thrombin resulted in the generation of several fragments, whose C- and N-terminal sequencing confirmed the predicted thrombin cleavage sites. Thrombin treatment of endoglin expressing cells, including endothelial and mesenchymal stem cells, led to the release of sEng concomitantly with a decrease of cell surface endoglin. These findings suggest the existence of multiple cleavage sites sequentially targeted by various proteases, leading to sEng composed by different fragments, which could reflect endothelial dysfunction and their involvement in disease progression of PE.

[0121] Accordingly, in a fourth aspect, the present invention relates to a method for diagnosing pre-eclampsia (PE) in a subject comprising the steps of: i) detecting endoglin fragments in a biological sample obtained from said subject; and ii) concluding that the subject suffers from pre-eclampsia (PE) when the presence of endoglin fragments is detected.

[0122] In a particular embodiment, the invention relates to a method for diagnosing pre- eclampsia (PE) in a subject comprising the steps of: i) measuring the level of endoglin fragments in a biological sample obtained from said subject; ii) comparing the level measured at step i) with its predetermined reference value, and iii) concluding that the subject suffers from PE when the level of endoglin fragments is higher than its predetermined reference value or concluding that the subject does not suffer from PE when the level of endoglin fragments is lower or similar than its predetermined reference value.

[0123] As used herein term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and / or prospects of recovery.

[0124] As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a human who is susceptible to have PE. As used herein, the term “pre-eclampsia” refers to a placental disease characterized by insufficiency of the uteroplacental circulation and which affects 10-12% of all pregnancies and is a major factor in the perinatal mortality rate. Pre-eclampsia is a severe complication of human pregnancy characterized by development of hypertension and proteinuria and it affects maternal and foetal morbidity and mortality worldwide.

[0125] As used herein, the term “endoglin fragments” refers to different fragments of endoglin released by a proteolysis from thrombin. In a particular embodiment, the endoglin fragments refers to circulating soluble endoglin (sEng) in a biological sample.

[0126] In a particular embodiment, the endoglin fragments consisting of but not limited to : ETVHC (SEQ ID NO: 4), CGGRLQTS (SEQ ID NO: 5), CTSKG (SEQ ID NO: 6), GGRLQT (SEQ ID NO: 7), CGGRLQT (SEQ ID NO: 8), SFFLH (SEQ ID NO: 9), AAKGN (SEQ ID NO: 10), SAYSS (SEQ ID NO: 11), GDPRFSFLLH (SEQ ID NO: 12), PRSFLL (SEQ ID NO: 13), FSFLLH (SEQ ID NO: 14), RFSFLL (SEQ ID NO: 15), FSFLLHFYTV (SEQ ID NO: 16)

[0127] As used herein, the term “biological sample” refers to a sample obtained from a subject, for example blood, saliva, breast milk, urine, semen, blood plasma, synovial fluid or serum.

[0128] In a particular embodiment, the biological sample is blood sample. The term “blood sample” means any blood sample derived from the subject. Peripheral blood is preferred, and mononuclear cells (PBMCs) are the preferred cells. Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.

[0129] In another embodiment, the biological sample is serum.

[0130] In another embodiment, the biological sample is plasma.

[0131] In another embodiment, the measure of the level of endoglin fragments is carried out by immunological detection. Typically, the immunological detection or quantification of the endoglin fragments is achieved by any methods known in the art using at least one antibody that binds specifically to endoglin fragments. Examples of said methods include, but are not limited to, standard electrophoretic and immunodiagnostic techniques such as western blots, immuno -precipitation assay, radioimmunoassay, ELISA (enzyme- linked immunosorbent assay), "sandwich" immunoassay, gel diffusion precipitation reaction, immunodiffusion assay, precipitation reaction, agglutination assay (such as gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, protein A assay, immunoelectrophoresis assay, high performance liquid chromatography, size exclusion chromatography, solid-phase affinity, etc. In a particular embodiment, the expression level of endoglin fragments is measured by ELISA.

[0132] Typically, Quantitative analysis of sEng in cell supernatants, as well as in plasma and serum samples was carried out via the sandwich enzyme-linked immunosorbent assay (ELISA) principle. Human Endoglin / CD105 quantikine ELISA immunoassay 96-well kits (R&D Systems, USA, MNDG00) were used according to the manufacturer’s instructions. The ensuing product of the sandwich was read spectrophotometrically at 450nm using a Spectrostar Nano- 96 micro-well reader (BMG-266 Labteck).

[0133] As used herein, the term “predetermined reference value” refers to a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the expression level of the selected peptide in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0134] In the context of the invention, inventors have performed assay comparing plasma from non-pregnant donors and plasma from pregnant women without PE with plasma from PE patients. The gel analysis clearly shows that sEng is undetectable in non-pregnant subjects, while pregnant women and PE patients exhibit increasing levels of sEng. sEng levels in PE patient plasma are higher than those in pregnant and non-pregnant controls, suggesting that sEng and the derived shorter forms may serve as markers of disease progression. In addition, the increased presence of bands of 40 and 20 kDa in some pregnant women could be used to monitor these women during pregnancy to early detect the possible evolution in pre-eclampsia.

[0135] In another embodiment, the invention relates to a method for monitoring the progress of pre-eclampsia in a subject comprising the steps of: i) measuring the level of endoglin fragments in a biological sample obtained from said subject in a first time; ii) measuring the level of endoglin fragments in a biological sample obtained from said subject in a second time ( few days, weeks or months later); iii) comparing the two measurements; and iv) concluding that the subject would be at high risk of PE when the level of endoglin fragments increases between the two measurements.

[0136] In a particular embodiment, the invention relates to a method for treating a subject diagnosed as having or is at risk to have PE according to diagnosis method as described above and treating said subject with a classical treatment.

[0137] As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat PE.

[0138] In the context of the invention, the classical treatment is selected from the group consisting of but not limited to: anti-hypertensive drugs such as Priniviln, anti-convulsant medication such as magnesium sulfate, corticosteroids such as Prednisone.

[0139] Therapeutics methods To validate the absence of differences in bleeding time due to pSCR treatment, Eng(+ / +) mice were subjected to the tail-clip bleeding model, where the distal tip of the tail (3 mm) is excised, followed by tail immersion in saline (PBS) with or without 50 pM of pSCR. Mice were monitored for 10 minutes post-injury, and total bleeding was quantified as the cumulative bleeding time over this interval.

[0140] Importantly, pEng significantly reduced both bleeding time and blood loss across all genotypes, underscoring its potential therapeutic value in managing bleeding disorders and suggesting that pEng can effectively enhance hemostasis even in the absence of full Endoglin functionality.

[0141] Accordingly, in a fifth aspect, the present invention relates to the peptide derived from endoglin or the vector of the invention for use as drugs.

[0142] In other words, the present invention relates to the peptide derived from endoglin or the vector of the invention for use in therapy.

[0143] In more particular, the invention relates to peptide derived from endoglin or the vector of the invention for use in the treatment of bleeding disorder.

[0144] In other words, the present invention relates to a method of treating a bleeding disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the peptide derived from endoglin of the invention or the vector of the invention.

[0145] In a particular embodiment, the peptide derived from endoglin or the vector of the invention for use in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of bleeding disorder.

[0146] As used herein, the term "subject" refers to a human or another mammal (e.g., mouse, rat, rabbit, hamster, dog, cat, cattle, swine, sheep, horse or primate). In some embodiments, the subject is a human being.

[0147] Typically, the subject is affected or likely to be affected with a disease affecting the blood system.

[0148] Typically, the subject is affected or likely to be affected with a bleeding disorder.

[0149] In a particular embodiment, the subject is affected or likely to be affected with one of the bleeding disorders as disclosed above.

[0150] Typically, the subject is affected or likely to be affected with a bleeding disorder.

[0151] In a particular embodiment, the subject is affected or likely to be affected with hereditary hemorrhagic telangiectasia (HHT). As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0152] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., the peptide of the invention) into the subject, such as by systemic, topical, intranasal, oral, sublingual, parenteral, mucosal, local, rectal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In a particular embodiment, the peptide derived from the endoglin or the vector according to the invention is administered intranasally or topically.

[0153] In a particular embodiment, the peptide derived from the endoglin or the vector according to the invention is administered intranasally.

[0154] In a particular embodiment, the peptide derived from the endoglin or the vector according to the invention is administered topically.

[0155] As used herein the term “intranasal administration” refers to a composition that is administered to the nose or by way of the nose for delivery across the mucosal membrane inside the nasal cavity. Typically, the intranasal administration is performed with a gel, ointment, lotion, emulsion, cream, foam, mousse, liquid, paste, jelly, or tape, that is applied to the nasal cavity.

[0156] In a particular embodiment, the intranasal administration is performed with a nasal spray.

[0157] As used herein, term “nasal spray” refers to a product that is intended to be delivered from a spray or aerosolizing device, which can for example be in the form of a liquid, powder, gel, foam, cream, ointment, or other sprayable composition.

[0158] In a particular embodiment, the intranasal administration is performed with a nasal spray inhaler.

[0159] In a particular embodiment, the intranasal administration is performed with mucoadhesive sprayable fluid gel.

[0160] In a particular embodiment, the intranasal administration is performed with powdered composition.

[0161] In a particular embodiment, the intranasal administration is performed with an aqueous composition.

[0162] In a particular embodiment, the intranasal administration is performed with nanoparticles, such as lipid-based nanoparticles, including nano / microemulsions, liposomes, solid lipid nanoparticles, or nanostructured lipid carriers.

[0163] As used herein the term “topical formulation” refers to a formulation that may be applied to skin. Topical formulations can be used for both topical and transdermal administration of substances.

[0164] As used herein, “topical administration” is used in its conventional sense to mean delivery of a substance, such as a therapeutically active agent, to the skin or a localized region of a subject's body. As used herein, “transdermal administration” refers to administration through the skin. Transdermal administration is often applied where systemic delivery of an active is desired, although it may also be useful for delivering an active to tissues underlying the skin with minimal systemic absorption. Typically, the topical pharmaceutically acceptable carrier or excipient is any substantially nontoxic carrier conventionally usable for topical administration of pharmaceuticals in which the voriconazole or any one of its pharmaceutically acceptable derivatives of the present invention will remain stable and bioavailable when applied directly to skin surfaces.

[0165] As used herein, “topical vehicle” has its general meaning in the art and refers to carriers (or excipients) such as those known in the art effective for penetrating the keratin layer of the skin into the stratum corneum and useful in delivering the voriconazole or any one of its pharmaceutically acceptable derivatives of the present invention to the area of interest. Suitable topical pharmaceutically acceptable carriers include water, buffered saline, petroleum jelly (vaseline), petrolatum, mineral oil, vegetable oil, animal oil, organic and inorganic waxes, such as microcrystalline, paraffin and ozocerite wax, natural polymers, such as xanthanes, gelatin, cellulose, collagen, starch, or gum arabic, synthetic polymers, alcohols, polyols, and the like. The topical vehicle will be any substantially non-toxic vehicle conventionally usable for topical administration in which peptide derived from the endoglin or the vector according to the invention or any one of its pharmaceutically acceptable derivatives of the present invention will remain stable and bioavailable when applied directly to the skin surface. Suitable cosmetically acceptable carriers or excipients are known to those of skill in the art and include, but are not limited to, cosmetically acceptable liquids, creams, oils, lotions, ointments, gels, or solids, such as conventional cosmetic night creams, foundation creams, suntan lotions, sunscreens, hand lotions, make-up and make-up bases, masks and the like. Any suitable vehicle effective for topical administration to a patient as known in the art may be used, such as, for example, a cream base, creams, liniments, gels, lotions, ointments, foams, solutions, suspensions, emulsions, pastes, oils such as Crisco®, soft-soap, as well as any other preparation that is pharmaceutically suitable for topical administration on human and / or animal body surfaces such as skin or mucous membranes. Topical acceptable carriers may be similar or identical in nature to the above described topical pharmaceutically acceptable carriers.

[0166] In a particular embodiment, the administration of peptide derived from the endoglin or the vector according to the invention is performed by systemic delivery. As used herein, the term “systemic delivery” refers to an administration of peptide derived from the endoglin or the vector according to the invention systemically in the body of the subject, including by intravenous, subcutaneous, oral or pulmonary administration.

[0167] In another embodiment, the peptide derived from the endoglin or the vector according to the invention is administered orally. In another embodiment, the peptide derived from the endoglin or the vector according to the invention is formulated as pills to be used for oral administration.

[0168] A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0169] In a particular embodiment, the therapeutically effective amount is 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,

[0170] 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

[0171] 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,

[0172] 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,

[0173] 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,

[0174] 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,

[0175] 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 mg / kg of body weight per day.

[0176] In a particular embodiment, the therapeutically effective amount comprises between 25mg / kg and 250 mg / kg of body weight per day.

[0177] In a particular embodiment, the therapeutically effective amount is at least 25mg / kg of body weight per day.

[0178] In a particular embodiment, the therapeutically effective amount is 250 mg / kg of body weight per day.

[0179] In a particular embodiment, the therapeutically effective amount is: 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 mg / kg of body weight per day.

[0180] In a sixth aspect, the invention relates to the peptide derived from endoglin for use according to the invention, and a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of bleeding disorder in a subject in need thereof

[0181] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

[0182] As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat bleeding disorder.

[0183] In the context of the invention, the classical treatment is selected from the group consisting of but not limited to: Factor Replacement Therapies, Non-factor Replacement Therapies, Antifibrinolytics, Anti -Angiogenic or Gene Therapy.

[0184] In a particular embodiment, the classical treatment is Factor Replacement Therapies. As used herein, the term “Factor Replacement Therapies” refers to use of a molecule that is either similar to natural factor found in humans (recombinant) or use an actual human molecule (plasma derived). This type of therapy increases the amount of factor in the body to levels that lead to better clotting, and therefore less bleeding.

[0185] In a particular embodiment, the Factor Replacement Therapies is recombinant Factor

[0186] VIII Products. Typically, recombinant Factor VIII Products such as Advate, Adynovate, Afstyla, Altuviiio, Eloctate, Esperoct, Jivi, Kovaltry, NovoEight, NUWIQ, Recombinate, Xyntha.

[0187] In a particular embodiment, the Factor Replacement Therapies is Recombinant Humanized Bispecific FIXa and FX directed monoclonal antibody for use in patients with inherited hemophilia A such as Hemlibra.

[0188] In a particular embodiment, the Factor Replacement Therapies is Human Plasma- Derived Immunoaffinity -purified Factor VIII Concentrates such as Hemofil M.

[0189] In a particular embodiment, the Factor Replacement Therapies is Human Plasma- derived Concentrates that Contain Factor VIII and Von Willebrand Factor such as Alphanate, Humate-P or Koate-DVI.

[0190] In a particular embodiment, the Factor Replacement Therapies is Recombinant Factor

[0191] IX Concentrates such as Alprolix, Benefix, Idelvion, Ixinity, Rebinyn or Rixubis.

[0192] In a particular embodiment, the Factor Replacement Therapies is Human Plasma- derived Coagulation Factor IX Concentrates such as AlphaNine SD, Mononine.

[0193] In a particular embodiment, the Factor Replacement Therapies is Desmopressin Formulations.

[0194] In a particular embodiment, the Factor Replacement Therapies is Recombinant Von Willebrand Factor such as Vonvendi.

[0195] In a particular embodiment, the Factor Replacement Therapies is Human Plasma- derived Concentrates that Contain Factor VIII and Von Willebrand Factor such as Alpanate, Humate-P, Wilate.

[0196] In a particular embodiment, the Factor Replacement Therapies is Human Plasma- derived Activated Prothrombin Complex Concentrate such as Feiba.

[0197] In a particular embodiment, the Factor Replacement Therapies is Recombinant Factor Vila Concentrate such as NovoSeven RT or SEVENFACT.

[0198] In a particular embodiment, the Factor Replacement Therapies is Human Plasma- derived Concentrate such as Fibryga, RiaSTAP. In a particular embodiment, the classical treatment is Non-factor Replacement Therapies.

[0199] As used herein, the term “Non-factor Replacement Therapies” refers to products that help prevent bleeding or assist in better clotting using other methods in the body besides replacing low factor levels.

[0200] In a particular embodiment, the Non-factor Replacement Therapies is Emicizumab (Hemlibra), Desmopressin (DDAVP), Aminocaproic acid (Amicar), Hormone therapy such as birth control pills, or oral contraceptives.

[0201] In a particular embodiment, the classical treatment is antifibrinolytics therapy.

[0202] As used herein, the term “fibrinolysis” refers to a process that prevents blood clots from growing.

[0203] As used herein, the term “antifibrinolytics” refers to a compound or an agent that inhibit the activation of plasminogen to plasmin, prevent the break-up of fibrin and maintain clot stability.

[0204] In a particular embodiment, the antifibrinolytics therapy is Tranexamic Acid, Aminocaproic Acid, Nafamostat or Aprotinin.

[0205] In a particular embodiment, the classical treatment is anti -angiogenic therapy.

[0206] As used herein, the term “Angiogenesis” refers to the formation of new blood vessels. This process involves the migration, growth, and differentiation of endothelial cells, which line the inside wall of blood vessels.

[0207] As used herein, the term “anti-angiogenic therapy” refers to compounds or agents that block the growth of blood vessels. In a particular embodiment, the anti -angiogenic therapy is Axitinib (Inlyta®), Bevacizumab (Avastin®), Cabozantinib (Cometriq®), Everolimus (Afinitor®), Lenalidomide (Revlimid®), Lenvatinib mesylate (Lenvima®), Pazopanib (Votrient®), Ramucirumab (Cyramza®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Thalidomide (Synovir, Thalomid®), Vandetanib (Caprelsa®), Ziv- aflibercept (Zaltrap®).

[0208] In a particular embodiment, the peptide derived from endoglin according to the invention can be combined with VAD044 (Vaderis Therapeutics).

[0209] In a particular embodiment, the classical treatment is gene therapy. As used herein, the term “gene therapy” refers to provide copies of the gene to correct the disease or disorder. There are different approaches to gene therapy, including gene transfer and gene editing.

[0210] In a particular embodiment, the gene therapy drug is Hemgenix or BEQVEZ.

[0211] As used herein, the term “administration simultaneously” refers to administration of at least 2 or 3 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of at least 2 or 3 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of at least 2 or 3 active ingredients at different times, the administration route being identical or different.

[0212] Pharmaceutical composition

[0213] The peptide derived from endoglin alone or combined with a classical treatment, as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0214] Accordingly, in a seventh aspect, the invention relates to a pharmaceutical composition comprising a peptide derived from endoglin for use in the treatment of bleeding disorder as described above.

[0215] In a particular embodiment, the invention relates to a pharmaceutical composition comprising a peptide derived from endoglin for use in the treatment of HHT.

[0216] In a particular embodiment, the invention relates to a pharmaceutical composition comprising pEng.

[0217] In a further embodiment, the invention relates to a pharmaceutical composition comprising i) a peptide derived from endoglin and ii) a classical treatment as described above as a combined preparation to treat a bleeding disorder.

[0218] In a particular embodiment, the invention relates to a pharmaceutical composition comprising a peptide derived from endoglin as described above for an intransal administration.

[0219] In a particular embodiment, the invention relates to a pharmaceutical composition comprising a peptide derived from endoglin as described above for topical administration.

[0220] In other embodiments the present invention provides a pharmaceutical composition comprising a peptide derived from endoglin as described above in a nasal spray. In other embodiments the present invention provides a pharmaceutical composition comprising a peptide derived from endoglin as described above in an aqueous composition.

[0221] In other embodiments the present invention provides a pharmaceutical composition comprising a peptide derived from endoglin as described above in a powdered composition.

[0222] In other embodiments the present invention provides a pharmaceutical composition comprising a peptide derived from endoglin as described above in a mucoadhesive sprayable fluid gel.

[0223] In one embodiment, the pharmaceutical compositions or formulations of the present invention comprise peptide derived from endoglin or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof and a pharmaceutically acceptable carrier. These formulations can be made using standard formulation and mixing techniques familiar to one of ordinary skill in the art of pharmaceuticals and formulations.

[0224] In another embodiment, the pharmaceutical composition is selected from a solution, suspension, or dispersion for administration as a spray or aerosol.

[0225] In other embodiment, the formulation can be delivered as drops by a nose dropper or applied directly to the nasal cavity. Other pharmaceutical compositions are selected from the group consisting of a gel, ointment, lotion, emulsion, cream, foam, mousse, liquid, paste, jelly, or tape, that is applied to the nasal cavity.

[0226] In other embodiment, the formulation can be delivered with nanoparticles such as lipid- based nanoparticles, including nano / microemulsions, liposomes, solid lipid nanoparticles, and nanostructured lipid carriers.

[0227] As used herein, the terms "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.

[0228] A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local, intransal or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.

[0229] These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0230] In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0231] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intranasal, topical, intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0232] Kits

[0233] In another aspect, the invention relates to a kit comprising the peptide derived from endoglin according to the invention for use as a diagnostic tool for PE.

[0234] In a particular embodiment, the kit according to the invention comprises the peptide derived from endoglin according to the invention and instructions to detect endoglin fragments in a subject suffering or is susceptible to suffer from PE.

[0235] In particular embodiment, the invention relates to a kit for performing the method for diagnosing pre-eclampsia (PE) according to the invention, wherein said kit comprises means to detect endoglin fragments as described above in a sample In another aspect, the invention relates to a kit comprising the peptide derived from endoglin according to the invention for use as a drug.

[0236] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention and instructions for use as a drug.

[0237] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention and instructions for use in the treatment of bleeding disorder.

[0238] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention and instructions for use in the treatment of HHT.

[0239] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention and instructions for use in a topical administration.

[0240] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention and instructions for use in a nasal administration.

[0241] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention, instructions and a nasal spray inhaler.

[0242] In a particular embodiment, the invention relates to a kit comprising the peptide derived from endoglin according to the invention, instructions for use in a nasal administration with nanoparticles, such as lipid-based nanoparticles, including nano / microemulsions, liposomes, solid lipid nanoparticles, and nanostructured lipid carriers.

[0243] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0244] FIGURES:

[0245] Figure 1: Plasma and serum analysis of patients with preeclampsia. The levels of sEng in a plasma and in b serum were quantified using an ELISA kit assay in a cohort of 60 patients. This cohort comprised 20 non-pregnant women controls, 20 pregnant women controls, and 20 cases diagnosed with preeclampsia, c Plasma and d serum samples from preeclampsia patients diluted to ratios of 1 :5, 1 : 10, or 1 :20 and reduced with DDT, were subjected to analysis through SDS-PAGE and Western blot with Endoglin / CD105 polyclonal rabbit antibody (ProteinTech). rEng at 100 ng / mLwas used as control. The data is presented as mean ± S.D. Statistical significance was determined at *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. NS indicates a non-specific band. Figure 2 : Human platelet aggregation in microplates and PARI inhibition / desensitization. Washed human platelets (3 x 108platelets / mL) were incubated with increasing concentration of a,b pEng or c,d pSCR (5, 10, 25 and 50 pM), with TRAP6 at 50 pM serving as a positive control. Aggregation kinetics were measured by monitoring OD (405 nm) variations of a platelet suspension under stirring in a microplate reader set at 37 °C. Results are expressed as the percentage of aggregation from n=4 different donors after peptide addition. The graphs show a significant dose-related induction of platelet aggregation with pEng, particularly at a,b 50pM with approximately 69% aggregation after lOmin incubation (p = 0.0002), while no significative aggregation is observed in the presence of pSCR (p = 0.0805), c,d with a maximum aggregation of approximately 14% at 50pM after lOmin of incubation.

[0246] Figure 3: Effect of pENG on platelet activation, secretion and calcium mobilization. Washed human platelets (3 x 108 platelets / mL) were stimulated with a range of pENG concentrations (5 to 50 pM). Platelet stimulation was then halted, and a,b integrin allbp3 activation (PAC-1) and c,d P-selectin exposure (CD62-P) were measured by flow cytometry. The results are expressed as a,c percentage of platelets and and b,d mean fluorescence intensity (MFI), e-k SCH-79797 (3pM; PARI inhibitor) or DMSO (control) were incubated for 10 min before platelet stimulation. Additionally, washed platelets were preincubated with a calcium fluorophore, and calcium mobilization induced by pENG (1 to 10 pM) or TRAP6 (10 pM) was recorded in real time by flow cytometry, i The Histogram represents respectively the inhibition of TRAP6 and pENG by the PARI inhibitor SCH-79797. -j-k The histograms rappresents Calcium mobilisation and inhibition by inhibitor SCH-79797 in presence of TRAP6 or pEng.

[0247] Figure 4 : Validation of phenotypes by tail-clips assays: First bleeding and total bleeding, a First bleeding time was assessed in Eng(+ / +) mice by tail tip clip, followed by tail immersion in saline with or without 50 pM of pSCR. b Total bleeding was quantified as the cumulative bleeding times over a 10 minute interval. No significant difference was observed in first bleeding time or total bleeding between mice treated with PBS and those treated with pSCR. To confirm the bleeding phenotype, c first bleeding time and d total bleeding time were assessed in Eng(+ / +), Eng(+ / -), and Eng iKO mice in the presence of pSCR. While first bleeding time failed to reveal significant differences, total bleeding time d was notably lower in Eng(+ / +) mice compared to Eng(+ / -) mice and Eng iKO. Eng(+ / +) : n=l 1 treated with PBS and n=22 treated with pSCR; Eng(+ / -) : n=9 treated with pSCR; Eng iKO : n=12 treated with pSCR. Data is presented as mean ± SEM.

[0248] Figure 5 : Total bleeding and hemoglobine dosage. Total bleeding was assessed and quantified in a Eng iKO, b Eng(+ / -) and c Eng(+ / +) mice by-tail-tip clip, followed by tail immersion in saline with or without 50 pM of pSCR or pEng. Haemoglobin loss was quantified as the cumulative bleeding times over a 10 minute interval, in d Eng iKO, e Eng(+ / -) and f Eng(+ / +) mice.

[0249] Figure 6: a. Platelet aggregation was assessed by light transmission aggregometry in the presence of TRAP6 (5 pM, positive control) or each of the three variants (50 pM). Results are displayed as platelet aggregation of 4 donors. Effect of pVl, pV2 on endothelial cell proliferation and viability. ECFCs (40,000 cells / well) were treated with peptides or control medium. Proliferation was assessed at 24, 48, and 72 h by cell counting (b) and pNPP assay (c). Ki67 expression was evaluated by immunostaining after 24 h (d). Data are mean ± SD; statistical analysis was performed using an unpaired t-test.

[0250] Figure 7: Tail clip assay and murine platelet aggregation with pVl and pV2.(A) The procedure applied in the mouse study included the assessment of bleeding time, blood loss volume, and hemoglobin evaluation. This technique was applied to wild-type (non- pathological) mice, to inducible endoglin-knockout (IKO) mice (ongoing experiments), to the mouse model of the hemorrhagic disorder Von Willebrand disease (VWF), and to the mouse model of hemophilia (HA). (B) Tail clip assay on WT mice: first bleeding time, total bleeding time, hemoglobin loss, and estimated blood loss. (C) Maximal aggregation at 8 min in washed platelets. Results include both males and females. For the tail clip assay: control (n = 21; 10 males, 11 females), VI (n = 19; 9 males, 10 females), and V2 (n = 20; 10 males, 10 females). For platelet aggregation experiments, at least 21 mice (both sexes) were analyzed per condition (C), DF- HT iKO mice ongoing. (D) Platets aggregation in microplate (n=5) WT and inducible endoglin-knockout mice (IKO-Eng) were used. Platelet aggregation induced by the pVl variant was assessed in the blood of these mice at different concentrations, confirming its effectiveness at 50 pM (lower dose compared to PAR4ap used as the positive control). (E) White bars (PBS), grey bars (treatment with the peptide pV2), WT (healthy controls) HA (Hemophilia model): Here in HA mice considering total bleeding, hemoglobin (Hb) loss and bleeding volume we found that 3 out of 4 mice treated with pV2 show a difference compared with mice treated with placebo (PBS).

[0251] EXAMPLES:

[0252] EXAMPLE 1:

[0253] Material & Methods

[0254] Patient plasmas In this study, we used samples from three groups of patients: pregnant women with PE, normotensive pregnant women without PE and healthy non pregnant women. The patients were recruited in 2 studies. Pregnant women with PE or without PE were enrolled in the ECLAXIR study23, which is a multicenter case-control study (research grant from the regional Programme Hospitalier de Recherche Clinique P020925). The cases were pregnant women with PE at time of diagnosis, PE being defined according to the definition of the American College of Obstetricians and Gynecologists (ACOG) as blood pressure >140 / 90 mm Hg occurring after 20 weeks of gestation with previously normal blood pressure, associated with proteinuria > 0.3 g in a 24-hour urine specimen. The controls were pregnant normotensive women without PE. The ECLAXIR study was approved by the Ethics Committee (Comite de Protection des Personnes dans la Recherche Biomedicale, CCPPRB) of the Bichat-Claude Bernard hospital (Paris). Non pregnant women were enrolled in the SERCOB study24, which is a case-control study (research Grant CRC 10018 Contrat de Recherche Clinique from Assistance Publique-Hopitaux de Paris, clinical trials NCT00632671), where the controls were healthy women with age > 18 and < 60 years old, a body mass index (BMI) > 18.5 and < 25 kg / m2 and without weight variation > 5 kg in the last 3 months. The SERCOB study was approved by the ethics committee of Saint- Louis University Hospital, Paris (France) (Institutional Review Board). For both studies, all patients gave written informed consent before enrolment and blood sampling and all samples were stored at -80 °C until testing. sEng was quantified by quantikine ELISA immunoassay kit performed on plasma (n=20) and serum (n=20) samples.

[0255] Sequence alignment

[0256] Alignments of human endoglin sequence with those of ten other mammal species were done online using Clustal Omega (vl.2.4)25. This is a multiple sequence alignment program that uses seeded guide trees and Hidden Markov Model profile techniques to generate the alignment. The amino acid sequences of different mammal species were obtained from the UniProt / SwissProt database.

[0257] Algorithm of binding sequence prediction

[0258] Bioinformatic analysis in search of possible cleavage sites on the Eng sequence by Thr through a Profile Specific Scoring Matrix (PSSM) analysis was performed. In ref26 53 peptide sequences were reported to be substrates for Thr cleavage and aligned around a central arginine from position P3 to position P4’. This alignment constitutes the dataset to extract a profile characterizing the cleavage motif. In a PSSM a score is associated with the presence of a given amino acid at a specific position of the motif, generating a scoring matrix. In our case the matrix is of dimension 20 x 8, i.e. 20 amino acids times 8 positions in the motif. The score is calculated computing the probability of occurrence of each amino acid at a given position from the known sequence alignments, normalized by its relative abundance in nature. Based on this scoring matrix it is then possible to analyze a new sequence in search for subsequences of the size of the motif. For the new sequence a score is computed adding the values of the PSSM corresponding to the amino acids of the subsequence at the position in which they appear in the motif. In our case, we scanned the whole endoglin sequence and for each arginine we computed the score of motifs P3 to P4’.

[0259] Docking analysis

[0260] X-ray crystallographic information on the orphan region and the ZP module that constitute the structure of the ectodomain of sEng is available in the Protein Data Bank with accession numbers 5104 (orphan region, 2.42 A resolution), 5HZW (orphan region / BMP complex, 4.45 A resolution) and 5HZV (ZP module, 2.7 A resolution)26; based on these experimental structures, a model of BMP9 ligand-bound homodimeric ENG was suggested, where the molecule adopts a Y-shaped open conformation. A low resolution negative stain EM map of unbound endoglin also exist, suggesting that the free molecule can also exist in a more compact, closed form27. Given the only structure available at atomistic resolution is that of the open ENG, we performed docking analysis using this model which also has the advantage of allowing Thr to approach ENG at the various sites identified by sequence analysis. The Thr structure used for docking was extracted from the high-resolution complex of Thr with an extracellular fragment of PARI receptor (PDB id 3LU9), in which Thr is found in an activated form, as the one it sould adopt for cleavage. Rigid protein-protein docking was performed using the program ClusPro28 searching for positions of interaction between the two molecules that optimizes physical interactions (electrostatic interactions, hydrogen bonds, Van der Waals interactions). Because of the large size of the proteins and the fact that the possible binding sites on ENG identified by sequence analysis are at multiple distant regions, rigid docking seemed an appropriate compromise between accuracy and speed of the search29,30.All figures to illustrate docking results were produced using the Chimera software31.

[0261] Recombinant endoglin (rEng) and thrombin

[0262] Recombinant endoglin (rEng), corresponding to human sEng containing the entire extracellular region (Glu26-Gly586), was obtained from R&D system (1097-EN-025 / CF). rEng (20 pg / mL) was diluted in Tris-buffered saline polyethylene glycol lx buffer (TBS-PEG) and treated with human > -thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human P-thrombin (Thr- C) (Cambridge ProteinWorks, #10108). Dose responses were performed with Thr-H from 0.01 to 1 pM for Ih at 37°C and kinetic with 1 pM of Thr-H during 1, 5, 15, 30 and 60 min at 37°C. Reaction was stopped by adding 50 pM of Phe-Pro-Arg-chloromethylketone, an irreversible thrombin inhibitor (PPACK, Merck, 520222-5MG). Samples were then analysed by different methodologies.

[0263] Western blot and blue Coomassie staining

[0264] Samples (Thr-treated rEng, plasma and serum) were homogenized in an appropriate volume of lx NuPAGE LDS Sample Buffer (Invitrogen, NP0007) supplemented with lx NuPAGE Reducing Agent (Invitrogen, NP0009). Samples were then heat-denatured for 5 min at 80°C and loaded on a NuPAGE 4-12% Bis-Tris polyacrylamide gel (Invitrogen, NP0323BOX). After running, the gels were then either stained with Coomassie brilliant blue (InstantBlue, Abeam, abl 19211), de-stained for 2h in distilled water and scanned before protein spot excision for further sequencing or the gels were transferred to nitrocellulose membranes for western blotting (WB). The membranes were saturated with 5% BSA in TBS-Tween lx and incubated overnight at 4°C with endoglin polyclonal rabbit antibody (1 / 1000, ProteinTech, #10862-l-AP) or endoglin monoclonal mouse antibody (1 / 500, R&D, MAB1097). Membranes were washed in TBS-Tween lx and incubated for Ih at room temperature with a fluorescent- labeled secondary antibody, Dylight 800-conjugated secondary antibody (1 / 10000, Thermo Fisher Scientific, SA5-10036). Fluorescent immunoblot images were acquired using an Odyssey scanner (Li-Cor Biosciences, Lincoln, NE, USA) and quantified using Imaged or Image Studio Lite (Li-Cor) software.

[0265] Capillary electrophoresis immunoassay

[0266] Recombinant endoglin cleavage products and cells supernatant (MSCs and HUVECs) were analyzed after treatment with 1 pM of Thr-H or Thr-C using a capillary electrophoretic based immunoassay (WES; ProteinSimple, San Jose, CA), following the manufacturer’s instructions. Briefly, the WES measurement was performed using a 2-40 kDa separation module (8 x 13 mm capillary cartridge, ProteinSimple Co., SM-W009) and endoglin polyclonal rabbit antibody (1 / 100).

[0267] N-terminal amino acid sequence analysis

[0268] The spots of interest were excised and incubated in a buffer to extract the protein from the acrylamide gel. After overnight incubation under shaking, the solution was incubated on a ProSorb Filter (Applied Biosystems) to fix the protein on a PVDF disc. The N-terminal sequences of proteins were determined by introducing the PVDF disc into an Applied Biosystems 494 automated protein sequencer (Applied Biosystems). Runs of Edman degradation (15 cycles of pulsed-liquid chemistry) were carried out. The sequences obtained were matched to public protein sequence databases with PATTINPROT, a software developed at the Pole Bio-Informatique Lyonnais (PBIL) in Lyon, France (http: / / npsa-pbil.ibcp.fr), and with MS-PATTERN on the protein prospector web site (http: / / prospector.ucsf.edu). For inconclusive searches, sequences were matched against microbial genomes at the NCBI using the more general tBLASTn algorithm.

[0269] C-terminal protein characterization by mass spectrometry

[0270] Gel bands of proteolytic fragments were cut and subjected to in-gel digestions with different endoproteases (Trypsin, Asp-N or chymotrypsin) before submission to mass spectrometry analysis. Peptides mixtures were analyzed by nanoLC-MSMS using a nanoElute liquid chromatography system (Bruker) coupled to a timsTOF Pro mass spectrometer (Bruker). Peptides were loaded with solvent A on a trap column (nanoEase Cl 8, 100 A, 5 pm, 180 pm x 20 mm) and separated on an Aurora analytical column (ION OPTIK, 25 cm x 75 pm, C18, 1.6 pm) with a gradient of 0-35% of solvent B for 30 minutes. Solvent A was 0.1% formic acid and 2% acetonitrile in water and solvent B was acetonitrile with 0.1% formic acid. MS and MS / MS spectra were recorded from m / z 100 to 1700 with a mobility scan range from 0.6 to 1.4 V s / cm2. MS / MS spectra were acquired with the PASEF (Parallel Accumulation Serial Fragmentation) ion mobility-based acquisition mode using a number of PASEF MS / MS scans set as 10. MS and MSMS raw data were processed and converted into mgf files with DataAnalysis software (Bruker). Identification and C-terminal characterization of protein were performed using the MASCOT search engine (Matrix Science, London, UK) with semi-specific cleavages against endoglin sequence. Carbamidomethylation of cysteines was set as fixed modification and oxidation of methionines as variable modification. Peptide and fragment tolerances were set at 10 ppm and 0.05 Da, respectively.

[0271] Cell isolation, culture and thrombin treatment

[0272] Endothelial Colony Forming Cells (ECFCs) were isolated as described32, expanded on fibronectin (FN)-coated plates (1 pg / cm2; Millipore, Billerica, MA, USA) using EGM-2 medium (without hydrocortisone; Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Pooled Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (C-2519A) and seeded in a complete culture medium as described above for ECFCs. Mesenchymal Stem Cells (MSCs) were purchased from Lonza (PT-2501) and seeded in minimum essential medium a (MEM a) with GlutaMAX™ supplement, no nucleosides medium (Life Technologies, Gibco™, 32561094) supplemented with 10% of FBS (Dutscher, S1810-500), 10 ng / mL of fibroblast growth factor (FGF, R&D Systems®, 233-FB-500), and 1% of antibiotic / antimycotic (Thermo Fisher Scientific, Gibco™, 15240062). ECFCs, HUVECs, and MSCs were used at passages P2-8 and day <40. All cells were cultured in appropriate basal medium (controls) vs basal medium with Thr-H (concentrations 0.01, 0.1 and 1 pM, treatment Ih at 37°C). We consider for the study n=6 ECFC (± Thr-H), n=4 HUVEC (± Thr-H) cultured in EBM-2 and n=8 MSC (± Thr-H) cultured in basal MEMa. Supernatant was recollected to perform ELISA assays, while cells were fixed with 4% paraformaldehyde (PF A) in PBS for 15 minutes at room temperature (RT) to perform immunofluorescence assays.

[0273] Quantitative assay of human endoglin by ELISA

[0274] Quantitative analysis of sEng in cell supernatants, as well as in plasma and serum samples was carried out via the sandwich enzyme-linked immunosorbent assay (ELISA) principle. Human Endoglin / CD105 quantikine ELISA immunoassay 96-well kits (R&D Systems, USA, MNDG00) were used according to the manufacturer’s instructions. The ensuing product of the sandwich was read spectrophotometrically at 450nm using a Spectrostar Nano- 96 micro-well reader (BMG-Labteck).

[0275] Immunofluorescence

[0276] Cells incubated with or without Thr-H during Ih were fixed with 4% PFA in medium for 15 min at room temperature (RT). Cells were then washed with Dulbecco’s phosphate buffered saline (DPBS, ThermoFischer) and blocked in 2% BSA in PBS for 30 min at RT, incubated for Ih at RT under agitation with Endoglin / P4 A4 monoclonal mouse antibody (1 / 500, Santa Cruz, sc-20072). Cells were then washed three times with PBS and then incubated with an anti-mouse fluorescein-labeled secondary antibody (1 / 200, Vector Laboratories, FL2000- 1.5) for Ih at RT under agitation. Cells were washed three times with PBS, and the vectashield mounting medium for fluorescence with DAPI (1 mg / mL, Vector Laboratories, H-1200) was used. Cover slips were mounted onto slides that were stocked at 4°C. Observations were performed on confocal microscopy at 20x (Confocal Laser Scanning Microscope, CLSM, Leica TCS SP5).

[0277] Statistics

[0278] Data were subjected to statistical analysis, and the results are shown as mean ± SD. Normality was analyzed with the Shapiro-Wilk test. When the test did not indicate normality, we performed a nonparametric analysis using the Mann-Whitney test. Otherwise, 2-group comparison was performed with parametric Student’s t-test (unpaired).

[0279] Results

[0280] Plasma and Serum Levels of sEng in preeclampsia (PE)

[0281] Plasma and serum levels of sEng were measured in 60 subjects, including 20 nonpregnant controls, 20 pregnant controls and 20 PE patients. sEng concentration (ng / mL) in both plasma and serum was significantly higher in PE than in control plasma [57.35 ± 18.2 (PE) versus 3.47 ± 0.68 (non-pregnant controls) or 7.05 ± 1.83 (pregnant controls) ng / ml], and control serum [47.90 ± 24.50 (PE) versus 3.70 ± 0.77 (non-pregnant controls) or 7.74 ± 1.95 (pregnant controls) ng / mL] (Fig. la,b). Plasma and serum analyzed by WB for Eng showed different molecular weight bands of sEng: 60, 40 and 20 kDa (Fig. lc,d). The visualization of these bands was possible thanks to a new antibody against endoglin that targets over 50% of the Eng sequence (residues 331-658) (Proteintech, #10862-l-AP), at variance with the P4A4 antibody that only targets -10% of the sequence (residues 270-330) (data not shown). These previously undescribed Eng fragments suggested the existence of novel cleavage sites in the endoglin sequence.

[0282] Computational analysis revealed potential Thr cleavage sites in endoglin

[0283] When searching for potential new cleavage sites, amino acid sequence analysis of endoglin highlighted the presence of a GDPRFSFLLH sequence (residues 526-534, SEQ ID NO: 12). Of note, a clear similarity was found (in bold) to the protease activated receptor PAR- 1 site PRSFLL (SEQ ID NO: 13), a sequence known to be cleaved by Thr33, a protease involved in PE22’23. Alignment of the human endoglin amino acid sequence with those of 10 other mammal species revealed that this sequence (amino acids 528 to 534) is highly conserved among different species (data not shown), potentially suggesting that this may be an important recognition site for a putative protease. Given the high sequence similarity to the Thr cleavage site of PARI, we hypothesized that Thr might also be able to cleave endoglin. The crystal structure of the ZP module of ENG29 shows that the GDPRFSFLLH (SEQ ID NO: 12) sequence is not highly exposed within the context of the ZP-C module and is thus unlikely to be readily accessible to Thr or other proteases. However, it could become accessible upon structural rearrangements of ENG, possibly triggered by cleavage at other sites occurring prior. Indeed, a Profile Specific Scoring Matrix analysis identified several other potential cleavage sites with relatively high scores and accessibility (data not shown). As a proof of principle, computational docking of Thr to the site 329-CGGRLQTS-338 (SEQ ID NO: 5) (data not shown) suggests that the latter may be cleaved by the protease. Looking at the crystal structure of ENG, this site appears to be the most exposed to the solvent, and it could be a plausible first target for the protease, since it is easily accessible. These analyses suggest that 1) several possible cleavage sites are present on the ENG sequence, 2) the approach of Thr to at least one of the identified cleavage sites is plausible, or at least not impossible. These results encouraged a further experimental exploration for cleavage of fragments of different sizes (data not shown).

[0284] Thrombin cleaves endoglin in vitro and generates different fragments

[0285] To address the above hypothesis, we investigated whether Thr can target Eng in vitro. Physiologic concentrations of free Thr generated during coagulation are estimated to vary from 1 nM to over 500 nM34,35, whereas pathological concentrations can reach up to 10 pM36. Thus, we tested the ability of a-thrombin (Thr-H) to cleave rEng within this concentration range (10 nM-1 pM) during Ih of treatment. Using a polyclonal antibody against Endoglin, we detected different rEng cleavage products (data not shown). Indeed, bands at « 60, 40, 20 and 10 kDa were detected in a Thr-H concentration-dependent manner (data not shown) simultaneously with a decrease of the total rEng (non-cleaved, 70 kDa) (data not shown). All bands were detectable from the lowest concentration of Thr-H (10 nM). Of note, in the absence of Thr-H no cleavage of rEng was observed after Ih of incubation at 37°C. A kinetic study performed at a concentration of 1 pM of Thr-H (from 1 min to Ih) revealed that the bands appeared already at 1 minute and progressively increased up to Ih of treatment (data not shown). Conversely, a progressive decrease of the total endoglin was observed (data not shown). The smallest fragment (10 kDa) was the last one to be detected and appeared after 15 minutes (data not shown). Kinetic- and Thr-H concentration-dependence of the generation of small bands (40, 20 and 10 kDa) was confirmed in a Simple Western (WES) assay (data not shown). Of note, P-thrombin (Thr-C) cleaved rEng in fragments of similar molecular weights as a-thrombin (Thr-H), and a supplemental smaller band at around 2 kDa was observed for both Thr (data not shown). Altogether, our data demonstrate that Thr (both a- and P-thrombin) cleaves Eng in vitro to generate different Eng fragments in a time- and concentration-dependent manner.

[0286] Identification of the endoglin sequences cleaved by thrombin

[0287] We then investigated Thr cleavage sites in rEng by sequencing each fragment. To this end, we fixed the experimental conditions: 1 pM of Thr-H, during Ih at 37°C, followed by detection and isolation of each fragment from Coomassie blue-stained gels and their N- and C- terminal (N-t and N-c, respectively) amino acid sequencing (data not shown). By Coomassie blue staining of SDS-PAGE gels, we identified all the bands detected by WB (60, 40, 20 and 10 kDa) as well as an extra band at 8 kDa (data not shown). In agreement with our previous data (data not shown), we observed a significant 50% decrease of the total rEng (70 kDa) upon Thr digestion (data not shown). The most abundant fragments were the bands of 40 kDa and 20kDa (data not shown). As a preliminary control, we analyzed the sequence of the initial rEng used in the study. According to N-t sequence analysis, the primary structure of recombinant mature Endoglin of 70 kDa starts at Glu 26 (ETVHC, SEQ ID NO: 4) and ends in Gly 586 (CTSKG, SEQ ID NO: 6), as expected from the commercial datasheet provided by the manufacturer and similar to the described cleavage produced by MMP147,37.

[0288] N-t analysis demonstrated that both the 60 kDa and 40 kDa fragments start with ETVHC (SEQ ID NO: 4) (data not shown), but C-t analysis was not able to identify with precision the corresponding C-terminal (data not shown). However, immunoblot with the monoclonal antibody MAB1097 (data not shown) that specifically recognizes the endoglin orphan region (data not shown) suggests that the 40 kDa fragment ends with CGGR (Fig. 2 and 4), as also supported by predictive studies (data not shown). Interestingly, this cleavage site (GGRLQT, SEQ ID NO: 7) maps within the 18-residue linker between the OR and the ZP module26, and it can be hypothesized that this precise location, right in the boundary between those two different functional regions, has a potentially relevant biological role. Considering the fragment of 20 kDa, N-t starts with SAYSS (Ser 407), (SEQ ID NO: 11) (data not shown), whereas the C-t ends with the fragment corresponding to CTSKG, (SEQ ID NO: 6) (Gly 586) (data not shown). Of note, the N-t cleavage site for the 20kDa fragment, between residues 406 and 407, is exactly the last residue previously mapped in sENG purified from PE patients’ plasmal6. The 10 kDa fragment presents the N-t starting with AAKGN, (SEQ ID NO: 10) (Ala 511) (data not shown) and the C-t ends with the peptide corresponding to CTSKG, (SEQ ID NO: 6) (Gly 586) (data not shown. The N-t starting sequence of 20kDa and 10 kDa fragments confirmed the prediction of Thr cleavage suggested by modelling (data not shown). The fragment of 8 kDa (data not shown) was also analyzed by N-t analysis and starts with SFLLH, (SEQ ID NO: 52 (Ser 531) (data not shown), but its C-t was not detectable (data not shown). However, by WES we identified the presence of a 2 kDa fragment after incubation (at different times from 1 min to Ihour) with Thr-H (data not shown), suggesting that the 10 kDa fragment can be further cleaved, leading to two smaller fragments of 2 and 8 kDa. This result was also confirmed using Thr-C (data not shown).

[0289] These above data allow us to speculate that rEng is cleaved in shorter forms by a treatment of 1 min of Thr first releasing the 40 kDa fragment, and then those of 20 kDa and 10 kDa, as suggested by the kinetics assays (data not shown). It can be speculated that after an initial cleavage, the molecule will be open and will become more accesible for further cleavages. Furthermore, the fragment of 40 kDa starting with ETVHC (SEQ ID NO: 4) also suggests that it can be monomeric because it does not contain Cys residues involved in intermolecular disulfide bonds (data not shown). By clustal alignment sequence, it is evident that in mouse there are different amino acids in the specific sequence where the cleavages are predicted (human CGGRLQTS (SEQ ID NO: 5), FVLRSAYS (SEQ ID NO: 53), IQGRAAKG (SEQ ID NO: 54)), while the sequence FSFLLH (SEQ ID NO: 14) is conserved, (data not shown). To confirm the specificity of the cleavage in the predicted sequences we treated Human and mouse rEng at a concentration of 20 pg / mL with increasing concentrations of Thrombin-H (0.01-0.05-0.1-0.5-1 pM) at 37°C for 1 hour. Gels were run simultaneously using MOPS buffer, optimal for revealing the bands at the expected cleavage sites (mutated in mice). Human rEng confirmed the cleavages at 60, 40, and 20 kDa as expected and showed the uncleaved monomeric form at 75 kDa. The uncleaved murine endoglin appears as a double band around 95-100 kDa, due to varying levels of glycosylation as informed by the supplier. Consequently, the cleaved bands that may originate from thrombin cleavage at the conserved motif DPRFSFLLH (SEQ ID NO: 55) might also appear as a double band with a 5 kDa difference (70-65 kDa). (data not shown)

[0290] Endoglin is cleaved by thrombin at the cell surface

[0291] The above data demonstrate that soluble rEng can be cleaved by Thr. We then investigated the physiological capacity of Thr to cleave endoglin from the cell surface of different cell types expressing surface Eng: ECFCs, HUVECs and MSCs6’38. ECFCs were incubated with different concentrations of Thr-H (10 nM, 100 nM, and 1 pM) and HUVECs and MSCs were treated with 1 pM of Thr-H. Importantly, Thr-H treatment did not affect ECFC viability, and cells conserved their monolayer appearance (data not shown). Cellular endoglin cleavage by Thr was analyzed by immunofluorescence. Endoglin-specific fluorescence was reduced in ECFCs after Thr-H treatment compared to control, in a dose-dependent manner (data not shown). This result was confirmed with the two other cell types, HUVECs and MSCs, that showed a decrease of endoglin staining on cell surface upon Thr treatment, without any effect on cell viability (data not shown). The specificity of Thr cleavage was supported by using a thrombin inhibitor PPACK in assays with ECFC and HUVEC supernatants (data not shown). Then, we quantified the level of sEng in cell supernatants by ELISA and WES techniques. Interestingly, sEng levels were significantly increased in all cell lines treated with Thr-H compared to controls (data not shown). These results were also replicated with Thr-C (data not shown). Importantly, the 60, 40 and 20 kDa bands were observed by WES in MSCs and HUVECs supernatants (data not shown), confirming the results obtained in vitro using rEng. Since the mature cell surface endoglin carries the standard O- and N-glycosylation8, while rEng which is less glycosylated (R&D, datasheet), the above findings may rule out the possible interference of sEng glycosylation in the Thr cleavage. Supporting this conclusion, endoglin glycosylation occurs in a region of the protein different from those of the Thr cleavage sites (data not shown). Hence, our data demonstrate that Thr is able to cleave Eng from cell surface, leading to the release in the extracellular medium of different fragments of sEng.

[0292] EXAMPLE 2:

[0293] Material & Methods

[0294] In silico modeling

[0295] The methodology employed for modelling the PARI peptide complex comprised several sequential steps. Initially, sequence selection involved obtaining the relevant amino acid sequences from UniProt (The UniProt Consortium, 2023) (id:P25116), with the signal peptide (1-21) omitted for PARI, as it is cleaved in the mature receptor (Zampatis et al., 2012) , which we will refer to as matPARl from now on. All scripts for deep learning models are obtained via ColabFold (Mirdita et al., 2022), which is a package that offers a user-friendly interface to run deep learning models for protein’s 3D predictions such as ESFMFOLD, Alphafold 2 and Alphafold 2 multimer, and then executed on Google Colab (Bisong, 2019), providing access access to GPU-accelerated computing. Refinement of predicted structures via steepest descent optimization was performed in UCSF Chimera software (Pettersen et al., 2004). All intermediate and final results were visualised using UCSF ChimeraX software (Goddard et al., 2018).

[0296] We used ESMFOLD (Rives et al., 2021) to make the first structure prediction, which was subsequently minimised. ESMFOLD’ s prediction was used as a template for the Alphafold2 (AF2) (Jumper et al., 2021) with MMseqs2 (Steinegger and Sbding, 2017). The third step used the prediction of AF2 as template for AF2 multimer (Evans et al., 2022), where both the sequence of PARI and peptide of interest were given as input to make a prediction of the PARl / peptide complex‘s 3D structure. For the last step, the peptide poses were refined using HADDOCK (van Zundert et al., 2016), a method computing physical interactions to perform peptide / protein docking. All residues of the peptide of interest and PARl’s residues within a 5-angstrom of the peptide in the predicted complex were defined as active residues. Active residues are used to define the Ambiguous Interaction restraint (AIR) of HADDOCK. Based on an energy score, HADDOCK predicted several optimal structures for the complex that were grouped by structural similarity (clustered). All clusters were then analysed using Protein Ligand Profiler (PLiP) (Salentin et al., 2015) to detect and quantify the contacts between PARI and peptide of interest.

[0297] Peptide synthesis In this study, we synthesized two peptides, namely pEng and pSCR, comprising a sequence of 10 amino acids: H2N-FSFLLHFYTV-COOH (pEng, SEQ ID NO: 17) and H2N- VTFSLHFLFY-COOH (pSCR, SEQ ID NO: 18), respectively. It is noteworthy that while pSCR contains the same amino acids as pEng, they are arranged in a different order. Additionally, pSCR was used as the control peptide in subsequent experiments. To ensure that any observed outcomes were attributable to the inherent properties of the peptides rather than variations in the synthesis process, peptide synthesis was conducted at three distinct facilities: Sigma-Aldrich, Altergen, and Polypeptide. Control experiments were conducted to confirm the absence of contaminants or impurities that could affect the observed effects of the peptides.

[0298] Human blood donors

[0299] Venous blood, sourced from informed and healthy donors who abstained from antiplatelet medications for a minimum of 2 weeks, was provided by the French Blood Bank Institute (EFS) through an agreement with the Paris Descartes University (C CPSL UNT N° 12ZEFS / 064). The blood was collected in Vacutainer tubes (Ozyme, France) containing ACD- A, with final concentrations of 13 mM citric acid, 12.6 mM sodium citrate, and 11 mM D- glucose. To obtain diluted platelet-rich plasma (PRP), the collected blood was subjected to centrifugation at 210 g for 11 minutes after dilution in washing buffer (3V / 1 V). Subsequently, washed platelets were meticulously prepared by centrifuging the diluted PRP at 1240 g for 12 minutes, incorporating 0.2 pM PGE1 and 0.06 U / mL apyrase in the process. The resulting platelet pellet was then re-suspended in washing buffer and subjected to centrifugation under the same conditions as mentioned earlier. Finally, the platelets were re-suspended in the assay buffer and 2 mM of calcium chloride.

[0300] Human platelet aggregation in microplates and aggregometry

[0301] Human washed platelets (3 x 108 / mL) were preincubated in the presence of vehicle alone or sEng at 0.1, 1, or 5 pg / mL (R&D Systems, #1097-EN / CF) for 2 minutes at 37 °C under stirring in the wells of a 6-well microplate (half-area flat bottom; Greiner Bio-one). Then, aggregation was induced by adding 5 pL of pEng or pSCR at concentrations of 5, 10, 25, and 50 pM, or PAR4-ap at 100 pM (Bachem, Germany), serving as a positive control for mouse platelets or 50 pM TRAP6 (Bachem, Germany) or 0.5 pM of the thromboxane-prostanoid receptor agonist U46619 (Calbiochem, Merck), used as the positive control. The extent of platelet aggregation was expressed as the percentage of maximal aggregation calculated as the change in absorbance at 405 nm relative to the absorbance under resting condition and after full platelet aggregation obtained with 10 pM thrombin receptor-activating peptide-6 (TRAP-6; Bachem), as previously described (Decouture B et al 2015). Platelet aggregation, performed under the same conditions as described above, was also measured using light transmission aggregometry (ChronoLog Aggregometer Model 700, Chronolog Corporation) (Martin AC et al Eur J Pharmacol. 2020) to confirm the microplate results.

[0302] For inhibition experiments with human platelets, prior to the aggregation assay, platelet suspensions were treated for 2 min under stirring with either assay buffer, DMSO (0.1%), SCH79797 (MedChemExpress MCE cat.# HY-14993 / CS) (Lei D et al. iScience 2021) at 5 pM or 10 pM. The extent of platelet aggregation was quantified as the percentage of maximal aggregation, calculated by measuring the change in absorbance at 405 nm relative to the absorbance in the resting condition (assay buffer) and after full platelet aggregation induced by TRAP6 or PAR4-ap.

[0303] Analysis of integrin «IIbp3 activation and P-selectin exposure in platelets

[0304] Integrin allbp3 activation and P-selectin exposure were evaluated by flow cytometry (BD AccuriC6Plus), using a specific antibody, PAC1, which recognizes the active conformation of the integrin, and a specific antibody, CD62-P. Washed platelets (30 pL; 3 x 108 / mL) in Tyrode’s buffer were stimulated or not for 10 minutes without stirring by a range of pENG (5 to 50 pM). Platelet stimulation was then stopped by adding 300 pL Tyrode’s buffer containing 2 mM CaCL before incubation of activated platelets (20 pL) with fluorescein isothiocyanate (FITC) anti-human-activated allbp3 integrin (clone PAC-1; Becton Dickinson; 20 pL of FITC-PAC1 for 5 x 105platelets) and CD62-P (Miltenyi Biotech, 2 pL of APC- CD62-P for 5 x io5platelets) for 20 minutes at room temperature. SCH-79797 (3 pM; PARI inhibitor) or DMSO as control were incubated for 10 minutes before stimulation.

[0305] Assessment of calcium mobilization in platelets

[0306] Calcium mobilization was assessed by flow cytometry. Washed platelets (3 x 108 / mL) were preincubated with the calcium fluorophore Oregon-green 488 BAPTA-1 AM (1 pM, Invitrogen) for 30 minutes at 37°C, then diluted to 3 x 106 / mL in Tyrode’s buffer. Calcium mobilization induced by pENG (1 to 10 pM) or TRAP6 (10 pM, Bachem) in the absence of external Ca2+(supplemented with 0.1 mM EGTA) was recorded in real time by flow cytometry as previously described (Feng M, Elaib Z, Borgel D, et al. Circ Res. 2020) The graphs represent Ca2+mobilization defined as the ratio of MFI of activated vs resting platelets (set as 1) as a function of time in seconds. The arrow indicates the time of platelet stimulation by pENG. SCH-79797 (3 pM; PARI inhibitor) or DMSO as control were incubated for 10 minutes before stimulatin.

[0307] Cell culture Endothelial Colony Forming Cells (ECFCs) were isolated from the adherent mononuclear cell (MNC) fraction as described (Smadja et al. Arterioscler Thromb Vase Biol 2008). Then, ECFCs were expanded on 0.2% gelatin-coated plates (1 pg / cm2; Millipore, Billerica, MA, USA) using an endothelial cell growth medium (EGM-2 MV) bulletkit (CC- 3156 & cc-4147, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). In parallel, pooled Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (C-2519A) and subcultured on 0.2% gelatin-coated plates in a complete culture medium as described above for ECFCs. Three different lots of HUVECs were used. Mesenchymal Stem Cells (MSCs) were also purchased from Lonza (PT- 2501) and seeded in minimum essential medium a (MEM a) with GlutaMAX™ supplement, no nucleosides medium (Life Technologies, Gibco™, 32561094), supplemented with 10% of FBS, 10 ng / mL of fibroblast growth factor (FGF, R&D Systems®, 233-FB-500), and 1% of antibiotic / antimycotic (Thermo Fisher Scientific, Gibco™, 15240062). ECFCs, HUVECs and MSCs were used at passages P2-6 and day <40. All cell lines underwent initial authentication by the vendor (Lonza) through assessments of cell morphology and cell-type specific markers using flow cytometric analyses. Subsequent authentication was not conducted. The vendor tested the cell lines for mycoplasma contamination, but no further testing for mycoplasma contamination was performed thereafter. Additionally, none of the cell lines utilized in this study were identified in the database of commonly misidentified cell lines (ICLAC and NCBI Biosample). siRNA transfections and validation by flow cytometry

[0308] Endoglin-specific siRNA (siRNA-Eng; sc-35302, Santa Cruz Biotechnology, CA, USA) was used to silence human Eng in ECFCs and HUVECs. Briefly, lOpM siRNA solution was mixed with Dhermafect reagent (SO-2511539G Dharmacon, USA) to form transfection complexes. These complexes were then added to the cells in their respective medium, excluding antibiotics, either in IBIDI chambers for flow-mediated behavior analysis or in P24 wells for flow cytometry analysis. For the control group, cells were transfected with scrambled siRNA (Scramble, Allstars Neg. control siRNA, Qiagen, Cambridge, MA, USA) designated as siRNA- SCR. Simultaneously, the efficiency of Eng suppression was assessed using flow cytometry with a Fortessa flow cytometer (BD Biosciences, France). Briefly, cells in suspension (150,000-200,000 cells / mL) were first incubated with 1% BSA / PBS for 30 min at 4°C and then with a mouse monoclonal antibody against human Eng (CD 105-Al exa488, #MHCD 10520 Invitrogen; dilution 1 :50) for 1 h at 4°C. After two washes with PBS at 4°C, the mean fluorescence intensity (MFI) was measured with a Fortessa flow cytometer (BD Biosciences, France).

[0309] Fibrin gel bead assay

[0310] Three-dimensional fibrin gel assays were performed as previously described (Nakatsu MN, et al. J Vis Exp JoVE. 2007). ECFCs were seeded (1 x 106 cells per 2500 beads) onto Cytodex beads (Sigma-Aldrich) and embedded in a 2.5 mg / mL fibrin gel in the presence of EGM-2 medium in chamber slides (Millicell EZ slide, Millipore). Mesenchymal stem cells (80,000 cells / well) were plated on the top of the gel as feeders. After 5 days of culture, feeders were trypsinized and the fibrin gel was fixed with 4% PF A. Gels were stained with Alexa Fluor- 488-conjugated phalloidin and TO-PRO-3 (Invitrogen — Thermo Fisher Scientific, MA, USA). Images were acquired with a Leica Confocal laser scanning microscope TCS SP8. The number of sprouts and cumulative tube length per bead was measured using the Image J macro as described (Eglinger J, et al Inflamm Regen. 2017). Images were taken using a confocal microscope (Leica SP-5) and video recording (Leica Las AF Lite). At least 150 beads per sample (n=3) were evaluated.

[0311] Co-culture and immunofluorescence

[0312] The co-culture of endothelial colony-forming cells (ECFCs) and mesenchymal stem cells (MSCs) was conducted to investigate their interactive behavior. ECFCs and MSCs were separately cultured in their respective growth media until reaching approximately 70-80% confluency. Following this, the cells were detached using trypsin-EDTA and counted. For the co-culture experiments, ECFCs and MSCs were mixed at a defined ratio and seeded together in appropriate culture vessels. The co-culture was maintained in a suitable medium that supported the growth and functionality of both cell types. The cell mixture was incubated under standard culture conditions, typically in a humidified atmosphere with 5% CO2 at 37°C.

[0313] Cells incubated with or without pEng / pSCR for 1 week (?) , then fixed with 4% PFA in medium for 15 min at room temperature (RT). Cells were then washed with Dulbecco’s phosphate buffered saline 2 (DPBS, ThermoFischer) and blocked in 2% BSA in PBS for 30 min at RT, incubated for Ih at RT under agitation with vWF monoclonal mouse antibody (1 / 500, Dako, #M0616). and rabbit Calponin (1 / 200, Abeam, ab46794) .Cells were then washed three times with PBS and then incubated with an anti -mouse Texas-Red labeled secondary antibody (1 / 200, Vector Laboratories, TI-2000, 1.5) and antirabbit FITC vector (Ref) for Ih at RT under agitation. Cells were washed three times with PBS, and the vectashield mounting medium for fluorescence with DAPI (1 mg / mL, Vector Laboratories, H-1200) was used. Cover slips were mounted onto slides that were stocked at 4°C. Observations were performed on confocal microscopy at 20x (Confocal Laser Scanning Microscope, CLSM, Leica TCS SP5).

[0314] EC Flow-mediated behaviour analysis

[0315] Cells were seeded at 5 * 105 cells / mL and cultured for 24 h to reach confluency in full medium before the transfection procedure, as described previously. For ECFCs and HUVECs gelatin-coated p-Slide I 0.4 Luer (ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized - ibidi GmbH) were used. After reaching 36 hours of transfection (siRNA-SCR or siRNA-Eng), pEng or pSCR were added to the cells in the p-slide at concentrations of 1, 5, 10, 25, and 50 pM for a duration of 1 hour. Shear stress was then applied using the ibidi pump system (ibidi GmbH) with the associated software (V.1.6.1). One pump system was used to the application of the unidirectional laminar shear stress (7.5 dyn / cm2) and the chamber was placed on microscope (equipped with cell culture system) for imaging (Widefield Zeiss 515 Roussy). Pre-warmed EGM-2 MV medium to which we added 10 mM of HEPES solution (Sigma- Aldrich) and a respective concentration of peptide were pumped through the flow chamber. Cells were allowed to adapt to increasing levels of shear stress in an adaption phase (2.5 dyn / cm2 for 15 min, 5 dyn / cm2 for 15 min and then 7.5 dyn / cm2 for 5 h). For this shear stress, we used ORANGE perfusion sets (DI de 1,6mm et length de 100cm, ibidi GmbH). Image acquisition were performed on IMAG’IC Facility of the National Infrastructure France BioImaging (ANR-10-INBS-04). Phase images were taken every 3 minutes for 5 hours. Cellular migration was analysed using MTrackJ in Imaged.

[0316] Cell size analysis with Maastricht chamber

[0317] Cell size under flow was evaluated using the Maastricht flow chamber, which has dimensions of 3 mm width, 30 mm length, and a depth of 50 pm. The experiment involved perfusing EGM-2 MV medium (800 pl) for 10 minutes at room temperature, applying a wall shear stress of 7.5 dyn / cm2. The infusion rate was maintained at 80 pL / min. ECFCs were subjected to this flow condition. Continuous observation of the same field was conducted throughout the perfusion period, with phase images captured at lOx magnification every 50 seconds. Cell size analysis was performed using Imaged software.

[0318] PNPP assay for ECFC viability

[0319] The viability of ECFCs after treatment with different concentrations of pEng (0, 1, 5, 10, 25 and 50 pM) for 24, 48, and 72 hours was assessed using the p-nitrophenyl phosphate (PNPP) assay. PNPP is a chromogenic substrate for most phosphatases. The reaction yields para-nitrophenol, which forms an intense yellow soluble product under alkaline conditions, conveniently measured at 405 nm on a spectrophotometer. Following the treatment periods, the medium was removed, and the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, ThermoFischer). PNPP solution (5 mM PNPP in 100 mM sodium acetate buffer, pH 5.5) was added to each well and incubated at 37°C for 30 minutes. The reaction was stopped by adding 0.1 M NaOH, and the absorbance was measured at 405 nm using a microplate reader.

[0320] Wound healing cell migration assay

[0321] A wound healing cell migration assay was performed to evaluate the migratory capacity of cells transfected with siRNA-SCR or siRNA-Eng in the presence or absence of various concentrations of pEng and pSCR. Cells were seeded at a density of 20,000 HUVECs / well in complete EGM-2 medium on a 96-well ImageLock® plate (Sartorius, reference #4379) and cultured until reaching confluence. Subsequently, a scratch was introduced in the cell monolayer using a sterile pipette tip. Wounded cell layers were washed with DPBS to eliminate cellular debris, and fresh culture medium was introduced. For experimental conditions, pEng and pSCR were added to the culture medium. The Incucyte® S3 Live-Cell Analysis system (Sartorius) was used to continuously record phase-contrast images at lOx magnification at 1- hour intervals over a 48-hour period, providing a real-time assessment of cell migration kinetics. Quantitative analysis of the wound closure rate, measured as the distance migrated by cells from the wound edges, was conducted using the IncuCyte software.

[0322] Animals

[0323] Endoglin heterozygote mice (Eng+ / -), mice depleted for endoglin in endothelial cells (Eng iKO) and control littermates (Eng+ / +) were provided by Dr. Franck Lebrin (Department of Internal Medicine (Nephrology), Leiden University Medical Center, The Netherlands) (Tual- Chalot et al, 2019 ; Arthur et al, 2000). For all conducted experiments, male and female C57BL / 6J mice, aged 8 to 12 weeks, of Eng + / +, Eng + / -, and Eng iKO genotypes were used. They were age- and gender-matched, and housed under standard conditions with a 12-hour light / dark cycle, ensuring ad libitum access to water and standard rodent chow. Anesthesia was induced through intraperitoneal injection of ketamine (80 mg / kg) combined with xylazine (10 mg / kg). This study complied to the guidelines of the French governments, as well as Directive 2010 / 63 / EU of the European Parliament. All efforts were made to minimize the number of animals used and alleviate any potential suffering. Ethical approval for all animal procedures was obtained from the Ethics Committee on Animal Resources at Paris Descartes University APAFIS #45614-2023100610496222v3.

[0324] Tail bleeding assay

[0325] The bleeding time was assessed using a standardized tail-tip bleeding model (Ferriere S, et al. Blood. 2020;136(6):740-748.). Briefly, tails of anaesthetized mice were pre-incubated in a 37°C saline solution during 5 min to homogenize vessel dilatation between animals. Mice were then positioned in lateral decubitus on a support. A distal 3 mm segment was amputated from the tail clip using a scalpel, and the tail was immediately immersed in a 15 mL conical tube containing 5 mL of Dulbecco’s phosphate buffered saline (DPBS, ThermoFischer), prewarmed to 37 °C, and supplemented with 50 pM of peptide. Experimental groups, including control (pSCR or PBS) and test groups (pEng), were established to assess the influence of specific variables on bleeding time. The tail was positioned vertically, with the tip about 2 cm below the body horizon. The duration from the incision until bleeding cessation was recorded using a stopwatch. After bleeding stopped, a 3 minute interval was observed to detect any rebleeding. In cases of bleeding on / off cycles, the sum of bleeding times within a 10 minute period was used (Total bleeding). The experiment was terminated after 10 minutes of total bleeding to prevent lethality, as required by the local animal ethics committee (End-point). Mice remained under anesthesia throughout the entire procedure. Hemoglobin levels in the saline were measured to assess blood loss accurately.

[0326] Hemoglobin assay

[0327] Hemoglobin levels were assessed using the Drabkin method (CROSBY WH, et al Blood. 1957;). Following the tail bleeding assay, blood samples in the saline solution were collected. The samples were then centrifuged at 2400 rpm for 12 minutes at room temperature to separate blood cells. Following centrifugation, the supernatant was removed, and erythrocytes were re-suspended in distilled water before undergoing re-centrifugation at 13000 rpm for 10 minutes at room temperature. The Drabkin reagent, consisting of potassium cyanide and potassium ferricyanide, was added to the samples. This reagent forms cyanmethemoglobin in the presence of hemoglobin, producing a stable compound with a characteristic color. Subsequently, the absorbance of the resulting solution was measured at a wavelength of 540 nm using a microplate spectrophotometer at 550 nm (BioRad). To quantify hemoglobin levels in the experimental samples, standard solutions of known hemoglobin concentrations were utilized to construct a calibration curve. The assay was executed in duplicate for each sample to ensure precision, and the average value was calculated.

[0328] Mouse cardiac blood sampling method

[0329] Blood collection was performed through cardiac puncture into ACD-C tubes using a closed-chest method. A horizontal line was meticulously drawn across the mid-torso of each mouse, with a complementary line positioned beneath the armpits. Employing a fine 23-gauge needle, a precise puncture was made directly through the chest wall, targeting the right-angle formed in the upper quadrant and entering the left ventricle of the heart. Blood naturally flowed into the needle, to which a syringe has then been added for optimal retrieval without additional pressure. Approximately 500-1000 pL of blood was collected from each mouse.

[0330] Mouse platelet preparation

[0331] Platelet-rich plasma (PRP) was obtained by centrifuging at 170g for 7 minutes blood diluted in washing buffer (2V / 1V; 36mM citric acid, 5mM D-glucose, 5mM potassium chloride, 2mM calcium chloride, ImM magnesium chloride, 103mM sodium chloride, pH 6.5) containing 0.03 U / mL apyrase (Agro-bio, France) and 1 pM prostaglandin El (PGE1, Sigma- Aldrich, France). As already described washed platelets were prepared by further dilution and centrifugation at 750 g for 10 minutes. The platelet pellet was resuspended, and experiments were conducted in an assay buffer (lOmM HEPES, 140mM sodium chloride, 3mM potassium chloride, 5mM sodium bicarbonate, 0.5mM magnesium chloride, lOmM D-glucose buffer, pH 7.35), with calcium chloride (2mM).

[0332] Statistics

[0333] All analysis was done on log-transformed data, using a linear mixed effects model: In Y = po + PAge Age + pWeight Weight + SSex Sex + SPeptide Peptide + ZSeries + additional terms + a where po is the expectation for the reference group, \beta_{ Age} is the increase for a unit increase in age, \beta_{Poids} is the increase for a unit increase in weight, \delta_{Sexe} is the difference between the two sexes, \delta_{Peptide} is the difference between the two peptides, Z_{ Series} a centered random variable modeling changes between experimental series (summarized by its standard deviation, \sigma_{ Series}), and \epsilon is the error term, a centered random variable of variance \sigma2. Both Z_{ Series} and \epsilon are assumed Gaussian, and independent. The reference group is for WT female mice, receiving PBS (for the PBS vs pSCR comparison) or the control peptide (for the pSCR vs pEng comparison). All models were fitted using maximum likelihood (Imer function of the lme4 R package). Effects of variables were tested using the likelihood ratio test for nested models, with the asymptotic chi-square law. Between groups comparisons were done using suited contrast, accounting for multiple comparison issues with the multcomp package for R. Model hypothesis, especially the Gaussian assumption, were checked graphically.

[0334] Blood stability studies and forced degradation studies

[0335] Stability of pEng in human and mouse blood samples, and forced degradation studies under a variety of conditions that are relevant for the pharmacological formulation of therapeutic peptides were conducted.

[0336] The pEng was solubilized in a mixture of 90% (v / v) ethanol and 10% (v / v) DMSO to achieve a stock concentration of 10 mg / mL, corresponding to 7.8514 mM based on a molecular weight of 1273.6615 Da. Subsequently, 4.5 pL of the pEng stock was combined with 1 mL of blood, resulting in a final concentration of 35 pmol / L. The mixture was incubated at 37 °C and 750 rpm using a Thermomixer (Eppendorf AG, Hamburg, Germany). At predet ermined time points (0, 5, 15, 30, and 60 min), aliquots of 100 or 200 pL were withdrawn and mixed with trichloroacetic acid (TCA) to obtain a final concentration of 3% (w / v). These samples were then ncubated on ice for 10 min. Following centrifugation at 12,000 * g for 5 min, the supernatant was neutralized with sodium hydroxide (1 mol / L) and stored at -80 °C until further analysis.

[0337] In the context of the forced degradation studies, an identical concentration of peptide (35 pmol / L) was subjected to four distinct conditions, as outlined below. The extent of degradation and the resulting products were subsequently analyzed using liquid chromatography -mass spectrometry (LC-MS). The degradation conditions encompassed: 0.1 M HC1 (70 °C for 1 h), 0.01 M NaOH (25 °C for 1 h), 0.3% (v / v) H2O2 (25 °C for 1 h), and H2O (70 °C for 3 h). Samples underwent flash-freezing in liquid nitrogen and were kept at -80 °C until further analysis. Peptide isolation and quantification in both techniques were performed by liquid chromatography-mass spectroscopy (LC-MS).

[0338] Results

[0339] Interaction Modeling of pSCR and pENG with PARI

[0340] In order to substantiate the hypothesis that the peptide of interest generated from the cleavage of Endoglin by Thrombin (pENG) interacts with PARI in a manner similar to that of TRAP6 we have modeled the structure of the PARl / pENG complex. The potential poses of control peptide (pSCR) and (pENG) were evaluated using our in-house protocol for modelling peptide protein interaction (as described above, Example 2). The methodology employed comprised several sequential steps as done previously for the PAR1 / TARP 6 complex. The goal of this pipeline is to use deep learning techniques to make a prediction of the matPARl / peptide complex’s three-dimensional (3D) structure (data not shown). We first predict an initial structure for PARI using ESMFOLD, refine it using Alphafold 2, and then make a prediction of the PARl-peptide complex. The combination of both EFSMFOLD and AF2 allows us to take advantage of both model strengths, with ESMFOLD providing reasonable structure for sequences with low MSA coverage and AF2 providing very accurate prediction for sequences with high MSA coverage. At last we use HADDOCK, a physics based method, to confirm or invalidate the prediction made by the neural networks. Specifically, this step is used to prevent the false positive binding of peptide to the binding site of PARI. A protocole that considered several poses for pENG-PARl and pSCR-PARl complexes was used (as described above Example 2). This shows the most relevant and representative cluster for both pSCR and pENG. pENG is found in the binding pocket of PARI that was previously determined by X-ray. The analysis of interactions with PLIP showed extensive interactions between pENG and PARI, including multiple Van Der Waals interaction, multiple hydrogen bonds, with 2 pi-stacking interaction (data not shown). The proposed binding site for pSCR is in the membrane far from the the actual binding pocket. The bound pSCR doesn't share any hydrogen bonds with PARI but only one pi-staking interaction and one pi-Cation interaction. This results is coherent with the notion that pSCR does not bind PARI (data not shown).

[0341] Taken together these modeling resultas are coherent with the hypothesis that pENG, but not pSCR, bind specifically to PARI. pEng induces strong platelet aggregation, synergizes with platelet agonists and target PARI receptor

[0342] To address the above hypothesis, we investigated whether pEng and pSCR can target human platelet-aggregation in vitro. Human washed platelets (3^ 108 platelets / mL), preincubated with buffer, were activated by TRAP6 (50 pM) or various concentrations of pEng and pSCR (5, 10, 25 and 50 pM), and aggregation was monitored using a previously described microplate-based technique (Marie Lordkipanidze et al, 2014). Compared to buffer-treated platelets, pEng induced strong dose-related platelet aggregation (Figure 2a, b), reaching at least 69% with all donors tested at the concentration of 50 pM after 10 minutes incubation (p = 0.0002). However, no significant aggregation (p = 0.0805) was observed with pSCR, with a maximum aggregation of approximately 14% at 50 pM after 10 minutes of incubation (Figure 2c, d)

[0343] To further explore the platelet aggregation effect seen with pEng, the synergistic effect of pEng on the percentage of human platelet aggregation in the presence of different platelet agonists was measured (data not shown). Thrombin Receptor Activating Peptide 6 (TRAP6) alone at I pM did not induce platelet aggregation at the concentration used (data not shown). On the contrary, pEng induced a dose-dependent synergy of TRAP6-induced aggregation that was statistically significant at 5pM of pEng (p = 0.0003) as well as at lOpM (p < 0.0001; data not shown).

[0344] To rule out that the pEng-induced synergy of aggregation was restricted to TRAP6- induced activation, the experiment was reproduced using the thromboxane-prostanoid (TP)- receptor agonist U46619 (0.07 pM). Similar to TRAP6 activation, the weak aggregation (<15%) induced by U46619 was significantly increased with 5pM of pEng (p = 0.0339) as well as with lOpM of pEng (p = 0.0286 ; data not shown). Hence, our data demonstrate that pEng induces strong platelet aggregation in human platelets compared to controls, while pSCR does not induce aggregation in human platelets.

[0345] To examinate the role of PARI in mediating the pEng induced human plateletaggregation in vitro, we performed the previous aggregation assay with or without SCH79797, a PARI (Ho-Sam Ahn et. Al 2000). We chose the dose of 3uM of the competitive inhibitor as it did not seem to induce any toxicity in the vehicule of SCH79797 being DMSO, we carried the experiments by incubating the platelets with SCH79797 or its DMSO equivalente dose (0.1%) for 2 minutes under stearing prior to the addition of TRAP6 or pEng. Strickingly, both TRAP6 and pEng achieved more than 50% of the maximal aggregation at 4 minutes in absence of inhibitor. At 8 minutes in presence of SCH79797, pEng induced aggregation higher than TRAP6 but not statistically significant, suggesting that both peptides target PARI receptor. (Figure 3i).

[0346] All together, these results advocate that pENG might activate platelet aggregation through PARI binding but we can not exclude the participation of another receptor. pEng-induced platelet activation., secretion., and calcium mobilization via PARI and additional pathways

[0347] To investigate further, the activation of integrin allbp3, a key receptor involved in platelet aggregation, was evaluated by flow cytometry. Herein, washed human platelets (3 x 108 platelets / mL) were stimulated with a range of pEng concentrations (1, 5, 10, 25, and 50 pM). Platelet stimulation was then halted, and we checked pEng’s ability to induce integrin allbp3 activation and P-selectin exposure using flow cytometry and FITC-PAC1 mAb directed against the activated epitope of allbp3, plus anti -P-selectin mAb (CD62-P) for monitoring dense granule secretion. The results showed that pEng induced integrin allbp3 activation at 25 pM (29.5 ± 4.48% in platelet percentage and 4386 ± 562.3 A.U. in MFI) and at 50 pM (51.57 ± 4.52% in platelet percentage and 13290 ± 1123.9 A.U. in MFI) (Figure 3 a,b), indicating platelet activation. Additionally, pEng induced P-selectin exposure at 25 pM (73.7 ± 0.46% in platelet percentage and 24657 ± 508.1 A.U. in MFI) and at 50 pM (81.3 ± 0.58% in platelet percentage and 49325 ± 1655.1 A.U. in MFI) (Figure 3c, d), indicating platelet secretion.

[0348] Following this, SCH-79797 (3 pM; a PARI inhibitor) or DMSO as a control was incubated for 10 minutes before stimulation with pEng. The activation signal induced by 50 pM pEng was partially inhibited by the PARI inhibitor SCH-79797 (from 51.6 ± 4.51% to 24.7 ± 1.27% in platelet percentage, p = 0.006 ; and from 13290 ± 1124 A.U. to 3396 ± 272 A.U. in MFI, p = 0.0028 ; Figure 3e,f), reflecting allbp3 inactivation, suggesting that pEng acts through the PARI pathway. Additionally, P-selectin expression was partially inhibited by the PARI inhibitor SCH-79797 (from 81.3 ± 0.58% to 42.1 ± 0.49% in platelet percentage, p < 0.0001 ; and from 49324.7 ± 1655 A.U. to 25435 ± 794 A.U. in MFI, p = 0.0003 ; Figure 3g, h), reflecting granule secretion inactivation, further suggesting pEng’s action through the PARI pathway. However, the partial reduction rather than complete inhibition with the addition of the inhibitor suggests the involvement of another pathway besides PARI . Integrin activation and subsequent PAR-dependent platelet aggregation require the involvement of signaling pathways, such as calcium signaling (Varga-Szabo D, et al J Thromb Haemost. 2009.) The rapid and transient Ca2+ mobilization observed with TRAP6 was totally abolished with the addition of the PARI inhibitor (Figure 3j). While the PARI inhibitor SCH-79797 effectively inhibited the peak calcium mobilization induced by pEng, reducing Ca2+ mobilization but suggesting that pEng might also act on another receptor involved in calcium mobilization (Figure 3k).

[0349] Overall, these findings suggest that pEng induces platelet activation, secretion, and calcium mobilization through multiple pathways, including PARI . pEng is not affecting endothelial behaviours nor angogenic properties.

[0350] To test whether pEng and pSCR can affect negatively or positively angiogenic activity, we examined whether it stimulates endothelial cell sprouting in vitro. To this end, ECFC cells were treated with different concentrations of peptides (0, 25, 50 and 100 pM) and sprouting was measured with the microcarrier bead-based sprouting assay with the MSCs used as feeders (Nehls V, 1995). Sprouting began around day 2 and continued until day 5, at which point the culture was fixed. ECFCs were stained with Alexa Fluor-488-conjugated phalloidin to visualize the cytoskeleton and TO-PRO-3 to stain the nuclei (data not shown). In the presence of pEng, no significant difference was found in the average sprout width (pm) (p = 0.1917), in the cumulative tube length (pm) (p = 0.1917) nor in the number of sprouts per bead as compared with the control (p = 0.4478 ; data not shown). ECFCs treated with pEng sprout identically to not treated cells (data not shown) as well as pSCR treatment that showed no significant difference in sprouting (Figure 4a). This result suggested that ECFCs treatment with pEng does not affect negatively their ability to participate in vessel formation, likely through increased sprouting and endothelial sprout extension.

[0351] As well, to study the direct effect of pEng and pSCR on angiogenesis, we evaluated the ability of MSCs to express pericyte markers such as calponin by immunofluorescence after 7 days of in vitro cocultures with ECFCs (Rossi et al, CMLS 2016) after treatment with 1, 5, 10, 25 or 50 pM of peptides (data not shown). The fluorescence intensity of MSCs differentiated into pericytes following coculture with ECFCs and treatment with either pEng or pSCR, along with the number of nuclei relative to the surface area, were quantified. No significant differences were observed in the presence of either pSCR or pEng compared to the control (data not shown). pEng does not influence tube formation nor MSC differentiation in mural cells in coculture, even at high concentrations. This indicates that pEng does not have a significant impact on the ability of MSCs to differentiate into pericytes or on the formation of vascular structures in coculture with ECFCs, even at elevated concentrations. pEng promotes the directional upstream migration of endothelial cells in response to flow and Wound Healing

[0352] During vascular development, several studies have shown that endothelial cells polarize and migrate based on flow direction and magnitude, typically against the flow. However, the significance of this behavior in vascular patterning has not been fully explored (Udan et al., 2013; Jakobsson et al., 2010). Additionally, it has been demonstrated that ENG controls flow- directed migration in a dose-dependent manner and is essential for proper vascular morphogenesis (Jin et , 2017). To investigate this, fully confluent endothelial cells transfected with siRNA-SCR as a control and siRNA-Eng to target CD 105 were subjected to laminar flow at 7.5 dyne / cm2and imaged by time-lapse microscopy (data not shown). The efficiency of Eng suppression was assessed using flow cytometry, with mean fluorescence intensity (MFI) measured at 24, 38, 48, and 72 hours (data not shown). The analysis revealed a 68% reduction in CD105 expression at 24 hours in HUVEC cells and a 58% reduction in CD105 expression at 24 hours in ECFC cells (data not shown). The highest suppression levels were observed at 38 hours, reaching 83% in HUVECs and 84% in ECFCs (data not shown). However, CD105 suppression gradually decreased over time, declining to 77% in HUVECs and 71% in ECFCs at 48 hours (data not shown) and 73% in HUVECs and 65% in ECFCs at 72 hours (data not shown). Therefore, all subsequent experiments were conducted at the 38-hour mark, the point of maximal transfection efficiency.

[0353] Tracking of cell migration showed that while most control cells moved against the flow, the majority of siRNA-Eng cells migrated with the flow (data not shown). The relative positions of the cells after 5 hours of flow are represented in the circular graph, with each dot representing an individual cell starting at (0,0) (data not shownjv To investigate whether pEng affects flow-induced polarization in siRNA-SCR and siRNA-Eng cells, it was added at 25 and 50 pM, and cell tracking was performed. The displacement of siRNA-SCR and siRNA-Eng ECFCs towards the direction of flow was analyzed, with n=50 cells pooled from 3 independent experiments. While all siRNA-SCR cells polarized against the flow direction regardless of pEng treatment (data not shown), a significant difference was found in siRNA-Eng cells after pEng treatment with 25 and 50 pM, where the fraction of cells showing altered polarization decreased significantly (p < 0.0001 ; data not shown). However, no significant difference was observed between siRNA-SCR and siRNA-Eng cells treated with pEng at 25 pM (p = 0.0688) and at 50 pM (p = 0.0874), suggesting that pEng at 25 and 50 pM restored the control condition of migration against the flow direction.

[0354] We also conducted a cell size analysis using the Maastricht Chamber. Phase images of siRNA-SCR and siRNA-Eng ECFCs under laminar flow (7.5 dyne / cm2) were acquired every minute for 10 minutes, with or without treatment with 50 pM of pEng and pSCR. Individual cell sizes were measured for n=30 cells pooled from 3 independent experiments. No significant differences in cell size were found between the different conditions tested (p = 0.3806 for siRNA-SCR + pEng, p = 0.7204 for siRNA-SCR + pSCR, p = 0.5630 for siRNA-Eng + pEng, and p = 0.4336 for siRNA-Eng + pSCR) (data not shown).

[0355] We also assessed the effect of pEng and pSCR on the migration of siRNA-SCR and siRNA-Eng cells in the case of a lesion using the cell scratch assay with videomicroscopy (data not shown). Cells were cultured to confluence, and a scratch was made in each culture. The cells were then cultured further in the presence of pEng and pSCR, and the scratched areas were quantified every 2 hours.

[0356] The results showed that Eng knockdown led to decreased migration of ECFCs and slower wound closure compared to siRNA-SCR cells (96.84 ± 0.78 vs 26.2 ± 1.27 relative wound area / 48h ; data not shown. However, treatment with pEng showed no significant difference in migration of siRNA-SCR ECFCs, even at 25pM (93.97 ± 2.48 vs 87.31 ± 7.11 relative wound area / 48h) (data not shown) and siRNA-Eng ECFCs (26.2 ± 1.27 vs 26.31 ± 8.85 relative wound area / 48h) (data not shown). As well, treatment with pSCR showed no significant difference in migration of siRNA-SCR ECFCs, even at 25pM (96.84 ± 0.78 vs 89.76 ± 7.82 relative wound area / 48h) and siRNA-Eng ECFCs (33.08 ± 1.14 vs 39.39 ± 3.23 relative wound area / 48h) (data not shown). These results indicated that pEng and pSCR does not affect negatively cell migration and wound healing in both siRNA-SCR and siRNA-Eng cells.

[0357] To determine if the pEng had effect on cell viability / proiferation, the phosphatase activity of cells was assayed using the substrate pNPP in a time-dependent manner (data not shown). Reactions with different doses of pEng (0, 1, 5, 10, 25 and 50 pM) were carried out and the absorbance was measured at 405 nm. The results indicated no significant difference in ECFC viability with respect to both the concentration of pEng and the duration of exposure. At 24 hours, cells treated with various concentrations of pEng (1, 5, 10, 25, and 50 pM) showed no significant difference in viability compared to the control, even at the highest concentration of 50pM (0.33 ± 0.01 vs 0.27 ± 0.03 ; p = 0.9714). Similarly, at 48 and 72 hours, no dosedependent or time-dependent differences in cell viability were observed (0.69 ± 0.2 vs 0.52 ± 0.05 ; p = 0.1049 and 0.79 ± 0.48 vs 0.87 ± 0.52 ; p = 0.3282 respectively). These findings suggest that pEng, within the tested concentration range, does not significantly impact ECFC viability over the course of 24, 48, and 72 hours.

[0358] The therapeutic potential of pEng in reducing bleeding time and blood loss across genotypes

[0359] To validate the absence of differences in bleeding time due to pSCR treatment, Eng(+ / +) mice were subjected to the tail-clip bleeding model, where the distal tip of the tail (3 mm) is excised, followed by tail immersion in saline (PBS) with or without 50 pM of pSCR (Figure 4)v Mice were monitored for 10 minutes post-injury, and total bleeding was quantified as the cumulative bleeding time over this interval. No significant difference was observed in first bleeding time between mice treated with PBS (168 ± 101s) and those treated with pSCR (190 ± 78s ; p = 0.07 ;Figure 4a). Similarly, total bleeding time was 242 ± 78s for PBS-treated mice and 262 ± 156s for pSCR-treated mice, showing no significant difference (p = 0.0502; Figure 4b)

[0360] Clinical symptoms similar to those of HHT patients can occur in up to 72% of Eng(+ / -) mice, highly dependent on the presence or absence of modifier genes in the background strain. To address these challenges and determine the role of Eng in late developmental and adult life, a floxed Eng mouse for conditional knockout studies was generated (Allinson et al., 2007).

[0361] To gain insight into the bleeding phenotype, first and total bleeding times were assessed in Eng(+ / +), Eng(+ / -), and Eng iKO mice in the presence of pSCR. First bleeding time was minimal across all phenotypes, with no significant difference observed, not exceeding 204 ± 115s in most mice (p = 0.7967 for Eng(+ / +) vs Eng(+ / -) mice ; p = 0.5185 for Eng(+ / +) vs Eng iKO mice ; p = 0.3327 for Eng(+ / -) vs Eng iKO mice ; Figure 4c). In contrast, total bleeding time was significantly more pronounced in Eng iKO mice (497 ± 390s ; p = 0.0024) than in Eng(+ / +) mice (262 ± 156s) (Figure 4d) while no significant difference was found with Eng(+ / -) mice (370 ± 272s ; p = 0.1226) and between Eng(+ / +) andEng(+ / -) mice (p = 0.7407).

[0362] Total bleeding was assessed and quantified in Figure 5a Eng iKO, b Eng(+ / -) and c Eng(+ / +) mice by-tail-tip clip, followed by tail immersion in saline with or without 50 pM of pSCR or pEng. Haemoglobin loss was quantified as the cumulative bleeding times over a 10 minute interval, in d Eng iKO, e Eng(+ / -) and f Eng(+ / +) mice. Both bleeding time and blood loss (hemoglobin loss) were significantly reduced in Eng iKO mice treated with pEng compared to those treated with pSCR (from 536 ± 384s to 257 ± 154s; p = 0.013 for total bleeding time, Figure 5a; and from 7.89 ± 5.56mg / mL to 3.56 ± 5.46mg / mL; p = 0.0197 for blood loss), as well as in Eng(+ / +) mice (from 262 ± 156s to 160 ± 76s; p = 0.0048 for total bleeding time, Figure 5c; and from 3.33 ± 2.59mg / mL to 1.63 ± 2.13mg / mL; p < le-4 for blood loss ; Figure 5f). Blood loss was also significantly reduced in Eng(+ / -) mice (from 10.04 ± 6.85mg / mL to 2.95 ± 2.92mg / mL; p = 0.003 ; Figure 5e), while total bleeding time showed no significant difference (from 370 ± 270s to 192 ± 89mg / mL; p = 0.1442 ; Figure 5b). Similarly, age did not show significant effects on the first bleeding (p = 0.9002), total bleeding (p = 0.9201), and hemoglobin levels (p = 0.5380), respectively (data not shown).

[0363] These results suggest that pSCR does not significantly affect bleeding time in Eng(+ / +) mice, validating its use as a control peptide. The observed differences in bleeding phenotypes among Eng(+ / +), Eng(+ / -), and Eng iKO mice highlight the critical role of Endoglin in hemostasis and validate the phenotype iKO as the most critical. Importantly, pEng significantly reduced both bleeding time and blood loss (quantified by Hb loss) across all genotypes, underscoring its potential therapeutic value in managing bleeding disorders and suggesting that pEng can effectively enhance hemostasis even in the absence of full Endoglin functionality. pEng induces strong platelet aggregation in murine models

[0364] To assess the impact of pEng on murine platelets, we investigated whether pEng and pSCR could target murine platelet aggregation in vitro. Washed platelets (3 * 108 platelets / mL) from Eng iKO, Eng(+ / -), and Eng(+ / +) mice were preincubated with buffer, then activated by PAR4-ap (100 pM) or various concentrations of pEng and pSCR (5, 10, 25, and 50 pM). Aggregation was monitored using a microplate-based technique. Compared to buffer-treated platelets, pEng induced robust dose-dependent platelet aggregation, reaching 88% in all mouse genotypes, especially at 25 pM after 10 minutes of incubation p = 0.0079 for Eng iKO mice (data not shown); p < 0.0001 for Eng(+ / -) (data not shown); p = 0.0286 for Eng(+ / +) mice (data not shown); Conversely, minimal, non-significant aggregation was observed in the presence of pSCR, with a maximum aggregation of 43% obtained with 50 pM in all mouse genotypes p = 0.0952 for Eng iKO mice (data not shown); p = 0.1333 for Eng(+ / -), p = 0.1 for Eng(+ / +) mice (data not shown). These findings suggest that pEng is capable of inducing platelet aggregation via its binding to PARI and probably PAR4, while pSCR exhibits minimal aggregation effects in comparison. Forced degradation studies reveal pEng’s high sensitivity to alkaline and oxidative conditions

[0365] We have conducted forced degradation studies under a variety of conditions relevant for the pharmacological formulation of therapeutic peptides to assess their stability and potential degradation pathways. In this context, pEng was subjected to five distinct conditions (data not shown), and the extent of degradation and resulting products were subsequently analyzed using liquid chromatography-mass spectrometry (LC-MS). In the control condition, pEng was dissolved in double-distilled water and analyzed immediately at room temperature, showing no degradation with a peak area of 707.916 mAU2Under thermal stress (70 °C for 60 minutes), the peak area was reduced by 10.37%, indicating reasonable stability. In acidic stress (0.1 N HC1 at 70 °C for 60 minutes), there was no reduction in mass, suggesting high tolerance. In fact, there is a slight increase in total mass that could be attributed to an increased solubility in acidic pH with respect to water. These stressors are frequently encountered during manufacturing or shipping and for oral formulations of therapeutic peptides. Conversely, in alkaline stress (0.01 N NaOH at 25 °C for 60 minutes), the mass decreased by 86.75%, demonstrating extreme sensitivity, as mild alkaline conditions can transiently occur in oral formulations. Finally, under oxidative stress (0.3% H2O2 at 25 °C for 60 minutes), the mass reduced by 53.82%, showing high sensitivity. Based on these results, it can be concluded that pEng is very sensitive to both alkaline and oxidative stress conditions while it seems to be stable a high temperature and acid conditions.

[0366] Variant pVl and pV2 induces platelet aggregation in murine models

[0367] Our results suggest that pEng (SEQ ID NO: 16), pVl (SEQ ID NO:21), and pV2(SEQ ID NO:23) are capable of reducing bleeding in both WT (Figure 7A and 7E) and iKO-endoglin mice, a model of HHT. pEng and pV2 also appear to be well tolerated by cells and non-toxic (Figure 6). pV2 (50pM) shows a strong platelets aggregation as the positive control PAR4ap (150pM) but at lower concentration (Figure 7C). Platelet aggregation induced by the pVl variant was assessed in the blood of these mice at different concentrations, confirming its effectiveness at 50 pM (lower dose compared to PAR4ap used as the positive control) (Figure 7D). The fact that these peptides are effective in stopping tail-clip-induced bleeding not only in HHT models (iKO-eng and Eng+ / -) but also in healthy controls (WT) led us to consider that pEng and its variants could potentially be applied to other types of bleeding disorders. For this reason, we tested them in mouse models of von Willebrand disease and hemophilia. Preliminary results show partial efficacy in hemophilic mice (Figure 7E). EXAMPLE 3:

[0368] Material & Methods

[0369] The goal is to analyze the stability of peptide 1 derived from endoglin (sequence: FSFLLHFYTV, SEQ ID NO: 16) in human and mouse blood samples. As a secondary goal, we have also conducted forced degradation studies under a variety of conditions that are relevant for the pharmacological formulation of therapeutic peptides.

[0370] Blood stability studies

[0371] The peptide was solubilized in a mixture of 90% (v / v) ethanol and 10% (v / v) DMSO to achieve a stock concentration of 10 mg / mL, corresponding to 7.8514 mM based on a molecular weight of 1273.6615 Da. Subsequently, 4.5 pL of the peptide stock was combined with 1 mL of human or mouse blood, resulting in a final concentration of 0.0448 mg / mL (35.1 pmol / L). The mixture was incubated at 37 °C and 750 rpm using a Thermomixer (Eppendorf AG, Hamburg, Germany). Aliquots of 300 pL were withdrawn at predetermined time points (0, 10 and 30 min) and centrifuged at 2000 * g for 15 min at 4 °C. The supernatant (100 pL) was then transferred to a fresh high-quality plastic tube and flash frozen at -80 °C until further analysis.

[0372] A total volume of 1 pL of each plasma sample, equivalent to 60-80 pg of total protein (including 44.8 ng of peptide 1), was processed on the day of analysis. The samples were digested with trypsin under denaturing conditions. After digestion, salts were removed using Ziptip Cl 8 tips, and the digestion product was quantified using the Qubit method. Subsequently, an equivalent of 500 ng of the digest from each sample (with a theoretical maximum amount of peptide 1 of 320 pg) was monitored using targeted proteomics in Parallel Reaction Monitoring (PRM) format. The liquid chromatography system used was a Thermo Ultimate 3000 RSLC nano-LC, coupled to a Thermo Orbitrap Exploris OE240 mass spectrometer, configured for targeted scan data acquisition. The analysis focused on the m / z value of 637.334, corresponding to the +2 charge state of the peptide FSFLLHFYTV (SEQ ID NO: 16). Separation was performed on a reversed-phase Cl 8 column with dimensions of 15 cm in length and 75 pm in internal diameter, using a 60-minute elution gradient.

[0373] Forced degradation studies

[0374] In the context of the forced degradation studies, an identical concentration of peptide (35 pmol / L) was subjected to four distinct conditions, as outlined below. The extent of degradation and the resulting products were subsequently analyzed using liquid chromatography -mass spectrometry (LC-MS). The degradation conditions encompassed: 0.1 M HC1 (70 °C for 1 h), 0.01 M NaOH (25 °C for 1 h), 0.3% (v / v) H2O2 (25 °C for 1 h), and H20 (70 °C for 3 h). Samples underwent flash-freezing in liquid nitrogen and were kept at -80 °C until further analysis. Peptide isolation and quantification were performed by liquid chromatography-mass spectroscopy (LC-MS).

[0375] Results

[0376] The time points for the blood stability studies have been prepared and collected, and they have been analyzed by targeted proteomics in RPM format. The results show that peptide 1 can be detected and quantified against an internal reference of 5 pg, and that the peptide abundance diminishes with time until becoming approximately zero at time 30 min. Fitting the data to an exponential decay curve results in half-life times for peptide 1 of 8.1 ± 1.7 min in human blood and 6.3 ± 1.5 min for mouse blood. These values are in line with the half-lives determined for other unmodified linear peptides. Regarding the forced degradation studies, which have been already completed, they reveal that peptide 1 is reasonably stable under thermal stress or in presence of a strong acid. These stressors are frequently encountered during manufacturing or shipping and for oral formulations of therapeutic peptides. Conversely, peptide 1 appeared to be very unstable under strong basic (alkaline) conditions (with nearly a 90% reduction in peptide 1 mass after 1 h at room temperature); milder alkaline stress conditions can be encountered transiently for oral formulations of therapeutic peptides. Finally, peptide 1 was found to be very sensitive to oxidative stress conditions implemented as exposure to the strong oxidant water peroxide.

[0377] REFERENCES:

[0378] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A peptide derived from endoglin (pEng) comprising at least the amino acid sequence RFSFLL (SEQ ID NO: 15).

2. The peptide derived from endoglin (pEng) according to claims 1 comprising the amino acid sequence: FSFLLHFYTV (SEQ ID NO: 16).

3. The peptide derived from endoglin (pEng) according to claim 1 to 2, wherein the derived peptide from endoglin (pEng) comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.

4. A peptide derived from endoglin (pEng) comprising at least the amino acid sequence LVX3HX5LSTFF (SEQ ID NO:57), wherein X3is F or Y and X5is F or Y.

5. The peptide derived from endoglin (pEng) according to claims 4, wherein the derived peptide from endoglin (pEng) comprises or consists of SEQ ID NO:21 or SEQ ID NO:23.

6. The peptide derived from endoglin (pEng) according to claims 4, wherein the derived peptide from endoglin (pEng) comprises at least 10, 11, 12, 13, 14, 15 or 16 amino acids.

7. A vector comprising the peptide derived from endoglin (pEng) according to claim 1 to 6.

8. The peptide derived from endoglin (pEng) according to claim 1 to 6 or the vector of claim 7 for use in therapy.

9. The peptide derived from endoglin (pEng) according to claim 1 to 6 or the vector of claim 7 for use in reducing or preventing bleeding in a subject in need thereof.

10. The peptide derived from endoglin (pEng) or the vector for use according to claim 9, wherein the subject suffers from bleeding disorders.

11. The peptide derived from endoglin (pEng) or the vector for use according to claim 10 wherein the bleeding disorder is selected from the group consisting of but not limited to: excessive bleeding, such as a congenital coagulation disorder, an acquired coagulation disorder, administration of an anticoagulant, or a trauma induced hemorrhagic condition. Bleeding disorders may include, but are not limited to hereditaryhemorrhagic telangiectasia (HHT), hemophilia A, hemophilia B, von Willebrand disease, idiopathic thrombocytopenia, a deficiency of one or more contact factors, such as Factor XI, Factor XII, prekallikrein, and high molecular weight kininogen (HMWK), a deficiency of one or more factors associated with clinically significant bleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II (hypoprothrombinemia), and von Willebrand factor (vWD Type 1, vWD Type 2 A, vWD Type 2B, vWD Type 2N, vWD Type 2M, vWD Type 3, and acquired vWD), a vitamin K deficiency, a disorder of fibrinogen, including afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia, an alpha2-antiplasmin deficiency, and excessive bleeding such as caused by liver disease, renal disease, thrombocytopenia, platelet dysfunction, hematomas, internal hemorrhage, hemarthroses, bleeding is associated with surgery, e.g. in a subject with a type of hemophilia, bleeding is associated with a medical procedure, e.g., a dental procedure, trauma, hypothermia, menstruation, pregnancy, Bernard- Soulier syndrome, Glanzmann’s thrombasthenia, and platelet storage pool deficiencies.

12. The peptide derived from endoglin (pEng) or the vector for use according to claim 11, wherein the bleeding disorders is hereditary hemorrhagic telangiectasia (HHT).

13. The peptide derived from endoglin (pEng) or the vector according to claims 8 to 121 wherein the peptide derived from endoglin (pEng) is used in combination with classical treatment of bleeding disorders.

14. The peptide derived from endoglin (pEng) for use according to claims 8 to 13 wherein the peptide derived from endoglin (pEng) is administered intranasally or topically.

15. A pharmaceutical composition comprising the peptide derived from endoglin (pEng) according to claims 1 to 6 for use in reducing or preventing bleeding in a subject in need thereof.

16. A method of treating bleeding disorders in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the peptide derived from endoglin (pEng) according to claims 1 to 6 or the vector of claim 7.

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