Peptide inhibitors of fibrinolysis, methods, and uses thereof

Abstract

Provided herein are synthetic peptide inhibitors of fibrinolysis, pharmaceutical compositions comprising at least one of the presently disclosed peptides, or a pharmaceutically acceptable salt thereof, and uses thereof.

Description

PEPTIDE INHIBITORS OF FIBRINOLYSIS, METHODS, AND USES THEREOFBACKGROUND

[0001] Antifibrinolytics are a class of drugs that help blood clot by preventing the breakdown of fibrin, a protein that is the main component of blood clots. Many patients depend on antifibrinolytics to treat any of a number of wide-ranging conditions, including acute conditions (such as heavy menstrual bleeding and bleeding from traumatic injury) to long-term bleeding (such as bleeding associated with conditions such as inherited bleeding disorders).

[0002] Plasmin is a primary effector of the fibrinolytic pathway and a clinically validated target for such bleeding disorders. Tranexamic acid (TXA) is an antifibrinolytic that prevents plasmin generation and is clinically approved for some bleeding disorders. However, TXA has a short half-life and is unsuitable for long-term prophylaxis.

[0003] Thus, there remains a need for improved antifibrinolytic therapeutics.SUMMARY

[0004] Provided herein are peptides for inhibition of fibrinolysis and their methods of use.

[0005] Disclosed herein, in certain aspects, are peptides comprising an amino acid sequence GRCYKSKPPICX1X2D (SEQ ID NO: 1), wherein Xi and X2 are any amino acid or amino acid analogue, provided that if Xi is phenylalanine, then X2 is not proline. In some aspects, the peptide’s amino acid sequence consists of the amino acid sequence of SEQ ID NO: 1, wherein Xi and X2 are any amino acid or amino acid analogue, provided that if Xi is phenylalanine, then X2 is not proline. In some aspects, Xi is tyrosine and X2 is 4-amino proline (4NHPgam). In some aspects, Xi is selected from the group consisting of: phenylalanine, tyrosine, tryptophan, and lysine. In some aspects, X2 is selected from the group consisting of: 4-amino proline (4NHPgam), a-Aminoisobutyric acid (Aib), or lysine. In some aspects, the peptides comprises an amino acid sequence selected from the group consisting of: (a) GRCYKSKPPICYXD, wherein X is 4NHPgam (SEQ ID NO: 2); (b) GRCYKSKPPICYXD, wherein X is a-Aminoisobutyric acid (Aib)(SEQ ID NO: 9); (c) GRCYKSKPPICXPD, wherein X is lysine (SEQ ID NO: 10); (d) GRCYKSKPPICX1X2D, wherein Xi is lysine and X2 is a- Aminoisobutyric acid (Aib) (SEQ ID NO: 11); (e) GRCYKSKPPICFXD, wherein X is lysine (SEQ ID NO: 12); (f) GRCYKSKPPICYXD, wherein X is lysine (SEQ ID NO: 13); and (g) GRCYKSKPPICX1X2D, wherein Xi is lysine and X2 is Pro, Leu, Thr, He, Gly, Vai, or Nle (SEQ ID NO: 14). In some aspects, the peptides consist of an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOS: 2, 9, 10, 11, 12, 13, and 14. In some aspects, the peptides consist of the amino acid sequence of SEQ ID NO: 2. In some aspects, the peptides comprise a protractor group. In some aspects, if the protractor group is attached to a lysine, the lysine is Keps. In some aspects, the protractor group is conjugated to the amino acid residue at position 7, 8, 12, or 13 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 13 of SEQ ID NO: 1. In some aspects, the protractor group comprises a fatty acid comprising between 13 and 21 carbons. In some aspects, the protractor group comprises a saturated diacid. In some aspects, the saturated diacid is selected from the group consisting of: tetradecanedioic acid (C14 diacid), hexadecanedioic acid (Cl 6 diacid), octadecanedioic acid (Cl 8 diacid) and icosanedioic acid (C20 diacid). In some aspects, the saturated diacid is hexadecanedioic acid (Cl 6 diacid) or octadecanedioic acid (Cl 8 diacid). In some aspects, the saturated diacid is octadecanedioic acid (Cl 8 diacid). In some aspects, the protractor group comprises a linker, and the fatty acid is attached to an amino acid residue of the peptide via the linker. In some aspects, the linker comprises [8-amino-3,6-dioxaoctanoic acid (OEG)]m, wherein m is an integer greater than or equal to 1. In some aspects, m is between 2 and 12. In some aspects, m is 2. In some aspects, m is 6. In some aspects, the linker comprises a peptide linker. In some aspects, the peptide linker comprises [gGlu]n, wherein n is an integer greater than or equal to 1. In some aspects, n is 2. In some aspects, n is 6. In some aspects, the peptide has an amino acid sequence according to SEQ ID NO: 2, wherein: the saturated diacid is octadecanedioic acid, and the linker comprises [gGlu]n[OEG]m, wherein m is 2 or 6, and n is 2. In some aspects, n is 2. In some aspects, n is 6. In some aspects, the linker comprises the amino acid sequence A(EAAAK)kA, where k is an integer greater than or equal to 1. In some aspects, k is 3. In some aspects, the peptides comprise a disulfide linkage between the amino acid residues at positions 3 and 11 ofSEQ ID NO: 1. In some aspects, the peptides are cyclized in that the amino acid residue at position 1 is linked to the amino acid residue at position 14 of SEQ ID NO: 1.

[0006] Disclosed herein, in certain aspects, are peptides comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein m and n are independently integers greater than or equal to 1. In some aspects, the saturated diacid is octadecanedioic acid. In some aspects, m is 2 and n is 2. In some aspects, m is 6 and n is 2. In some aspects, m is 12 and n is 2. In some aspects, m is 6 and n is 1. In some aspects, the saturated diacid is hexadecanedioic acid. In some aspects, m is 2 and n is 2. In some aspects, m is 6 and n is 2. In some aspects, m is 12 and n is 2. In some aspects, the saturated diacid is tetradecanedioic acid. In some aspects, m is 6 and n is 2. In some aspects, m is 2 and n is 2. In some aspects, the peptides comprise a disulfide linkage between the amino acid residues at positions 3 and 11 of SEQ ID NO: 1. In some aspects, the peptides are cyclized in that the amino acid residue at position 1 is linked to the amino acid residue at position 14 of SEQ ID NO: 1.

[0007] Disclosed herein, in certain aspects, are peptides comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a peptide linker comprising the amino acid sequence A(EAAAK)k A, where k is an integer greater than or equal to 1. In some aspects, the peptides comprise a disulfide linkage between the amino acid residues at positions 3 and 11 of SEQ ID NO: 1. In some aspects, the peptides are cyclized in that the amino acid residue at position 1 is linked to the amino acid residue at position 14 of SEQ ID NO: 1.

[0008] Disclosed herein, in certain aspects, are peptides comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2) and an acetyl group linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein m and n are independently integers greater than or equal to 1. In some aspects, m is 6 and n is 2. In some aspects, m is 2 and n is 2. In some aspects, the peptides comprise a disulfide linkage between the amino acid residues at positions 3 and 11 of SEQ ID NO: 1. In some aspects, the peptides are cyclized in that the amino acid residue at position 1 is linked to the amino acid residue at position 14 of SEQ ID NO: 1.

[0009] In some aspects, the peptide of the disclosure comprises a linker length greater than or equal to about 5 A. In some aspects, the peptide of the disclosure comprises a linker length of about 5 A to about 50 A. In some aspects, the peptide of the disclosure comprises a linker length of about 5 A, about 6 A, about 7 A, about 8 A, about 9 A, about 10 A, about 11 A, about 12 A, about 13 A, about 14 A, about 15 A, about 16 A, about 17A, about 18 A, about 19 A, about 20 A, about 21 A, about 22 A, about 23 A, about 24 A, about 25 A, about 26 A, about 27 A, about 28 A, about 29 A, about 30 A, about 31 A, about 32 A, about 33 A, about 34 A, about 35 A, about 36 A, about 37 A, about 38 A, about 39 A, about 40 A, about 41 A, about 42 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 49 A, or about 50 A.

[0010] In some aspects of the disclosure, the peptide inhibits clot lysis with IC50 values from about 1 to 5 pM. In some aspects, the peptide inhibits clot lysis with IC50 values from about 1 to 2 pM.

[0011] In some aspects, the peptide comprises an amino acid sequence GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [0EG]m and [gGlu]n, wherein the saturated diacid is octadecanedioic acid, m is 6, and n is 2. In some aspects, the peptide comprises an amino acid sequence GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [0EG]m and [gGlu]n, wherein the saturated diacid is octadecanedioic acid, m is 2, and n is 2. In some aspects, the peptide comprises an amino acid sequence GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [0EG]m and [gGlu]n, wherein the saturated diacid is hexadecanedioic acid, m is 6, and n is 2.

[0012] In some aspects of the disclosure, the peptide comprises the structure set forth in Figure 5A. In some aspects, the peptide comprises the structure set forth in Figure 5B.

[0013] Disclosed herein, in certain aspects, are pharmaceutically acceptable salts of a peptide provided here.

[0014] Disclosed herein, in certain aspects, are pharmaceutical compositions comprising (a) a peptide of provided herein or a pharmaceutically acceptable salt thereof, and (b) pharmaceutically acceptable carrier.

[0015] Disclosed herein, in certain aspects, are methods of promoting blood clot formation in a subject in need thereof comprising administering to the subject an effective amount of a peptide provided herein or a pharmaceutical composition provided herein. In some aspects, the subject is human. In some aspects, the subject has a blood clotting disorder. In some aspects, the subject has hemophilia. In some aspects, the subject has a disorder of hyperfibrinolysis. In some aspects, the subject has hereditary hemorrhagic telangiectasia. In some aspects, the subject is suffering from heavy menstrual bleeding. In some aspects, the subject is bleeding from a traumatic injury or from a surgical wound. In some aspects, the methods comprise administering the effective amount of the pharmaceutical composition to the subject at regular intervals. In some aspects, the regular intervals are longer than a week. In some aspects, the regular intervals are longer than two weeks. In some aspects, the regular intervals are longer than three weeks. In some aspects, the regular intervals are at least a month. In some aspects, the step of administering comprises systemic administration. In some aspects, the systemic administration comprises subcutaneous administration.BRIEF DESCRIPTION OF THE DRAWING

[0016] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings, where:

[0017] FIG. 1 is a schematic depiction of the primary structure of SFTIv3, a peptide inhibitor of plasmin, which has the sequence of SEQ ID NO: 3.

[0018] FIGs. 2A and 2B show plots depicting batch-corrected plasmin inhibition values (pKi) for peptides having amino acid substitutions at each position within SFTIv3. The dotted line represents the value for SFTIv3.

[0019] FIGs. 3A and 3B show plots depicting clot lysis time coefficients in human plasma for peptides having amino acid substitutions at each position within SFTIv3. The dotted line represents the value for SFTIv3.

[0020] FIGs. 4A and 4B include SHAP plots showing the effect on clot lysis of lipidation handles, lipidation linkers and lipidations across the library of peptide variants explored. FIG. 4A shows the effect on clot lysis according to lipidation handle and position as indicated. FIG. 4B shows the effect on clot lysis according to lipidation linkerand lipidation. Delta SHAP is the difference in average SHAP value between the modified sequence and SFTIv3.

[0021] FIGs. 5A and 5B include structures of exemplary modified peptides.

[0022] FIG. 6 includes plots showing plasma concentrations (mean ± SD) in mouse after administration of the indicated peptides at 50 nmol / kg by intravenous (IV) or subcutaneous (SC) injection.

[0023] FIGs. 7A and 7B are graphs showing the PK profiles of SFTIv3, Compound 105, and compound 102 after intravenous (IV) or subcutaneous (SC) administration (50 nmol / kg) in mouse. FIG. 7A shows the PK profiles after IV injection, and FIG. 7B shows the PK profiles after SC injection. Data are shown as mean ± SD (n=l-3).

[0024] FIG. 8 is a graph showing the plasma concentration of SFTIv3 and Compound 102 at 5 min, 6 hrs and 48 hrs post dosing in mouse as quantified by LC-MS / MS. Data are shown as mean ± SD (n=2).

[0025] FIG. 9 is a graph showing the plasma concentration of Compound 102 after IV (1075 nmol / kg) or SC administration (25nmol / kg) in minipigs. Data are shown as mean ± SD (n=2).

[0026] FIG. 10 is a graph showing the residual fibrinolytic activity (%) in minipig plasma samples after IV administration (1075 nmol / kg) of Compound 102.

[0027] FIG. 11 is a graph showing the fibrin accumulation at site of injury measured as fibrin fluorescence (AU) in mice after receiving 5 mg / kg tPA 5 min before injury, either alone or with Compound 102 (1, 2, 4, 6, or 8 mg / kg) administered 2 min before injury. Results are compared to control animals not receiving tPA or compound.

[0028] FIGs 12A and 12B are graphs showing the inhibition of active plasmin generation by compound 102 in human platelet-rich plasma (PRP) using a plasmin generation assay. FIG. 12A demonstrates representative curves of active plasmin generation in presence of indicated concentrations of Compound 102. FIG. 12B shows peak active plasmin generation (nM) as a function of Compound 102 concentration. Results are shown as mean ± SD based on two independent experiments conducted with plasma samples from two separate donors.

[0029] FIGs. 13A and 13B are graphs showing the degree of fibrinolysis (%) at different concentrations of Compound 102 (FIG. 13A) or TXA (FIG. 13B) in human whole blood perfused over a surface coated with collagen and tissue factor (TF) in a microfluidic device.

[0030] FIG. 14 shows an overlay of representative SEC-HPLC chromatograms of AF647-labeled Compound 102 with increasing concentrations of HSA.

[0031] FIG. 15 shows a structural model of a ternary complex of Compound 102 with full-length plasmin and human serum albumin (HSA). Plasmin is shown in shades of grey, with domain boundaries indicated by variations in tone and the lightest grey representing the protease domain. HSA is displayed as a uniformly colored dark-grey structure. Compound 102 is docked into three fatty-acid binding sites of HSA, and one representative configuration illustrating the plasmin:Compound 102 :HS A ternary complex is shown with the linker indicated between the two dotted grey lines.

[0032] FIGs. 16A-16C are graphs showing the thrombin generation initiated by different triggers in human platelet-rich plasma (PRP) in presence or absence of 8 pM compound 102 or appropriate controls. FIG. 16A shows the thrombin generation of in PRP human donor plasma supplemented with 1 pM tissue factor (TF). FIG. 16B shows the thrombin generation of in PRP human donor plasma supplemented with 1 pM TF and 3 nM thrombomodulin (TM). FIG. 16C shows the thrombin generation of in PRP human donor plasma supplemented with 2 pg / ml kaolin.

[0033] FIGs. 17A-17C are representative platelet activation and aggregation curves triggered by different agonists in the presence or absence of 8 pM Compound 102 or the platelet antagonist RGDW. FIG. 17A shows the platelet activation and aggregation (%) after platelet activation with 10 pM adenosine diphosphate (ADP). FIG. 17B shows the platelet activation and aggregation (%) after platelet activaiton with 50 pM TRAP-6. FIG. 17C shows the platelet activation and aggregation (%) after platelet activation with 1 pg / ml CRP-XL.DETAILED DESCRIPTION

[0034] The present disclosure provides, in part, synthetic peptide inhibitors of fibrinolysis. These peptides of the present disclosure have several biological advantages including, without limitation, increased circulation half-life, increased activity, and improved chemical and physical stability, selectivity, and solubility.

[0035] Various components and aspects of the disclosure are described in further detail in the subsections below.Definitions

[0036] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0037] The articles “a” and “an” are used in this disclosure to refer to one or more than one (z.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element.

[0038] As used herein, the term “about” or “approximately” refers to a ±10% variation from the recited quantitative value (and includes the recited quantitative value itself) unless otherwise indicated or inferred from the context.

[0039] The term “acyl” denotes a group of the formula -C(=O)-R. Where R is a methyl group, the structure is referred to as “acetyl,” having the formula -C(=O)CH3.

[0040] As used herein, the term “analogue” or “analog” of a peptide refers to a modified peptide, wherein one or more amino acid residues of the peptide have been substituted by another amino acid residue and / or wherein one or more amino acid residues have been deleted from the peptide and / or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues are contemplated at any place within the peptide.

[0041] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “is,” “are” or any other variation thereof, are intended to cover a nonexclusive inclusion. They are to be interpreted synonymously with the phrases “having at least” or “including at least.” The term “consisting of’ refers to including, and being limited to, whatever follows the phrase “consisting of.” As used herein, the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise. Similarly, the term “consisting essentially of’ is intended to include embodiments encompassed by the term “consisting of.”

[0042] As used herein, the term “isolated” nucleotide, polynucleotide, peptide, or polypeptide is one which is substantially separated from other cellular components thatnaturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated. For instance, an isolated molecule is one that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polynucleotide or polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a nucleotide, polynucleotide, peptide, or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A nucleotide, polynucleotide, peptide, or polypeptide may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that any molecule so described has been physically removed from its native environment. In some embodiments, as used in reference to an isolated construct, isolated means in the absence of a pharmaceutically acceptable salt.

[0043] As used herein, the term “Ki” (M) refers to the inhibition constant of a given entity and a target (e.g., a particular peptide-protease interaction). As used herein, “pKi” refers to -log(Ki).

[0044] As used herein, the term “kd” (s'1) refers to the dissociation rate constant between a given entity and a target (e.g., of a particular peptide-target interaction). This value is also referred to as the koff value.

[0045] As used herein, the term “ka” (M'1x s'1) refers to the association rate constant of a given entity and a target (e.g., a particular peptide-target interaction). This value is also referred to as the konvalue.

[0046] As used herein, the term “KD” (M) refers to the equilibrium dissociation constant of a given entity and a target (the particular interaction between an entity and its target (e.g., a polypeptide-target interaction)). KD = kd / ka.

[0047] As used herein, the term “KA” (M'1) refers to the equilibrium association constant of a given entity and a target (e.g., a particular peptide-target interaction). KA = ka / kd.

[0048] The affinity of a molecule X for its target Y can be represented by the equilibrium dissociation constant (KD). The kinetic components that contribute to the equilibrium dissociation constant are as described above. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0049] As used herein, the term “protractor group” refers to a group which upon conjugation to a protein or peptide increase the circulation half-life of said protein or peptide, when compared to the unmodified peptide (e.g., SFT-v3). As used herein, the term “protractor groups” refers to a lipid group and linker.

[0050] As used herein, the term “linker” refers to a moiety that is used to conjugate a lipid to a peptide as disclosed herein.

[0051] As used herein, the term “lipidation” refers to the covalent attachment of a lipid group, or a modified lipid group, to an amino acid in a peptide disclosed herein through a linker. Without being limited to the mechanism of action, lipidation of peptides can be used to, for example, improve pharmacokinetic properties, improve metabolic stability, reduce enzymatic degradation, lower excretion and metabolism, and extend in vivo halflife.

[0052] The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0053] The term “polypeptide” refers to a chain of amino acids bonded together by peptide bonds. A polypeptide can be cyclic, one chain or may be composed of more than one chains, which are held together by covalent bonds, e.g., disulfide bonds and / or non- covalent bonds. In some aspects, the peptides provided herein are cyclic (e.g., head-to-tail backbone cyclized). Polypeptides can have a length, e.g., of at least three, at least four, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid residues. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100, less than about 50, less than about 20, or less than about 10 amino acid residues.

[0054] In some aspects, the peptides provided herein have disulfide bonds connecting their cysteine residues.

[0055] As used herein, the term “subject” refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, minipigs, pigs and sheep. In certain aspects, the subject is a human. In some aspects the subject has a disease, disorder, or condition that can be treated with an peptide as provided herein. In some aspects, the disease, disorder, or condition is heavy menstrual bleeding. In some aspects, the disease, disorder, or condition is a blood clotting disorder.

[0056] As used herein, the term “residue” refers to a position in a protein and its associated amino acid identity.

[0057] As used herein, the term “synthetic” is used to refer to an entity that is lab-created and not naturally produced or isolated without modification from a naturally occurring source. A recombinant polymer, such as a recombinant nucleotide, polynucleotide, peptide, or polypeptide, may be synthetic. Synthetic polymers such as polynucleotides, peptides or polypeptides may be produced by any method known to those of skill in the art, including but not limited to solid phase synthesis, solution phase synthesis, biological synthesis by, e.g., host cells, etc.

[0058] As used herein, the terms “treatment,” “treat,” and “treating” refer to partially or completely alleviating, inhibiting, delaying onset of, preventing, ameliorating and / or relieving a disorder or condition, or one or more symptoms of the disorder or condition, as described herein. In some aspects, treatment may be administered after one or more symptoms have developed. In some aspects, the term “treating” includes preventing or halting the progression of a disease or disorder. In other aspects, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. Thus, in some aspects, the term “treating” includes preventing relapse or recurrence of a disease or disorder.

[0059] As used herein, the terms “effective amount” or “therapeutically effective amount” are used interchangeably and refer to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response. In some aspects, a therapeutically effective amount of a substance is an amountthat is sufficient, when administered as part of a dosing regimen to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of a provided compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition.Peptides

[0060] As described further below, provided are peptides having plasmin inhibitory properties. In certain aspects, provided peptides are chemically modified, e.g., by conjugation to a protractor group (e.g., as described herein) that comprises a linker and lipid group as described herein. In some aspects, provided peptides have an extended half-life compared to that of a reference peptide. In some aspects, the reference peptide is SFTI1, SFTIv3, or another control peptide described in Table 5 below. In some aspects, when the provided peptide is chemically modified, the reference peptide is a similar peptide having the same amino acid sequence as the provided peptide, except that the reference peptide lacks the chemical modification.

[0061] Half-lives can be measured by a suitable method of the art, e.g., in serum, in plasma, or in vivo in a suitable animal model (e.g., mice, rabbits, minipig, dog, nonhuman primates). In some aspects, provided peptides are characterized by a half-life that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18- fold, at least 19-fold, at least 20-fold, at least 21 -fold, at least 22-fold, at least 23 -fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29- fold, or at least 30-fold greater than that of a reference peptide.

[0062] In certain aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting plasmin with a Ki value comparable to that of a reference peptide. For example, in some aspects, provided peptides are capable of inhibitingplasmin with a Ki of about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, or about 15 nM in the presence of bovine serum albumin (BSA) that is fatty acid free. In some aspects, provided peptides are capable of inhibiting plasmin with a Ki of about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM or about 35 nM in the presence of BSA that is fatty acid free. In some aspects, provided peptides are capable of inhibiting plasmin with a Ki of about 75 nM, about 70 nM, about 65 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, or about 20 nM in the presence of BSA that is fatty acid free. In some aspects, provided peptides are capable of inhibiting plasmin with a Ki of about 10-60 nM, about 10-50 nM, about 10-40 nM, about 10-30 nM, about 10-25 nM, about 10-20 nM, about 20-60 nM, about 20-50 nM, about 30-50 nM, about 40-50 nM, about 30-60 nM, about 40-60 nM, or about 50-60 nM in the presence of BSA that is fatty acid free. In certain aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting plasmin with a better (smaller) Ki value relative to that of a reference peptide. For example, in some aspects, provided peptides are capable of inhibiting plasmin with a Ki of less than about 75 nM, less than about 70 nM, less than about 65 nM, less than about 60 nM, less than about 55 nM, less than about 50 nM, less than about 45 nM, less than about 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, or less than about 20 nM in the presence of BSA that is fatty acid free. In some aspects, provided peptides are capable of inhibiting plasmin with a Ki of less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, or less than about 3 nM in the absence of BSA that is fatty acid free.

[0063] In some aspects, the ability of the provided peptides, including peptides with extended half-lives, to inhibit clot lysis is comparable to that of a reference peptide. For example, in some aspects, the provided peptides can inhibit clot lysis with IC50 values of less than about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting clot lysis with IC50 values of less than about 10 pM. In some aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting clot lysis with IC50 values of less than about 15 pM. In some aspects, provided peptides, including peptides withextended half-lives, are capable of inhibiting clot lysis with IC50 values of less than about 20 pM. In some aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting clot lysis with IC50 values in the range of about 1-20 pM, about 1-10 pM, about 1-5 pM, about 1-4 pM, about 1-3 pM, about 1-2 pM, about 3-7 pM, about 5-20 pM, about 5-15 pM, about 5-10 pM, about 10-20 pM, or about 15-20 pM.

[0064] In some aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting clot lysis with IC50 values of about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 8 pM, about 9 pM, or about 10 pM. In some aspects, provided peptides, including peptides with extended half-lives, are capable of inhibiting clot lysis with IC50 values of about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some aspects, the peptides, including peptides with extended half-lives, are capable of inhibiting clot lysis with IC50 values of about 1 pM or about 2 pM.

[0065] Methods of assessing plasmin inhibition are known in the art and include in vitro methods such as those described further in the Examples.

[0066] In some aspects, the present disclosure provides a peptide consisting of the sequence of any one of the peptides disclosed herein.Sunflower Trypsin Inhibitor

[0067] Sunflower trypsin inhibitor 1 (SFTI1) is a disulfide-bridged cyclic plasmin inhibitory peptide in the Bowman-Birk inhibitor (BBI) family. SFTI1 is a 14-amino acid backbone-cyclized peptide containing a single disulfide bond and is naturally found in the sunflower seeds (Helianthus annuus).

[0068] Disclosed herein, in certain aspects, are synthetic plasmin inhibitory peptides incorporating the SFTIv3 scaffold_that, among other things, improve and sustain the biological activity of plasmin inhibitory peptides. For example, presently disclosed peptides may exhibit improved half-life, activity, selectivity, stability, and / or solubility and which may be useful, for example, as antifibrinolytic agents.Amino acid sequences

[0069] In certain aspects, provided peptides have an amino acid sequence of GRCYKSKPPICX1X2D (SEQ ID NO: 1), wherein Xi and X2 are any amino acid or amino acid analogue, provided that if Xi is phenylalanine, then X2 is not proline. In some aspects, Xi is selected from the group consisting of phenylalanine, tyrosine, tryptophan,or lysine. In some aspects, X2 is selected from the group consisting of 4-amino proline (4NHPgam), a-Aminoisobutyric acid (Aib), and lysine. In some aspects, wherein a protractor group is conjugated to the lysine, the lysine is a Keps. In some aspects, Xi is tyrosine and X2 is 4NHPgam.

[0070] For example, in some aspects, provided are peptides having a sequence selected from the group consisting of GRCYKSKPPICYXD, wherein X is 4-amino proline (4NHPgam) (SEQ ID NO: 2); GRCYKSKPPICYXD, wherein X is a-Aminoisobutyric acid (Aib) (SEQ ID NO: 9); GRCYKSKPPICXPD, wherein X is lysine (SEQ ID NO: 10); GRCYKSKPPICX1X2D, wherein Xi is lysine and X2 is a-Aminoisobutyric acid (Aib) (SEQ ID NO: 11); GRCYKSKPPICFXD, wherein X is lysine (SEQ ID NO: 12); GRCYKSKPPICYXD, wherein X is lysine (SEQ ID NO: 13), and GRCYKSKPPICX1X2D, wherein Xi is lysine and X2 is Pro, Leu, Thr, He, Gly, Vai, Aib, or Nle (SEQ ID NO: 14). In some aspects, provided peptides have the amino acid sequence of SEQ ID NO: 2.

[0071] In some aspects, wherein a protractor group is conjugated to the lysine, the lysine is a Keps.

[0072] In some aspects, provided are peptides which have the amino acid sequence of a reference peptide (e.g., that of SEQ ID NO: 3) and comprise one or more chemical modifications (e.g., conjugation to a protractor group) as described herein. In some aspects, the protractor group is conjugated to the amino acid residue at position 2, 7, 8, 9, 10, 12, or 13 of SEQ ID NO: 3. In some aspects, the protractor group is conjugated to the amino acid residue at position 13 of SEQ ID NO: 3. In some aspects, the peptide is modified by introduction of an unnatural amino acid analog, to which a protractor group is conjugated. For example, 4NHPgam can be introduced in place of proline, thereby allowing attachment of a protractor group to the 4NHPgam residue. In some aspects, Keps is introduced at positions 2, 7, 10, 12, or 13 of SEQ ID NO: 3. In some aspects, 4NHPgam is introduced at positions 8, 9, or 13 of SEQ ID NO: 3.Protractor groups

[0073] In some aspects, a peptide of the present disclosure comprises a protractor group (also referred to herein as a “protractor”). As used herein, a protractor comprises a lipid group (e.g., fatty acid) and a linker moiety. The protractor group can be conjugated to an amino acid residue in a peptide of the present disclosure. For example, in some aspects,the protractor group is conjugated to the amino acid residue at position 8, 12, or 13 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 7 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 2 or 10 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 8 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 12 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 13 of SEQ ID NO: 1. In some aspects, the protractor group is conjugated to the amino acid residue at position 8, 12, or 13 of SEQ ID NO: 3. In some aspects, the protractor group is conjugated to the amino acid residue at position 7 of SEQ ID NO: 3. In some aspects, the protractor group is conjugated to the amino acid residue at position 2 or 10 of SEQ ID NO: 3. In some aspects, the protractor group is conjugated to the amino acid residue at position 8 of SEQ ID NO: 3. In some aspects, the protractor group is conjugated to the amino acid residue at position 12 of SEQ ID NO: 3. In some aspects, the protractor group is conjugated to the amino acid residue at position13 of SEQ ID NO: 3.A. Lipid groups

[0074] In some aspects, the lipid group in a protractor provided herein comprises a fatty diacid. In some aspects, the fatty acid comprises between 13 and 21 carbons. For example, in some aspects, the fatty diacid comprises 12 carbons (C 12). In some aspects, the fatty diacid comprises 13 carbons (Cl 3). In some aspects, the fatty diacid comprises14 carbons (C14). In some aspects, the fatty diacid comprises 15 carbons (Cl 5). In some aspects, the fatty diacid comprises 16 carbons (Cl 6). In some aspects, the fatty diacid comprises 17 carbons (Cl 7). In some aspects, the fatty diacid comprises 18 carbons(Cl 8). In some aspects, the fatty diacid comprises 19 carbons (Cl 9). In some aspects, the fatty diacid comprises 20 carbons (C20). In some aspects, the fatty diacid comprises 21 carbons (C21). In some aspects, the fatty diacid comprises 22 carbons (C22). Additional examples of fatty acid protraction are known to those of ordinary skill in the art.

[0075] A lipid group of the present disclosure can comprise a saturated diacid. As used herein, the term "diacid" refers to a hydrocarbon-based compound comprising two - COOH carboxyl functions. A diacid is referred to as “saturated” when the hydrocarbonbased chain from which it is constituted lacks an unsaturated group, e.g., a carbon-carbondouble bond. In some aspects, the lipid group can comprise a single diacid or a mixture of several different diacids.

[0076] In some aspects, the saturated diacid is selected from the group consisting of tetradecanedioic acid (Cl 4 diacid), hexadecanedioic acid (Cl 6 diacid), octadecanedioic acid (Cl 8 diacid), and eicosanodi oic acid (C20 diacid). In some aspects, the saturated diacid is hexadecanedioic acid (Cl 6 diacid) or octadecanedioic acid (Cl 8 diacid). In some aspects, the saturated diacid is octadecanedioic acid (Cl 8 diacid).

[0077] Non-limiting examples of saturated diacids include ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, decanedioic acid, tetradecanedioic acid (Cl 4 diacid), hexadecanedioic acid (Cl 6 diacid), heptadecanedioic acid (Cl 7 diacid), octadecanedioic acid (Cl 8 diacid), nonadecanedioic acid (Cl 9 diacid), eicosanodi oic acid (C20 diacid), heneicosanodioic acid (C21 diacid), docosanodi oic acid (C22 diacid), tetracosanedioic acid (C24 diacid), malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid (or 1,10-decanedioic acid), octadecamethylenedicarboxylic acid, eicosadicarboxylic acid, including branched or substituted derivatives thereof, or mixtures thereof.

[0078] Further non-limiting examples of diacids contemplated herein include phthalic anhydride, phthalic acid, isophthalic acid, terephthalic acid, tetrahydrophthalic acid, a halophthalic anhydride, adipic acid, their alkyl esters, including branched or substituted derivatives thereof, or mixtures thereof.B. Linkers

[0079] In some aspects, a lipid group in a protractor provided herein is attached to an amino acid residue of a provided peptide via a linker in the protractor. In some aspects, that linker comprises (OEG)m, wherein m is an integer greater than or equal to 1. In some aspects, m is between 2 and 12. In some aspects, m is between 1 and 12. In some aspects, m is between 6 and 12. In some aspects, m is 2. In some aspects, m is 6. In some aspects, m is 12.

[0080] As used herein, “OEG” is 8-amino-3,6-dioxaoctanoic acid and has the following structure:

[0081] In some aspects, the linker comprises the amino acid sequence A(EAAAK)k A, where k is an integer greater than or equal to 1. In some aspects, k is 3.

[0082] In some aspects, the linker comprises [OEG]mand [gGlu]n, wherein m and n are as in any of the aspects provided herein. In some aspects, the linker comprises [gGlu]n[OEG]m, wherein n and m are as in any of the aspects provided herein. In some aspects, m is 6 and n is 2. In some aspects, m is 5 and n is 2. In some aspects, m is 4 and n is 2. In some aspects, m is 3 and n is 2. In some aspects, m is 2 and n is 2. In some aspects, m is 1 and n is 2. In some aspects, m is 0 and n is 2. In some aspects, m is 0 and n is 1. In some aspects, the lipid group is conjugated to [gGlu]n. In some aspects, [OEG]n is conjugated to an amino acid residue of a provided peptide. In some aspects, the lipid group is conjugated to [gGlu]n, [gGlu]n is conjugated to [OEG]n, and [OEG]n is conjugated to an amino acid residue of a provided peptide.

[0083] In some aspects, the linker comprises eLysl,2. As used herein, “eLysl,2” refers to lysine, wherein the epsilon (s) amino group in the lysine side chain is covalently connected to a lipid.

[0084] In some aspects, the linker is a flexible linker.

[0085] In some aspects, the length of the linker is about 1 A. In some aspects, the length of the linker is about 2 A. In some aspects, the length of the linker is about 3 A. In some aspects, the length of the linker is about 4 A. In some aspects, the length of the linker is about 5 A. In some aspects, the length of the linker is about 6 A. In some aspects, the length of the linker is about 7 A. In some aspects, the length of the linker is about 8 A. In some aspects, the length of the linker is about 9 A. In some aspects, the length of the linker is about 10 A. In some aspects, the length of the linker is about 11 A. In some aspects, the length of the linker is about 12 A. In some aspects, the length of the linker is about 13 A. In some aspects, the length of the linker is about 14 A. In some aspects, the length of the linker is about 15 A. In some aspects, the length of the linker is about 16 A. In some aspects, the length of the linker is about 17 A. In some aspects, the length of the linker is about 18 A. In some aspects, the length of the linker is about 19 A. In some aspects, the length of the linker is about 20 A. In some aspects, the length of the linker isabout 21 A. In some aspects, the length of the linker is about 22 A. In some aspects, the length of the linker is about 23 A. In some aspects, the length of the linker is about 24 A. In some aspects, the length of the linker is about 25 A. In some aspects, the length of the linker is about 26 A. In some aspects, the length of the linker is about 27 A. In some aspects, the length of the linker is about 28 A. In some aspects, the length of the linker is about 29 A. In some aspects, the length of the linker is about 30 A. In some aspects, the length of the linker is about 31 A. In some aspects, the length of the linker is about 32 A. In some aspects, the length of the linker is about 33 A. In some aspects, the length of the linker is about 33 A. In some aspects, the length of the linker is about 34 A. In some aspects, the length of the linker is about 35 A. In some aspects, the length of the linker is about 36 A. In some aspects, the length of the linker is about 37 A. In some aspects, the length of the linker is about 38 A. In some aspects, the length of the linker is about 39 A. In some aspects, the length of the linker is about 40 A. In some aspects, the length of the linker is about 41 A. In some aspects, the length of the linker is about 42 A. In some aspects, the length of the linker is about 43 A. In some aspects, the length of the linker is about 44 A. In some aspects, the length of the linker is about 45 A. In some aspects, the length of the linker is about 46 A. In some aspects, the length of the linker is about 47 A. In some aspects, the length of the linker is about 48 A. In some aspects, the length of the linker is about 49 A. In some aspects, the length of the linker is about 50 A.

[0086] In some aspects, the length of the linker is from about 1 to about 50 A. In some aspects, the length of the linker is from about 2 to about 49 A. In some aspects, the length of the linker is from about 3 to about 48 A. In some aspects, the length of the linker is from about 4 to about 47 A. In some aspects, the length of the linker is from about 5 to about 46 A. In some aspects, the length of the linker is from about 6 to about 45 A. In some aspects, the length of the linker is from about 7 to about 44 A. In some aspects, the length of the linker is from about 8 to about 43 A. In some aspects, the length of the linker is from about 9 to about 42 A. In some aspects, the length of the linker is from about 10 to about 41 A. In some aspects, the length of the linker is from about 11 to about 40 A. In some aspects, the length of the linker is from about 12 to about 39 A. In some aspects, the length of the linker is from about 13 to about 38 A. In some aspects, the length of the linker is from about 14 to about 37 A. In some aspects, the length of the linker is from about 15 to about 36 A. In some aspects, the length of the linker is from about 16 to about 35 A. In some aspects, the length of the linker is from about 17 to about34 A. In some aspects, the length of the linker is from about 18 to about 33 A. In some aspects, the length of the linker is from about 19 to about 32 A. In some aspects, the length of the linker is from about 20 to about 31 A. In some aspects, the length of the linker is from about 21 to about 30 A. In some aspects, the length of the linker is from about 22 to about 29 A. In some aspects, the length of the linker is from about 23 to about 28 A. In some aspects, the length of the linker is from about 24 to about 27 A. In some aspects, the length of the linker is from about 5 to about 50 A. In some aspects, the length of the linker is greater than or equal to about 5 A.Amino Acid Residues and Amino Acid Substitutions

[0087] Suitable amino acids incorporated into peptides of the present disclosure include, without limitation, natural alpha-amino acids such as D- and L-isomers of the 20 common naturally occurring alpha-amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as provided in Table 1 depicted below), non-canonical alpha-amino acids (as depicted in Tables 2 and 3 below), natural beta-amino acids (e.g., beta-alanine), and unnatural beta-amino acids.

[0088] Amino acids used in the construction of peptides of the present disclosure may be prepared by organic synthesis, or obtained by other means, including, but not limited to, degradation of or isolation from a natural source and automated peptide synthesis. Additional examples of amino acids and methods for synthesis are described in U.S. Patent No. 11,279,734, the relevant disclosures of which are herein incorporated by reference.

[0089] Any of a variety of non-canonical amino acids may be included in peptides of the present disclosure. Non-limiting examples of non-canonical amino acids include a- Aminoisobutyric acid (Aib), norleucine (Nle), 4-hydroxyproline, (2S, 4R)-4-amino-L- proline (4NHPgam), 2-amino-3-[(4-aminophenyl)formamido]propanoic acid (Dapbeta) desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)- methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1 -amino-cyclopropanecarboxylic acid, l-amino-2-phenyl-cyclopropanecarboxylic acid, 1 -amino- 1 -cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3 -amino-cyclohexanecarboxylic acid, 4- piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4- (aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and para-substitutedphenylalanines (e.g., substituted with — C(=0)C6Hs; — CF3; — CN; -halo; — NO2; CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with — C(=0)CeH5; — CF3; — CN; -halo, — NO2; CH3), and statine. As used herein, “4NHPgam” refers to a (2S, 4R)-4-amino-L-proline, wherein the 4-amino group is covalently connected to a protractor. As used herein, “Dapbeta” refers to 2-amino-3-[(4- aminophenyl)formamido]propanoic acid, wherein the 4-amino group is covalently connected to a protractor. Additionally, amino acids suitable for use in the present disclosure may be derivatized to include amino acid residues that are hydroxylated, phosphorylated, sulfonated, acylated, and glycosylated, to name a few.

[0090] In some aspects, a peptide of the present disclosure comprises a substituted amino acid. In some aspects, the substituted amino acid is a canonical amino acid. Canonical amino acids for use in substitutions are listed in Table 1.Table 1. Canonical amino acids

[0091] As used herein, “Keps” refers to a lysine (K) residue wherein the epsilon (a) amino group in the lysine side chain is covalently connected to a protractor.

[0092] In some aspects of the disclosure, disclosed peptides comprises one or more non- canonical amino acids (also known as “unnatural amino acids”). Examples of noncanonical amino acids include, but are not limited to, a-Aminoisobutyric acid (Aib), norleucine (Nle), O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3 -methylphenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl- GlcNAcbeta-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo- phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, and an isopropyl-L- phenylalanine, 4-(Boc-amino)-L-Phe-OH, 3-(4’-pyridyl)-L-Ala-OH, L-a-aminoadipic acid(StBu), (4-aminomethyl) benzoic acid, L-2-aminooctanoic acid, P-cyclobutyl-L-Ala- OH, P-cyclohexyl-L-Ala-OH, P-cyclopropyl-L-Ala-OH, 2-(Boc)aminobutyric acid, 3,4- difluoro-L-Phe-OH, 3,4-dimethoxy-L-Phe-OH, Nco-(Pbf)-L-homoArg-OH, homoPhe-OH (hPh), L-hydroxyproline(tBu)-OH, 4-fluoro-L-Phe-OH, L-Met sulfone-OH, 3-(2- naphtyl)-L-Ala-OH, L-ornithine(Boc)-OH, 4-phenylpiperidine-4-carboxylic acid, 3,4,5- trifluoro-L-Phe-OH, 3-(4-thiazolyl)-L-Ala-OH, 4-phenyl phenylalanine, napthylalanine, quinolylalanine, 6-C1 tryptophan, cyclohexylglycine, cyclopentylglycine, d-Alanine, amino isobutyric, homocysteic acid, L-2, 3 -diaminopropionic acid, norvaline, phenylglycine, hydroxyproline, (2S, 4R)-4-amino-L-proline (4NHPgam), Keps, and the like. In some aspects, as used herein, the protecting groups (e.g., Boc, tBu, Pbf groups) are removed during peptide cleavage.

[0093] In some aspects, the amino acids of a presently disclosed peptide are mixed canonical and non-canonical amino acids.

[0094] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are known to those of ordinary skill in the art. For example, the groups of amino acids provided in Tables 2-4 are considered conservative substitutions for one another.Table 2: Selected groups of amino acids that are considered conservative substitutions for one another, in certain aspects.(Hydrophilic Uncharged Residues(Aliphatic Uncharged Residues(Non-polar Uncharged Residues(Aromatic Residues f"Y?and W ITable 3: Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain aspects.Table 4: Further selected groups of amino acids that are considered conservative substitutions for one another, in certain aspects.

[0095] A peptide generated by making one or more conservative substitutions of amino acid residues is referred to as a “conservatively modified variant.”Reference peptides

[0096] In certain aspects, certain properties (e.g., activity in a particular assay) of peptides of the present disclosure may be compared to that of a reference peptide. In some aspects, activity or values may be “decreased” or “increased” relative to that of a reference peptide. Non-limiting examples of reference peptides include any of the following peptides in Table 5. Reference peptides may have the sequences as shown in Table 5 without any protractor group. The reference peptides as provided in Table 5 are head-to-tail backbone cyclic peptides and each comprise an internal disulfide bond connecting their two cysteines.Table 5: Examples of reference peptides and their sequences.* within the sequences:• Bold indicates the P2 position according to Schechter and Berger nomenclature.• Underline indicates the Pl position according to the Schechter and Berger nomenclature.• Italics indicates the PT position according to the Schechter and Berger nomenclature,indicates the P2' position according to the Schechter and Berger nomenclature.Compositions

[0097] Provided herein are compositions (e.g., pharmaceutical compositions) comprising peptides of the present disclosure.

[0098] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0099] The present disclosure also contemplates salts of peptides disclosed herein — in some aspects, pharmaceutically acceptable salts, for example, salts as acid adduct with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid and boric acid; or with organic acid such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, benzenesulfonic acid, p-toluenesulfonicacid, naphthalenesulfonic acid and sulfanilic acid; and salts with metals such as alkali metal, e.g. sodium, potassium, lithium, zinc, and aluminum.

[0100] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0101] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N(Ci-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

[0102] Combinations of substituents and variables envisioned by this disclosure are those that result in the formation of stable compounds. The term “stable”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and whichmaintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0103] For therapeutic use, a peptide disclosed herein preferably is combined with a pharmaceutically acceptable carrier and / or excipient.

[0104] The terms “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” “pharmaceutically acceptable excipient,” and “pharmaceutically acceptable vehicle” are used interchangeably herein and refer to a non-toxic carrier, excipient, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound(s) with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the compounds disclosed herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0105] A “pharmaceutically acceptable derivative” refers to any non-toxic salt, ester, salt of an ester or other derivative of a compound of this disclosure that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this disclosure or an inhibitorily active metabolite or residue thereof.Methods of TreatmentSubjects

[0106] For therapeutic applications, peptides of the present disclosure are administered to a subject (e.g., a mammal, generally a human) in a pharmaceutically acceptable dosage form such as those known in the art.

[0107] In some aspects, provided herein is a method of treating a disease, disorder, or condition in a subject in need thereof by administering an effective amount of a peptide as disclosed herein. In some aspects, the presently disclosed peptides are used to treat a disease, disorder, or condition that can benefit from treatment with a disclosed peptide.Peptides provided herein may be useful for the treatment of a subject having a blood clotting disorder. In some aspects, the subject may be suffering from, exhibits at least one symptom of, is diagnosed with, and / or is identified as at risk of a blood clotting disorder.

[0108] In some aspects, peptides provided herein are provided for use as a medicament. In some aspects, peptides provided herein are provided for use in the manufacture or preparation of a medicament. In some aspects, the medicament is for the treatment of a disease, disorder, or condition that can benefit from treatment with a disclosed peptide.

[0109] Non-limiting examples of blood clotting disorders that are treatable with a peptide of the present disclosure include hemophilia, hyperfibrinolysis, hereditary hemorrhagic telangiectasia, and heavy menstrual bleeding.

[0110] In some aspects, the subject is suffering from heavy menstrual bleeding. In some aspects, the subject is suffering from a blood clotting disorder (e.g., hemophilia, hyperfibrinolysis, hereditary hemorrhagic telangiectasia, or heavy menstrual bleeding). In some aspects, the subject is suffering from a traumatic injury or has a surgical wound.Routes of Administration[OHl] In some aspects, peptides or pharmaceutical compositions of the present disclosure are administered systemically. Systemic routes of administration include parenteral routes and enteral routes. In some aspects, peptides or pharmaceutical compositions thereof are administered by a parenteral route, for example, intravenously, intraarterially, intraperitoneally, subcutaneously, or intradermally. In some aspects, peptides or pharmaceutical compositions thereof are administered intravenously. In some aspects, peptides or pharmaceutical compositions thereof are administered subcutaneously. In some aspects, peptides or pharmaceutical compositions thereof are administered by an enteral route of administration, for example, trans-gastrointestinal, or orally.

[0112] In some aspects, peptides or pharmaceutical compositions thereof are administered locally, e.g., to the site of a wound.Dosing

[0113] In some aspects, peptides of the present disclosure are administered in a therapeutically effective amount (e.g., within a pharmaceutical composition) to the subject at regular intervals. In some aspects, peptides of the present disclosure areadministered prophylactically to a subject (e.g., at regular intervals). In some aspects, the regular intervals are longer than a week. In some aspects, the regular intervals are longer than two weeks. In some aspects, the regular intervals are longer than three weeks. In some aspects, the regular intervals are at least a month. In some aspects, e.g., wherein the peptides of the present disclosure are administered to treat, prevent or ameliorate heavy menstrual bleeding, the regular intervals are one menstrual cycle.Outcomes

[0114] In many embodiments, methods disclosed herein result in a measurable improvement in the subject, e.g., in amelioration or resolution of symptoms. For example, such improvement may include an improvement in a clinical score or a score from a survey or questionnaire associated with, or suitable for assessing the disease, disorder, or condition being treated.Methods of Making

[0115] A peptide of the present disclosure may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said peptides or polypeptides, by standard techniques for production of polypeptides, even if non-natural amino acids are used. For instance, peptides of the present disclosure can be synthesized using well- known solid phase methods, in particular using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California, Gyros Protein Technology, Tucson, Arizona or CEM corporation, Matthews, North Carolina) and following the manufacturer's instructions.Kits

[0116] In some aspects, peptides disclosed herein are assembled into a pharmaceutical or diagnostic or research kit to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing any of the systems or vectors disclosed herein and instructions for use.

[0117] The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, whereapplicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration.EXAMPLES

[0118] Below are examples of specific aspects for carrying out what is disclosed herein. The examples are offered for illustrative purposes only and are not intended to limit scope.Materials and Methods for Examples 1, 2, 4, and 5:General procedure for solid phase peptide synthesis.

[0119] Peptides were synthesized using a SyroII fully automated parallel peptide synthesizer (MultiSynTech GmbH, Germany), equipped with heating block. The resin (resin info below) was prepared with MeDbz-Gly, coupled using amino acids in a 4-fold molar excess preactivated with 3.9-fold excess HATU (hexafluorophosphate azabenzotriazole tetramethyl uronium) and 8-fold excess DIPEA (N,N- diisopropylethylamine). Na-Fmoc deprotection was performed in two stages by treating the resin with 20% piperidine / DMF (N,N-dimethylformamide) (0.1 M HOBt (1- hydroxybenzotriazole)) for 3 min at 25°C followed by 15 min at 25°C. The coupling chemistry was DIC (N,N'-diisopropylcarbodiimide) / Oxyma (ethyl cyano(hydroxyimino)acetate) in DMF using amino acid solutions of 0.5 M in DMF and a molar excess of 5-fold. Standard Fmoc-protected amino acids were used. Couplingcondition was single or double couplings for 15 min at 50°C. Amino acids coupled after Aib (2-Aminoisobutyric acid) were double coupled. Fmoc-amino acids were dissolved at 0.5 M in DMF containing 0.5 M Oxyma. The resin was washed 5x with DMF after Na- Fmoc deprotection and 3x after couplings.

[0120] The lipidation handle was incorporated as an orthogonally protected attachment amino acid, such as lysine, 4-amino proline or 2-amino-3-[(4- aminophenyl)formamido]propanoic acid, here Lys(Mtt), Pro(4-NH-Mtt) or Dap(Mtt) coupled to Amino-benzoic acid after Mtt-removal. The Mtt group (4-methyl-trityl) was removed by treating the resin with 75% HFIP (l,l,l,3,3,3-hexafhioro-propan-2-ol) in DCM (di chloromethane) plus 5% TIPS (triisopropylsilane) for 10 minutes. Procedure repeated 3 times. The resin was washed with 10% DIPEA in DCM, followed by 3x DMF wash.

[0121] The lipidated peptides optionally contain linker residue(s). The linker residue(s), when used, were coupled to the amino group of the deprotected attachment-amino acid prior to coupling a fatty acid, such as tert-butyl protected fatty diacids, such as such as tBu-C14-diacid, tBu-C16-diacid, tBu-C18-diacid, tBu-C20-diacid etc. The linker residues were double- or triple coupled using standard conditions. The fatty acid was double coupled using 2 eq. building block.

[0122] To convert the MeDbz linker to MeNbz, the resin was washed with 3x DCM, then treated with 2x 2.5 eq 4-nitrophenyl chloroformate in DCM for 30 min. The resins were washed with 3x DCM and 3x DMF, then treated with 2x 25 eq DIPEA in DMF for 15 min. The resins were then washed with 3x DMF and 3x DCM.For library peptide synthesis:

[0123] Peptides were prepared using Tentagel XV NH2 resin with a loading of 0.35 mmol / g (Rapp polymer GmbH, Germany).

[0124] After synthesis, the resin was dried, and the protecting groups were removed on- resin by a 45-60 min treatment with TFA (trifluoroacetic acid) / TIPS (triisopropylsilane) / DODT(2,2'-(ethylenedioxy)diethanethiol) / water (92.5 / 2.5 / 2.5 / 2.5) at 40°C, then washed with lx DCM, lx DMF, 3x DCM, then dried. For the on-resin head- to-tail cyclization, the resins were treated with 50% acetonitrile in 100 mM phosphate buffer, facilitating a final pH of 6.8, and heated for 3 h at 50°C. The filtrate (nowcontaining the cyclized peptide) was collected, and the resin was washed with the same pH 6.8 buffer and filtrate collected and combined.

[0125] To form a disulfide bond, peptide solutions were treated with 200 mM iodine in acetic acid until pale yellow, then shaken for 20 min and quenched with ascorbic acid. Peptide solutions were loaded onto a Strata-X solid phase extraction plate (Phenomenex, Denmark) and eluted using 55% ACN / water. Peptides were characterized by LC-MS (Waters, Denmark) and quantified by LC-CAD (ThermoFisher scientific, Denmark), and lyophilized using a Telstar benchtop freeze drier.For peptides to be purified:

[0126] Peptides were prepared using Tentagel S RAM with a loading of 0.22 mmol / g (Rapp polymer GmbH, Germany).

[0127] After synthesis, the resin was washed with DCM and dried, and the polypeptide was cleaved from the resin by a 120 min treatment with TFA (trifluoroacetic acid) / TIPS(triisopropylsilane) / DODT(2,2'-(ethylenedioxy)diethanethiol) / water (92.5 / 2.5 / 2.5 / 2.5) at 40°C, followed by precipitation with cold diethyl ether, further washing with diethyl ether, dissolution in 50% ACN (acetonitrile) / water, and left to dry.

[0128] For the head-to-tail cyclization, dried peptides were dissolved in 50% ACN / water and added dropwise to 50% acetonitrile in 100 mM phosphate buffer, facilitating a final pH of 6.8, with stirring and heating to 50°C. To form a disulfide bond, peptides were subsequently treated with 200 mM iodine in acetic acid until pale yellow, then stirred for 5 min and quenched with ascorbic acid.

[0129] Peptides were purified by reverse phase (RP) HPLC using a Waters preparative HPLC with C18 column (Reprosil Phoenix, C18, 5 pm, 30 mm x 250 mm), preparative pumps (waters 2545), UV / VIS detector (Waters 2489) and a Waters fraction collector III. The mobile phase was run with a gradient of buffer A (0.1% TFA in H2O) and buffer B (0.1% TFA in acetonitrile) at a flow rate of 20 mL / min at room temperature. Relevant fractions were analysed, pooled, and lyophilized using a Telstar benchtop freeze drier.

[0130] Peptide purity and mass were determined by analytical RP-HPLC-MS on a ACQUITY UPLC Peptide CSH C18 column (Waters, ACQUITY UPLC Peptide CSH, C18, 130 A, 1.7 pm, 2.1 mm x 100 mm) using a Waters Acquity HPLC System equipped with 3100 Mass Detector. Analysis was performed by gradient elution with buffer A(0.3% TFA in H2O) and buffer B (0.3% TFA in acetonitrile) at a temperature of 40°C (gradients used 40-60%B over 14 min, or 50-70% B over 14 min).Peptide concentrations were determined by LC-CAD or absorbance measurements.Clot lysis assay

[0131] Pooled normal human plasma (George King Biomedical, cat. no. 0010-1) was incubated with graded concentrations of test compound in a 384 well plate for 10 min at 37°C while shaking. Tissue plasminogen activator (tPA; Kem-en-tec, cat. no. 30C- CP1109-22) was added, and then clot formation was triggered by addition of lipidated tissue factor in the presence of 20 mM CaCh. Immediately afterward, the plate was thoroughly shaken, and absorbance at 405 nm was measured at 37°C every minute for 3 h. Curves were split into clot formation and clot lysis, and a sigmoidal curve was fitted to each part of curve. The time to 50% clot formation and time to 50% lysis of the clot were extracted, and the difference between them was used to obtain the clot lysis time. Clot lysis time was normalized using the ECO (i.e., absence of test compound) wells on each plate. Clot lysis time vs. concentration was fitted to a logarithmic function.For peptide libraries:

[0132] For each peptide sample, five activity readings of progressively diluted samples were measured by sampling clot formation and subsequent clot lysis at 450 nm in 1 min intervals. The clot lysis curves were split at the max value of the clot peak into the two clot modes: clot formation eformation (first part of the curve) and clot lysis eiysis (last part of the curve). One sigmoidal curve was fitted for each of the clot modes. For each clotting mode the time corresponding to 50% of clot formation and 50% of clot lysis was determined from the fitted functions. The clot lysis time (CLT) was then obtained by subtracting the two eiysis-eformation. The resulting clot lysis time was normalized by subtracting the mean CLT of non-inhibited wells with the CLT of the test compound. A linear regression model is fitted to the normalized CLT values plotted against the logarithm of the concentrations for each sample. The slope of the linear regression model representing the CLT Coefficient was used as final endpoint for SHAP modelling.For purified peptides:

[0133] Concentration-response evaluation of compounds was performed with 11 concentrations of agonist peptides and IC50 values were calculated by nonlinear regression using sigmoid concentration-response with variable slope.Plasmin inhibition assay

[0134] Human plasmin (Prolytix, cat. no. HCPM-0140) was incubated with graded concentrations of test compound in a 384 well plate for 10 min at room temperature (RT) while shaking. The chromogenic substrate pNAPEP-1751 (Cryopep, cat. no. 6101-1751) was then added, and absorbance at 405 nm was immediately measured every minute for 10 min while shaking at room temperature (RT). Assays were performed either in the presence of 0.05% casein or 1.2% BSA (Sigma- Aldrich, cat. no. A6003). However, in both cases, the test compound serial dilution was performed in buffer with casein, instead of BSA.For peptide libraries:

[0135] Concentration-response evaluation of compounds was performed with 5 concentrations of peptide and Ki values were calculated by nonlinear regression using the Morrison equation for tight binding competitive inhibitors with the enzyme concentration (Et) set to 5 nM, chromogenic substrate concentration (S) set to 600,000 nM and Km set to 300,000 nM. The dataset was normalized by setting Vo = 1. Ki values were transformed to pKi = -log(Ki) values for the random forest regression models and subsequent SHAP analysis.For purified peptides:

[0136] Concentration-response evaluation of compounds was performed with 11 concentrations of agonist peptides and Ki values were calculated by nonlinear regression using the Morrison equation for tight binding competitive inhibitors with Et set to 5 nM, S set to 600,000 nM and Kmset to 300,000 nM.General procedure for determination of solubility of purified peptides.

[0137] Solubility was tested in the following vehicle: 100 mM phosphate buffer facilitating a target pH of 7.0. For the desired concentration, such as a concentration of 10mg / mL, a suitable amount of peptide was dissolved in 400 pL vehicle. Samples were incubated for at least 1 h at room temperature on a rocking table. The pH was measured and adjusted. Samples were left on the rocking table for an additional hour before a second pH measurement and adjustment. Visual inspection was performed and noted before the filter was pushed. The concentration of peptide in the filtrate was determined using CAD (Charged Aerosol Detection), double determination was done for each sample. The measured concentration was reported in mg / mL. Furthermore, the pH of the filtrate was measured and reported. Peptides with a measured concentration within + / - 20% of target concentration are considered fully soluble.General procedure for determination of chemical stability of purified peptides.

[0138] Chemical stability was tested at a concentration of 267 pM peptide in the following vehicle: 50 mM phosphate buffer pH 7.0. Dissolved samples were incubated for at least 2 h at room temperature on a rocking table. The main peak purity at time zero (TO) was determined using reversed phase chromatography. The samples were incubated at 40°C for 14 days (T14). The samples were analyzed with reversed phase chromatography and size exclusion chromatography.

[0139] Reversed Phase chromatography was performed on a Thermo Dionex Ultimate UHPLC system with UV detection at 215 nm and equipped with a Kinetex column (1.7 pm, C8, 100 A, 150 x 2.1 mm (00F-4499-AN)). Analysis was performed by gradient elution with buffer A (0.1% TFA in 95:5 H2O:acetonitrile) and buffer B (0.1% TFA in 5:95 H2O: acetonitrile) at a column temperature of 50 °C. Gradient used 25-55% buffer B over 40 min. Flowrate was 0.5 mL / min. The main peak purity was determined from the integrated chromatogram as the area under the main peak relative to the total peak area. The purity loss was calculated as the difference between the TO and T14 time point.

[0140] Size exclusion chromatography (SEC) was used as an analysis for the formation of high molecular weight products (HMWP, covalent dimers, trimers etc.,). SEC analyses was performed on Thermo Dionex Ultimate UHPLC system with UV detection at 215 nm equipped with a SEC column (Waters BEH 125 , 1.7 pm, 300 mm x 4.6). Analysis was performed by isocratic elution using 60% buffer B (0.1% TFA in 5:95 H2O: acetonitrile) in buffer A (0.1% TFA in 95:5 H2O: acetonitrile) at a column temperature of 60°C over 20 min. Flowrate was 0.3 mL / min.

[0141] Chromatograms are integrated as HMWP (all peaks eluting before the main peak), main peak (assuming this is monomeric peptide) and, if relevant, LMWP (low molecular weight products, all peaks eluting after main peak). The amount of HMWP was reported as area relative to total area in percent.PK in mice

[0142] Lean male C57BL / 6JRj mice were obtained from JanVier (JanVier Labs, France) at 6 weeks of age. The animals were single housed under a 12 / 12 h dark-light cycle, light off at 3 PM. Room temperature was controlled to 22°C ± 1°C, with 60 % ± 20% humidity. Animals had ad libitum access to regular rodent chow (Altromin 1324, Brogaarden, Denmark) and tap water.

[0143] After minimum 2 weeks of acclimatization, the animals were randomized into treatment groups (n=9 per group) based on body weight. The animals were co-dosed with two test compounds; one compound was dosed intravenously, and the other was dosed subcutaneously. In a parallel group, the same two test compounds were dosed with opposite routes of administration. Blood samples were collected in EDTA tubes for PK analysis at time points: 0.083, 0.5, 1, 2, 4, 6, 24, 48, 72 and 96 hours post dosing in a sparse sampling design with 3 mice per group per timepoint. Plasma was obtained by centrifugation of the blood samples (3000xg, 10 min 4 °C), and the plasma samples were frozen at -70 °C until analysis.

[0144] Plasma samples were analyzed using LC-MSMS and PK parameters were estimated by Non-Compartmental Analysis (NCA) using the log-linear trapezoidal method for the estimation of AUC.Example 1 - Deep mutational scan of a peptide plasmin inhibitor

[0145] To identify positions and substitutions that can improve plasmin inhibition, a deep mutational scan was performed on SFTIv3 as a starting peptide. SFTIv3 is a plasmin inhibitor based on the circular Sunflower Trypsin Inhibitor 1 (SFTI1) peptide scaffold and has the sequence of SEQ ID NO: 3 (shown below) and is also depicted in FIG. 1.

[0146] GRCYKSKPPICFPD (SEQ ID NO: 3)

[0147] A peptide library based on the SFTIv3 backbone was designed and synthesized, wherein each amino acid residue was changed into all of the remaining natural amino acids (except Cys and Met) or 2-Aminoisobutyric acid (Aib) one at a time or incombination. A library of 570 peptides was generated in this manner, each peptide having a SFTIv3 backbone with 1-3 substitutions. A random forest model describing the relationship between peptides sequence and plasmin inhibition and clot lysis (endpoints) was constructed. The random forest model was used to compute the batch corrected effects of changing an amino acid residue based on a large number of peptides from the library.

[0148] The peptides’ turbidity and propensity to form fibrils were assessed and found to be low.

[0149] Direct plasmin inhibition and clot lysis were assessed in vitro as described above. Plasmin inhibition and CLT coefficients for each of the single-substitution variants in the deep mutational scan were batch-corrected to correct for batch effects. Results are summarized in FIGs. 2A-3B.

[0150] Results from these experiments show that substitutions at positions 7, 12, and 13 of SEQ ID NO: 3 may improve plasmin inhibition and clot lysis.Example 2: Lipidation scan of a peptide plasmin inhibitor

[0151] A lipidation scan was performed by introducing lipidation handles at each position in SEQ ID NO: 3 except positions 3 and 11 containing cysteines. Lipidation handles used included Keps, 2-amino-3-[(4-aminophenyl)formamido]propanoic acid (Dapbeta), and (2S, 4R)-4-amino-L-proline (4NHPgam). The following lipid and linker combinations that were attached via the lipidation handles incorporated at each position in the peptide:• C16DA-gGlu• C18DA-gGlu• C20DA-gGlu• C16DA-gGlu-2xOEG• C18DA-gGlu-2xOEG• C20DA-gGlu-2xOEG• C16DA-eLysl,2• C18DA-eLysl,2• C20DA-eLysl,2

[0152] “C16DA” refers to hexadecanedioic acid; “C18DA” refers to octadecanedioic acid; “C20DA” refers to icosanedioic acid; “OEG” refers to 8-amino-3,6-dioxaoctanoic acid; “gGlu” refers to glutamic acid wherein the sidechain acid is covalently connected to the lipidation handle or the linker / spacer. The respective structures are provided below:el_y * s1, ’2 C18DA and , C20DA

[0153] A total of 190 peptides were screened in this scan. In some peptides, an acetyl group (Ac) was attached as a control.

[0154] The peptides’ turbidity and propensity to form fibrils were assessed and found to be low.

[0155] The pKi values for inhibition of plasmin (in presence or absence of BSA) and CLT coefficients were determined for the peptide library (190 peptides) and Shapley additive explanations (SHAP) values calculated from a random forest model, where pKi values and CLT coefficients were fitted to the peptide amino acid sequences. SHAP values were used to determine the level of contribution of each modification on the endpoints pKi and CLT coefficients. A unified approach to interpreting model predictions. Advances in neural information processing systems, 30.). The contributions of each substitution was determined as the difference in average SHAP value between the substitution and the reference (i.e. SFTIv3). Substitutions with positive SHAP values increased the endpoint (i.e. increased plasmin inhibition or increased CLT coefficient), while negative SHAP values decreased the endpoint (i.e. decreased plasmin inhibition or decreased CLT coefficient).

[0156] Results from the clot lysis assays are shown in FIGs. 4A and 4B.

[0157] The results revealed that a limited set of positions in the peptides could accommodate lipidation (2, 7, 8, 10, 12, and 13). There were advantages to placement of Keps at positions 2, 7, 10, 12, and 13 of SEQ ID NO: 3. There were advantages to placement of 4NHPgam at positions 8 and 13 SEQ ID NO: 3. The results also showed improvement with longer linkers in the protractor.Example 3 - Preformulation and pharmacokinetic analysis of certain peptides

[0158] Preformulation and pharmacokinetic studies were also performed on the parental molecule (SFTIv3) and on a lipidated form of SFTIv3. Results from these studies indicated favorable chemical and pharmacokinetic characteristics. Solubility of at least 10 mg / mL was observed for both molecules, with high chemical stability and no fibrillation observed.

[0159] The half-life of a lipidated form of SFTIv3 with a C18DA-2xgGlu-2xOEG protractor conjugated to Keps introduced at position 12 (referred to as “Peptide A” in Table 6) was compared against that of reference compounds SFTI1 (SEQ ID NO: 4) and SFTIv3 (SEQ ID NO: 3) in mice dosed intravenously (IV) or subcutaneously (SC) with the peptide. Table 6 presents the parameters and calculated values from this experiment.Table 6 - Pharmacokinetic characteristics for the indicated peptides.

[0160] While SFTI1 and SFTIv3 showed rapid clearance and a short ti / 2 life (similar to values reported in the literature), the lipidated version of SFTIv3 exhibited a half life of12-13 hours. Bioavailability was high for all tested compounds. These data demonstrate that lipidation in the context of the SFTI scaffold is able to extend in vivo half-life.Example 4 - Exploration of substitutions and lipidations at defined positions to identify improved peptide-based plasmin inhibitors

[0161] Plasmin inhibition and clot lysis assays were conducted to investigate the activity for ten candidate peptides Table 7 summarizes the sequences, lipidation positions, lipidation handle, linker and lipids as well as activity in plasmin inhibition and clot lysis assays of ten candidate peptides. These data show that Tyr at position 12 with lipid attachment at position 13 using 4NHPgam handle provided the best activity in the plasmin inhibition assay with BSA present and in clot lysis assay.Table 7: Exemplary candidate peptidesKey: Bold underlining indicates lipidation position; P* = 4NHPgam; K* = KepsExample 5 - Further exploration of substitutions and lipidations at defined positions to identify improved peptide-based plasmin inhibitors

[0163] With the preference for Tyr at position 12 and a 4NHPgam lipidation handle at position 13 (SEQ ID NO: 2), additional combinatorial scans were performed to further explore linker and lipid compositions. Table 8A summarizes the sequences, lipidation handle, lipidation positions, lipids, linkers, and plasmin inhibition Ki values in conditions with or without BSA in ten candidate peptides. Table 8B shows the clot lysis IC50 values for the ten candidate peptides shown in Table 8A; characteristics of the peptides presented in Table 8A are repeated in Table 8B for ease of viewing. Values for the reference peptide (SFTIv3 without lipidation) are provided for comparison.Table 8A: Exemplary candidate peptides. The protractor (lipid and linker) had the composition of Lipid-(gGlu)v(OEG)x or Lipid-(gGlu)vA(EAAAK)3A, where X and Y refer to the number of each unit in the linker.Key: Bold underlining indicates lipidation position; P* = 4NHPgamTable 8B: Plasmin inhibition and clot lysis values of exemplary candidate peptides. The protractor (lipid and linker) had the composition of Lipid-(gGlu)y(OEG)x or Lipid-(gGlu)vA(EAAAK)3A, where X and Y refer to the number of each unit in the linker.Key: Bold underlining indicates lipidation position; P* = 4NHPgam

[0164] The structure for compound 101 in Tables 8A and 8B is shown in FIG. 5A. The structure for compound 102 in Tables 8A and 8B is shown in FIG. 5B.Summary of key findings from experiments described in Examples 1-5

[0165] Lipidation resulted in improved half-life in vivo, which supports dosing at longer intervals.

[0166] All of the libraries of peptides exhibited good solubility, and stability in formulation.

[0167] Linkers of intermediate length, e.g., 2xgGlu-2xOEG and 2xgGlu-6xOEG, exhibited lower IC50 values compared to those of longer length.

[0168] Lipidation at positions 8, 12, and 13 was preferred.Example 6 - Pharmacokinetic studies of half-life extended SFTIv3 in mice

[0169] Pharmacokinetic studies were performed in mice as described in the Materials and Methods section above. The studies were conducted to evaluate the half-life modified SFTIv3 peptides. Briefly, the following peptides were administered to mice at 50 nmol / kg by IV or SC injection:• Peptide 9-1 (also referred to herein as compound 102): SFTIv3 12Y, 4NHPgam at position 13 with C18DA-2xgGlu-6xOEG attached. The structure for which is shown in FIG. 5B.• Peptide 9-2: SFTIv3 12Y, 4NHPgam at position 8 with C18DA-2xgGlu-6xOEG attached. Plasmin inhibition (absent BSA) and clot lysis assays on this peptide revealed a Ki of 5.22 nM in absence of BSA and a IC50 of 10.66 uM.• Peptide 9-3 (also referred to herein as compound 105): SFTIv3 12Y, 4NHPgam at position 13 with C16DA-2xgGlu-6xOEG attached• Peptide 9-4 (also referred to herein as compound 101): SFTIv3 12Y, 4NHPgam at position 13 with C18DA-2xgGlu-2xOEG attached. The structure for which is shown in FIG. 5A.

[0170] The results are shown in Table 9 and FIG. 6.Table 9: PK results after administration of the indicated peptides at 50 nmol / kg IV or SC.Example 7 - Pharmacokinetics and duration of effect in mice

[0171] The objective of the first part of this study was to characterize the pharmacokinetic (PK) properties of SFTIv3, Compound 105, and Compound 102 following intravenous (IV) and subcutaneous (SC) administration in male C57BL / 6JRjmice over a four-day period.

[0172] Male C57BL / 6JRjmice were randomized into six study groups (n=9 per group) based on absolute body weight on the day before administration. Following IV and SC administration of 50 nmol / kg of SFTIv3, Compound 105 or Compound 102, respectively, intermittent plasma samples were collected from subgroups of three mice per group at predetermined time points (5 min, and 0.5, 1, 2, 4, 6, 24, 48, 72, and 96 hours postdosing). Plasma drug concentrations were quantified using liquid chromatographytandem mass spectrometry (LC-MS / MS). Pharmacokinetic parameters were calculated using non-compartmental analysis of mean composite concentration-time profiles.

[0173] Measured PK profiles are shown in Figures 7A and 7B. Following IV administration, the estimated terminal elimination half-lives (t’A) were 0.4 hours, 2.2 hours, and 19.4 hours for SFTIv3, Compound 105, and Compound 102, respectively. Subcutaneous absorption proceeded with time to maximum plasma concentration (Tmax) ranging from 1 to 6 hours. The calculated SC bioavailability was high, ranging from 95% to 111%. Values exceeding 100% may be attributed to minor variability in actual administered doses.

[0174] The second part of the study was designed to characterize the PK properties and assess ex vivo pharmacodynamic (PD) effects on fibrinolysis of SFTIv3 and Compound 102 following single IV administration in male C57BL / 6JRj mice in comparison to tranexamic acid (TXA).

[0175] Male C57BL / 6JRj mice were randomized into three study groups (n=4 per group) based on absolute body weight on the day before administration. The treatment groups were assigned as follows: Group 1 received SFTIv3 at 4.102 mg / kg (IV), Group 2 received Compound 102 at 7.861 mg / kg (IV), and Group 3 received TXA at 100 mg / kg (IV). Following administration, blood samples were collected via retro-orbital puncture at 5 minutes, 6 hours, and 48 hours post-dose. At the 48-hour time point, all animals were euthanized following the final blood collection. Plasma was isolated for pharmacokinetic analysis.

[0176] Plasma concentrations of the test compounds (n = 2 per group) were quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). The lower limit of quantification (LLOQ) for all compounds was 0.5-1 nM; as the plasma samples were diluted 100-fold prior to analysis, the effective LLOQ was 50 nM. Additional plasma samples (n = 4 per group) were collected for further evaluation of the fibrinolytic activity in a clot lysis assay.

[0177] The lysis of a tissue factor (TF)-induced clot by exogenous tPA was studied by tracking turbidity changes during clot formation and subsequent lysis of plasma samples from the study. Plasma (final concentration of 20 % v / v) was added to a microtiter plate and supplemented with human fibrinogen (Enzyme Research, catalog no. 1-574-288- 2268; final concentration of 1.2 mg / ml). After incubation for 10 min at 37°C, tPA (Fitzgerald, catalog no. 30C-CP1109; final concentration of 15 nM) was added, followed by human tissue factor (TF) incorporated in 25% phosphatidyl serine, 75% phosphatidyl choline vesicles (final concentration of 0.2 pM) and CaCh (final concentration of 20 mM). The TF incorporated in vesicles was prepared according to the method described by Morrissey et al (Smith SA & Morrissey JH (2004) Rapid and efficient incorporation of tissue factor into liposomes. J. Thromb. Haemost. 2: 1155-1162). After thorough mixing, turbidity was measured at 405 nm for 180 min at 37°C using a Spectramax 340 kinetic microplate reader (Molecular Devices Corporation). From the turbidity profile, the 50% clot lysis time was determined for each mouse plasma sample. This parameter is defined as the time interval between the midpoint of the increase from clear to maximum turbidity, indicating clot formation, and the midpoint of the decrease from maximum to clear turbidity, representing clot lysis.

[0178] Analysis of plasma samples by LC-MS / MS demonstrated that compound SFTIv3 was detectable only at the 5 minutes time point, whereas Compound 102 remained detectable for up to 48 hours post-dose as shown in Figure 8.

[0179] Measurement of fibrinolytic activity using the clot lysis assay demonstrated full inhibition of fibrinolysis in plasma samples taken 5 minutes after administration of Compound 102 and TXA, and partial inhibition after administration of SFTIv3 (Table 10). At 6 hours post-dosing, only Compound 102 significantly extended the clot lysis time compared to the clot lysis time measured in plasma from vehicle-treated mice (6.3 ± 0.7 minutes, n = 12).

[0180] Table 10 shows the estimated clot lysis time in minutes (mean ± SD, n=4) at 5 min, 6 hours and 48 hours post dosing. The clot lysis time in vehicle-treated mouse plasma was estimated to be 6.3 ± 0.7 minutes (n = 12). Assay duration was 180 min. Statistically significant differences (p < 0.05) from the vehicle value are indicated (*).Table 10: The estimated clot lysis time in minutes at 5 min, 6 hours and 48 hours post dosing.Example 8 - Duration of effect in minipigs

[0181] The objective of this study was to determine the pharmacokinetics (PK) of Compound 102 dosed subcutaneously (SC) and intravenously (IV) in a 6-week study in female Gottingen minipigs and to evaluate the time-dependent effects on the fibrinolytic system in plasma samples.Methods:

[0182] Female Ellegaard Gottingen minipigs were maintained on a standard chow diet (Altromin 9069) for a 3-week acclimation period prior to study initiation. On Day 1, subjects in Group 1 (n=2) received a single SC dose of Compound 102 of 25 nmol / kg, while subjects in Group 2 (n=2) received a single IV dose of 1075 nmol / kg. Body weight measurements were recorded biweekly through week 6, with a final measurement taken prior to the terminal blood collection. Blood samples for plasma collection were obtained at predetermined timepoints after administration. Dose accuracy was verified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of the administered formulation, and the stock solution used for preparation was analyzed by liquid chromatography with charged aerosol detection (LC-CAD).

[0183] Plasma samples for PK measurements were collected in K2EDTA tubes and stored at -70°C until analysis. Plasma proteins were removed by addition of organic solvent followed by centrifugation. The supernatant after protein precipitation was analyzed by LC-MS / MS. For Compound 102, a dedicated LC-MS / MS method was developed, and a calibration curve was prepared and analyzed along with the plasmasamples. Pharmacokinetic parameters were determined by non-compartmental analysis of individual concentration-time profiles for both IV and SC administration routes.

[0184] In addition, fibrinolytic activity was determined in citrate-anticoagulated plasma samples collected up to 408 hours post-dosing from IV-administered animals using the clot lysis assay. In this assay, turbidity changes are used to track clot formation (induced by tissue factor (TF)) and subsequent clot lysis due to the action of the fibrinolytic system in the plasma sample (induced by tPA). Plasma (final concentration of 30 % v / v) was added to a microtiter plate. After incubation for 10 min at 37°C, tPA (Fitzgerald, catalog no. 30C-CP1109; final concentration of 150 nM) was added, followed by human tissue factor (TF) incorporated in 25% phosphatidyl serine, 75% phosphatidyl choline vesicles (final concentration of 0.2 pM) and CaCh (final concentration of 20 mM). The TF incorporated in vesicles was prepared according to the method described by Morrissey et al (Smith SA & Morrissey JH (2004) Rapid and efficient incorporation of tissue factor into liposomes. J. Thromb. Haemost. 2: 1155-1162). After thorough mixing, turbidity was measured at 405 nm for 90 min at 37°C using a Spectramax 340 kinetic microplate reader (Molecular Devices Corporation). From the turbidity profile, the 50% clot lysis time was determined for each minipig plasma sample. This parameter is defined as the time interval between the midpoint of the increase from clear to maximum turbidity, indicating clot formation, and the midpoint of the decrease from maximum to clear turbidity, representing clot lysis.Results:

[0185] Compound 102 was successfully quantified in plasma following IV and SC administration with PK profiles shown in Figure 9. The mean plasma terminal elimination half-life (t’ ) was determined to be 153 hours following IV administration and 154 hours following SC administration (Table 11). Following SC administration, compound 102 demonstrated a bioavailability of 113% (100%). The dose accuracy of the administered solution was confirmed to be within 15% of the nominal concentration when measured by LC-MS / MS.Table 11: Pharmacokinetic parameters of Compound 102 following IV and SC administration in Gottingen minipigs.

[0186] The fibrinolytic activity in the plasma samples was evaluated using a clot lysis assay. Following IV administration of Compound 102 (1075 nmol / kg), fibrinolytic activity was fully inhibited for seven days followed by partial inhibition at day 10 and a return to pre-dose fibrinolytic activity at day 17 (Figure 10). The relative fibrinolytic activity (%) in each sample was determined based on the estimated clot lysis time. The activity measured in the pre-dose sample served as the baseline, defined as 100% fibrinolytic activity. In contrast, samples exhibiting complete inhibition of fibrinolysis (when the clot lysis time exceeds assay duration) were assigned a value of 0% activity, representing the absence of detectable fibrinolytic activity within the assay limits.Example 9 - In vivo hemostatic effect in mouse injury model

[0187] The dose-dependent effect of Compound 102 on the fibrinolytic system was determined in a mouse electrolytic injury model.Methods:

[0188] Male, 10-14 week-old C57B1 / 6 mice were subjected to a femoral vein electrolytic injury model of venous thrombosis. Mice were placed, supine, on a warming pad prior to removal of fur from the inner surface of the left hind limb with depilatory cream. A 1-2 cm incision was made in the inguinal region to expose the femoral vein just prior to intravenous infusion of 4 mg / kg GR-144053, a potent aiibbs inhibitor (Tocris Bioscience), Alexa Fluor 488-labeled anti-GPIX antibody (3.75 mg / mouse, clone Xia.B4, Emfret Analytics), Alexa Fluor 647-labeled anti-fibrin antibody (2 mg / mouse, clone 59D8, in-house) to label platelets and fibrin, respectively. Mice were administered 5 mg / kg tPA (alteplase, Genentech) 5 minutes prior to injury followed by infusion of Compound 102 (1, 2, 4, 6 or 8 mg / kg) 2 minutes prior to injury. Electrolytic injuries were achieved using a 100 pm diameter stainless steel wire to apply a 1.5V direct current (0.02 Amps) generated by a linear power supply (model: DP832, Rigol Technologies) to the ventral surface of the femoral vein for 30 seconds. Accumulation of platelets and fibrin was monitored by intravital video-microscopy using a stereo microscope (SMZ25, Nikon Corp.) coupled to an ORCA Flash 4.0 digital camera (Hamamatsu, Japan). Data were analyzed using NIS-Elements software (Nikon, Corp.).Results:

[0189] The ability of Compound 102 to prevent tPA-mediated fibrinolysis was evaluated using an electrolytic femoral vein injury model in mice. Animals received vehicle control (n=24), tPA alone (5 mg / kg, n=6), or tPA combined with Compound 102 at 1, 2, 4, 6 or 8 mg / kg (n=3-5 per group) prior to electrolytic injury (1.5 V, 0.02 A, 30 seconds). Platelet and fibrin accumulation was monitored in real-time by intravital microscopy (SMZ25, Nikon) with an ORCA Flash 4.0 camera and analyzed using NIS-Elements software. Statistical analysis was performed using ANOVA (n = 3-6 per group).

[0190] As shown in Figure 11, Compound 102 dose-dependently restored fibrin accumulation in the presence of tPA. Untreated control animals exhibited normal fibrin deposition over the 10 minute observation window, while tPA treatment prevented fibrin accumulation, maintaining levels near baseline. Administration of Compound 102 dose- dependently rescued fibrin formation in tPA-treated mice with noticeable effect already at 1 mg / kg and slightly increased fibrin accumulation relative to untreated controls animals at 8 mg / kg.

[0191] These findings demonstrate that Compound 102 effectively down-regulates tPA- mediated fibrinolysis in a dose-dependent manner, supporting its utility as an antifibrinolytic agent.Example 10 - Determination of antifibrinolytic activity in plasmin generation assay in human plasma

[0192] The objective of this study was to characterize the activity of Compound 102 in platelet-rich human plasma using a plasmin generation assay.

[0193] Plasmin generation was assessed using a calibrated assay based on the cleavage of a plasmin-specific fluorogenic substrate. To prepare the samples, 3.2% (v / v) citrated whole blood from healthy human donors was processed to obtain both platelet-rich plasma (PRP) and platelet-poor plasma (PPP). These two plasma fractions were then combined to achieve a final platelet concentration of 250 * 109platelets / L prior to use in the assay.

[0194] For each sample, two parallel measurements were carried out. The first measurement assessed endogenous plasmin generation in designated reaction wells. The second measurement utilized an a2-macroglobulin-plasmin calibrator in separate calibrator wells, which served as a reference for accurate quantification. In the reaction wells, citrate-stabilized PRP (with platelet count set to 250 * 109plt / L prior to dilution with 1 :3 (v / v) final dilution in the assay) was supplemented with tissue factor (TF, Innovin, Dade Behring; 5 pM final concentration), tissue plasminogen activator (tPA, Actilyse, Boehringer; 18 nM final concentration) and Compound 102 (0-64 pM final concentration). Samples were incubated at 37°C for 10 min to allow for equilibration before plasmin generation was initiated by addition of CaCh (16.7 mM final concentration) and Boc-Glu-Lys-Lys-AMC substrate (Bachem, catalog no. 4007739; 500 pM final concentration). The reaction was monitored for 60 min at 37°C using Thrombinoscope software (Diagnostica Stago) with fluorescence emission detection at 460 nm and excitation at 390 nm. To correct for potential inner filter effects due to plasma color and to account for substrate consumption during the reaction, each plasma sample’s fluorescence signal was compared with its corresponding calibrator well. Data were transformed into plasmin generation curves, from which quantitative parameters were derived.

[0195] As shown in Figure 12A, Compound 102 demonstrated a concentration-dependent reduction of peak active plasmin generation and endogenous plasmin potential (EPP) confirming its plasmin inhibitory activity. From the relationship between peak active plasmin generation and compound concentration, an ICso value of 1.4 pM was estimated for Compound 102 (Figure 12B).Example 11 - Activity in human whole blood flow model

[0196] To determine the antifibrinolytic activities of Compound 102 and tranexamic acid (TXA) in a system recapitulating key elements of the hemostatic system, i.e., primary andsecondary hemostasis, and fibrinolysis, a whole blood flow model was employed. In this model, human whole blood, supplemented with tPA, is perfused over a surface coated with collagen and tissue factor. This allows for platelet capture, initiation of coagulation, and subsequent lysis of the formed fibrin-rich clot.

[0197] Coverslips were degreased using chromosulfuric acid (Jones reagent; Sigma- Aldrich), thoroughly rinsed with deionized water, and subsequently incubated for 1 hour at room temperature (RT) with a 1 : 100 dilution of tissue factor (TF; Dade® Innovin®) (approximately 150 pM) and a 1 : 10 dilution of collagen solution derived from bovine skin (Sigma) (approximately 300 pg / mL) in HEPES-buffered saline (HBS). Following incubation, the coverslips were blocked with 1% bovine serum albumin (BSA) in HBS overnight at 4 °C. In parallel, polydimethylsiloxane (PDMS) microfluidic devices were cleaned by sequential sonication in hydrochloric acid, acetone, and ethanol for 15 minutes each, rinsed with water, and subsequently blocked with 1% BSA in HBS overnight at 4 °C. On the following day, coverslips were rinsed with HBS, PDMS devices were rinsed with water, and both components were assembled using vacuum. Human citrated whole blood was incubated with a 1 :2000 dilution of MitoTracker™ Orange CMTMRos for 30 minutes at RT to fluorescently label platelets. The blood was then treated with 30 nM tissue plasminogen activator (tPA; Actilyse), varying concentrations of Compound 102 or tranexamic acid (TXA), 8.4 pg / mL Alexa Fluor™ 488-conjugated anti-fibrin VHH (clone B12; UMC Utrecht), and recalcified with 3.08 mM MgCE and 6.60 mM CaCk The prepared blood was perfused over the TF / collagen-coated surface at a shear rate of 300 s ' for 25 minutes. Fibrin formation and platelet accumulation were visualized using a Zeiss Z1 Observer microscope at 200* magnification, capturing images at a frame rate of three per minute. Fluorescent signal of Alexa Fluor 488 was quantified using ZEN Pro software (version 2.0). For every run, maximum Alexa Fluor 488 signal was set to 100%. The endpoint was set at the timepoint that had complete fibrinolysis in the control condition (30 nM tPA only) of the same donor. For every concentration of Compound 102 or TXA, the degree of lysis (%) was calculated as the Alexa Fluor 488 signal at the determined endpoint relative to the maximum signal.

[0198] The resulting concentration-response curves were fitted by non-linear regression to an inhibitor vs normalized response (variable slope) function in Graphpad Prism. From this analysis ICso values of Compound 102 and TXA were estimated to 1.4 pM and 41 pM, respectively (Figures 13 A and 13B). Data are shown as mean ± SD (n = 3).Example 12 - Binding of Compound 102 to human serum albumin using SEC HPLC

[0199] The objective of this study was to characterize the binding interaction between Compound 102 and human serum albumin (HSA) using size exclusion chromatography- high performance liquid chromatography (SEC-HPLC). Compound 102 was labeled with AlexaFluor647 (AF647) at a single site between the 3rd and 4th oligoethylene glycol (OEG) unit of the protractor handle, yielding AF647-labeled Compound 102 (-93% purity by HPLC). The AF647-labeled Compound 102 retained functional activity comparable to unlabeled Compound 102, as demonstrated in a clot lysis assay, confirming that the fluorescent label did not interfere with the peptide's biological properties. HSA (>99% purity, Sigma-Aldrich, catalog number A3782) was extensively dialyzed against phosphate-buffered saline buffer, pH 7.4 (PBS). For the binding experiments, HSA was serially diluted in PBS to generate final concentrations of 0, 1, 2, 5, and 10 mg / mL, spanning the range from complete absence to near-physiological levels. Each HSA dilution was mixed with AF647-labeled Compound 102 to achieve a final peptide concentration of 1.5 pM, and samples were incubated at room temperature for 30 minutes.

[0200] SEC-HPLC analysis was performed using an AdvanceBio SEC 300A 2.7 pm, 4.6 x 300 mm (Agilent, Catalog number PL1580-5301) installed on an Agilent HPLC 1260 Infinity II equipped with a fluorescence detector. Detection was performed at 650 nm excitation wavelength to specifically monitor the AF647-labeled Compound 102. The mobile phase consisted of PBS, pH 7.4, delivered isocratically at a flow rate of 0.35 mL / min. The column was maintained at room temperature (approximately 20-25°C), and 10 pL of each sample was injected for analysis. Control samples of AF647-labeled Compound 102 alone (0 mg / mL HSA) and HSA alone (detected at 280 nm excitation wavelength) were analyzed to establish baseline elution profiles and confirm the absence of interfering peaks.

[0201] As expected, in the absence of AF647-labeled Compound 102 (0 pM), HSA was not detected at 650 nm, whereas, in the absence of HSA (0 mg / mL), AF647-labeled Compound 102 eluted as a single, well-defined peak at a retention time of 11.4 minutes. As the HSA concentration was systematically increased from 0 to 10 mg / mL, concentration-dependent changes in the elution profile of AF647-labeled Compound 102 were observed. A new peak emerged at an earlier retention time of 8.7 minutescorresponding to the retention time of free HSA (detected separately using a 280 nm excitation wavelength). Simultaneously, the intensity of the free peptide peak at 11.4 minutes progressively decreased, indicating a shift in equilibrium from unbound to bound AF647-labeled Compound 102. At 1 mg / mL HSA, the chromatogram displayed two distinct peaks - a minor peak at 11.4 minutes representing residual free AF647-labeled Compound 102 and a major peak at 8.7 minutes representing the AF647-labeled Compound 102-HSA complex, demonstrating that substantial binding occurs even at HSA concentrations well below physiological levels. As the HSA concentration was further increased to 2 mg / mL and 5 mg / mL, the free AF647-labeled Compound 102 peak continued to diminish while the bound complex peak increased proportionally. At HSA concentrations of 10 mg / mL, the bound AF647-labeled Compound 102 peak at 8.7 minutes dominated the chromatogram with minimal to no detectable free AF647-labeled Compound 102 peak at 11.4 minutes, indicating near-complete saturation of AF647- labeled Compound 102 with HSA under these conditions. The retention time of the bound complex remained constant at 8.7 minutes across all HSA concentrations where binding was observed, confirming the formation of a stable complex. Results are shown in Figure 14, which shows representative SEC-HPLC chromatograms at each HSA concentration tested.

[0202] Qualitative analysis of chromatographic peak areas shows that at physiologically relevant HSA concentrations (i.e., 35 - 55 mg / mL), more than 95% of AF647-labeled Compound 102 is albumin-bound. This supports the conclusion that Compound 102 will also circulate predominantly in the albumin-bound form in vivo. This high degree of binding to HSA is expected to confer significant pharmacokinetic advantages, including extended serum half-life through reduced renal clearance (as the ~67 kDa peptide-HSA complex exceeds the glomerular filtration threshold).Example 13 - Molecular modeling of Compound 102 bound to plasmin and albuminPredicting the minimum length of the linker between the fatty acid and peptide moieties allowing for simultaneous binding to albumin and plasmin:

[0203] The objective of this study was to generate a ternary molecular model comprising full-length plasmin, Compound 102, and human serum albumin (HSA) and from this predicting the minimum length of the linker between the fatty acid and peptide moieties allowing for simultaneous binding to albumin and plasmin.

[0204] Structural model of Compound 102 in complex with p-plasmin'. The structure of the SFTIv3 in complex with p-plasmin (PDB: 6D3X) served as the starting template for modeling the peptidic region of Compound 102. To match the Compound 102 sequence, two substitutions were introduced into the SFTI scaffold: (i) substitution of Phel2 with Tyrl2, and (ii) substitution of Prol3 with 4-amino-Prol3 (4NHPgam). These modifications were applied using PyMOL (PyMOL Molecular Graphics System v2.0, Schrodinger). From the modified 4-amino-Prol3 position, the model was extended using Maestro (Schrodinger) by sequentially adding six OEG (8-amino-3,6-dioxaoctanoic acid) units, two gGlu residues, and a terminal C18DA (diacid) moiety. In total, it corresponds to the C18DA-2xgGlu-6xOEG protractor of Compound 102. The resulting complex of Compound 102 with p-plasmin underwent energy minimization followed by a 0.5-ps molecular dynamics (MD) relaxation.

[0205] Structural model of Compound 102 bound to HSA'. To model the interaction between free Compound 102 and HSA, the crystal structure of HSA bound to a Cl 8 fatty acid (PDB: 1E7I) was used as the docking template for the C18DA moiety. The 1E7I structure contains seven C18 fatty-acid binding pockets. Three of these pockets were populated with Compound 102 by aligning its terminal C18 moiety to the corresponding Cl 8 ligands in the template. Energy minimization and a subsequent 0.5-ps MD relaxation showed that the protractor moiety of Compound 102 formed stable interactions within the albumin binding pocket. Throughout the simulations the peptidic region of Compound 102 remained solvent-exposed and sterically accessible for binding to p-plasmin.

[0206] Structural model of Compound 102 with HSA and plasmin'. To generate a ternary complex of Compound 102 bound to albumin and plasmin, an initial model of the ternary complex was constructed following the same methodology as outlined above. Subsequently, the X-ray structure of full-length human plasminogen (PDB: 4DUU) was aligned to the p-plasmin protease domain using PyMOL to incorporate full-length plasmin in the ternary complex model. A 2-ps MD simulation of this ternary assembly demonstrated that Compound 102 could simultaneously engage plasmin while remaining bound to HSA.

[0207] In one observed conformation (Figure 15), the six OEG units adopt a semiextended arrangement spanning 28.3 A between the backbone nitrogen of the first OEG unit (N3, proximal to the second gGlu residue) and the terminal backbone carbon of the sixth OEG unit (C24 of C=OH, proximal to plasmin). In addition, the distance betweenthe carboxyl carbons of the C18DA moiety and the second gGlu residue is 8.5 A. In this conformation, both gGlu side chains are fully visible and solvent-exposed, while the C18DA is completely inserted into the fatty-acid binding pocket of HSA. The two additional C18DA modeled at the other HSA fatty-acid binding pockets are likewise fully inserted, leaving all gGlu side chains exposed to solvent. Although the gGlu residues lie near HSA residues due to their proximity to the C18DA, they remain fully solvent- exposed, and no component of the OEG chain inserts into any HSA fatty-acid binding pocket. While OEG units are formally neutral, each unit contains two ether oxygens and a carbonyl group with a polarized C=O bond, where the oxygen carries a partial negative charge and the adjacent carbon a partial positive charge. In the model, however, the ether oxygen and carbonyl oxygen of the first OEG unit is positioned >3.5 A away from the nearest HSA atom, a distance at which electrostatic effects arising from weakly polar oxygen are expected to be strongly attenuated under physiological ionic strength. Moreover, in all three modeled HSA fatty-acid binding pockets, most atoms within the first OEG unit lie outside van der Waals contact distance (>3.5 A). Given the high flexibility and strong solvation of the OEG units / chain, these atoms are expected to contribute at most only weak, transient interactions with HSA. Taken together, the following truncations of Compound 102 are sterically plausible while still allowing formation of a ternary complex between plasmin, the modified Compound 102, and HSA:• Removal of the first OEG unit: five OEG units remain, with a separation of 26.7A between the backbone nitrogen of the second OEG unit (N4) and C24 of the sixth OEG unit.• Removal of the first two OEG units: four OEG units remain, with a separation of23.3 A between the backbone nitrogen of the third OEG unit (N5) and C24.• Removal of the first three OEG units: three OEG units remain, with a separation of 17.9 A between the backbone nitrogen of the fourth OEG unit (Nl) and C24.• Removal of the first four OEG units: two OEG units remain, with a separation of12.4 A between the backbone nitrogen of the fifth OEG unit (N2) and C24.• Removal of the first five OEG units: one OEG unit remains, with a separation of 6.5 A between the backbone nitrogen of the sixth OEG unit (N3) and C24.• Removal of all six OEG units: direct linkage of 4-amino-Prol3 to the carboxyl carbon (C28) of the second gGlu, resulting in an 9.3 A distance between 4-amino- Prol3 and the C18DA carboxyl carbon.• Removal of all six OEG units and the second gGlu: direct linkage of 4-amino- Prol3 to the carboxyl carbon (C23) of the first y-Glu, resulting in a 5 A distance between 4-amino-Prol3 and the C18DA carboxyl carbon. This configuration maintains a minimum linker length of at least 5 A while still permitting the modified Compound 102 to engage both plasmin and HSA. However, because the three components are brought into close spatial proximity and approaching typical van der Waals contact distances, the structural model indicates that the ternary complex can still form and allow inhibition of plasmin, but with reduced flexibility and suboptimal geometry. Consequently, this configuration supports the use a linker length of 5 A.Example 14 - Effect on thrombin generation in human platelet rich plasma

[0208] The objective of this study was to investigate the effect of Compound 102 on the intrinsic and extrinsic coagulation pathways. The study was performed in human platelet rich plasma using thrombin generation as a readout.

[0209] Thrombin generation in plasma was assessed using a calibrated assay based on the cleavage of a thrombin-specific fluorogenic substrate. To prepare the samples, 3.2% (v / v) citrated whole blood from healthy human donors was processed to obtain both plateletrich plasma (PRP) and platelet-poor plasma (PPP). These two plasma fractions were then combined to achieve a final platelet concentration of 250 * 109platelets / L prior to use in the assay.

[0210] For each sample, two parallel measurements were carried out. The first measurement assessed endogenous thrombin generation in designated reaction wells. The second measurement utilized an a2-macroglobulin-thrombin calibrator in separate calibrator wells, which served as a reference for accurate quantification. In the reaction wells, thrombin generation was triggered in 2:3 (v / v) diluted PRP by either 1 pM tissue factor (TF, Innovin; Dade Behring) and 3 nM recombinant thrombomodulin (TM;produced by Biointron), or 2 pg / ml kaolin (Sigma Aldrich, catalog no. KI 512) in presence or absence of Compound 102 (8 pM final concentration), anti -F VIII antibody (Sheep anti -Human Factor VIII, #PAHFVIII-S, Prolytix; 100 pg / ml final concentration)or Asundexian (BAY- 2433334, MedChemExpress catalog no. HY-137431; 80 pM final concentration). Samples were incubated at 37°C for 10 min to allow equilibration.Thrombin generation was then initiated with CaC12 (16.7 mM final concentration) and Z- Gly-Gly-Arg-AMC substrate (Bachem, catalog no. 4002155; 417 pM final concentration). Reactions were monitored for 120 minutes at 37°C with fluorescence detection at 460 nm and excitation at 390 nm and conversion of the measured signal to thrombin activity (nM) based on the calibrator using Thrombinoscope software (Diagnostica Stago).

[0211] As shown in Figures 16A-16C, Compound 102 did not affect thrombin generation triggered with TF in presence or absence of TM (extrinsic pathway; Figure 16A and 16B). Similarly, Compound 102 did not affect thrombin generation or with kaolin as trigger (intrinsic pathway; Figure 16C). In contrast, the presence of a polyclonal anti- FVIII antibody inhibited TF-triggered coagulation (Figures 16A and 16B) and a FXIa inhibitor (Asundexian) inhibited kaolin-triggered coagulation (Figure 16C) serving as controls for the sensitivity of thrombin generation to inhibition of components of the intrinsic and extrinsic coagulation pathways. The absence of any detectable inhibition or enhancement of thrombin generation in the presence of a high concentration of Compound 102 indicates that Compound 102 is unlikely to interfere with the normal physiological mechanisms of thrombin generation.Example 15 - Effect on platelet activation and aggregation in human platelet rich plasma

[0212] The objective of this study was to investigate the effect of Compound 102 on human platelet activation and aggregation.

[0213] Citrated whole blood from healthy human donors was centrifuged at 220g for 15 min at room temperature to obtain platelet-rich plasma (PRP). The remainder of the blood was centrifuged at 2,840g for 10 min at room temperature to obtain platelet-poor plasma (PPP) to serve as a blank in the aggregometer. Platelet count was adjusted to 250* 109plt / L platelet by combining PRP and PPP plasma fractions.

[0214] PRP was either untreated or spiked with Compound 102 (8 pM final concentration) or RGDW peptide (BIOSYNTH, catalog no. #PEP-00003932, 8 pM or 100 pM final concentration), which is an allbpill receptor antagonist. After incubation for 10 min at 37°C, platelet aggregation was initiated by either adenosine diphosphate(ADP; Diagnostica Stago, catalog no. 86923, 10 pM final concentration), thrombin receptor activating peptide-6 (TRAP-6; Diagnostica Stago, catalog no. 86926, 50 pM final concentration), or cross-linked collagen related peptide (CRP-XL, Triple Helical Peptides, 1 pg / ml final concentration). Platelet aggregation was measured by light transmission aggregometry on a TA-8 Thrombo-Aggregometer (Diagnostica Stago). The TA-8 aggregometer was calibrated using PPP as the reference for 100% light transmission and PRP as 0% transmission.

[0215] As shown in Figures 17A-17C, Compound 102 did not impact platelet activation and aggregation triggered by ADP (Figure 17A), TRAP-6 (Figure 17B), or CRP-XL (Figure 17C). In comparison, the antagonist peptide RGDW demonstrated a concentration-dependent inhibition of platelet aggregation. These findings show that Compound 102 is unlikely to disrupt the normal physiological processes of platelet activation and aggregation.OTHER EMBODIMENTS

[0216] While the present disclosure has been described in connection with specific embodiments and aspects thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the present disclosure following, in general, the principles of the present disclosure and including such departures from the present disclosure that come within known or customary practice within the art to which the present disclosure pertains and may be applied to the essential features set forth herein.

Claims

WHAT IS CLAIMED IS:

1. A peptide comprising an amino acid sequence GRCYKSKPPICX1X2D (SEQ ID NO: 1), wherein Xi and X2 are any amino acid or amino acid analogue, provided that if Xi is phenylalanine, then X2 is not proline.

2. The peptide of claim 1, wherein the peptide’s amino acid sequence consists of the amino acid sequence of SEQ ID NO: 1, wherein Xi and X2 are any amino acid or amino acid analogue, provided that if Xi is phenylalanine, then X2 is not proline.

3. The peptide of claim 1 or 2, wherein Xi is tyrosine and X2 is 4-amino proline (4NHPgam).

4. The peptide of claim 1 or 2, wherein Xi is selected from the group consisting of: phenylalanine, tyrosine, tryptophan, and lysine.

5. The peptide of any one of claims 1 to 4, wherein X2 is selected from the group consisting of: 4- amino proline (4NHPgam), a- Aminoisobutyric acid (Aib), or lysine.

6. The peptide of claim 1, comprising an amino acid sequence selected from the group consisting of: (a) GRCYKSKPPICYXD, wherein X is 4NHPgam (SEQ ID NO: 2); (b) GRCYKSKPPICYXD, wherein X is a-Aminoisobutyric acid (Aib) (SEQ ID NO: 9); (c) GRCYKSKPPICXPD, wherein X is lysine (SEQ ID NO: 10); (d) GRCYKSKPPICX1X2D, wherein Xi is lysine and X2 is a-Aminoisobutyric acid (Aib) (SEQ ID NO: 11); (e) GRCYKSKPPICFXD, wherein X is lysine (SEQ ID NO: 12); (f) GRCYKSKPPICYXD, wherein X is lysine (SEQ ID NO: 13); and (g) GRCYKSKPPICX1X2D, wherein Xi is lysine and X2 is Pro, Leu, Thr, He, Gly, Vai, or Nle (SEQ ID NO: 14).

7. The peptide of claim 1, consisting of an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOS: 2, 9, 10, 11, 12, 13, and 14.

8. The peptide of claim 7, consisting of the amino acid sequence of SEQ ID NO: 2.

9. The peptide of any one of claims 1 to 8, comprising a protractor group.

10. The peptide of claim 9, wherein if the protractor group is attached to a lysine, the lysine is Keps.

11. The peptide of claim 9, wherein the protractor group is conjugated to the amino acid residue at position 7, 8, 12, or 13 of SEQ ID NO: 1.

12. The peptide of claim 9, wherein the protractor group is conjugated to the amino acid residue at position 13 of SEQ ID NO: 1.

13. The peptide of any one of claims 9-12, wherein the protractor group comprises a fatty acid comprising between 13 and 21 carbons.

14. The peptide of claim 13, wherein the protractor group comprises a saturated diacid.

15. The peptide of claim 14, wherein the saturated diacid is selected from the group consisting of: tetradecanedioic acid (Cl 4 diacid), hexadecanedioic acid (Cl 6 diacid), octadecanedioic acid (Cl 8 diacid) and icosanedioic acid (C20 diacid).

16. The peptide of claim 14, wherein the saturated diacid is hexadecanedioic acid (Cl 6 diacid) or octadecanedioic acid (Cl 8 diacid).

17. The peptide of claim 14, wherein the saturated diacid is octadecanedioic acid (Cl 8 diacid).

18. The peptide of any one of claims 9 to 17, wherein the protractor group comprises a linker, and the fatty acid is attached to an amino acid residue of the peptide via the linker.

19. The peptide of claim 18, wherein the linker comprises [8-amino-3,6-dioxaoctanoic acid (OEG)]m, wherein m is an integer greater than or equal to 1.

20. The peptide of claim 19, wherein m is between 2 and 12.

21. The peptide of claim 20, wherein m is 2.

22. The peptide of claim 20, wherein m is 6.

23. The peptide of any one of claims 18 to 22, wherein the linker comprises a peptide linker.

24. The peptide of claim 23, wherein the peptide linker comprises [gGlu]n, wherein n is an integer greater than or equal to 1.

25. The peptide of claim 24, wherein n is 2.

26. The peptide of claim 24, wherein n is 6.

27. The peptide of claim 24, comprising SEQ ID NO: 2, wherein: the saturated diacid is octadecanedioic acid, and the linker comprises [gGlu]n[OEG]m wherein m is 2 or 6, and n is 2.

28. The peptide of claim 27, wherein m is 2.

29. The peptide of claim 27, wherein m is 6.

30. The peptide of any one of claims 27 to 29, wherein the octadecanedioic acid is conjugated to [gGlu]n.

31. The peptide of claim 23, wherein the linker comprises the amino acid sequence A(EAAAK)kA, where k is an integer greater than or equal to 1.

32. The peptide of claim 31, wherein k is 3.

33. The peptide of any one of claims 18 to 32, wherein the length of the linker is greater than or equal to about 5 A.

34. The peptide of any one of claims 18 to 33, wherein the length of the linker is from about 5 A to about 50 A.

35. The peptide of any one of claims 18 to 34, wherein the length of the linker is about 5 A, about 6 A, about 7 A, about 8 A, about 9 A, about 10 A, about 11 A, about 12 A, about 13 A, about 14 A, about 15 A, about 16 A, about 17 A, about 18 A, about 19 A, about 20 A, about 21 A, about 22 A, about 23 A, about 24 A, about 25 A, about 26 A, about 27 A, about 28 A, about 29 A, about 30 A, about 31 A, about 32 A, about 33 A, about 34 A, about 35 A, about 36 A, about 37 A, about 38 A, about 39 A, about 40 A, about 41 A, about 42 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 49 A, or about 50 A.

36. A peptide comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4- amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein m and n are independently integers greater than or equal to 1.

37. The peptide of claim 36, wherein the saturated diacid is octadecanedioic acid.

38. The peptide of claim 37, wherein m is 2 and n is 2.

39. The peptide of claim 37, wherein m is 6 and n is 2.

40. The peptide of claim 37, wherein m is 12 and n is 2.

41. The peptide of claim 37, wherein m is 6 and n is 1.

42. The peptide of claim 36, wherein the saturated diacid is hexadecanedioic acid.

43. The peptide of claim 42, wherein m is 2 and n is 2.

44. The peptide of claim 42, wherein m is 6 and n is 2.

45. The peptide of claim 42, wherein m is 12 and n is 2.

46. The peptide of claim 36, wherein the saturated diacid is tetradecanedioic acid.

47. The peptide of claim 46, wherein m is 6 and n is 2.

48. The peptide of any one of claims 36 to 47, wherein the length of the linker is greater than or equal to about 5 A.

49. The peptide of any one of claims 36 to 48, wherein the length of the linker is from about 5 to about 30 A.

50. The peptide of any one of claims 36 to 49, wherein the peptide inhibits clot lysis with IC50 values from about 1 to 5 pM.

51. The peptide of any one of claims 36 to 50, wherein the peptide inhibits clot lysis with IC50 values from about 1 to 2 pM.

52. A peptide comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4- amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a peptide linker comprising the amino acid sequence A(EAAAK)k A, where k is an integer greater than or equal to 1.

53. A peptide comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4- amino proline (4NHPgam) (SEQ ID NO: 2) and an acetyl group linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein m and n are independently integers greater than or equal to 1.

54. The peptide of claim 53, wherein m is 6 and n is 2.

55. The peptide of claim 53, wherein m is 2 and n is 2.

56. The peptide of any one of claims 1-55, comprising a disulfide linkage between the amino acid residues at positions 3 and 11 of SEQ ID NO: 1.

57. The peptide of any one of claims 1-56, which is cyclized in that the amino acid residue at position 1 is linked to the amino acid residue at position 14 of SEQ ID NO: 1.

58. The peptide of any one of claims 53 to 57, wherein the length of the linker is greater than or equal to about 5 A.

59. The peptide of any one of claims 53 to 58, wherein the length of the linker is from about 5 to about 30 A.

60. The peptide of any one of claims 53 to 59, wherein the peptide inhibits clot lysis with IC50 values from about 1 to 5 pM.

61. The peptide of any one of claims 53 to 60, wherein the peptide inhibits clot lysis with IC50 values from about 1 to 2 pM.

62. A peptide comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4- amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein the saturated diacid is octadecanedioic acid, m is 6, and n is 2.

63. A peptide comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4- amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein the saturated diacid is octadecanedioic acid, m is 2, and n is 2.

64. A peptide comprising an amino acid sequence GRCYKSKPPICYXD, wherein X is 4- amino proline (4NHPgam) (SEQ ID NO: 2) and a saturated diacid linked to the 4NHPgam at position 13 of SEQ ID NO: 2 via a linker comprising [OEG]mand [gGlu]n, wherein the saturated diacid is hexadecanedioic acid, m is 6, and n is 2.

65. A peptide comprising the structure set forth in Figure 5 A.

66. A peptide comprising the structure set forth in Figure 5B.

67. A pharmaceutically acceptable salt of a peptide of any one of claims 1-66.

68. A composition comprising the peptide of any one of claims 1-66 or the pharmaceutically acceptable salt of claim 67 and pharmaceutically acceptable carrier.

69. A method of promoting blood clot formation comprising a step of administering to a subject in need thereof an effective amount of the composition of claim 68.

70. The method of claim 69, wherein the subject is human.

71. The method of claim 69 or 70, wherein the subject has a blood clotting disorder.

72. The method of claim 71, wherein the subject has hemophilia.

73. The method of claim 71, wherein the subject has a disorder of hyperfibrinolysis.

74. The method of claim 71, wherein the subject has hereditary hemorrhagic telangiectasia.

75. The method of claim 69 or 70, wherein the subject is suffering from heavy menstrual bleeding.

76. The method of claim 69 or 70, wherein the subject is bleeding from a traumatic injury or from a surgical wound.

77. The method of any one of claims 69-76, comprising administering the effective amount of the pharmaceutical composition to the subject at regular intervals.

78. The method of claim 77, wherein the regular intervals are longer than a week.

79. The method of claim 78, wherein the regular intervals are longer than two weeks.

80. The method of claim 79, wherein the regular intervals are longer than three weeks.

81. The method of claim 79, wherein the regular intervals are at least a month.

82. The method of any one of claims 69-81, wherein the step of administering comprises systemic administration.

83. The method of claim 82, wherein the systemic administration comprises subcutaneous administration.

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