Hemostatic elastin-like polypeptide

Abstract

The present invention relates to hemostatic elastin-like polypeptides containing glutamine incorporated into the Q block sequence and, optionally, lysine incorporated into the K block sequence. Under physiological conditions, the Q block and K block sequences are recognized by human transglutaminase factor XIIIa and cross-linked to the fibrin network. The present invention also relates to medical uses of the polypeptides, nucleic acid sequences encoding the polypeptides, expression vectors containing the nucleic acid sequences, and cells containing the nucleic acid sequences or the expression vectors.

Description

[Technical Field] 【0001】 This application claims priority to European Patent Application No. 20191629, filed on 18 August 2020, which is incorporated herein by reference. 【0002】 The present invention relates to a hemostatic elastin-like polypeptide comprising glutamine incorporated into a Q block sequence and lysine optionally incorporated into a K block sequence. Under physiological conditions, the Q block sequence and the K block sequence are recognized by human transglutaminase factor XIIIa and crosslinked with a fibrin network. The present invention also relates to the medical use of the polypeptide, a nucleic acid sequence encoding the polypeptide, an expression vector comprising the nucleic acid sequence, and cells having the nucleic acid sequence or the expression vector. [Background technology] 【0003】 Severe trauma is a leading cause of death for people under 45, and is projected to account for as many as 8.4 million deaths annually in 2020. Many of these deaths are due to the inability to control bleeding. In cases of massive bleeding, clotting factors are rapidly depleted at the site of injury, leading to a condition called trauma-induced coagulopathy (TIC) in as many as 25% of trauma patients, which is associated with an increased mortality rate. 【0004】 Hemostasis occurs in two stages. In the first stage, circulating platelets are activated and aggregated at the injury site, causing the formation of a platelet plug. In the second stage, fibrin (Fb) polymerizes to form an insoluble protein hydrogel (=clot), which provides structural support and obstructs blood flow. In this second stage, activated thrombin cleaves fibrinopeptides from the precursor protein fibrinogen (Fg), and when sequences called knobs A and B appear, an Fb network is formed. These knobs covalently bind to sites called holes A and B at the distal ends of adjacent Fb / Fg, allowing Fb to self-associate in a half-staggered structure to form protofibrils. These protofibrils then bundle together to form fibers, ultimately creating an insoluble Fb network. Subsequently, the Fb network is stabilized by covalent crosslinks formed by the reaction of lysine and glutamine residues catalyzed by activated, aggregate-associated transglutaminase FXIIIa, and further hardened by the contractile force exerted by platelets distributed throughout the network. Therefore, both Fb and FXIIIa are important players in hemostasis and can be considered effective molecular targets of the hemostatic control system. 【0005】 Targeting Fb using synthetic systems is difficult because it is challenging to obtain specific high-affinity binders that can distinguish between gelled Fb and circulating Fg. Since Fb clots and soluble Fg share sequence and structural homology, only a very small number of three-dimensional epitopes exist that serve as the basis for molecular identification. Nevertheless, we have succeeded in isolating Fb-specific binders using phage display. This has led to the development of Fb-targeted hemostatic agents by grafting Fb-binding peptides or nanobodies onto synthetic polymers or particles to support in vivo clot formation. Alternatively, Fb has also been targeted by engaging with endogenous hole a and b binding pockets via knob A and B mimetic compounds. By attaching these peptide mimetic compounds to larger molecules such as polyethylene glycol (PEG) polymers or proteins, various structures have been realized that allow for alteration of Fb's mechanical properties, modification of the Fb network structure, or targeted delivery of therapeutic agents to Fb gels. [Overview of the project] [Problems that the invention aims to solve] 【0006】 Based on the latest technologies described above, the object of the present invention is to provide means and methods for achieving hemostasis. [Means for solving the problem] 【0007】 This objective is achieved by the subject matter of the independent claims herein. [Brief explanation of the drawing] 【0008】 [Figure 1]Figure 1 schematically shows the design of hELP and its integration into Fb clot. (a) hELP was designed as a triblock copolymer containing a Q block, a phase separation block, and a K block. (b) Above the lower critical solution temperature (LCST), hELP phase-separates to form coacervates, which can be covalently cross-linked by FXIIIa. (c) When mixed with fibrinogen, thrombin, and FXIII, the coacervates of hELP are covalently integrated into the Fb network, leading to a mutually dependent improvement in mechanical strength, gelation kinetics, a decrease in the plasmin degradation rate, and a decrease in the pore size of the Fb network. [Figure 2] Figure 2 shows the cloud point and cross-linking via FXIIIa of hELP and control ELP (conELP). (a) The cloud point was measured for a 30 μM solution of hELP or conELP in 20 / 150 mM HEPES / NaCl + 20 mM CaCl2. The cloud point was defined as the temperature at which the normalized transmittance was less than 0.5. Data are mean ± SD (shaded) (n = 2). (b) SDS-PAGE gels are shown after incubating 50 μM of hELP or conELP with 10 μg mL−1 of human FXIIIa at 37 °C for 1 h. The arrow indicates the position corresponding to the hELP dimer. [Figure 3] Figure 3 shows the structural morphology of the hELP-Fb clot. (a) Two-color confocal fluorescence microscopy observations of Fb clots formed at 22 °C or 37 °C with f-conELP, f-hELP, or HEPES buffer as additives are shown. The scale bar is 30 μm. (b) Comparison of Pearson's correlation coefficients quantifying the spatial co-localization of signals from the Fb (green) or ELP (red) channels in confocal fluorescence images is shown. (c) Pore size measurements of Fb clots containing hELP, conELP, or HEPES buffer are shown. Statistical significance was determined using one-way ANOVA and Tukey's post hoc test. *P < 0.05, ***P < 0.001. All data in panels b and c are shown as mean ± SD (n = 3). [Figure 4]Figure 4 shows the gelation kinetics of Fb clots in the presence of hELP or conELP. a) Absorbance over the wavelength range of 500–800 nm over 37°C for gelation of 2.2 mg mL-1 Fb clots containing 30 μM hELP, conELP, or HEPES buffer. Measurements were taken at 1-minute intervals over 1 hour. b) Coagulation onset time (R) measured by thromboelastography at 37°C, c) Alpha angle, and d) Maximum amplitude (MA) of Fb clots containing 30 μM hELP, conELP, or HEPES buffer. Data for panels b, c, and d are shown as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001: One-way ANOVA and Tukey's post-hoc test. [Figure 5] Figure 5 shows the mechanical properties and in-vitro thromboelastography properties of hELP-Fb clots. a) Mean shear storage modulus of Fb gels containing either 30 μM hELP, conELP, or the same amount of HEPES buffer is shown. After forming the gel between the rheometer cone and plate at either 22°C or 37°C, frequency sweeps were performed at 0.1–3 Hz with a 1% strain. The dotted line shows the stiffness of the critical physiological threshold corresponding to the mean shear storage modulus of a 2.2 mg mL-1 Fb HEPES control gel. b) Strain sweeps of 0.1–100% (f=1 Hz) of a 2.2 mg mL-1 Fb gel containing 30 μM hELP, conELP, and HEPES buffer at 37°C are shown. All panel data are shown as mean ± SD (n=3). **P<0.01, ***P<0.001; One-way ANOVA and Tukey's post-hoc test. [Figure 6]Figure 6 shows the degradation of Fb clot by plasmin. a) Time-lapse confocal images after exposing 1.5 mg mL-1 Fb clot containing 30 μM hELP, conELP, or HEPES buffer to 10 μg mL-1 plasmin at 37 °C. b) Quantification of the percentage of lysed clot over time measured from confocal images of multiple clots (n = 3) for 1.5 mg mL-1 Fb clot containing 30 μM hELP, conELP, or HEPES buffer. [Figure 7] Figure 7 shows the size distribution of coacervates of hELP in 1.5 mg mL-1 Fb clot formed at 37 °C. The particle size was determined from images of three different hELP-containing Fb clots. [Figure 8] Figure 8 shows the differences in the volume flow rate of isotonic HEPES buffer through Fb clots containing HEPES buffer, 30 μM conELP, or 30 μM hELP at 22 °C or 37 °C. Statistical significance was determined by one-way analysis of variance (ANOVA) and Tukey's post hoc test. ***P < 0.001. [Figure 9] Figure 9 shows the storage modulus (G') and loss modulus (G'') measured over 1 hour at 37 °C for 2.2 mg mL-1 Fb clot containing (a) HEPES, b) 30 μM conELP, c) 30 μM hELP, d) 20 μM hELP, e) 10 μM hELP, and f) 5 μM hELP. The clot was subjected to a constant oscillatory shear stress (γ = 0.1%; f = 1 Hz). The shaded area indicates one standard deviation from the mean (n = 3). [Figure 10] Figure 10 shows the cell viability of human dermal fibroblasts (neonatal; HDFn) after 24-hour exposure to various concentrations of purified hELP and conELP under physiological conditions, compared to a control containing only medium. Data are shown as mean ± SD (n = 4). **P < 0.01; one-way analysis of variance and Tukey's post hoc test. [Figure 11]Figure 11 shows the survival curves of rats after intravenous administration of approximately 140 mg kg-1 of hELP (4Tg-4Tg) or inactive conELP (see Example 10) in a femoral artery injury bleeding model. The first 15 minutes of the experiment (after the release of the clamp surrounding the femoral artery injury) were allowed as a free bleeding period, after which fluid resuscitation was performed to maintain the MAP above 60 mm Hg for as long as possible. Animals were euthanized when the MAP fell below 20 mm Hg. The hELP polymer was effective compared to conELP at a significance level of p=0.0554 (log-rank Cox-Mantel test). [Modes for carrying out the invention] 【0009】 Summary of the Invention A first aspect of the present invention is: a. i. DQMMLPWPAVAL(Sequence ID 003), ii. WQHKIDLRYNGA (Sequence ID 004), iii. SQHPLPWPVLML (Sequence ID 005), iv. EQFPIAFPRYSI (Sequence ID 006), v. SEQHLLKWPPWH (Sequence ID 007), vi. WQIPVDWPPLPP(Sequence ID 008), vii. DQWMMAWPSLTL (Sequence ID 009), and / or viii. SQIPMAWPLLSL (Sequence ID 010), A Q-block amino acid sequence selected from, b. Multiple spacer amino acid sequences VPGXG (SEQ ID NO: 012); c. Optionally, a K-block sequence containing at least one lysine residue, Regarding polypeptides including [specific polypeptides]. 【0010】 Each X within any single spacer amino acid sequence can be independently selected from any nascent protein amino acid except Pro. 【0011】 A second aspect of the present invention relates to a polypeptide according to the first aspect for use in the treatment or prevention of hemostatic disorders, excessive bleeding, or coagulation disorders. 【0012】 A third aspect of the present invention relates to a nucleic acid sequence encoding a polypeptide according to the first aspect. 【0013】 Another aspect of the present invention relates to an expression vector comprising a nucleic acid sequence according to a third aspect. 【0014】 Yet another aspect of the present invention relates to isolated cells comprising a nucleic acid sequence according to a third aspect of the present invention, or an expression vector according to a fourth aspect of the present invention. 【0015】 Detailed description of the invention Terms and Definitions For the purposes of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular form also include the plural form, and vice versa. In the event of any conflict between the following definitions and any documents incorporated herein by reference, the defined definition shall prevail. 【0016】 The terms “comprising,” “having,” “containing,” “including,” and other similar forms, as used herein, and their grammatical equivalents, are intended to be semantically equivalent and open-ended, in that one or more items following any one of these words do not exhaustively list or limit the one or more items listed. For example, an item “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or it may contain one or more other components in addition to components A, B, and C. Thus, it is intended and understood that “comprise” and its similar forms, and their grammatical equivalents, include disclosures of embodiments that “consisting essentially of” or “consisting of.” 【0017】 Where a range of values ​​is provided, unless otherwise explicitly indicated in the context, each intermediary value up to one-tenth of the lower limit between the upper and lower limits of that range, and each of the other stated or intermediary values ​​within that range, are understood to be included in this disclosure, subject to the limits specifically excluded within the stated range. If the stated range includes one or both of the limit values, the range excluding one or both of the limit values ​​that they include is also included in the disclosure. 【0018】 In this specification, any reference to a value or parameter using the term "about" includes (and describes) variations relating to that value or parameter itself. For example, any description referring to "about X" includes any description of "X". 【0019】 As used herein, including in the attached claims, the singular “a,” “or,” and “the” include plural references unless otherwise clearly indicated by the context. 【0020】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (generally Sambrook et al., Molecular Cloning: A Laboratory Manual, Part 4 (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002), Part 5, John Wiley & Sons, Inc.) and chemical methods. 【0021】 In the context of this specification, the term "ELP" refers to elastin-like polypeptides. 【0022】 In the context of this specification, the term "Fb" refers to fibrin. 【0023】 In the context of this specification, the term "hELP" refers to hemostatic ELP. 【0024】 In the context of this specification, the term "polypeptide" refers to a molecule consisting of 30 or more amino acids that form a linear chain linked by peptide bonds. The amino acid sequence of a polypeptide may also represent the amino acid sequence of an entire protein or a fragment thereof (as found physiologically). The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. 【0025】 In the context of this specification, the term "peptide" refers to a molecule consisting of up to 50 amino acids, more particularly 8 to 30 amino acids, and more specifically 8 to 15 amino acids, in which amino acids form a linear chain linked by peptide bonds. 【0026】 The sequence of amino acid residues is written from the amino terminus to the carboxyl terminus. Uppercase letters indicating the sequence position refer to the single-letter code for the L-amino acid (Stryer, Biochemistry, Part 3, p. 21). Lowercase letters indicating the position of the amino acid sequence represent the corresponding D- or (2R)-amino acid. The sequence is written from left to right, from the amino terminus to the carboxyl terminus. Following standard nomenclature, the sequence of amino acid residues is represented by either a three-letter or one-letter code, as follows: Alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). 【0027】 In the context of this invention, the term "specific binding" refers to the properties of a ligand, which binds to its target with a certain affinity and target specificity. Such ligand affinity is indicated by the ligand's dissociation constant. A ligand that reacts specifically has a dissociation constant of 10 when bound to its target. -7 Although the dissociation constant is less than mol / L, interactions with molecules that have the same overall chemical composition as the target but different three-dimensional structures exhibit a dissociation constant at least three orders of magnitude higher. It is noteworthy that, since the polymer according to the present invention is covalently bonded, it does not possess "reversible" bonds to Fb / Fg and therefore is not characterized by a dissociation constant. The affinity of the enzyme (FXIIIa) to the polymer, characterized by the enzyme's Km value (Mikeliss-Menten constant), can be formulated. 【0028】 A "polymer" of a given group of monomers is a homopolymer (composed of multiple identical monomers; these monomers have either a Q-block sequence or a K-block sequence), and a copolymer of a given selection of monomers is a heteropolymer composed of monomers from at least two groups. 【0029】 As used herein, the term “pharmaceutical composition” refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, accompanied by at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral, or infusion administration. 【0030】 As used herein, the term “pharmaceutically acceptable carrier” includes, as is known to those skilled in the art, any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic, absorption retarder, salt, preservative, drug, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, etc., and combinations thereof (see, for example, Remington: The Science and Practice of Pharmacy, ISBN 0857110624). 【0031】 As used herein, the terms “treatment” or “to treat” any symptom, disease, or disorder (e.g., impaired hemostasis) mean, in one embodiment, improving the disease or disorder (e.g., delaying, preventing, or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, “treatment” or “to treat” means alleviating or improving at least one physical parameter, including one that may not be identifiable by the patient. In yet another embodiment, “treatment” or “to treat” means modulating the disease or disorder physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for evaluating the treatment and / or prevention of diseases are generally known in the art unless specifically described below. 【0032】 This invention relates to an intrinsically disordered protein based on an elastin-like polypeptide (ELP) sequence that specifically binds to fibrin and modulates its mechanical properties. The inventors designed a hemostatic ELP (hELP) containing peptide tags at the N-terminus and C-terminus by introducing glutamine and lysine residues at the N-terminus and C-terminus of the ELP, respectively. The peptide tags (Q-block sequence, K-block sequence) are selectively recognized by human transglutaminase factor XIIIa and covalently linked to the fibrin network via the innate coagulation cascade. Phase separation of the hELP above the lower critical solution temperature (LCST) resulted in rigidification under conditions simulating dilutional coagulation disorders, leading to the salvage of the biophysical properties of the clot. In addition to phase-dependent hardening, the resulting hELP-Fb network exhibited resistance to plasmin degradation, reduced pore size, and accelerated gelation rate after coagulation initiation. 【0033】 A first aspect of the present invention relates to a polypeptide comprising or consisting of one or more of the following components: a. i. DQMMLPWPAVAL(Sequence ID 003), ii. WQHKIDLRYNGA (Sequence ID 004), iii. SQHPLPWPVLML (Sequence ID 005), iv. EQFPIAFPRYSI (Sequence ID 006), v. SEQHLLKWPPWH (Sequence ID 007), vi. WQIPVDWPPLPP(Sequence ID 008), vii. DQWMMAWPSLTL (Sequence ID 009), and / or viii. SQIPMAWPLLSL (Sequence ID 010), Q block array selected from, b. Multiple spacer sequences of the VPGXG sequence (sequence number 012); c. Optionally, a K-block sequence containing at least one lysine residue. 【0034】 "Guest residue X" in the spacer sequence In principle, each X can be independently selected from any proteinogenesis amino acid other than Pro. 【0035】 In a particular embodiment, each X is independently selected from Ala, Val, and Glu. 【0036】 In certain embodiments, the Ala:Val:Glu ratio used for X is 1-3 Ala:7-10 Val:1 Glu. 【0037】 In certain embodiments, the Ala:Val:Glu ratio used for X is approximately 2:8:1 to 2:9:1. 【0038】 The Ala:Val:Glu ratio and the exact amino acids of the X residues are highly flexible and common. The ratio and order of X residues within the molecule can vary depending on how the polymer's temperature / pH responsiveness is tuned. The relative ratio of X residues is considered important in determining the pH and temperature-dependent phase transitions of the molecule. In certain embodiments, any of the classical substitutions of Glu, Ala, or Val may be made in a small number of cases. The classical substituent of Glu is Asp. The classical substitutions of Ala are Gly, Val, Ser, and Thr. The classical substitutions of Val are Ala, Leu, Thr, or Ile. In certain embodiments, a small number of cases means less than 30%. In certain embodiments, a small number of cases means less than 20%. In certain embodiments, a small number of cases means less than 10%. In certain embodiments, a small number of cases means less than 5%. 【0039】 Since the ELP transfer temperature is determined by both the length and composition of the guest residues, too many pentapeptides may result in an ELP that is too long to be effectively expressed. There is a range of feasible compositions and lengths in which the ELP can be expressed and transferred at temperatures above room temperature but below, at, or not significantly above, physiological temperature. 【0040】 Typical lengths of polypeptides according to the present invention include, but are not limited to, 90 to 1,340 amino acid lengths, which correspond to molecular weight values ​​of 10,000 to 150,000 grams / mol. 【0041】 The X parameter (composition of guest residues) does not affect the range of possible lengths. For a given X residue composition, both long and short ELPs can be produced. However, solubility is a crucial aspect, controlled by a combination of factors including the guest residue composition, length, and buffer composition. For example, when using hydrophobic guest residues, they cannot be made too long, as they will become insoluble in water and will not be expressed in E. coli (see below / next page for how this affects the transition temperature). 【0042】 In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 27°C and 47°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 32°C and 42°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 34°C and 40°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 35°C and 39°C. 【0043】 In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 27°C and 37°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 32°C and 37°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 35°C and 37°C. 【0044】 In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 37°C and 47°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 37°C and 42°C. In certain embodiments, the compounds of the present invention are characterized by a transition temperature between 37°C and 39°C. 【0045】 Those skilled in the art will recognize that there are clear rules that can be followed to adjust the transition temperature. For example, adding a large amount of hydrophobic amino acids (A, I, L, M, F, W, Y, or V) at the guest residue X position will lower the transition temperature. Adding charged or hydrophilic amino acids (R, H, K, D, E, S, T, N, Q) tends to raise the transition temperature. To determine the transition temperature, each composition is produced in E. coli and tested by a cloud point assay, in which absorbance is measured essentially as a function of temperature from about 15°C to 100°C (see Method 2 in the Examples below). 【0046】 Without being bound by scientific hypotheses, the inventors propose that the actual spacer arrangement is not very important, and that the defined ratio of Ala:Val:Glu used in X is one way to solve the problem underlying the invention. In addition to the polypeptide length, the Ala:Val:Glu ratio determines the phase separation with respect to physiological temperature. 【0047】 The inventors designed an hELP with a transition temperature of less than 37°C that drives aggregate / nanoparticle formation at physiological temperatures. 【0048】 The data obtained so far does not indicate that it is particularly important for all repeats to have the same sequence. Each may have a different ratio / composition of X residues. 【0049】 Q block arrangement In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 003. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 003, and additionally a spacer sequence identified by SEQ ID NO: 012. 【0050】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 004. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 004, and additionally a spacer sequence identified by SEQ ID NO: 012. 【0051】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 005. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 005, and additionally a spacer sequence identified by SEQ ID NO: 012. 【0052】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 006. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 006, and additionally a spacer sequence identified by SEQ ID NO: 012. 【0053】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 007. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 007 and an additional spacer sequence identified by SEQ ID NO: 012. 【0054】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 008. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 008, and additionally a spacer sequence identified by SEQ ID NO: 012. 【0055】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 009. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 009 and an additional spacer sequence identified by SEQ ID NO: 012. 【0056】 In certain embodiments, the polypeptide according to the present invention comprises a Q-block sequence identified by SEQ ID NO: 010. In particular, in certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 010, and additionally a spacer sequence identified by SEQ ID NO: 012. 【0057】 In certain embodiments, the polypeptide comprises two or more Q-block sequences. 【0058】 In certain embodiments, the polypeptide contains two or more identical Q-block sequences. 【0059】 In certain embodiments, the polypeptide comprises two or more different Q-block sequences. 【0060】 In certain embodiments, the polypeptide includes two or more Q-block sequences and spacer sequences, but does not include K-block sequences. 【0061】 In particularly specific embodiments of any of the embodiments disclosed herein and general embodiments, the Q block sequence is DQMMLPWPAVAL (Sequence ID 003). 【0062】 In certain embodiments, the polypeptide contains 2 to 50 Q-block sequences. In certain embodiments, the polypeptide contains 2 to 8 Q-block sequences. In certain embodiments, the polypeptide contains 3 to 6 Q-block sequences. In certain embodiments, the polypeptide contains 4 Q-block sequences. 【0063】 In certain embodiments, the polypeptide consists essentially of only a Q block sequence and a spacer sequence. The Q block sequence may be flanked by short (1-3, particularly 2 amino acid) framing sequences. In certain embodiments, these framing sequences are GS. Framing sequences, in particular GS spacers, are typically used in protein engineering as inert and soluble "flexible spacers." 【0064】 In more specific embodiments, the polypeptide consists essentially only of Q-block sequences and spacer sequences, and the polypeptide comprises the following: N-terminal Q-tract represented by -(VPGXG)n-[(Q-block)-(VPGXG)n]m - -[(Q block)-(VPGXG)n]m-(VPGXG)o C-terminal Q tract - A spacer array polymer [(VPGXG)n]p that separates the N-terminal Q-tract and the C-terminal Q-tract, in the formula, - Each n is an integer between 8 and 14, especially between 10 and 12, independently of any other n; - Each m is an integer between 2 and 8, especially between 3 and 6, independent of any other m, and more particularly m is 4; - o is an integer between 0 and 10; - p is an integer between 3 and 6, and in particular p is 4 or 5. 【0065】 In more specific embodiments, the polypeptide is a sequence having at least 95% sequence identity to or from SEQ ID NO: 16, and possessing at least 80% of the biological activity as defined elsewhere herein. 【0066】 Q-block array and K-block array In certain embodiments, the polypeptide comprises two or more Q-block sequences, spacer sequences, and K-block sequences. 【0067】 The Q-block sequence and the K-block sequence are selectively recognized and crosslinked by human transglutaminase factor XIIIa. Crosslinking occurs between two polypeptides of the present invention (also known as hELPs), or between one polypeptide of the present invention and a fibrin molecule. This integrates the polypeptides of the present invention into a fibrin network. Both the Q-block sequence and the K-block sequence may be crosslinked with fibrin. 【0068】 In certain embodiments, the polypeptide essentially consists of the above-described Q-block sequence, K-block sequence, and multiple spacer sequences. In certain embodiments, the Q-block sequence is located at the N-terminus of the polypeptide, and the K-block sequence is located at the C-terminus of the polypeptide. In certain embodiments, the K-block sequence is located at the N-terminus of the polypeptide, and the K-block sequence is located at the C-terminus of the polypeptide. 【0069】 In certain embodiments, the polypeptide contains a K-block sequence. In certain embodiments, the polypeptide contains the K-block sequence GSKGS (sequence number 011). In certain embodiments, the polypeptide contains two or more K-block sequences GSKGS (sequence number 011). 【0070】 In certain embodiments, the polypeptide contains 2 to 50 K-block sequences. In certain embodiments, the polypeptide contains 2 to 8 K-block sequences. In certain embodiments, the polypeptide contains 3 to 6 K-block sequences. In certain embodiments, the polypeptide contains 4 K-block sequences. 【0071】 In certain embodiments, the polypeptide comprises 2 to 8 Q-block sequences and 2 to 8 K-block sequences independently of each other. In certain embodiments, the polypeptide comprises 3 to 6 Q-block sequences and 3 to 6 K-block sequences independently of each other. In certain embodiments, the polypeptide comprises 4 Q-block sequences and 4 K-block sequences independently of each other. 【0072】 In certain embodiments, the polypeptide consists essentially of a Q block sequence and a spacer sequence. In certain embodiments, the polypeptide consists essentially of a Q block sequence, a spacer sequence, and a K block sequence. 【0073】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q-block sequences identified by SEQ ID NO: 003, a K-block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0074】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 004, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0075】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 005, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0076】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 006, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0077】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 007, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0078】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 008, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0079】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 009, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0080】 In certain embodiments, the polypeptide according to the present invention comprises a plurality of Q block sequences identified by SEQ ID NO: 010, a K block sequence identified herein, and an additional spacer sequence identified by SEQ ID NO: 012. 【0081】 In certain embodiments, the spacer sequence forms a continuous amino acid chain without the interposition of sequences that are neither Q-block nor K-block sequences. In other words, a spacer sequence is followed by another spacer sequence, interrupted only by a Q-block or K-block sequence. Essentially, there are no other components of the polypeptide other than the Q-block, spacer, and K-block sequences. 【0082】 The number of K blocks and Q blocks does not need to be the same. In some embodiments, the number of K blocks and Q blocks may be different. 【0083】 In one embodiment, the number of K-block arrays and Q-block arrays are the same. 【0084】 In certain embodiments, each Q-block array and each K-block array is separated from any other Q-block array and K-block array by at least two spacer arrays. In certain embodiments, each Q-block array and each K-block array is separated from any other Q-block array and K-block array by at least three or four spacer arrays. In certain embodiments, each Q-block array and each K-block array is separated from any other Q-block array and K-block array by 10 to 14 spacer arrays. In certain embodiments, each Q-block array and each K-block array is separated from any other Q-block array and K-block array by 12 spacer arrays. 【0085】 In some embodiments, Q-block sequences and K-block sequences are mixed in that order, meaning that not all sequences of one type are at the N-terminus and all sequences of the other type are at the C-terminus. For example, a Q-block sequence may be followed by a K-block sequence, and then another Q-block sequence. In other words, the adjacency of Q-block sequences and K-block sequences within a sequence is a valid design. 【0086】 In one embodiment, all Q-block sequences contained in the polypeptide are included in the Q-sequence tract, and all K-block sequences are included in the K-sequence tract. 【0087】 In certain embodiments, the Q sequence tract is the N-terminus of the K sequence tract. 【0088】 In this invention, it is not necessary for the Q-block sequence tract to be at the N-terminus and the K-block sequence tract to be at the C-terminus. 【0089】 In certain embodiments, the polypeptide contains 50 to 1200 spacer sequences. In certain embodiments, the polypeptide contains 90 to 250 spacer sequences. In certain embodiments, the polypeptide contains 120 to 180 spacer sequences. 【0090】 In certain embodiments, the Q-sequence tract and the K-sequence tract are separated by at least 30 spacer arrays. In certain embodiments, the Q-sequence tract and the K-sequence tract are separated by at least 40 spacer arrays. In certain embodiments, the Q-sequence tract and the K-sequence tract are separated by at least 50 spacer arrays. 【0091】 In one embodiment, the spacer array is contained in a spacer array polymer containing 6 to 15 spacer arrays as a continuous array. In another embodiment, the spacer array is contained in a spacer array polymer containing 10 to 14 spacer arrays as a continuous array. 【0092】 In certain embodiments, each Q-block sequence is separated from any other Q-block sequence by a single spacer sequence polymer. 【0093】 In certain embodiments, each K-block sequence is separated from any other K-block sequence by a single spacer sequence polymer. 【0094】 In certain embodiments, the Q sequence tract is separated from the K sequence tract by 3 to 5 spacer sequence polymers. 【0095】 In certain embodiments, all spacer array polymers have the same sequence. In certain embodiments, the sequence of the spacer array polymer is or includes the sequence VPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGAG (Sequence ID 013). In certain embodiments, the spacer array polymer sequence is or includes the sequence VPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGAG (Sequence ID 014). 【0096】 In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)85% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)90% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)92% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)94% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)95% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)96% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)97% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001.In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)98% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by (≧)99% or more identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. In certain embodiments, the polypeptide comprises or is essentially derived from an amino acid sequence characterized by 100% identity with the polypeptide sequence of SEQ ID NO: 001, and is characterized by at least 85% of the biological activity of the polypeptide sequence of SEQ ID NO: 001. 【0097】 Bioactivity assay Rheological measurements of Fb clots containing the target protein: The mechanical properties of Fb clots in vitro containing 30, 20, 10, or 5 μM (μmol / L) of the target protein, 30 μM conELP, or an equal volume of HEPES buffer were evaluated using an Anton Paar MCR 302 rheometer with a cone-plate shape (d=25 mm; 1° angle). Frequency sweep measurements were performed at 1.5, 2.2, and 3.0 mg mL to measure the vibrational shear modulus. -1 Fibrinogen (Fg), the protein of interest, conELP, or HEPES buffer, 20 mM CaCl2, and 0.2 U mL -1The procedure was carried out by preparing a clot solution containing thrombin. Immediately after adding thrombin, 90 μL of the clot solution was transferred to a Peltier plate of a rheometer preheated to 22 or 37°C, the measuring cone was lowered onto the sample, and the cone was rotated at 60 rpm for 5 seconds to ensure proper mixing and sample distribution. Silicone oil (η=100 cSt) was applied to the edge of the sample to prevent evaporation, and after equilibrating the clot for 1 hour, a frequency sweep of 0.1–3 Hz (γ=1%; predetermined to fall within the linear viscoelastic region (LVE) of the material) was performed to determine the biological activity of the target protein. From the measurement results, the storage modulus (G') and loss modulus (G'') of the material could be obtained. These parameters are well known in materials science and are related to the stiffness (G') and viscosity (G'') of the material. When these parameters were compared between hELPs / Fb gel and Fb alone, an increase in stiffness (G') was observed, particularly at low strain values ​​(i.e., low force). In the absence of other parameters, the threshold for biological activity is an increase of G' > 200 Pascals. 【0098】 A second aspect of the present invention relates to a polypeptide according to the first aspect for use in the treatment or prevention of a condition selected from trauma, hemostatic disorders, excessive bleeding, or coagulation disorders. 【0099】 In certain embodiments, the coagulation disorder is either a dilutive (dilutional) coagulation disorder or a trauma-induced coagulopathy. 【0100】 In addition to traumatic coagulation disorders, the treatment according to the present invention may also be useful in cases of internal bleeding where the patient's coagulation response is insufficient due to the size of the wound, even though the patient has sufficient endogenous coagulation factors. 【0101】 Dilutional coagulation disorder refers to coagulation disorders seen during massive blood transfusions for major trauma and / or bleeding. Major trauma and bleeding lead to coagulation abnormalities due to the consumption of coagulation factors and platelets. Dilutional coagulation disorder is caused by the consumption and dilution of platelets during massive blood transfusions. Large amounts of crystalloid fluid used for resuscitation in these cases can also contribute to thrombocytopenia. If packed red blood cells are stored for more than 24 hours, they contain very few platelets, and the platelets contained in packed red blood cells are usually damaged and removed from circulation during transfusion. Platelet levels during massive transfusions are typically 50,000-75,000 / mm³. 3 Thrombocytopenia during this period should be treated with platelet concentrate. The number of transfused packed red blood cells does not accurately predict the degree of thrombocytopenia or the need for platelet transfusions. 【0102】 A coagulation disorder (also known as a hemorrhagic disorder) is a condition in which the blood's ability to clot (form a clot) is impaired. Coagulation disorders can cause uncontrolled internal or external bleeding. If left untreated, uncontrolled bleeding can damage joints, muscles, or internal organs and can be life-threatening. Coagulation disorders can be caused by decreased or absent levels of blood clotting proteins known as clotting factors or coagulation proteins. Genetic disorders such as hemophilia and von Willebrand disease can cause a decrease in clotting factors. 【0103】 In certain embodiments, impaired hemostasis, excessive bleeding, or impaired coagulation is related to or caused by: a. Platelet disorders, coagulation disorders, vascular defects, and / or thrombocytopenia, b. Excessive anticoagulation, particularly anticoagulation caused by the administration of warfarin, heparin, or direct oral anticoagulants (e.g., apixaban, edoxaban, rivaroxaban); c. Liver disease (defective production of coagulation factors), d. Von Willebrand disease, e. hemophilia, f. Trauma. 【0104】 Further details on excessive bleeding can be found below: https: / / www.msdmanuals.com / professional / hematology-and-oncology / hemostasis / excessive-bleeding. 【0105】 A third aspect of the present invention relates to a nucleic acid sequence encoding a polypeptide according to the present invention, as described in any of the aspects and embodiments described herein. 【0106】 Another aspect of the present invention relates to an expression vector comprising a nucleic acid sequence according to a third aspect. This may be an expression vector or an expression construct. 【0107】 A fifth aspect of the present invention relates to a cell comprising a nucleic acid sequence according to the third aspect or an expression vector according to the fourth aspect. 【0108】 Medical supplies, dosage forms, and salts Similarly, within the scope of the present invention is the treatment of hemostatic disorders, excessive bleeding, or coagulation disorders in patients who require such treatment, including administering the polypeptide described above to the patient. 【0109】 Similarly, a dosage form for the prevention or treatment of hemostatic disorders, excessive bleeding, or coagulation disorders is provided, comprising a non-agonist ligand according to any of the above aspects or embodiments of the present invention. 【0110】 Those skilled in the art will recognize that any of the drugs specifically mentioned may exist as pharmaceutically acceptable salts thereof. Pharmaceutically acceptable salts include ionized drugs and reversed counterions. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetates, benzoates, besilates, bitatrates, bromides, carbonates, chlorides, citrates, edetates, edisylates, embonates, estolates, fumarates, gluceptates, glucons, hydrobroms, hydrochlorides, iodides, lactates, lactobionates, malates, maleates, mandelates, mesylates, methyl bromides, methyl sulfates, mucinates, napsylates, nitrates, pamosates, phosphates, diphosphates, salicylates, disalicylates, stearates, succinates, sulfates, tartrates, tosylates, triethiozides, and valersates. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc. 【0111】 The administration method may be enteral, such as nasal, oral, rectal, transdermal, or oral administration, or as an inhalant or suppository. Alternatively, parenteral administration such as subcutaneous, intravenous, intrahepatic, or intramuscular injection may be used. Optionally, pharmaceutically acceptable carriers and / or excipients may be present. 【0112】 Topical administration is also within the scope of the advantageous use of the present invention. Those skilled in the art will be familiar with a wide range of possible formulations for providing topical formulations, as exemplified by the following: Benson and Watkinson (eds.), Topical and Transdermal Drug Delivery: Principles and Practice (Part 1, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (Part 2, CRC Press 2002, ISBN-13: 978-0824708610); and Osborne and Amann (eds.), Topical Drug Delivery Formulations (Part 1, CRC Press 1989; ISBN-13: 978-0824781835). 【0113】 Local administration can be achieved using a two-barrel syringe as a two-component precursor solution that can be extruded onto a local wound or used as an additive to wound dressings such as bandages. 【0114】 Pharmaceutical composition and administration Another aspect of the present invention relates to a pharmaceutical composition comprising the compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. 【0115】 In certain embodiments of the present invention, the compounds of the present invention are typically formulated into pharmaceutical dosage forms that provide an easily controllable dose of the drug and give the patient a product that is simple and easy to handle. 【0116】 In embodiments of the present invention relating to the topical use of the compounds of the present invention, the pharmaceutical composition is formulated in a manner suitable for topical administration, such as an aqueous solution, suspension, ointment, cream, gel, or sprayable formulation, for delivery by an aerosol, and contains the active ingredient together with one or more solubilizers, stabilizers, isotonic enhancers, buffers, and preservatives known to those skilled in the art. 【0117】 The pharmaceutical composition can be formulated for oral, parenteral, or rectal administration. Furthermore, the pharmaceutical composition of the present invention may be in solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or liquid form (including, but not limited to, solutions, suspensions, or emulsions). 【0118】 The administration regimen of the compounds of the present invention will vary depending on known factors such as the pharmacodynamic properties of the particular drug and the mode and route of administration: the recipient's species, age, sex, health status, medical condition, and weight; the nature and severity of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect. In certain embodiments, the compounds of the present invention may be administered in a once-daily dose, or the total daily dose may be divided into two, three, or four doses per day. 【0119】 In certain embodiments, the pharmaceutical compositions or combinations of the present invention may have a unit dose of approximately 1 to 1000 mg of active ingredient(s) per subject weighing approximately 50 to 70 kg. The therapeutically effective dose of a compound, pharmaceutical composition, or combination thereof depends on the species, weight, age, and individual condition of the subject, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian with ordinary skill can easily determine the effective amount of each active ingredient necessary for the prevention, treatment, or slowing of the progression of the disorder or disease. 【0120】 The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical procedures such as sterilization, and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. These can be produced by standard processes, such as conventional mixing, granulation, dissolution, or freeze-drying processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art; see, for example, L. Lachman et al., The Theory and Practice of Industrial Pharmacy, Vol. 4, 2013 (ISBN 8123922892). 【0121】 Where, in this specification, alternative forms of a single separable feature, such as an isotype protein or a medical indication, are described as “embodiments,” it should be understood that such alternative forms may be freely combined to form separate embodiments of the invention disclosed herein. Accordingly, any alternative embodiment relating to an isotype protein may be combined with any alternative embodiment of a medical indication referred to herein. 【0122】 The present invention is further illustrated by the following embodiments and figures, from which further embodiments and advantages can be derived. These embodiments are for illustrative purposes only and do not limit the scope of the present invention. [Examples] 【0123】 Example 1: Design and characterization of hemostatic ELP (hELP) The inventors designed an hELP with an ABC triblock structure (Figure 1a). The repeating ELP components present in all three blocks contained 11 VPGXG (SEQ ID NO: 012) pentapeptides with alanine, valine, and glutamic acid residues in a 2:8:1 (A2V8E1) ratio at the guest position. The N-terminal hELP block, called the Q block, further contains four transglutaminase tags (Figure 1a) and contextual sequences. JPEG0007873497000001.jpg13153(D Q MMLPWPAVAL (Sequence ID 003)) Each contains one glutamine residue embedded within it. These transglutaminase tags have been previously reported to be highly specific to human FXIIIa recognition. We hypothesized that by embedding these FXIIIa-sensitive sequences into a broader hELP sequence, hELP would be selectively integrated into the Fb network at the FXIII-activated wound site while avoiding off-target interactions with soluble fibrinogen. The intermediate hELP block confers phase separation ability and consists of four consecutive A2V8E1 units, totaling 48 pentapeptide repeats. This stimulus-responsive intermediate block induced phase separation of hELP in response to physiological temperature (37°C). Finally, the C-terminus of hELP contains four lysine blocks. It contained JPEG0007873497000002.jpg11140, which functioned as a complementary partner to glutamine in the reaction catalyzed by FXIIIa. The control ELP (conELP) was also prepared with the same sequence as hELP, except that the glutamine and lysine residues were mutated to glycine so that conELP would not be crosslinked by FXIIIa. 【0124】 The inventors cloned, expressed, and purified hELP and conELP by inverse transition cycling (ITC), and measured LCST using a cloud point assay (C. Boutris et al., Polymer (Guildf). 1997, 38, 2567). At a working concentration of 30 μM, both hELP and conELP showed cloud points below 37°C (32.7°C and 34.1°C, respectively), indicating that both ELPs aggregated at physiological temperatures (Figure 2a). Next, the inventors confirmed the functionality of the Q and K blocks by testing the ability of FXIIIa to crosslink hELP in the absence of Fg using SDS-PAGE (Figure 2b). The decrease in the intensity of the band corresponding to a single hELP polymer (approximately 69.5 kDa) and the appearance of high molecular weight bands corresponding to the dimer and polymer of hELP confirmed that FXIIIa was able to crosslink hELP. On the other hand, the conELP sample incubated with FXIIIa did not crosslink. The molecular weights of hELP and conELP were confirmed by mass spectrometry. 【0125】 Example 2: Integration of hELP into the Fb network The inventors N-terminally labeled hELP and conELP with Atto647-N-hydroxysuccinimide (red channel) and confirmed that they were still crosslinked by FXIIIa. Next, fluorescent-hELP (f-hELP) or fluorescent-conELP (f-conELP) was incorporated into Fb clots doped with 1% AlexaFluor 488-labeled fibrinogen (Fg-488, green channel). The inventors characterized the network morphology of hELP and Fb, the degree of colocalization of hELP and Fb, and the effect of the hELP phase transition on the clot structure using a two-color confocal fluorescence microscope. At 22°C, all three clots (HEPES-Fb, conELP-Fb, hELP-Fb) showed a clear Fb network when imaged in the green Fb channel (Figure 3a, left). When imaging was performed using the red hELP channel, f-hELP fluorescence similarly showed an ordered network with a high degree of spatial colocalization between the hELP signal and the Fb signal (Figure 3a, left, hELP). The inventors confirmed that when only HEPES buffer or f-conELP was added during Fb network formation, the fluorescence intensity of the red channel was almost zero (Figure 3a, left, HEPES and conELP). 【0126】 To quantify the spatial colocalization of hELP and Fb, the inventors calculated Pearson's correlation coefficient (PCC) using ImageJ's Coloc2 (J. Adler, I. Parmryd, Cytom. Part A 2010, 77, 733). In f-hELP-Fb clots at 22°C, the PCC between the f-hELP channel and the Fb channel was 0.69 ± 0.1, indicating a high spatial correlation. In f-conELP-Fb clots, the PCC value was 0.05 ± 0.09, indicating no spatial correlation (Figure 3b). These results indicate that when hELP is mixed with Fb and FXIIIa below LCST, it is specifically crosslinked and localized to Fb fibers. 【0127】 At 37°C, exceeding the LCST of hELP (Figure 3a, right), the structures observed in conELP-Fb and hELP-Fb clots were consistent with the expected phase separation. High ELP density punctate spots indicated the formation of ELP-rich coacervates at T>LCST. The density of ELP-rich coacervates was higher in f-hELP-Fb clots than in f-conELP-Fb clots, which is attributed to conELP lacking the Q and K residues necessary for covalent integration. F-hELP coacervates were not randomly distributed relative to the Fb network, and some co-localization with Fb fibrils was observed. Image analysis of hELP-Fb clots at 37°C yielded a PCC value of 0.163±0.04. For f-conELP-Fb clots formed at 37°C, the inventors measured a PCC value of -0.04±0.06, indicating no spatial correlation. The average radius of hELP coacervates was determined to be 0.57 ± 0.17 μm from threshold (processed) images of three separate clots (Figure 7). The unimodal distribution of hELP coacervate size, with a clear central peak, suggests an energy balance that limits the growth of hELP coacervates within the Fb gel. Previous studies on enzymatically crosslinked ELP hydrogels or the enzymatic integration of ELP into collagen networks have reported that enzyme-mediated crosslinking was not inhibited by LCST excess. Our results are also consistent with these findings, indicating that coacervation did not inhibit hELP association with the Fb network. In fact, polyvalent hELP-rich coacervates with locally increased concentrations of Q-blocks and K-blocks may promote FXIIIa-mediated crosslinking. 【0128】 Example 3: Quantification of pore size Pore ​​size is an important structural feature that contributes to the rigidity and resistance to enzymatic degradation of Fb clots. Covalent crosslinking of Fb fibrils with FXIIIa reduces pore size, and in vitro supplementation with FXIIIa increases rigidity and resistance to fibrinolysis. The inventors investigated the effect of hELP using a gravimetric perfusion assay in which they measured the liquid flow rate through hELP-Fb clots, and then estimated the pore size using Darcy's law and a model developed by Carr and Hardin (LWChan et al., Sci. Transl. Med. 2015, 7, 277ra29; MECarr et al., Am. J. Physiol. 1987, 253, H1069). 【0129】 At 22°C, the flow rates through the HEPES-Fb and conELP-Fb control clots were approximately 100 times higher than those through the hELP-Fb clot (see Figure 8). These flow rates corresponded to average pore sizes of 686.6±39.3, 761.2±41.8, and 73.6±5.4 nm for the HEPES-Fb, conELP-Fb, and hELP-Fb clots, respectively (Figure 3C). The approximately 10-fold reduction in pore size observed in the hELP-Fb clots represented a significantly larger reduction than previously reported in clots treated with either FXIII (2.1-fold reduction) or synthetic fibrin-binding polymer (1.5-fold reduction). Vacuum-based SEM analysis of HEPES-Fb and hELP-Fb also qualitatively agreed with the reduction in pore size. 【0130】 At 37°C, the pore sizes of the HEPES-Fb and conELP-Fb control clots were smaller than those at 22°C (390.3±28.9 nm and 504.3±51.3 nm, respectively), which the inventors believe is due to the temperature dependence of FXIIIa activity. However, in the hELP-Fb clot, the pore size at 37°C was 124.8±11.5 nm, which was slightly larger than the radius at 22°C, but the difference was not statistically significant (Figure 3c). Therefore, the temperature increase from 22°C to 37°C did not result in a significant change in the apparent pore size of the hELP-Fb clot as measured by gravimetric perfusion, as observed in these controls. This suggests that the hELP-Fb clot is already maximally crosslinked at 22°C. 【0131】 Example 4: Gelation Dynamics The inventors investigated the effect of hELP on gelation dynamics using a UV-Vis spectrophotometer and a turbidity assay (LWChan et al., Sci. Transl. Med. 2015, 7, 277ra29; ASWolberg, Blood Rev. 2007, 21, 131; E. Mihalko, AC Brown, Semin. Thromb. Hemost. 2019) (Figure 4a). The absorbance of gelling Fb clots was measured in the range of 500-800 nm over 1 hour at 37°C. Multiple distinct gelation profiles appeared among the different groups. The HEPES-Fb buffer control clot showed a steady increase in absorbance at all wavelengths over time. The ConELP-Fb clot showed a maximum absorbance 5 minutes after gelation, after which the absorbance remained constant. The HELP-Fb clots exhibited a two-stage gelation profile, with absorbance rapidly increasing within the first three minutes, then slowly rising to a final maximum at eight minutes. While analysis of the turbidity of Fb clots as a function of wavelength has previously been used to estimate the fiber mass / length ratio, such analysis has not been straightforward in this system due to unknown refractive index differences and the turbidity of the coacervates of hELP within the hELP-Fb composite hydrogel. 【0132】 The inventors further measured the gelation dynamics of hELP-Fb clots using low-strain vibrational shear rheology under physiological conditions, focusing particularly on the shear storage modulus (G') and loss modulus (G''). In the case of HEPES-Fb and conELP-Fb clots, a period of rapid gelation followed an initial lag period, and a second stage of slow asymptotic growth of G' (Figure 9). The gelation time (defined as the point where G'=G'') for both conELP-Fb and HEPES-Fb clots occurred at approximately 270 seconds for both samples, while the gelation point for hELP-Fb clots occurred significantly later at approximately 510 seconds. After the onset of gelation, G' in hELP-Fb clots increased more rapidly than in HEPES-Fb or conELP-Fb clots. The maximum values ​​of the first derivatives are 0.15, 0.14, and 0.39 Pa s for HEPES-Fb, conELP-Fb clot, and hELP-Fb, respectively. -1 Therefore, the presence of coacervates in hELP had an inhibitory effect on the time to coagulation initiation, but had a positive effect on the coagulation growth rate and maximum stiffness after initiation. 【0133】 Example 5: Effects of hELP on Thromboelastography Thromboelastography (TEG) is a clinical technique for measuring the coagulation ability of blood (D. Whiting, J.A. DiNardo, Am.J. Hematol. 2014, 89, 228). Here, the inventors evaluated three TEG parameters: R, which indicates the time to the start of clot formation; alpha angle, which indicates the rate of clot formation; and maximum amplitude (MA), which indicates the stiffness of the clot. Here, as observed in the turbidity and rheology experiments described above, a two-stage diagram of hELP-Fb clot formation was obtained. At both low and high Fb concentrations, hELP-Fb clots required a longer time to start coagulation than conELP-Fb clots or HEPES-Fb clots (Figure 4b). However, after the start of coagulation, 1.5 mg mL -1The hELP-Fb clot containing Fb had a significantly higher alpha angle (Figure 4C) than the HEPES-Fb or conELP-Fb controls (27.83 ± 1.95° and 25.93 ± 3.26°, respectively). When the Fb concentration was increased to 3.0 mg mL -1 , the increase in the alpha angle of the hELP-Fb clot relative to the control was more gradual. In this case, the alpha angle of the hELP-Fb clot was 58.2 ± 1.6°, while the alpha angles of the HEPES-Fb clot and conELP-Fb clot were 47.9 ± 2.31° and 47.07 ± 4.04°, respectively. In previous studies, FXIII has been found to be important in the second stage of clot stiffening during gelation. Since hELP is incorporated into the Fb clot by FXIII, this could be a case where its presence enhances the rate of this second stage. 【0134】 The effect of hELP on the MA value was similar to that observed for R and the alpha angle. In hELP-Fb clots containing 30 μM hELP and 1.5 mg mL -1 of Fb, there were increases in MA of 62% and 59% relative to the HEPES-containing clot and conELP-containing clot, respectively. In hELP-Fb clots formed with 3.0 mg mL -1 of Fb, the MA was 16% higher than the HEPES-Fb clot and 24.5% higher than the conELP-Fb clot (Figure 4d). Overall, these TEG results were consistent with the shear rheology and suggested that hELP had a more significant positive effect on the clot properties of clots with a low threshold concentration below the critical value of Fg. 【0135】 Example 6: Effect of the coacervate of hELP on Fb clot mechanics Approximately 2.3 mg mL -1Fb concentrations below a certain threshold are associated with increased mortality in clinical hemostasis. The inventors used vibrational shear rheology to measure the effect of hELP on clot stiffness (G') at Fb concentrations above and below this threshold. At 22°C, no significant difference in G' was observed for any Fb concentration of HEPES-Fb clots, conELP-Fb clots, or hELP-Fb clots (Figure 5a). However, at 37°C, the inventors found that G' significantly increased for all Fb concentrations in the hELP-Fb clots. The lowest Fb concentration (1.5 mg mL) -1 The HELP-Fb clot showed a G' of 201.3±16.4 Pa, which was significantly higher than the conELP-Fb (71.0±11.6 Pa) clot and the HEPES-Fb (45.0±12.8 Pa) clot. 1.5 mg mL -1 The G' of the hELP-Fb clot formed with Fb was 2.2 mg mL -1 The G' value was equivalent to that of the control clot formed at the physiological Fb concentration. This indicates that, in T>LCST, the coacervate of hELP restored the clot's rigidity to physiological levels under simulated conditions of TIC and Fg depletion. 【0136】 Example 7: Effect of hELP on strain stiffening of Fb clots Strain-stiffening of Fb clots is attributed to the multiscale structural organization of the Fb network, from single monomers to protofibrils, protofibril bundles, and fibers. According to this theory, when the Fb network is strained, the force is first dissipated entropically by minimizing thermal fluctuations in the flexible interfibril crosslinks, and then further dissipated by the expansion and contraction of the fibrils themselves. Ultimately, at higher tensions, the secondary, tertiary, and quaternary structural elements of the folded regions within the Fb domains denature, and strain-stiffening behavior rarely occurs in synthetic crosslinked polymer networks (IKPiechocka et al., Biophys.J.2010,98,2281; IKPiechocka et al., Soft Matter 2016,12,2145). 【0137】 To investigate the effect of hELP on the strain stiffening of Fb, the inventors performed vibrational rheology using strain gradients from 0.1 to 100% (Figure 5b). At low strain (0.1 to 1%), 30 μM hELP and 2.2 mg mL -1 hELP-Fb clots containing Fb showed increased G' compared to conELP-Fb or HEPES-Fb clots, consistent with observations in low-amplitude frequency sweep experiments (Figure 5a). At moderate strains (1–10%) and high strains (10–100%), all clots exhibited strain stiffening behavior; however, the strain at which stiffening began was greater for hELP-Fb (approximately 10%) compared to conELP-Fb or HEPES-Fb clots (approximately 2–3%). The HEPES-Fb and conELP-Fb clots showed a faster rate of strain stiffening than the hELP-Fb clot, as indicated by the maximum values ​​of the first derivatives of each curve (38.1 Pa, 38.8 Pa, and 23.1 Pa, respectively). At 100% strain, G' was approximately the same for all clots (approximately 1400–1500 Pa). Comparing G' for strains between 0.1% and 100%, the hELP-Fb clot was 4.6 times harder, while conELP-Fb and HEPES-Fb were 8.8 times and 8.4 times harder, respectively. Considering these results in the Fb hierarchical structure, it is likely that by bridging the protofibrils, hELP minimizes thermal fluctuations in the unstructured region of the network in the low strain range. Once the clot is sufficiently stretched, the elastic response is dominated by the stretching of individual protofibrils, and further bridging in the form of hELP coacervates no longer plays a role. Previous studies have shown that FXIII addition increases the modulus of Fb clots at low strains within the linear viscoelastic region of fibrin, but not in the nonlinear portion of the stress-strain curve, which is similar to what we have observed herein using hELP-added materials. 【0138】 The phase transition dependence of the hELP rigidification effect in Fb clots can be explained in several ways. Firstly, high local concentrations of hELP molecules in coacervates promote the formation of more intermolecular hELP-hELP crosslinks, potentially establishing a secondary network stiffer than Fb in the low-strain range. Similar effects have been observed in vivo in phase-separation-driven formation of biomolecular condensates, where increased local concentrations of substrates and enzymes can accelerate chemical reactions. This has been shown to increase reaction rates, for example, in actin polymerization and RNA catalysis. Secondly, aggregation of hELP beyond the LCST may drive the formation of a secondary network of crosslinks between Fb molecules, independently of the formation of inter-hELP crosslinks. The formation of secondary networks in hydrogels by thermal aggregation of ELPs has also been reported by Wang et al., who showed that aggregation of hydrazide-modified ELPs crosslinked in hyaluronic acid hydrogels results in mechanical rigidification of these materials. Finally, the phase separation of hELPs bound to Fb can exert mechanical forces on Fb fibers, producing a strain-stiffening effect even without external tension, summarizing the active cell-driven contractile strain-stiffening that occurs in fibrin networks embedded with fibroblasts and platelets. Strain-stiffening is a well-known property of Fb networks, and ELP coacervation is known to stiffen ELP hydrogels and exert mechanical forces. There is evidence that the mechanical forces of molecular aggregation are applied in other in vivo situations: Shin et al. recently demonstrated that the phase separation of chromatin-associated IDPs functions in a mechanism that regulates DNA transcription by physically attracting distal genomic elements while mechanically excluding others (Y. Shin et al., Cell 2018, 175, 1481). However, considering that a stiffening effect is observed in hELP that has been preheated / aggregated before Fb polymerization, it seems unlikely that the network of hELP is actively contracted / stiffened by coacervates. 【0139】 Example 8: Effect of hELP coacervate in plasminolysis In the body, clots are enzymatically degraded by plasmin, a protease produced from plasminogen upon activation of tissue plasminogen activator (tPa). The proteolytic activity of plasmin is spatiotemporally controlled by the binding of tPa and plasminogen to cryptic binding sites on exposed Fb clots. Crosslinking by FXIIIa has been previously shown to have an inhibitory effect on fibrinolysis in vivo. Since hELP coacervates are covalently incorporated into Fb clots by FXIIIa, we hypothesized that hELP could extend the lifespan of Fb clots in the presence of plasmin. To evaluate this, we performed time-lapse confocal microscopy. 【0140】 Fluorescent Fb clots containing 30 μM hELP, conELP, or an equal amount of HEPES were formed in chamber-type coverslips, and a physiologically concentrated plasmin solution was applied to the front surface of the clots. Images were taken at regular intervals and analyzed to evaluate the percentage of clots that dissolved over time. The results showed a significant difference in the rate of plasminolysis between hELP clots and control clots. In general, HEPES control clots were completely degraded from the microscopic field 4 minutes after plasmin application, while approximately 20% of conELP-Fb and approximately 85% of hELP-Fb remained (Figure 6). The dissolution rate of hELP-Fb clots was approximately three times slower than that of conELP-Fb clots and approximately five times slower than that of HEPES-Fb clots. Since HELP does not contain the sequence recognized by plasmin, further non-degradable components in the gel inhibited fibrinolysis. 【0141】 Example 9: Cytotoxicity of hELP Although ELPs are generally recognized as biocompatible and non-toxic, like many recombinant proteins produced from E. coli, purified protein products may retain bacterial endotoxins (i.e., lipopolysaccharides, LPS), which can trigger inflammatory and immune responses in the body. Here, we applied an in vitro resazurin-based cytotoxicity assay to neonatal human dermal fibroblasts (HDFn) to measure the cytotoxicity of hELPs. In this experiment, ELPs were subjected to further dialysis and lyophilization steps following ITC purification to remove low molecular weight and non-aggregated LPS. Compared to untreated control cells, no significant decrease in cell viability was observed after 24-hour exposure of HDFn cells to 30 μM standard operating concentrations of hELP and conELP (Figure 6). Exposure to 50 μM conELP resulted in a significant decrease in HDFn viability, while exposure to 80 μM ELP resulted in a significant decrease in cell viability with both hELP and conELP treatment. However, even at the highest test concentration of 80 μM, cell viability did not fall below 80% of the control. Therefore, hELP and conELP exhibit minimal cytotoxicity under these conditions. 【0142】 Example 10: In vivo study of coagulation-promoting hELP The efficacy of hELP as a hemostatic agent was evaluated using an in vivo rat bleeding model. Ten male CD rats were divided into two test groups: hELP (4Tg-4Tg; SEQ ID NO: 16) and conELP (control ELP). 4Tg refers to the "Q block" transglutaminase substrate sequence recognized by coagulation agent FXIIIa (SEQ ID NO: 003). In control ELP, the glutamine in the Q block sequence was mutated to glycine, making it unrecognizable by FXIII. On the day of surgery, the rats were anesthetized with isoflurane, placed on a warming bed, and two catheters were inserted: one into the carotid artery and the other into the jugular vein. After baseline levels of CO2, O2, and lactate were established, clamps were placed at the proximal and distal ends of the left femoral artery, and then a 3 mm incision was made in the artery. After controlling catheter bleeding and lowering the mean arterial pressure (MAP) of each animal to 40-60 mm Hg, the femoral artery clamp was removed, and the rats were administered 5 mL of the indicated therapeutic agent in kg. -1 A bolus was injected with the following volume (maximum 2 mL min). -1 ). Each rat received a blood volume of 64 mL / kg. -1 Assuming the above, the target final blood ELP concentration was 30 μM. This corresponds to an ELP dose of approximately 140 mg per kg of body weight. After removing the clamp, the animals were allowed to bleed freely for 15 minutes, and the amount of blood loss was measured using pre-weighed gauze. After the 15-minute free bleeding period, blood samples were taken, blood gases were measured, and prothrombin time was measured. Subsequently, to increase the MAP to over 60 mm Hg, physiological saline was administered to the animals as needed (at a rate of 3 mL / kg / min, total volume 60 mL / kg). -1 (Until) Blood loss and MAP were continuously monitored until MAP fell below 20 mm Hg or until the end of the experiment (t=75 minutes), at which point the animals were euthanized. This study design was approved by the Animal Care and Use Committee at Charles River Laboratories. 【0143】 method Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland). Plasmids containing the genes encoding ELP(A2V8E1), ELP(A2V8E1)-Tgf11, ELP(A2V8E1)-GSKGS (GSKGS module is SEQ ID NO: 11), ELP(A2V8E1)-Tgf11(Q65G), and ELP(A2V8E1)-GSGGS (GSGGS module is SEQ ID NO: 15) were synthesized at GeneArt (Thermo Scientific). Human fibrinogen (FIB 3, depleted of plasminogen, fibronectin, and von Willebrand factor), thrombin, and FXIIIa were purchased from Enzyme Research Laboratories (Rheinfelden, Switzerland). Fluorescently tagged fibrinogen (Fg-488) was purchased from Thermo Scientific (Basel, Switzerland). 【0144】 Method 1: Expression and purification of ELP hELP and conELP proteins were designed and fabricated using standard molecular cloning techniques. The gene encoding full-length ELP was fabricated starting from one of five 11-pentapeptide gene monomers: A2V8E1-Tgf11, A2V8E1, A2V8E1-GSKGS (SEQ ID NO: 11), A2V8E1-Tgf11 (Q65G), or A2V8E1-GSGGS. These were repeatedly digested and ligated together according to recursive directional ligation, a known technique for extending repetitive gene sequences. After preparing the gene encoding ELP of the desired length and composition, it was inserted into a pet28a expression vector, and the resulting plasmid was transformed into BL21(DE3)E.Coli. ELP was expressed in 1 L of Terrific Broth (TB) at 37°C for 24 hours without the addition of an inducer, relying instead on the leakage of the T7 promoter. After expression, the cell pellet was centrifuged and resuspended in 40 mL of 20 / 150 mM HEPES / NaCl, and lysed by 3 cycles of sonication. This lysate was centrifuged at 4°C to remove cell debris, and then ELP was purified by iterative transition cycling (ITC). Briefly, 1 M NaCl was added to the supernatant remaining after centrifuging the cell lysate. The sample was heated at 65°C for 10 minutes and centrifuged at 18000 g at 40°C for 15 minutes. The supernatant was discarded, and the resulting pellet was resuspended in 6 mL of cold HEPES buffer. The resuspended pellet was again centrifuged at 18000 g at 4°C for 15 minutes, and any contaminants that could not be resolubilized in the cold buffer were discarded. These steps were combined to form one ITC (Internal Cell Tuning), and this process was repeated two more times to obtain the final ELP solution, which was then aliquoted before use and stored at -20°C. The typical yield obtained from a single expression was in the range of 50–100 mg / L of culture medium. 【0145】 Method 2: Characterization of the cloud point of ELP The cloud points of hELP and conELP were measured at a concentration of 30 μM. ELP was dissolved in 20 / 150 mM HEPES / NaCl buffer (w / 20 mM CaCl2) at an appropriate concentration, transferred to a cuvette, and placed in a UV-Vis spectrophotometer (Evolution 260 Bio, Thermo Scientific) at 15°C. After equilibrating the sample to the starting temperature for 10 minutes, it was raised from 15 to 60°C at 1°C. min -1 A temperature gradient was performed at a rate of . Absorbance at 350 nm was measured every 0.25 minutes, and a corrected absorbance value was obtained by subtracting the blank reading of the cuvette containing only HEPES from this value. This was then converted to transmittance and normalized for the maximum and minimum absorbance values. The cloud point of each ELP was defined as the point where the normalized transmittance fell below 95%. 【0146】 Method 3: In vitro crosslinking of ELP with FXIIIa The ability of FXIII to crosslink hELP or conELP was evaluated by SDS-PAGE. hELP or conELP was diluted to a concentration of 50 μM with HEPES buffer, and 0.2 U was used. mL -1 Thrombin and 20 mM CaCl2 were also added to each sample to replicate the standard coagulation conditions used throughout this study. FXIIIa was added to a final concentration of 10 μg mL. -1 The experimental samples were treated with HEPES buffer, while the control samples were treated with the same amount of HEPES buffer. All samples were then incubated at 37°C for 1 hour, followed by non-reducing SDS-PAGE. The samples were stained with Coomassie-based instant blue stain and imaged using the ChemiDoc Mp imaging system (BioRad). 【0147】 Method 4: Rheological measurement of ELP-containing Fb clots The mechanical properties of in vitro Fb clots containing hELP, conELP, or an equivalent volume of HEPES buffer were evaluated using a cone-plate shaped (d=25 mm; 1° angle) Anton Paar MCR 302 rheometer. Frequency sweep measurements were performed to determine the vibrational shear modulus, at 1.5, 2.2, or 3.0 mg mL. -1 Fibrinogen (Fg), 30 μM hELP or conELP or HEPES buffer, 20 mM CaCl2, and 0.2 U mL -1 This was performed by preparing a clot solution containing thrombin. Immediately after adding thrombin, 90 μL of the clot solution was transferred to a Peltier plate of a rheometer preheated to 37°C, the measuring cone was lowered onto the sample, and the cone was rotated at 60 rpm for 5 seconds to ensure proper mixing and sample distribution. Silicone oil (η=100 cSt) was applied to the edge of the sample to prevent evaporation, and after equilibrating this clot for 1 hour, a frequency sweep of 0.1–3 Hz was performed (γ=1%; previously measured to be within the linear viscoelastic region (LVE) for this material). 【0148】 To evaluate the effect of temperature on the stiffness-modulating properties of ELP in Fb clots, 1.5 or 3.0 mg mL -1 Samples containing Fg, hELP, conELP, or HEPES buffer were formed on a Peltier plate of a rheometer preheated to either 22°C or 37°C, as described above. After equilibrating the clot as described above, a frequency sweep was performed. 【0149】 To evaluate the effect of ELP on the strain-hardening behavior of Fb clots, 2.2 mg mL -1 A sample containing Fg and 30 μM hELP, conELP, or HEPES buffer was formed between the cone and plate of a rheometer at 37°C as described above. After 1 hour of equilibration, vibration sweeps were performed from 0.1 to 100% strain (f=1 Hz). 【0150】 To track the gelation rate of Fb clots formed in the presence of ELP, clot solutions were prepared as described above and then placed between the cone and plate of a rheometer preheated to 37°C. Small amplitude vibrational shear stress was applied to the formed clots (γ=1%; f=1Hz), and the transitions between G' and G'' were measured. The gel point of each clot was defined as the point at which G' exceeded G'' and thereafter remained below G'' for the remainder of the experiment. 【0151】 Method 5: Turbidity measurement of gelation dynamics To study the gelation kinetics, the turbidity changes during gelation of Fb clots were measured at various wavelengths. In typical experiments, 2.2 mg mL -1 Fg, 20 mM CaCl2, 0.1U mL -1 A clot solution consisting of thrombin and one of the following: 30 μM hELP, 30 μM conELP, or HEPES was prepared in a cuvette and immediately transferred to an Evolution 260 Bio UV-Vis spectrophotometer (Thermo Scientific) preheated to 37°C. Absorbance was then measured at 5 nm intervals in the range of 500–800 nm, and this scan was repeated every minute for the duration of the 1-hour experiment. 【0152】 Method 6: Perfusion assay to determine pore size of Fb clots The pore size of Fb clots with and without ELP was evaluated using a perfusion assay adapted from a previous study by Carr and Hardin (Shin et al., Cell 2018, 175, 1481). To support the clot solution during gelation, clots were formed at the bottom of an upright gravity filtration column, which had its tip cut and sealed with Parafilm. In each experiment, 1.5 mg mL -1 20 mM CaCl2, 0.1 U mL -1A 1 mL clot solution consisting of thrombin and 30 μM hELP or conELP, or an equal volume of HEPES buffer, was used. The clots were allowed to form for 1 hour at 22°C or 37°C, after which 13 mL of isotonic and isothermal HEPES buffer was dispensed onto each clot and the clots were equilibrated for 10 minutes. The flow rate was then determined by gravimetric measurement by measuring the mass of the buffer passing through the clot every 10 minutes for 50 minutes. Next, the pore radius (r) of the clot containing ELP and the clot without ELP was compared. p The volumetric flow rate was calculated using Darcy's Law and a model developed by Carr and Hardin for determining the pore size of Fb clots containing embedded red blood cells (Shin et al., Cell 2018, 175, 1481): 【number】 In the formula, V is the volumetric flow rate, η is the viscosity of water (0.9544 mPa s at 22°C and 0.6913 mPa s at 37°C), h is the length of the clot, A is the cross-sectional area, t is time, and P is the average hydrostatic pressure exerted by the buffer on the clot during the experimental period. 【0153】 Method 7: Confocal imaging Using a confocal microscope, we investigated the integration of hELP into the Fb network and the degradation of the Fb network in the presence of plasmin. Fluorescent hELP (f-hELP) or conELP (f-conELP) were prepared by preferentially functionalizing the N-terminal amines of these proteins with the Atto-647-NHS dye. In a typical reaction, Atto-647-NHS was dissolved in DMSO and added to a solution of hELP or conELP at a ratio of 1.2 dye molecules per ELP molecule. The reaction was carried out at pH 8.0 to preferentially target the N-terminal amine, which has a lower pka than the ε-amino group of lysine (approximately 8 and 10, respectively). The reaction was allowed to proceed at room temperature for 1 hour, and then quenched by adding a 100-fold excess of TRIS-HCl. Subsequently, the functionalized ELP was purified from the reaction mixture by performing two ITCs as described above. 【0154】 For simple imaging experiments, use 1.5 mg mL -1 Fibrinogen (with 1% fluorescent Fg-488 added), 0.2 U mL -1 Clots were formed in the channels of an Ibidi μ-slide VI 0.5 (glass bottom) from a 40 μL clot solution consisting of thrombin, 20 mM CaCl2, and one of the following: 30 μM f-hELP, 30 μM f-conELP, or HEPES buffer. After forming the clots at either 22°C or 37°C for 1 hour, they were transferred to the imaging chamber of a Nikon Ti2-A1 confocal microscope preheated to the application temperature. To prevent water loss from the clots during the experiment, 40 μL of buffer was added to each port of the slide. A 5.06 μm, 5-slice Z-stack was imaged at three different positions on each clot using a laser, first at 488 nm (Fb channel) and then at 640 nm (ELP channel). Three different clots were imaged for each treatment group. 【0155】 In the decomposition experiment, 1.5 mg mL -1 Fibrinogen (with 1% Fg-488 added), 0.2 U mL -1 Clots were formed in μ-slide ibidi 8-well chamber coverslips from a 100 μL clot solution consisting of thrombin, 20 mM CaCl2, and one of the following: 30 μM hELP, 30 μM conELP, or HEPES buffer. After gelling the samples at 37°C for 1 hour, half of each formed clot was cut out from the coverslip well using a surgical scalpel. The coverslips were then placed in the imaging chamber of a microscope at 37°C, and the location of the ends of the Fb network was identified using a 488 nm laser. Next, 10 μg mL of preheated solution was used. -1 Plasmin solution was applied to the edge of the clot, and images were taken every 10 seconds until the clot was completely removed from the microscope field of view. Images were taken at three different locations on each clot, resulting in three different clots for each treatment group. 【0156】 Method 8: In vitro cell viability assay The effect of ELP coacervate on the viability of human dermal fibroblast (neonatal; HDFn) cells was investigated using a resazurin-based assay. HDFn cells were seeded at a density of 20,000 cells / well in wells of a 96-well tissue culture plate and incubated at 37°C and 5% CO2 for 24 hours. The cells were then treated with stock solutions of conELP or hELP dissolved in DMEM to a final ELP concentration of 30, 50, or 80 μM. Control cells were treated with the same volume of DMEM and then incubated for a further 24 hours at 37°C and 5% CO2. Finally, a stock solution of resazurin in 10 mM PBS was added to a final concentration of 10 μg mL. -1 The solution was applied to each experimental well and control well, and after incubating the plate at 37°C for 4 hours, the fluorescence of each well was measured using a Safire II plate reader (λ exc = 531 nm, λ emi (=572nm). The final cell viability in each treatment was determined by taking the average fluorescence intensity for each treatment (excluding blanks that did not contain cells) and dividing it by the average fluorescence intensity of the control that did not contain ELP. 【0157】 Method 9: Fluorescent labeling of hELP To study the FXIIIa-mediated incorporation of hELP into Fb clots, the inventors preferentially labeled hELP and conELP at the N-terminus of the protein using the fluorescent dye Atto647-N-hydroxysuccinimide (Atto647-NHS). By carrying out the reaction at pH 8, the inventors selectively targeted α-amino groups with lower pKas to preserve lysine ε-amino groups in the K-block for FXIIIa-mediated crosslinking after labeling. The extinction coefficient of hELP / conELP (ε 280 = 2.75 × 10 4 M -1 cm -1 ) and the extinction coefficient of Atto647 (ε 647 = 1.5 × 10 5 M -1 cm -1Using this method, the inventors determined that the average number of fluorescent dye molecules per ELP molecule is approximately 0.95. The inventors then used SDS-PAGE to test whether the fluorescent HELP (f-hELP) retained its ability to be crosslinked by FXIIIa. In the FXIIIa-containing sample, a single f-hELP band of approximately 69.5 kDa disappeared, suggesting that sufficient active lysine residues remained after fluorescent labeling with Atto647-NHS for f-hELP to be crosslinked by FXIIIa. 【0158】 array Elastin-like polypeptides (ELPs) are naturally occurring denatured protein-based polymers derived from the hydrophobic domain of the human extracellular matrix protein tropoelastin, and containing a repeating pentapeptide VPGXG sequence (where X is any amino acid except proline). VPGXG represents an essential portion of the endogenous sequence of human tropoelastin used in this invention. 【0159】 The inventors designed a hemostatic ELP (hELP) having an ABC triblock structure. The repeating ELP component was present in all three blocks and contained 11 VPGXG (SEQ ID NO: 012) pentapeptides with alanine, valine, and glutamic acid residues in a 2:8:1 ratio (A2V8E1) at the guest residue position. While the guest residues are theoretically modifiable from this composition, this design was chosen to produce an hELP with a transition temperature within the physiological temperature range. The N-terminal hELP block further contained four transglutaminase tags (referred to as the Q block), which contained glutamine residues embedded in a context peptide sequence (DQMMLPWPAVAL (SEQ ID NO: 003)) previously shown to be recognized with high specificity by human FXIIIa. By incorporating these sequences into a broader hELP sequence, the inventors designed an hELP that selectively integrates into the Fb network at the in vivo wound site where FXIII is activated, while simultaneously avoiding off-target interactions with circulating fibrinogen. The intermediate hELP block was a phase separation block consisting of four consecutive A2V8E1 units, totaling 48 pentapeptide repeats. This stimulus-responsive intermediate block induced phase separation of the hELP in response to physiological temperature (37°C). Finally, the C-terminus of the hELP contained four lysine blocks (K blocks; GSKGS (SEQ ID NO: 011)), which functioned as complementary partners to the glutamine residue in the FXIIIa-catalyzed reaction. 【0160】 JPEG0007873497000004.jpg234153JPEG0007873497000005.jpg26153

Claims

[Claim 1] a. i. DQMMLPWPPAVAL (Sequence ID 003), ii. WQHKIDLRYNGA (Sequence ID 004), iii. SQHPLPWPVLML (Sequence ID 005), iv. EQFPIAFPRYSI (Sequence ID 006), v. SEQHLLKWPPWH (Sequence ID 007), vi. WQIPVDWPPLPP (Sequence ID 008), vii. DQWMMAWPSLTL (Sequence ID 009), and / or viiii. SQIPMAWPLLSL (Sequence ID 010), 2 to 50 Q-block arrays selected from each of the following, b. A plurality of spacer arrays of sequence VPGXG (sequence number 012), wherein each X is independently selected from A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y, and V. A polypeptide containing or consisting of. [Claim 2] The polypeptide according to claim 1, wherein each X is independently selected from Ala, Val, and Glu. [Claim 3] The Ala:Val:Glu ratio used for X is: - 1-3 Ala: 7-10 Val: 1 Glu That is, The polypeptide according to claim 2. [Claim 4] The polypeptide according to any one of claims 1 to 3, wherein the Q block sequence is DQMMLPWPPAVAL (sequence number 003). [Claim 5] The polypeptide according to any one of claims 1 to 4, wherein the polypeptide comprises 2 to 8 Q-block sequences. [Claim 6] The polypeptide consists of a Q-block sequence and a spacer sequence. The aforementioned polypeptide is - (VPGXG) ｎ -[(Q block)-(VPGXG) ｎ ] ｍ N-terminal Q-tract described by - -[(Q block)-(VPGXG) ｎ ] ｍ - (VPGXG) ｏ C-terminal Q tract described by - Spacer sequence polymer [(VPGXG)] that separates the N-terminal Q-tract and the C-terminal Q-tract. ｎ ] ｐ It consists of, During the ceremony, - Each n is an integer between 8 and 14, independently of any other n; - Each m is an integer between 2 and 8, independent of any other m; - o is an integer between 0 and 10; - p is an integer between 3 and 6. The polypeptide according to any one of claims 1 to 3. [Claim 7] The polypeptide according to any one of claims 1 to 6, wherein the polypeptide is sequence number 16. [Claim 8] The polypeptide according to any one of claims 1 to 7, wherein the spacer sequence forms a continuous amino acid chain without interposing sequences that are not Q-block sequences. [Claim 9] The polypeptide according to any one of claims 1 to 8, wherein all Q-block sequences contained in the polypeptide are contained within a Q-sequence tract. [Claim 10] The polypeptide according to any one of claims 1 to 9, wherein the spacer sequence is contained in a spacer sequence polymer comprising 6 to 15 spacer sequences as a continuous sequence. [Claim 11] Each Q-block sequence is separated from any other Q-block sequence by a single spacer sequence multimer. The polypeptide according to claim 10. [Claim 12] The polypeptide according to claim 10, wherein all spacer array polymers have the same array. [Claim 13] The polypeptide according to claim 6 or any one of claims 7 to 12 as a reference to claim 6, wherein the sequence of the spacer array polymer is a sequence of VPGVGVPGAAGVPPGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGEGEGVPGAAG (sequence number 013), or VPGVGVGVGVGVGGAAGVPPGVGVGVGVGVGVGVGVGVGVGVGVGVGVGVGEGEGVPGAAG (sequence number 014), or includes the same. [Claim 14] The aforementioned polypeptide is a. Containing, or comprising, an amino acid sequence characterized by (≥) 90% identity with the polypeptide sequence of SEQ ID NO: 001 or SEQ ID NO: 16, and b. Characterized by the biological activity of at least 85% of the polypeptide sequence of Sequence ID No. 001 or Sequence ID No. 16, The polypeptide according to any one of claims 1 to 13. [Claim 15] A polypeptide according to any one of claims 1 to 14, for use in the treatment or prevention of hemostatic disorders, excessive bleeding, or coagulation disorders. [Claim 16] The polypeptide for use according to claim 15, wherein the coagulation disorder is dilution-induced coagulation disorder or traumatic coagulation disorder. [Claim 17] Hemostatic disorders, excessive bleeding, or coagulation disorders are, a. Platelet disorders, coagulation disorders, vascular defects, and / or thrombocytopenia, b. Excessive anticoagulation; c. Liver disease (deficient production of coagulation factors), d. Von Willebrand disease, e. hemophilia, f. trauma, A polypeptide for use according to claim 16, relating to or caused by the above. [Claim 18] A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 14. [Claim 19] An expression vector comprising the nucleic acid molecule described in claim 18. [Claim 20] A cell comprising the nucleic acid molecule described in claim 18 or the expression vector described in claim 19.

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