NZ740264B2 - Hemostatic material - Google Patents

Abstract

Disclosed is a hemostatic material, wherein a thrombin receptor activating agent is covalently coupled to a biocompatible matrix, and the thrombin receptor activating agent retains thrombin receptor activating activity after covalent coupling to the biocompatible matrix.

Description

Hemostatic Material The present invention relates to hemostatic material and s for producing and using such materials.

Uncontrolled bleeding is still the leading cause of mortality in traumatic and surgical injuries.u] Developing e""ective therapeutic approaches to control bleeding is therefore of paramount clinical and social values. In the last decades, a number of atic productsm] have been developed, ing fibrin-based glue or sealants,[3 &b] zeolite powders, [4 alb] crosslinked gelatin matrix [5 a,b] and so forth. r, each 0: these products has its respective limitations. Fibrin products suf .C .C er rom high cost, short shelf-life and weak mechanical s,reng,h.[m Zeolite minerals are prone to cause severe burns and are not able. [6] Crosslinked gelatin matrix could. halt bleeding within minutes only when ed with high doses of thrombin. [7] However thrombin is unstable in solution due to autoproteolysis. Highly concentrated thrombin is known to induce sis 0: human keratinocytes and can cause impairments in wound. healirg.[m Hence, there exists a strong need. to design alternative hemostatic als with improved safety. In particular, designing an effective strategy that avoids the use 0: highly concentrated thrombin is a desirable solution.

Thrombin is a serine protease that plays important roles in blood clotting (coagulation).[9 %b] As the key coagulation protease, thrombin converts soluble fibrinogen into fibrin networks inked by a transglutaminase (EXIll).[w] In addition, thrombin is the most potent tor of platelets by ating protease-activated. receptors [“” 1% Upon activation by in, platelets physically alter the conformation of GP IIb/IIIa receptors and provide high—affinity binding sites for fibrinogen, providing' fibrinogen—crosslinked platelet aggregation. Both PAR—l and PAR—4 are present on human platelets, yet activation of human platelets by thrombin is primarily mediated by ?AR-l.[n] The molecular mechanism of PAR—l activation by thrombin is ed in Fig. 7A. PAR—1 is highly expressed in platelets,[ 14 a, b] and PAR-l activation is initiated by proteolytic cleavage of part 0: the extracellular N—terminal domain of PAR—1 receptor by thrombin. lysis generates new inal ligand domains (SFLLRN (SEQ ID NO:l), a.k.a. thrombin receptor agonist peptide—6, TRAP6) that interact with the receptor within the ellular loop 2 and triggers the signaling pathway of PAR—l activation. It has been proven that short TRAP6 peptide (SFLLRN) could. work as a potent platelet activator separately and stimulates platelet aggregation via PAR—l signaling.[”] Multiplate® TRAP test has become a standard in vitro assay in whole blood or in platelet rich plasma for quantitative determination of platelet function triggered by TRAP6. TRAP test allows analysis of platelet function activated through PAR-l signaling without triggering fibrin formation, which otherwise occurs when thrombin is the agonist, because of using a thrombin inhibitor in the sample.

WO 96/40033 Al discloses a hemostatic material with hemostatic agents, including epsilon aminocaproic acid and a thrombin receptor activatirg peptide, wherein the hemostatic agents are sprayed or coated on a atic matrix so as to obtain a matrix wherein the agent is physically (but not covalently) adsorbed on the matrix. Such patches have the drawback that the hemostatic agents provided. therewith. easily release from the patch when contaCted to a bleeding area 0: a patient. Such there is the potential danger of inducing systemic thrombotic events, especially since there are no circulating antagonists in the circulation.

WO 03/057072 A2 discloses hemostatic compositions comprising cellulose and a polysaccharide covalently linked thereto. "t is an object o: the present invention to provide improved hemostatic al with thrombin receptor activating agents for control‘ing bleeding.

Therefore, the present invention provides a hemostatic al, wherein a thrombin receptor activating agent is covalently coupled to a biocompatible matrix.

With the present invention it is shown for the first time that a thrombin or ting agent can be covalently lized on a biocompatible matrix so as to obtain an ed hemostatic material suitable for administration to human patients in need f. As a preferred embodiment, covalent coupling of TRAP6, TQAP7 or TRAP8 to a synthetic hydrogel matrix (e.g. a polyvinyl alcohol based polymer) resulted in a suitable hemostatic material, maintaining the activity' for platelet tion in a safe, localized. manner over a considerable period 0: time so as to enable an improved hemostasis, ally via an induced et aggregation.

The biocompatible matrix ing to the present invention may' be any matrix that is useable for being adminiStered to human patients, especially for wound coverage or filling of volumetric defects (e.g. in organs) 0: a human patient.

According to the present invention, it is preferred to use the matrix materials that have been suggested in the prior art for such purposes. In general, a “biocompatible” matrix is a matrix that may be administered to human patients and that does not induce a negative effect in the course of this administration and contact with the patient. A mpatible” matrix is a matrix that does not contain materials or components that threaten, poison, impede, or ely a ect living tissue (e.g. human tissue that is exposed to the surface in ).

Examples for such matrices "II are “classical wound coverages, such as patches, sponges, but also flowable or sprayable matrices, powders, etc. such as FloSealTM (a cross-linked gelatin matrix), SurgifloPM (a bovine gelatin paste). Whereas patches are advantageous for l wound coverages, non—material hemOStats, most prominently flowable matrices, can be delivered within the same phase as opposed to liquid/solid ches or can be used. to r'exibly' fill es or provide a flexible scaffold (“volumetric defects”). The matrix should be chemically active or chemically activated. so that the thrombin receptor activating agent can be covalently coupled to the matrix according to the present invention. For example, the matrix may have tydroxyl groups, vinyl groups, yl groups, or amino groups to allow covalent attachment of the thrombin receptor acuivating agent to the matrix.

Preferably, the matrix is a hemostatic matrix, i.e. the matrix material as such has already hemostatic properties. Such materials are well available in the art and comprise e.g. collagen, gelatin or chitosan.

A preferred biocompatible matrix is selected from the group ting o: a erial, preferably a protein, a biopolymer or a polysaccharide matrix, especially a collagen, gelatin, fibrin, starch or chitosan matrix; and a synthetic r, preferably a polyvinyl alcohol, polyethylene glycol, poly(N— isopropylacrylamide), etc..

Preferably, the matrix 0; the present invention is biodegradable, i.e. it is naturally' absorbed. by the patient’s body after some time. In any way, the material (including the matrix) must be biocompatible, i.e. have no harming effect to the patient to whom the material is administered. Such biodegradable als are specifically suitable in situations where hemOStasis is achieved inside the body, i.e. in the course of surgery and the site is closed after surgery.

Accordingly, the matrix is preferably a biomaterial selected from biopolymers such as a protein, or a polysaccharide. al‘y preferred is aa biomaterial selected from the group consisting o: collagen, gelatin, fibrin, a polysaccharide, e.g. hyaluronic acids, an, and a derivative thereof, more preferred collagen and chitosan, especially preferred collagen.

Such collagen matrix used for the present invention can be derived from any collagen suitable to form a gel, including a material from liquid, pasty, librous.C or y collagenous als that can be sed to a porous or fibrous matrix as well as particles. The preparation. of' a collager gel for the production 0: a sponge or sheet may include acidificauion until gel formation occurs and subsequent pH neutralisation. To improve gel forming capabilities or solubility the collagen may be (partially) hydrolyzed or Hmdified, as long as the property to form a stable sponge or sheet when dried is not diminished.

The Hatrix used for ng the thrombin or activating agent can be a ymer, i.e., a lly occurring polymer or a derivative :, or can be a synthetic polymer. Examples 0: biopolymers useful in a hemostatic material ing to the present invention e polypeptides such as collagen, collagen derivatives such as gelatin, elastin, and elastin derivatives, and polysaccharides such as hyaluronic acids, starch, cellJlose, .C or a derivative thereof, lor example, oxidized ose. Preferably, the biopolymer is a human biopolymer, which can be isolated from an individual or can be a synthetic biopolymer, e.g., a recombinantly produced biopolymer.

In various embodiments, the matrix comprises a recombinant human r. Tn particular, the recombinant human polymer can be a recombinant human collagen, such as, for example, recombinant human collagen type I, recombinant human collagen type I, or a combination thereof. In one ment, the matrix comprises recombinant human collagen type III. ln another embodiment, the matrix comprises recombinan: human collagen type For example, the recombinant human gelatin can be derived from recombinant human collagen type I . n ye: r embodiment, the matrix comprises recombirant gelatin derived from recombinant human collagen type I. In further embodiments, the Hatrix comprises recombinant gelatin produced directly' by expression 0: encoding polynucleo:ide The polysaccharide used as a matrix in the present invention is preferably selected from the group consisting of cellulose, alkyl cellulose, methylcellulose, alkylhydroxyalkyl cellulose, hydroxyalkyl cellulose, cellulose e, salts of carboxymethyl cellulose, carboxymethyl cellulose, yethyl cellulose, chitin, carboxymethyl chitin, hyaluronic acid, salts o: hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin, pullulan, n, chondroitin, chondroitin sulfates, carboxymethyl dextran, ymethyl chitosan, chitosan, n, heparin sulfate, heparan, heparan sulfate, an sulfate, keratan sulfate, carrageenans, an, starch, amylose, amylopectin, poly-N-glucosamine, polymannuronic acid, polyglucuronic acid, polyguluronic acid, derivatives of said polysaccharides, or combinations thereOi.

The present matrix may also be based on a synthetic polymer.

The synthetic absorbable polymer can be an aliphatic polyester polymer, an aliphatic polyester copolymer, or combinations thereOi.

The present matrix may also be ed in the form 0; a woven or non—woven fabric made of fibers. Such fibers are preferably made of a biocompatible and/or biodegradable al. A. number of such fibers have been used. so far to provide hemostatic fabrics. In some embodiments, such nonwoven or woven fibers may comprise one or more polysaccharides such as pectin, acetylated. , hyaluronic acid. and derivatives of f, and the like. In some embodiments, the pectin and/or acetylated pectin may be d from. sugar beets. In other embodiments, the ccharide may be a non-cellulosic polysaccharide. The woven or nonwoven fibers may also include fibers comprising Other biodegradable polymers including po yg‘ycolide, polylaCtide, poly(lactide—co-glycolide), poly(tcaprolactone ), ioxanone), polycaprolactone, poly(3— hydroxybutyric acid), poly(3- hydroxybutyric acid-co-3 -hydroxy valeric acid), alginates, en, chitosan, gelatin, fibrinogen, elastin, polyethers, polyanhydrides, polyesters, polyorthoesters, polyphosphazenes, polyvinyl alcohol, polyvinylpyrrolidone, polytrimethylene carbonate, and the like.

In addition, l n fibers such. as cotton, silk and wool may also be used.

The matrix material ing to the present invention may preferably be provided as granules of various morphologies, including powder or matrices "or ”lowable hemostats. For example, the granules may have a (median particle) size of l to 1.000 um, preferably from 10 to ;.000 um, especially from 200 to 800 um. le matrices as flowable hemostats are disclosed e.g. in WO 98/08550 A or A.

According to a specifica'ly preferred embodiment, the present invention uses polyvinyl alcohol (PVA) as a matrix material. PVA is a water-soluble polymer originated from l hydrolysis of polyvinyl acetate. PVA-based hydrogels used as a matrix according to the present invention have been widely used in tissue engineering and drug delivery systems because of their superior biocompatibility (FiDA—approved).[16 Tb] In addition, PVA hydrogels are well known for being uniquely stronger than most other synthetic hydrogels.

The present invention uses thrombin receptor activating agents covalently' bound. to a atically' suitable matrix. ?referred embodiments of thrombin receptor aCtivating agents are in receptor activating peptides (TRAPs). TRAPs are a family of peptides of varying amino acid length which correspond to the new inal region. of the thrombin receptor. These synthetic or recombinant peptides mimic the activated form of the extracellular portion of the thrombin receptor protein and function as thrombin agonists.

US 766 A and WO 96/40033 A1 describe pharmaceutical compourds and atic patches containing TRAPs or "agonists" as usetul to encourage blood clotting, for example, in localized application at internal bleeding sites 0: hemophiliacs. The agonists are disclosed as ing thrombin's ability to stimulate fibroblast proliferation and, concomitantly, p'ate et aggregation. TRAPs thus can be useful in promoting hemostasis and wound hea ing. With the use of TRAPs, an effective remostatic material and hemostatic bandage can be provided which can be complete‘y free of biological compounds such as thrombin and fibrinogen and the concomitant dangers 0: viral contamination.

Representative TRAPs which may be incorporated into a al according to the present ion include peptides capable of activating' thrombin. receptor, such. as the agonists identified in the US 5,256,766 A by the formula AAx ——AAy —— (AAi)n——z.

Other TRAPs which have been disclosed which activate fibroblasts and are implicated in wound healing e peptides TRAP 508-530, amino acids AGYKPDEGKRGDACEGDSGGPFV (SEQ ID NO:2); and TRAP 517—530, amino acids RGDACEGDSGGPFV (SEQ ID NO:3).

Further suitable TRAPs are disclosed by Carney et al. J. Clin Invest. 89:14691477 (1992); Furman et al. PNAS 95 (1998), 3082— 3087 and Cromack et al., J. Surg. Res.53: 117 (1992).

Accordingly, suitable TRAPs useful in the present invention, for e, include peptides PNDKYEPF (SfiQ D NO:4), SFLLRRPNDKYEP (SEQ ID NO: 5) , SFLLRNPNDKYE (SEQ ID NO: 6) , SFLLRRPNDKY (SEQ ID NO:7), SFLLRN'9NDK (SEQ ID NO:8), SFLLRW?ND (SEQ -3 NOz9), SFLLRNPN (TRAP8 (SEQ ID ), SFLLRNP (TRAP7 (SEQ ID NO:ll)), SFLLRN (TRAP6), SFLLR, SFLL, and SFL, and the amidated forms thereof. Because TRAPs are small peptides, they are more stable than large proteinaceous et activating agents, such as thrombin. The stability of TRAPs contributes to the properties .C (1. the present Heterial which permit ii: to be stored t refrigeration.

Preferably, the thrombin receptor activating agents (preferably the TRAP) is provided with a linker so as to covalently c0iple the TRAP to the . Preferred linkers are amino acids (single amino acids, such as Cysteine, Arginine, Lysine, Serine, Glycine, etc. (preferably' Cysteine), or short amino acid linkers with e.g. 2 to 5 amino acid residues, preferably' comprising amino acids selected from the group of Cysteine, Arginine, Lysine, Proline, Asparagine, Glutamine, Serine and Glycine. red dipeptidic linkers may be y— (the terminal “-“ indicates the bond to the thrombin receptor activating agent), —Gly-Cys, Cys-Arg-, -Arg-Cys, -Asn-Cys, Cys— Asn—, —Pro-Cys, Cys-Pro-; red tripept‘dic linkers may be Cys-Gly-Gly-, -Gly-Gly-Cys, -Pro-Asn-Cys, Cys-Asn-Pro-, Cys-?ro— Asn-, —Asn-Pro-Cys etc., or any other ic linker sing 2 to 5 amino acid residues known for pharmaceutical peptide coupling to carriers or matrices.

According to a ical'y preferred embodiment, the thrombin or activating agent of the present material is a thrombin receptor activating peptide (TRAP), preferably TRA?8, TRAP7, TRAP6, TRAPl-4l, SLIGKV (for PAR-2 (human) (SEQ ID NO:l2)), TFRGAP (for PAR-3 (human) (SEQ ID NO:13)), GYPGQV (for PAR—4 (human) (SEQ ID NO:l4)), or ed. forms f, as well as mixtures of such agents.

The hemostatic material ing to the present invention may have any suitable form that is usable for the treatment of human patients in need 0: a hemostatic material, i.e. as a ‘lowable or sprayable form; as a two-dimensional form (where the third dimension extension is comparably small (e.g. less than 1/10 or 1/20) compared. to the other two dimensions; or as a three-dimensional form (e.g. a sponge, a paste, a cavity implant, etc.). A preferred two— or three—dimensional embodiment of the material according to the present ion may, for example, be a sponge, a woven or non-woven :abric, a preformed shape, preferably as a cylinder or cone (e.g. for tooth extraction) or as being used as a flexible or non—flexible scaffold, a particulate or ate material or a sheet. It is specifically' preferred. if the matrix is able to absorb fluid from the wound so as to attract further blood coagulation components from the wound once the Haterial is applied to the wound to achieve platelet aggregation. Furthermore, the material is preferably' flexible and suitable to be applied. on diverse tissues and ons with s shapes.

Further preferred embodiments of the hemostatic material according to the present invention comprise further ingredients, such as anti-bacterial agents, atively active , suppressive agents, anti-inflammatory agents, anti— fibrinolytic agents, such as aprotinin or ECEA, growth factors, vitamins, cells, etc.. In a preferred embodiment, however, the material according to the present invention may or may not have such further ingredients, provided that the material is free of ents which could have negative impact on the storability or administration of the atic material. Accordingly, the present hemostatic material is preferably essentially' free of any protein degrading activity, especially free of se activity, specifically' free of thrombin activity. in is added frequently' to hemostatic materials in order to promote fibrin cleavage and clot formation; however, it may also be proteolytic 11) the hemostatic Haterial, which.1nay be ted especially during production and storage of such material. With the present invention, the addition or presence of thrombin or comparable components is not required and may therefore be omitted without negative impact on the hemostatic ties of the hemostatic material.

Preferably, the hemostatic material according to the present invention is provided in a state wherein it is able to soak up liquid Haterial, such as blood. The ability to soak up blood (and the components therein promoting clot ion, bleeding termination and wound e) significantly enhances the overall efficacy of the hemostatic Haterial. For example, the hemostatic material according to the present invention is provided. in a dry form. or in a wet form still allowing the material to take up further liquid material (i.e. being soa<ed with liquid in an amount which amount is still under its soaking capacity). This allows blood entering and/or passing through the hemostatic material so as to provide the blood ents useful e.g. in the wound closure process in an enlarged volume or in the whole (or virtually the whole) volume Oi the al applied.

According to a further aspect, the present irvention relates to a method for producing the atic material accordirg to the present ion. This method for producirg a hemostatic material is characterised by the step of covalently coupling a in receptor activating agent to a hemostatic matrix.

Preferably, the thrombin receptor activating agent is a thrombin receptor activating peptide (TRAP), preferab'y TRA?8, TRAP7, TRAP6, TRAPl-4l, SLIGKV (for PAR-2 (human)), TF'RGAP (for PAR—3 (human)), GYPGQV (for PAR—4 )), or amidated. forms :hereo:, as well as mixtures of these agents. "t is, of course, important for the present invention that ,he in receptor activating agent keeps its thrombin receptor activating activity after covalent coupling to the biocompatible . Not any biomolecule can be simply bound to a biocompatible e and still retain its bio-functionality.

In the course of generating the present invention, it turned out that the usage of conventional binding techniques to covalently couple C— or inus of the thrombin receptor activating agents according to the present ion (e.g. TRAP6 peptides) usually results in the loss of said bioactivity. Accordingly, the provision. of covalent lization. ch. that indeed retains full bio—functionality of the thrombin receptor activating agent (which is a peptide) is not trivial and needs to rely on the disclosure to obtain such functional embodiments contained herein.

For example, use of traditional ng techniques for peptides, such as those using Lil DC (l—Ethyl—3—(3— dimethylaminopropyl)carbodiimide; reaction of C-terminus, Asp and Glu) or NHS (Nthdroxysuccinimide; reac:ion o: NFterminus, Lys), did not result in active immobilised thrombin or activating agent (because they are usually non-specific, prone to side reaction with acidic amino acids or lysine, deactivation of es that relies on N-terminus, etc. [291,[301,£311) Techniques using cysteine and/or l addition also carry the risk 0: loss of function due to possible side reactions with amine groups[%]. Accordingly, such traditional coupling techniques result in loss of activity. While a variety of chemical approaches for peptide immobilization have been reportedly], most of these methods rely on reactions with carboxyl groups at the C-terminus (e.g. EDC)[”], or with primary amines at the N-terminus (e.g. NHS) BOL[3H, or with ne residues via Michael—addition based on maleimide or vinyl sulfone groups[%]. Unfortunately, most reported reactions are non—specific and prone to side reactions, either with acidic/basic amino acids (for EDC/NHS) or with amine groups (for Michael addition), which induce unfavorable loss of peptide activity. For instance, N—hydroxysuccinimide (NHS) eSter conjugation is the most often used approach to covalently immobilize bioactive peptides (e.g. cell-adhesive RG3) onto polymer substrates through reacting with the N—terminus of peptide [35] Although primary amines are the most reactive groups for NHS esters, recent studies have demonstrated that a series 0: side reactions could occur with oth r p ptidc r sidu s ( .g. -0H for tyrosine, serine, threonine, guanidinium for arginine)[w]fl3n. Furthermore, previous studies on the ure— function relationship of TRAPs (SFLLRN—), highlighted the significance of the amino acids at the NFterminus to maintain peptide activity [36] With these considerations in mind, it has been found that it is important I: for the present invention to develop a peptide immobilization approach that circumvents side reactions with. the N—terminus of the in receptor activating agent (being a e, such as TRAP6), in order to retain its bioactivity.

The present invention therefore provides a thrombin receptor ting active agent in immobilised form with retained activity. This turned out to be not le to be provided by conventional state-or-the-art approaches for e immobilization. The present ion therefore also provides a selection of specific coupling' techniques red. to above) with no risk of side reactions with N- or inus or acidic or basic amino acids by using e.g. bioorthogonal reactions, such as photo-click conjugation of cysteine—containing TRAP6 onto PVA norbornenes, which offers a very high degree of conjugation efficiency (>95%), site-specificity and modularity. It is clear that the same ation approach is also applicable for other substrates g nene groups, such as naturally-derived molecules (gelatin, onan, alginate, etc.) and synthetic analogues such as PEG. In addition, the polymer—bound thrombin receptor activating active peptide conjugates have lower probability to be internalized by blood cells than soluble forms thereof through PAR—l receptor signalling, as the size of 2016/070619 r substrates applied as biocompatible solid matrix for the t invention, such as PVA substrates eds of repeating inits), is far larger than a short TRAP6 sequence. In all, the approach. ing' to the present invention. with. coupling' the thrombin receptor activating active agent with retained activity to a biocompatible solid matrix (e.g. by photo—click conjugation for TRAP6 peptide immobilization) provides significant practical values for local hemostasis as well as other medical applications.

According to a rred. embodiment of this method, the hemostatic matrix is functionalized with chemical groups (e.g., alkene, sulfhydryl, alkyne, azido, hydroazide, hydrazine), preferably groups that allow high—efficiency covalent binding of the thrombin receptor activating agent via bioorthogonal reactions (e.g., ene addition, alkyne-azide cycloaddition, Diels—Alder reaction, hydrazide—hydrazine reaction).

Preferably, the thrombin receptor activating agent is modified with peptide sequences that are engineered with bioorthogonal groups (e.g., alkene, suifhydryl, alkyne, azido, hydroazide, hydrazine), especially with a cysteine moiety with an —SH group. 211 a preferred ment o: the HBthOd according to the present invention, the hemostatic al is functionalized with an ene group, such as norbornene, maleimide, allyl, vinyl ester, acrylate, vinyl carbonate, methacrylate, etc.

According to a specifically advantageous embodiment o: the present method, the chemical ng is performed by' photo— induced reactions, especially by radical—mediated thiol- rn-)ene photo—click chemistry (reviewed in [18a]) or (other) photo-triggered click chemistry (reviewed in .

The present hemostatic material may be finished as a cial produCt by the usual steps performed in the present field, for example by appropriate sterilisation and. packaging steps. For example, the present material may be treated by UV/vis irradiation (200—500 nm), preferably' with the help of photoinitiators with different absorption wavelengths (e.g.

Irgacure 184, 2959), preferably soluble initiators (Irgacure 2959). Such irradiation is usually performed for an irradiation time 0" 1-60 min, but also longer irradiation times WO 37178 may be applied, depending on the specific method. The material according to the present invention may be finally sterile— wrapped so as to retain sterility until use and packaged (e.g. by the addition 0: specific t information leaflets) into suitable containers (boxes, etc.).

According to another aspect, the present invention aiso relates to the hemostatic material according to the present invention for use in surgery and/or in the treatment of injuries and/or wounds. The hemostatic material according to the present invention is specifica'iy suitab'e and effective for increasing the release 0: platelet-derived. growth factors and for accelerating wound healing.

This makes the atic material according to the t invention an excellent tool for sealing of anastomosis, for suture line sealing and to safeguard. hemostasis in resection sites.

According to another aspect of the present invention, the hemostatic material according to the t ion may aiso be provided in kit form combined with other components necessary for administration of the material to the patient. For exampie, if the hemostatic material .C may be provided in flowabie dry orm (e.g. as granules or as a ) or as a flowable paste, it is preferred to provide such material with a suitable buffer on which can be added shortly before administration to the patient. SuCh a buffer solution usually contains (besides the buffer components, such as ate, carbonate, TRIS, etc. buffer systems) divalent metal ions, preferably' Ca2+ ions, or other functional ents (if not already present on or in the ), such as anti-bacterial agents, coagulatively active agents, suppressive agents, anti-inflammatory' agents, anti—fibrinolytic agents, such as aprotinin or ECEA, growth factors, vitamins, cells, etc.. The kit may further contain means for stering or preparing administering the hemostatic material, such as syringes, tubes, catheters, forceps, scissors, sterilising pads or lotions, etc..

Accordingly, the present invention relates to a kit, preferably for use in surgery and/or in the treatment of es and/or wounds, comprising - a hemostatic material according to the present invention and - at least one administration device, preferably selected from the group buffer solution, ally a bu "er solution containing' Ca2+ ions, a syringe, a tube, a er, s, scissors, a sterilising pad or lotion.

Preferably, the buffer solution further comprises a component selected from. the group anti-bacteria" agent, coagulatively active agent, immunosuppressive agent, anti— inflammatory agent, anti—fibrinolytic agent, ally aprotinin. or ECEA, growth factor, vitamin, cell, or mixtures thereof. Alternatively, the kit may also further comprise a container with a component selected from the group anti— bacterial agent, coagulatively' active agent, immunosuppressive agent, anti—inflammatory agent, anti—fibrinolytic agent, especially aprotinin or ECEA, growth factor, vitamin, cell, or mixtures thereof.

The present invention is further illustrated by the following examples and the figures, yet without being restriCted thereto.

Fig. 1 shows (A) synthesis scheme of PVA—N3 and PVA—TRAP6 ion in abs. DMSO at 50 °C for 12 h, TsOH: p- esulfonic acid, 22959: Irgacure 2959, i.e. one commonly used water-soluble PI); (B) lH—NMR (D20) spectra of PVA, PVA—NB and AP6 ate; and normalized viability of C7Cl2 ce'ls after 24/48 h exposure to , (C) and PVA—TRAP6 solutions (3) with varying polymer concentrations ( %, 0.5%, and 0.;%) investigated by MTT assay (n>3).

Fig. 2 shows (A)—(C) ROTEM characterization of the ation process of whole blood. in response to the investigated materials (CT: clotting time in seconds, i.e. the latency until the clot reaches a firmness of 2 mm; MCF: maximum clot firmness in mm). (A) Plotted ROTEM curves stowing the coagulation process of whole blood. in response to TRAP6 (0.1 mVl), PVA-TRAPES (0.1 mM TRAP6-), PVA-NB, and 0.9% NaCl control; (a) Influence of unconjugated- and conjugated—TRAP6 (PVA—TRAP6) at g dosage (0.01, 0.1, 1 mM) on CT; and (C) comparative analysis on CT n TRAP6, PVA—TRAP6, and PVA—NB at optimal TRAP6— concentration (0.1 mM). (D) Multiplate analysis of platelet function in response to TRAP6 (0.1 mM), PVA—TQAP6 (0.1 mM TRAP6), and PVA—NB; each measurement was performed in duplicate. (L)._‘ Comparison. ,— 0: the key jparameter‘ in. Multiplate: ation area in Units.

Fig. 3 shows (A) FACS analysis of mediated platelet activation measured by determination o: CD62p/CD42 co—expression after 15 min incubation. Experiments were run in duplicate, data are presented as tage of platelets positive for both CD62p and CD41 epitopes +SD. (B) Histogram plots showing the value of the sample d with the specific CD41 PE and CD62p APC antibodies. (C) Representative dot-plots for the expression of CD62p and CD41 of a TRAP6 treated sample, a PVA-TRAP6 treated sample, and a PVA-NB treated control after 15 min incubation.

Fig. 4 shows (A) tic showing the preparation 0: PVA—WB hydrogels by UV-photocrosslinking of PVA—NB with dithiothreitol (DTT) through radical-mediated. photo-click chemistry. (3) Mechanical characterization of PVA hydrogels with varying thiol— to-NB ratios (0.4, 0.8, 1.0 and 1.2) using in situ oscillatory photo-rheometry: G’- gel storage moduli, 10% PVA-NB, 0.5% 22959, 60 s delay, light intensity: 20 mW cm”; 50 um gap ess, 10% strain, 10 2. (C) Representatives 0: photopolymerized. PVA—NB hydrogel pellets (scale bar: 1 cm). (D) Influence of thiol—to—NB ratio (N) or the G’-plateau value: A=0.4 (l), 0.8 ( ), 1.0 (III), 1.2 (IV). (E) Equilibrium mass swelling ratios of PVA—NB hydroge's ( — V) after swelling in PBS for 48 t.

Fig. 5 shows (A) schematics of the ation of PVA hydroge' (", -SH:-NB=0.4) particulates (PVA-NB-P) by tial Lyophilization and cryo—milling,; (B) schematics of the surface functionalization of PVA—NB—P with ne—containing TQAP6 peptide via triggered. thiol—NB conjugation, —SH:—NB=1.2, 0.1% P: (Li—TPO) in PBS, 20 mW cmfl; (C,D) SEM images of PVA- TRAP6—P, scale bars: 100 um (C), 10 um (D).

Fig. 6 shows ) ROTEM characterization of the coagulation process of whole blood in response to the suspension of PVA-NB-P and PVA-TRAP6-P (10 wt% in saline). (A) Plotted ROTEM curves showing the coagulation process of whole blood in response to PVA—NB—P and PVA—TRAP6—P suspensions; (B) comparative analysis on the CT between PVA—NB—P, PVA—TRAP6, and NaCl (control). (C)—(F) FACS analysis of particulated polymers.

(C) FACS analysis of TRAP6-mediated et activation measured by determination of CD41 co—expression after 15 min incubation. Experiments were repeated twice using blood samples from different donors (n=2), and data are presented as percentage of platelets positive for both CD62p and CD41 epitopes i8). (D,E,F) Representative dot-plots for the co— expression of CD62p and CD41 of a PVA-NB—P d sample and a PVA—TRAP6—P treated sample (positive control: 0.1 mM TRA?6, negative contro_: NaCl) after 15 min incubation.

Fig. 7 shows (A) lar mechanism of protease activated or—1 (PAR—l) activation. (B) TRAP6- e motifs are covalently immobilized within synthetic polyvinyl alcohol (PVA) hydrogels, i.e. TRAP6-presenting els, which are e of activating platelets in a highly localized manner.

Fig. 8 shows the influence of TRAP6 (non-conjugated, 1 mM) and. polymer-TRAP6 conjugates on blood. coagulation. measured. by rotational oelastometry (ROTEM). A, clotting time (CT); B, clot formation time (CFT). P3G-TRAP6 was prepared by reacting PEG—lOk—NHS with the NFterminus of TRAP6, while PVA—TRAP6 was prepared. by reacting' photo-clickable PVA. norbornenes (22 kDa) with the cysteine residue in TRAP6 (SFLLRNPNC). PEG—lOk—Glycine was used as the blank polymer control. All s were measured in triplicates.

Fig. 9 shows experimental proof of retained bio-activity in PVA—TRAP6 (1 mM) in comparison to 1 mM TRAP6, 1 mM PVA—N3 and saline. A, total platelet aggregation area measured by Multiplate—TRAP method; 3, level 0: CD4l/CD62P co-expression due to platelet activation measured by flow cytometry (FACS).

Examples: Development of tic Platelet—Activating Hydrogel Matrices to Induce Local Hemostasis The present examples demonstrate the present invention by way of 21 water-soluble PVA-TRAP6 conjugate as Hmdel platelet- activating polymers as well as insoluble (crosslinked) PVA—TRAP6 hydrogel particulates (PVA-TRAP6-P) for safe and localized acceleration of hemOStasis. In this work, it is demonstrated for the first time that TRAPs platelet-activating peptides, suck as TRAP6, can be covalently immobilized in synthetic hydrogel matrices (Fig. 7B) for hemorrhage control. With this hemostatic material the esis was tested that polymer-conjugated TRAP6 peptides can maintain their activity for platelet tion while rating hemostasis in a safe, localized manner and no systemic e of the platelet-activating agent can occur. The water-soluble PVA-TRA?6 conjugates was designed as model platelet-activating polymers as well as insoluble (crosslinked) AP6 hydrogel particulates (PVA—TRAP6—P) for safe and localized acceleration of asis. These new polymer-peptide conjugates were ed using highly ent norbornene photo-click try. The extent to which these materials could activate platelets was systematically characterized. using rotational thromboelastography (ROTEM), platelet aggregation assay (Multiplate) and flow cytometry (FACS).

Several hemostatic strategies rely on the use 0‘ b'ood components such as fibrinogen and thrombin, which suffer from high cost and short shel"—li"e. "n the present es, a cost— effective synthetic biomaterial is developed for rapid local hemostasis. Instead. of using thrombin, thrombin—receptor— agonist-peptide—6 (TRA?6) is covalently engineered in polyvinyl alcohol (PVA) hydrogels. Soluble ?VA-TRAP6 was firstly prepared by covalent ment of cysteine—containing TRAP6 onto the backbone of PVA-norbornenes (PVA-WB) through photo—conjugation.

Cytotoxicity studies using C2Cl2 Hyoblasts indicated that PVA—NB and PVA—TRAP6 are nontoxic. Thromboelastography ed. that hemostatic activity 0: TRAP6 was retained in conjugated form, which was comparable to free TRAP6 solutions with equal concentrations. A 0.1% PVA-TRAP6 solution can shorten the 2016/070619 clotting time (CT) to ~45% of the physiological CT. High platelet-activating efficiency was further confirmed by platelet aggregation assay and FACS. For potential clinical applications, TRAP6—presenting hydrogel ulates (PVA—TQAP6—P) were developed for local platelet activation and henostasis. PVA— P was prepared by biofunctionalization of photopolymerized PVA—N3 hydrogel particulates (PVA—NB—P) with TQAP6. It was demonstrated that PVA-TRAP6-P can effectively shorten the CT to ~50%. FACS showed that PVA-TRAP6-P can activate ets to a comparable extent as soluble TRAP6 l. Altogether, PVA— TRAP6—P represents a promising class 0: biomaterials for sa:e hemostasis and wound healing. 1.Experimental Section 1.1. Materials and Reagents.

All reagents were purchased from Sigma-Aldrich and used as received unless otherwise noted. 1.2. Synthesis of PVA-Norbornene (PVA-NB).

In a three—neck flask, 10 g of PVA (22 kDa) and 20 mg of p— toluenesulfonic acid were dissolved in 750 m1 of anhydrous DMSO at 60 °C for 1 l1 under‘ argon. atmosphere. 211 a . flask, under argon atmosphere 2 <3 of cisnorbornene-endo-2,3- dicarboxylic anhydride (O.l eq. to —OH ) was dissolved in 50 mL of ous DMSO. The obtained on was added dropwise into the firso flask containing PVA. The reac:ion was maintained at 50 °C for 12 h. After reaction, the crude product was ed by dialysis against 10 mM NaHCO3 solution for 24 h and subsequently against deionized (DI) water for 12 h. After lyophilization, PVA—NB was obtained. as ess solid. in 95% yield.lH—NMR (D20): 8(ppm): 6.2 (24, s, —CH=CH-), 3.3 (2H, s, — C=CCH-CH-), 3.1 (2H, s, -C=C-CF-CH-), 1.3 (2H, s, -CH2-).

Degree of substitution ()8): 7.5%. 1.3. Synthesis of PVA—TRAP6 conjugates.

PVA—TRAP6 was prepared by covalent attachment of a cysteine— containing TRAP6 peptide (N-C: SFLLRNPNC (SEQ ID NO:15), China Peptide Co.) onto the backbone of PVA-NB throagh thiol—ene photo-click conjugation. Specifically, 60 mg of PVA—NB was dissolved in PBS solution of 0.5% Irgacare 2959 (12959, BASF) to give a final macromer concentration of 5%. To this solution, l00 mg of TRAP6—Cys peptide (1.2 Eq. to N3 groups) was added. The obtained solution was stirred under argon and irradiated with filtered UV—light (320-500 nm) for 300 s at 20 mW cmfl. The UV— light was guided from an Omnicure $2000 lamp. 1.4. Preparation of PVA—NB hydrogels.

PVA—NB (DS—7.5%) was ved in 0.5% 22959 solution, achieving a final concentration. 10 %. Then, aliquots of this solution was mixed with appropriate amount of dithiothreitol (DT ), providing —SH/-kB ratios as 0.4 (I), 0.8 (II), 1.0, and 1.2 ( T), respectively. Hydrogel pellets were prepared by photopolymerization in a multi—well PDMS mold (well diameter: 6 mm). Specifically, 200 ifli o: macromer solutions were pipetted n two glass coverslips ted by the PDMS mold (thickness: 1.5 mm) and then exposed to ed UV light (20 mW cm&) for 600 s. P llcts wcr detached from the slides and washed with e PBS. 1.5. Preparation of PVA—NB hydrogel particulates (PVA—NB—P).

Hydrogel precursor solutions ( - II) were prepared as aforementioned. and. photopolymerized. at same conditions except using a 10 mL cylindrical glass vial as the mold. After photopolymerization, the hydrogel ers were transferred into a l00 mL beaker and washed with PBS (2 changes) for 12 h in order to remove ted polymer and PI. Afterward, the swollen hydroge's were frozen with liquid N2 and lyophilized. Finally, the dry PVA—NB matrix was grinded. into fine powders using a RETSCH Cryomill R8232. 1.6. ation of TRAP6—presenting PVA hydrogel particulates (PVA—TRAP6—P).

PVA—TRAP6—P was prepared by covalent attachnent of TRAP6-Cys peptide onto the residual NB groups on PVA—NB—P. Specifica'ly, l00 mg of PVA—NB was dispersed in PBS solution of 0.1% visible light PI (LAP)[U] to give a final polymer content of 5%. To this sion, specific amounts of TRAP6-Cys e (1.2 Eq. to NB groups) were added. The obtained suspension was stirred under argon and irradiated with UV-light (365 nm) for 300 s at 20 mW cm%. The UV—light was guided from an Omnicure LX400 LED lamp. 1.7. Photo—rheometry.

Photo-rheometry was performed on a Hmdular rheometer (Anton Paar MGR-302) as previously' reported.[N] Specifically, MCR302 was integrated with ed UV—light (320—500 nm) from a light guide (Omnicure S2000) to the bottom of the glass plate.

Specifically, plate-to-plate atory photo-rheometry was applied for real—time monitoring of the curing kinetics of hydrogel formulations during olymerization. Light intensity 2 at the plate surface was ~20 mW cm‘ as determined by an Ocean Optics US3 2000+ spectrometer. 30th storage moduli (G’) and loss Hoduli (G”) of the samples could be Hwnitored as a function of ation time. Gel point was determined in the vicinity of the G’ and G” ver. 1.8. Water—uptake.

Mass swelling ratios of PVA-N3 hydrogels were tested using a generic protocol. ma Hydroge' pel'ets (n == 3) were prepared as aforementioned and allowed to swell in DI HgO for 24 h at room temperature. The wet pellets were weighed to determine the equilibrium swollen mass (Ms) and then lyophilized to obtain the dry weight (Md). The equilibrium mass swelling ratio (Qm) was calculated as MS/Md. 1.9. MTT assay.

Cytotoxicities of PVA, PVA—NB and PVA—TRAP6 macromer solutions were evaluated via MTT assay. C2C12 cells were cultured in Dulbecco’s ed Eagles Medium (DMEM) supplemented with 5% fetal calf serum (FCS), 1% L-Glutamine, 1% Penicillin/Streptomycin (all from Sigma—Aldrich, Austria).

Macromer solutions with three concentrations ( %, 1% and 0.1%) were prepared. in DMEM culture ” C2C12 cells were then seeded in a l plate at a density 0: 5><103 cells per well in 200 uL of culture medium. After 24 h incubation (37 °C, 5% COfl, 100 uL of the respeCtive macromer solutions were added to the cells in triplicates. After 24 h incubation, cells were washed twice with sterile PBS before the addition 0: 100 uL thiazolyl blue tetrazolium bromide (MTT) working solution (5 mg/mL in ?BS). After 1 h incubation, the liquid was discarded and 100 ul of DMSO was added to dissolve the formazan crystals. Finally, the absorbance was measured at 540 nm using a microplate reader. 1.10. Rotational thromboelastometry ).

ROTEM (TfiM nnovation, Germany) was applied to monitor the interactions of platelet-activating polymers and whole blood over time. ROTEM system contains an ating sensor pin that is immersed iJ1 a temperature-controlled cuvette containing the blood sample. Four measurements can be performed in paralle' in the same device. Generally, coagulation of the citrated blood sample is initiated by re-calcification. The formation kinetics of a fibrin clot could be monitored mechanically and calculated by an integrated computer to the typical curves and numerical parameters .

Blood. was collected from. three un—medicated and healthy donors using minimal stasis from an antecubital vein through a ge needle. After discarding the first 3 ml, blood was collected in 3.5 ml tubes (Vacuette; Greiner e) containing 0.3 ml buffered 3.2 % sodium citrate. The samples were kept on a pre-warming stage at 37°C for at least 10 mir prior to is and were processed within 3 h. ROTEM analysis of the whole blood sample was initiated by recalcification with the addition 0: 20 ul of CaC12 (star-TEM®, 200 mM). Polymer solutions and/or polymer suspensions were added directly to thc cuvctt imm diat ly after recalcification of the citrated blood and mixed by gently pipetting up and down as previously reported.[ml The final reaction volume per ROTEM e was 370 ul, consisting of 300 ul citrated whole blood, 20 ul CaCl2 and 50 ul r solution or polymer suspension.

The ing ROTEM parameters were calculated from the signal and included in the statistical is: clotting time (CT) in seconds (sec), i.e. the y until the clot reaches a firmness o: 2 mm, which is a measure of initial fibrin lormation;.C clot formation time (CFT) in sec, i.e. the time from CT until the clot reaches a firmness o: 20 mm, which indicates platelet function and librinogen.C quality; d-angle, the angle (°) between the x-axis and the tangent of the forming curve starting 2016/070619 from CT point, which is comparable to CFT; maximum clot firmness (MCF) in mm, the maximum amplitude of the curve, which indicates the te strength of the clot; and A30 (mm), i.e. the clot firmness after 30 min. 1.11. Multiplate Analysis.

The principle of Multiplate test is based on ,he fact shat ets become sticky upon activation, and trus prone to adhere and ate on the metal sensor wires in the MJltiplate test cuvette. The sensor‘ wires are made of highly conductive copper, which. is silver-coated» As activated. platelets adhere and ate on the sensor wires, the electrical resistance between the wires rises, which can be Hmnitored in real time.

For each measurement, 300 uL of saline and 300 uL of hirudinized whole blood was added sequentially' to a Multiplate cuvette.

After 3 min incubation at 37 °C, 20 uL of sample solution was added and at the same time the program started to collect signals. The late device allows 4 measurments in parallel.

Each measurement was performed for 6 min and in duplicate. 1.12. FACS Analysis.

The blood of ,wo unmedicated and healthy donors was collected and stabilized by sodium citrate. To 110 uL of whole blood (W3), 20 uL of ?VA—TRAP6 solution (30 mg mL‘1 and 3 mg mL‘1 to obtain final TRAP6 concentrations in the WB of 2 mM and 0.2 mM) were added and ted for 15 min at 37 °C. Twenty microliter of TRAP6—C on (14 mg mL&) and/or 20 uL of NaCl solution (0.9%) were added as the positive control and the negative control, respectively. The incubated mixture was mixed with 300 uL o: 1% paraformaldehyde for 15 min at room temperature. After washing and centrifugation a double immuno— s:aining was performed using 20 uL of phycoerythrin (PR) labeled monoclonal antibody directed against CD41 and 20 uL of allophycocyanin (APC) labeled monoclonal antibody directed against CD62P. Isotype controls were incubated with mouse IgG dies conjugated with APC or PE dye, tively (all antibodies were purchased from @D %iosciences, Heidelberg, Germany). After 30 min incubation at room temperature the ac:ivation state of the platelets was determined 'using fluorescence flow cytometry. Expression of the platelet activation marker CD62P and the constitutively present platelet marker CD41 were ed using a Beckmann Coulter Cytomics FC— 500 equipped with Uniphase Argon ion laser, 488 nm, 20 mW output. Overall 100 000 platolcts wcr moasurcd per sample and analyzed with the Cytomics CXP software. The experiment was repeated twice. 1.13. Statistics.

A'1 error bars indicate the standard deviation. The statiStica; significance was determined by student’s t-test, where ‘*’, ‘**’, ‘***' te P < 0.05, P < 0.01, P < 0.001, respeCtiveLy. 2. Results & sion 2.1. Materials Design and Synthesis In this study, a linear synthetic r polyvinyl alcohol (PVA) was selected as the substrate for covalent immobilization o: the potent platelet-activating' peptide (TRAP6) due to the rollowing’.C reasons. First, PVAs are FDA—approved. polymers with superior cost-e "icacy and cytocompatibility, therefore intriguing for applications. [in many ical Second, the existence 0: a high. number of hydroxyl groups in PVA. o "ers significant freedom for tunable functionalization and presentation of ive ligands, which is not ble with other synthetic substrates such as pendent polyethylene glyco; (PEG).

To introduce TRAP6 peptides onto PVA, we chose the robust ene photo-click chemistry as the conjugation approach. On one hand, norbornene group was selected as the ene functionality due to its ultrahigh reactivity towards thiol-ene reaction as well as low cytotoxicity.fl8’l% On the other hand, we engineered a cysteine moiety with a free thiol group into the C—terminus of a TRAP6 peptide sequence (SFLLRNPNQ), since it is accepted that the N—terminus of TRAP6 sequence is critica1 For its ability to activate platelets.nm PVA—NB was synthesized through a facile esterification on between PVA and norbornene anhydride for 12 h at 50 °C in DMSO (Figure 1A). One notable age of this modification approach is that after modification a high number of carboxylate groups could be neutralized into the sodium salt form to provide the products with good solubility. The crude products were purified by sequential dialysis against 10 mM NaHCO3 for neutralization and later on against H20, and finally lyophilized (>95% yield). To confirm. the synthesis, PVA—NB was analyzed using lH—NMR in ison with unmodified. PVA. As shown in Figure 1B (bottom), the spectruH1 of unmodified. PVA. represents two major peaks at 4.0 ppm and 1.6 ppm, which are corresponding to the -CH- and methylene groups, respectively. The spectrum of PVA-NB (Figure 1B, middle) shows new peaks at 6.2 ppm (s, 2H, - CH=CH-), 3.3 ppm (s, 2}, -C=C-CH-CH-), 3.1 ppm (s, 2H, -C=C-CH— CH—) and 1.3 ple (s, 2H, -CH2—), respectively; The degree of substitution (D8) of BVA-NB was determined by comparing the integral values corresponding to signals (a, d, f). By changing either the stoichiometry between the reactants or reaction time, it was feasible to precisely control the DS in a wide range from %-50% (Table 81). Since PVA (22 kDa) is a linear polymer consisting o: ~500 repeating units, we selected PVA—NB with the lowest 38 (BS—7%) as the precursor, providing ~35 reaction sites for corjugation with cysteine-containing peptide.

AP6 conjugates were prepared. by conjugation of cysteine-containing TRAP6 peptide with the N3 groups of PVA—NB in PBS solution of 12959 as photoinitiator (PI). To confirm the conjugation efficiency, NMR model reactions were firstly performed. in D20. Based on the NMR reaction, Figure 1B (Top) ents the spectrum of PVA-TRAP6 conjugates. The significant decrease of NB proton signals (a) at 6.2 ppm indicates the success of conjugation. Besides, the spectrum of PVA—TRAP6 also shows a new peak at 7.4 ppm, corresponding' to the aromatic protons of phenylalanine (Phe or F) moieties in the TRAP6 sequence . 2.2. In—vitro Cytotoxicity To prove the ability of the prepared materials for biomedical applications, the in vitro patibility of PVA, PVA—NB and PVA—TRAP6 solutions was igated. by MTT assay using C2012 myoblasts. MTT assay showed. that PVA. and PVA—NB (Figure 1C) solutions were non—toxic at varying concentrations (0.1, 0.5, 1 %) after 24h and 48h incubation. For the AP6 conjugates, the Hetabolic activity of C2C12 cells (Figure 1D) after 24h incubation was significantly increased when mixed with 1% PVA—TRAP6 (P < 0.001), while not increased for 0.5% and 0.1% PVA—TRAP6. After 48h tion, the metabolic activity for all three concentrations was significantly increased compared to the l (P < 0.001). Several studies by other groups have shown that PAR—1 activating peptide such as TRAP6 can ate cytokine release from different cell types, including human girgival fibroblasts, endothelial cells, intestinal epithelial cells, and human muscle myoblasts. [20 a,b,c] Therefore, we assume that the increased metabolic activity of C2C12 myoblasts during MTT assay is attributed to TRAP6—induced PAR-1 activation.

Corsidering that a 1% PVA—TRAP6 solution gives a TRA?6— tration of 5 mM whereas the effective TRAP6- concentration for platelet tion. is in the range of 5-100 uM,[”] 'the toxicity results suggest that PVA-TRAP6 are cytocompatible materials within its effective range. 2.3. Hemostatic Activity 2.3.1. Thromboelastometry We next studied. the hemostatic efficacy of AP6 in comparison with TRAP6 and PVA—NB using rotational thromboelastometry (ROTEM),[”” ”] which is a clinical diagnostic tool allowing in situ characterization o: viscoelastic properties of blood clot during coagulation. Figure 2A shows the plotted ROTEM curves of the studied samples that were mixed with recalcified whole blood. Clotting time (CT) refers to the latency until the clot reaches a firmness o: 2 mm while maximum clot firmness (-CF) refers to the maximum. amplitude of the curve, which indicates the absolute strength of the clot.

From the ROTEM results, it was observed that the CT of ?VA— TRAP6 at 0.1 mM was very comparable to that of TRAP6 at 0.l mM while the MCF of PVA—TRAP6 was relatively' less than that of TRAP6 l. The lower MCF might be attributed to the sed accessibility of TRAP6- to platelets alter conjugation in PVA—TRAP6, which is a macromolecular conjugate (65 kDa) and icantly larger than TRAP6 (1 kDa). In comparison, the ?VA— NB control (Figure 2C) showed a curve very similar to the physiological curve (NaCl control), showing no hemostatic activity of the PVA-NB backbone.

To test whether the hemostatic activity' of PVA—TRAP6 is dose—dependent, we tested PVA-TRAP6 solttions in comparison with TRAP6 ons at three e concentrations (0.01, 0.1, 1 mM) in ROTE- (Figure 2B). It was found that the optimal hemostatic tration for PVA—TRAP6 was 0.1 mM while there was no significant dose influences for TRAP6 control in the chosen range. This may again imply the influence of differential molecular structure in PVA—TRAP6 ard TRAP6 peptide on the saturation level of TRAP6 for platelet activation. 2.3.2. Multiplate Analysis In order to quantify the extent 0: platelet activation, we utilized Multiplate assay to investigate the influence of PVA— TQAP6, TRAP6 and PVA—N3 on platelet aggregation. The principle 0: this method is based on the fact that ets become sticky upon activation, and thus prone to adhere and ate on the metal sensor wires in the Multiplate test cuvette. As activated platelets adhere and aggregate on the sensor wires, the electrical ance between the wires rises, which can be continuoasly monitored. A typical Multiplate curve (Figure 2D) represents the accumulation 0' e'ectronic signals corresponding to the extent of platelet aggregation. One key parameter of Multiplate assay is aggregation area (in Units), i.e. the area underneath the aggregation curve. It was observed that PVA—TRAP6 (0.1 mM) induced an aggregation curve (Figure 2D) that was able to that of TRAP6 (0.1 mM), while the PVA—NB control displayed negligible capability of platelet aggregation. The aggregation area value e 2E) for TRAP6 was 147 J whereas the area value for AP6 and PVA—NB was 130 U and. 2 U (p<0.001), respectively. Together, Multiplate assay proved that PVA—TRAP6 presented high efficiency for platelet tion while the substrate (PVA-NB) did not. 2.3.2. FACS of the Soluble System We next utilized flow cytometry (FACS) to quantify the extent 0: platelet activation. ets can be distinguished from other blood cells by the constitutive expression of the surface antigen CD41, which recognizes the et membrane glycoprotein Gp_"b which is non—covalently associated with GpIIIa (the integrir beta 3 chain) to form the GpIIb/ZIIa complex. antly, the CD62p (P—selectin) ne glycoprotein. is exclisively expressed. on activated. platelets.

The CD62p marker was used to identify the extent of activation in hunan platelets after incubation with the studied materials (?VA—T?AP6, TRAP6, PVA—NB, and. NaCl). The measurement of the CD41/CD62p co—expression (Figure 3 A-C) in blood samples treated with these materials for 15 min revealed that there was significant effect (~80%) of PVA-TRAP6 (0.1 mM) on the CD62p expression in CD41 positive cells. The percentage of activated platelet phenotype in terms of CD62p positive cells stayed at the same level 0: the control sample treated with TRAP6 (0.1 m“). By contrast, there was no significant effect (<10%) of PVA— N3 on the CD62p expression in CD41 positive cells, which was at the same level of the negative control samples treated with 0.9% NaCl. In all, FACS analysis “urther1: confirmed the high efficiency of PVA—TRAP6 for et tion. 2.4. Preparation and Characterization of PVA Hydrogels Since platelet—activating PVA—TRAP6 in solub1e Form. or in releasable form, such as in WO 96/40033 A1 has the ial to cause thrombotic risks in the circulation, we further ped an ble PVA—TRAP6 system for localized hemostasis whereby TRAP6 peptide were covalently' immobilized in photocrosslinked PVA hydrogel matrices. We selected radical-mediated thiol-NB photopolymerization as the approach to create PVA hydrogel matrices (Figure 4A). In contrast to tional crosslinking chemiStry of (meth)acrylates, thiol—NB photopolymerization offers several advantages, including robust kinetics, excellent spatiOtemporal l and cytocompatible conditions. [1% 2m instance, PEG—based thiol-N3 hydrogels have d in situ encapsalation o: mammalian cells with high viability (>90 %) [1% In this study, PVA—NB ers in combination. with a model crosslinker (dithiothreitol, DTT) were photopolymerized under UV irradiation in the presence of 12959 as a water-soluble and biocompatible PI. 2.4.1.Photo-rheometry We utilized in situ photo-rheometry to test the photo— reactivity and mechanical properties 0" PVA hydrogels. It was hypothesized that the chemo-physical properties 0; PVA hydrogels could be easily adjusted by tuning the thiol to NB ratio. Four PVA—NB/JTT formulations with equal O macromer content (10 6) but varying thiol to NB ratios (0.4, 0.8, ‘.0, 1.7) were screened in photo-rheometry (Figure 4B). After a 60s blank period (no UV), upon. UV irradiation. the storage moduli (G’) of PVA. hydrogels increased to di""erenL extents (8-120 kPa) in seconds until reaching' a G“-plateau. It was found that all of the photopolymerized PVA hydrogels were tOtally arent (Figire 4C). By increasing the thiol to NB ratio from 0.4 to 1.2, the G’-plateau values (Figure 4D) changed from 8, 22, 120 to 45 (Ba, respeCtively. The highest teau value was obtained for hydrogel (T ) whereby the thiol to NB ratio was 1:1, indicating the highest degree of inking. Notably, these teau values from rheometry measurements can only represent the temporal e moduli o: PVA hydrogels in the pre—swollen state, as the swelling process could affect the storage moduli o: the hydrogels.[2 Further investigation into the Hechanical properties of n PVA hydrogels is warranted by using alternative approaches such as AFM Nanoindentation. 2.4.2. water-uptake We r analyzed the water—uptake properties of PVA hydrogels (l—IV). Photopolymerized PVA hydrogel pellets were soaked in PBS for 48h to reach an equilibrium wet weight (mmt), which was compared to the polymer dry weight (mmy) after lyophilization and give the equilibriun mass swelling ratio (Qm).

As shown in Figure 4E, the Qm values of hydrogels (l-IV) changed from. 130, 45, 10 to 17. In combination with the G’-plateau values, these data suggest that the t crosslinking degree was ed. when the thiol to N3 ratio was 1:1 ( ). This observation correlates with previous studies on PEG-based thiol- [19, 25] N3 hydrogels by other groups. For instance, Lin et al. demonstrated. that thiol-NB photopolymerized. PEG els are hydrolytically degradable due to th pr sonc of stcr bonds.mm WO 37178 The degradation rate was dependent on the gel crosslinking density, which was dictated by thiol to N3 ratio and macromer content. Since presented PVA hydrogels also possess a number of ester linkages, we pate that these hydrogels are hydrolytically degradable. s DTT, alternative di-cysteine protease—sensitive peptides can also be used. as enzymatically cleavable crosslinker in order to foster cellular remodelling and wound g. Nevertheless, further investigation into the degradation behavior 0: presented PVA hydrogels in vitro and in vivo is needed. 2.5. Biofunctionalization 2.5.1. Preparation of TRAP6—Functionalized Hydrogel Particulates In order to prepare appropriate hydrogel matrices for TRAP6— tinctionalization, photopolymerized PVA hydrogels (I, —S{:- N3=O.4) were sequentially lyophilized and cryo—milled into fine particulates (Figure BA). Since excessive NB groups were present after photopolymerization, these residual groups were exploited for photo-click conjugation (Figure 5B) with cysteine—containing TRAP6 e. SEM analysis (Figure 5C) revealed that the length scale of PVA—TRAP6—P was in the range of 5—50 um. The l agglomeration. of PVA—TRAP6-P was presumably' due to the charge effects of NE groups. 2.5.2. ROTEM Analysis We tested the hemostatic ability of AP6—P in comparison with PVA—NB—P in ROTEM. Prior to test, these particulates were lly mixed with saline to form injectable slurries. As shown in Figure 6 A—3, the addition of PVA—TRAP6—P into whole blood induced a icant decrease 0: CT to ~50% of the physiological CT. Interestingly, the PVA—NB—? control also induced a decrease 0: CT to ~709. Since negatively charged surfaces are known to bute coagulation (i.e. the intrinsic )fll26 y we suppose that the observed hemostatic activity of PVA—NB—P is due to the charge effects 0: NB groups. 2.5.3. FACS Analysis In order to quantify the ability of these particulated materials to activate platelets, we analyzed whole blood samples that were pre—incubated with AP6—P and/or PVA—NB—P in FACS. FACS analysis e 6 C-F) revealed that the tage 0: activated platelets (CD4l+/CD62p+) for PVA—TRAP6—P was as high as ~55%, which. was comparable to the positive control (O.lmM TRAP6). By contrast, blood samples incubated with PVA-NB-P only exhibited a minimal amount of activated ets (<lO%). These results show that TRAP6-presenting hydrogel matrices (PVA-TRAP6- P) t good potency of activating platelets in a localized manner. 2.6. Comparison of conventional immobilization techniques with coupling techniques preserving activity of the peptidic thrombin receptor activating agent To show the importance of diligently' choosing' the suitable immobilization technique, a comparison. of conventional peptide immobilization methods (e.g. NHS conjugation) and methods which have been selected in the course or the present invention, such as the photo-click conjugation, was performed for the process of covalent binding of the TRAP6 peptide to polymer substrates. It turned out that conventional peptide lization methods lead to the loss of its bio—activity. This s in a lack of thrombin receptor activation and to no induction of b' ood coagulation, making the resultant materials not useful for local hemostasis.

To prepare r-TRAP6 via NHS conjugation, polyethylene glycol (10 kDa) with terminal NHS group (PEG-lOk-NHS) was used as the polymer ate. TRAP6 was linked to PEG-lOk-NHS through reaCtion at the N-terminus, and L nreacted. NHS gro ips were blocked with glycine. Furthermore, PEG—lOk—NHS reacted with excessive glycine was prepared as the negative control. A: ter dialysis against led water and lyophilization, the conjugates were obtained in high yields and subsequently analyzed in standard rotational thromboelastometry (ROTEM) to study their effects on blood ation. In order to compare the bioactivity of polymer—TRAP6 conjugates prepared by ent approaches, PVA—TRAP6 prepared by our claimed approach (i.e. photo-click conjugation) was included as the ve control. The samples consist of 1 mM TRAP6, 1 mM PEG—TRAP6, mM PEG—Glycine, 1 mM PVA—TRAP6 and saline.

As shown in Figure 8, non—conjugated TRAP6 (1 mM) induced a significant decrease o: clotting' time (CT) and. clot formaoion time (CFT) in comparison to the saline control. However, this effect was lost when TRAP6 was conjugated to the PEGLOk using the conventional NHS conjugation procedure. Similar effects can also be observed for the PEG-Glycine conjugate control, showing minimal effects 0: the polymer backbone and the preparation proccdurc. chcrthclcss, the PVA-TRAP6 conjugates prepared. by photo-click conjugation could still significantly shorten the CT and CFT, showing that the bioactivity of TRAP6 is retained. In summary these data shows that the traditional NHS immobilization approach fails to retain the bioactivity of TRAP6. By st, the covalent immobilization. approach. based. on thiol-norbornene photo-click conjugation can retain almost the full bio— functionality of the TRA?6 e.

To r demonstrate the ability of PVA-TRAP6 conjugates for platelet activation, we tested the samples in standard platelet ation assay (Multiplate) and flow try (FACS). As shown in Figure 9A, the total platelet aggregation area of PVA- TRAP6 is able to that of TRAP6 at equal peptide corcentration, while the PVA—NB substrate control and saline cortrol show negligible level 0: platelet aggregation. rmore, FACS s (Figure 9B) confirm. that PVA—TRAP6 corjugates prepared by photo-click conjugation can induce et activation (i.e. CD41/CD62P co-expression) to a very similar level as TRAP6 control (1 mM), while no significant platelet activation could be observed for PVA—NB and saline cortrol. These findings prove that our conjugation approach indeed. can retain the bioactivity' of TRAP6, even when it is covalently immobilized to PVA.

In contrast to state-or-the-art ches for peptide lization, the approach of the present invention is based on specific coupling techniques with no risk of side reactions with N— or C—terminus or acidic or basic amino acids by using e.g. bioorthogonal reactions, such as photo-click ation of cysteine—containing TRAP6 onto PVA norbornenes, which offers.C a very high degree 0: conjugation efficiency (>95%), site— specificity and modularity. The same conjugation approach is also applicable for other substrates bearing norbornene groups, 2016/070619 such as naturally—derived molecules (gelatin, hyaluronan, alginate, etc.) and synthetic analogues such as PEG. In addition, the polymer-bound TRAP6 conjugates have lower probability to be internalized by blood cells than soluble TRAP6 peptide through. PAR—1 receptor signaling, as the size of PVA substrates (hundreds o: repeating units) is far larger thar a short TRAP6 sequence. In all, the approach according to the t invention with coupling the thrombin receptor activating active agent with retained activity (e.g. by click conjugation for TRAP6 peptide immobilization) provides significant practical values for local hemostasis as well as other medical ations. 3.Conclusion In this work, we ped. a synthetic hemostatic system that can ently activate platelets and accelerate hemostasis in a zed manner. The use of highly potent protease, thrombin, is avoided. in this system. Instead, :he thrombin receptor agonist peptide (TRAP) was covalently engineered on cytocompatible PVA hydrogels via highly efficient thiol-norbornene photo—conjugation. The presented TRAP6- functionalization approach is also able, th not restricted to other synthetic materials/hydrogels such as PEG as well as naturally-derived hydrogels such as gelatin and onic acid. From a biological point of view, activated platelets are known to release platelet-derived growth factors (PDNF), which regulate cell proliferation and play a significant role in blood. vesse' formation (angiogenesis). Therefore, we anticipate that these platelet—activating hydrogel matrices are versatile biomaterials not only for safe hemostasis but also for potential applications in tissue regeneration and wound healing.

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Claims (15)

Claims:

1. Hemostatic material, wherein a in receptor activating agent is covalently coupled to a biocompatible matrix and n the thrombin receptor activating agent has a thrombin receptor activating activity after covalent coupling to the biocompatible

2. Hemostatic al according to claim 1, wherein the biocompatible matrix is a hemostatic matrix and selected from the group consisting of a biomaterial, preferably a protein, a biopolymer or a polysaccharide matrix, especially a collagen, gelatin, fibrin, starch or chitosan matrix; and a synthetic polymer, preferably a polyvinyl alcohol, polyethylene glycol, or poly(N-isopropylacrylamide).

3. Hemostatic material according to claim 1 or 2, wherein the thrombin receptor activating agent is a thrombin receptor activating e (TRAP), preferably TRAP8, TRAP7, TRAP6, TRAP1- 41, SLIGKV (for PAR-2 (human)), TFRGAP (for PAR-3 (human)), GYPGQV (for PAR-4 (human)), or amidated forms thereof, as well as es thereof.

4. Hemostatic material according to any one of claims 1 to 3, n the matrix is a sponge, a woven or non-woven fabric, a preformed shape, preferably as a er or cone for tooth extraction, a particulate or granulate material or a sheet.

5. atic material according to any one of claims 1 to 4, wherein the matrix comprises polyvinyl alcohol.

6. Method for producing a hemostatic material according to any one of claims 1 to 5, wherein a thrombin receptor activating agent is covalently coupled to a biocompatible matrix; wherein the thrombin receptor activating agent is activated with peptide sequences with bioorthogonal groups and/or n the biocompatible matrix is activated with an ene group.

7. Method according to claim 6, wherein the thrombin receptor activating agent is a in receptor ting peptide , preferably TRAP8, TRAP7, TRAP6, TRAP1-41, SLIGKV (for PAR-2 (human)), TFRGAP (for PAR-3 )), GYPGQV (for PAR-4 (human)), or amidated forms thereof, as well as mixtures thereof.

8. Method according to claim 6 or 7, wherein the thrombin receptor activating agent is activated with peptide sequences with bioorthogonal groups, and wherein the bioorthogonal group is alkene, sulfhydryl, alkyne, azido, hydroazide, or hydrazine, especially a cysteine moiety with an –SH group.

9. Method according to any one of claim 6 to 8, wherein the biocompatible matrix is activated with an ene group, and n the ene group is norbornene, maleimide, allyl, vinyl ester, te, vinyl carbonate, or methacrylate.

10. Method according to any one of claims 6 to 9, wherein the chemical coupling is performed by a photoreaction, preferably by photo-triggered click chemistry or photo-triggered biorthogonal reactions.

11. Method according to any one of claims 6 to 10, wherein the chemical coupling is performed by thiol-norbornene photo-click chemistry.

12. Hemostatic material according to any one of claims 1 to 5 for use in y and/or in the treatment of injuries and/or wounds.

13. Kit, ably for use in surgery and/or in the treatment of injuries and/or wounds, comprising - a hemostatic material according to any one of claims 1 to 5 - at least one administration device, preferably selected from the group comprising buffer solution, especially a buffer solution containing Ca2+ ions, a syringe, a tube, a catheter, forceps, scissors, a sterilising pad or lotion.

14. Kit according to claim 13, wherein the buffer on further comprises a component selected from the group anti-bacterial agent, coagulatively active agent, immunosuppressive agent, antiinflammatory agent, anti-fibrinolytic agent, especially aprotinin or ECEA, growth factor, vitamin, cell, or mixtures thereof.

15. Kit according to claim 13, further comprising a container with a component ed from the group anti-bacterial agent, coagulatively active agent, immunosuppressive agent, antiinflammatory agent, anti-fibrinolytic agent, especially aprotinin or ECEA, growth factor, vitamin, cell, or es thereof.