Polypeptide derived from tridegin

Abstract

The present invention relates to a polypeptide derived from natural tridegin by structural modification, which may serve as an inhibitor of plasma coagulation factor XIIIa, and to pharmaceutical compositions comprising such polypeptide. The present invention further refers to such polypeptide or pharmaceutical composition for use in a method for treating or preventing a thrombotic event in a patient. Moreover, the invention relates to an endoprosthesis or an enzyme-linked immunosorbent assay (ELISA) plate coated with a polypeptide of the present invention. Furthermore, the invention also encompasses a recombinant fusion protein comprising this polypeptide linked to a fibrinolytic enzyme such as streptokinase, nattokinase or tPAs via a flexible peptide linker containing a thrombin-cleavable site. This construct allows selective inhibition of FXIIIa within a thrombus, while enabling thrombin-triggered release of the fibrinolytic enzyme to induce localized fibrinolysis, thereby achieving targeted dual-action thrombolytic therapy.

Description

[0001]Rheinische Friedrich-Wilhelms-Universität Bonn 23 July 2025P74921EP JA / ZIT / staPolypeptide derived from trideginThe present invention relates to a polypeptide derived from natural tridegin bystructural modification, which may serve as an inhibitor of plasma coagulation factor XIII, and to pharmaceutical compositions comprising such polypeptide. The present invention further refers to such polypeptide or pharmaceutical composition for usein a method for treating or preventing a thrombotic event in a patient. Moreover, theinvention relates to an endoprosthesis or an enzyme-linked immunosorbent assay(ELISA) plate coated with a polypeptide of the present invention.Thrombotic events can still be health-threatening and even life-threateningpathological conditions. For many years, cardiovascular diseases which are oftenassociated with thrombotic events, including ischemic stroke and venous thromboembolism, have been the most common cause of death in Germany andworldwide (Germany 2020: 34.3%). The formation of thrombosis, which is due to acombination of dysregulated hemostasis, changes in vascular functions and changes in blood flow velocity, play an essential role in the development of such diseases. The development of anticoagulants for the prophylaxis and treatment ofthromboembolic diseases therefore remains the focus of research, although thereare some known anticoagulants. In the recent past, an interest has been oncompounds that specifically switch off a specific coagulation factor, such as thrombin (FIIa) and FXa inhibitors, in contrast to earlier, non-specific therapyprinciples such as the use of vitamin K antagonists or heparins.Compounds targeting the coagulation factors may play a major role in monitoringand treatment of excessive coagulation processes, and the focus in drug development is still on natural products, including venoms from various organisms,such as snakes and bees as well as saliva from leeches. Substances isolated fromleeches have been used in antithrombotic therapy for thousands of years, with the enzymatic and non-enzymatic reactions of the blood coagulation cascade and the factors involved playing a major role in the regulation and specific control of these processes. This may include cysteine-rich, anticoagulant peptides isolated fromleeches. In particular, compounds targeting the serine protease thrombin havealready been successfully used as lead structures for the development of the nowused active inhibitors bivalirudin and desirudin.In contrast to other blood coagulation factors, only a few useful inhibitors have been described for blood coagulation factor XIII (FXIIIa). In this context, fibrinpolymerization starts with the cleavage of fibrinogen into fibrin that is then finallycross-linked by FXIIIa. The catalytic mechanism of FXIIIa includes a distinct three-dimensional structure of the active site with the catalytic triad Cys314, His373, and Asp396. A technical drawback of many FXIIIa inhibitors is that many of these interact with a low potency towards FXIIIa and are non-specific, i.e. they simultaneously inhibit other enzymes such as the tissue transglutaminase TG2. It has been tried toprepare polypeptide inhibitors of FXIIIa, which contain a number of disulfide bondsin the 1990s (WO 96 / 34890). One of the very few if not the only polypeptide that hasa selective inhibitory effect on FXIIIa is tridegin (Finney et al., Biochemical Journal,1997, 324:797-805; Wallis et al., Blood Coagulation and Fibrinolysis, 1997, 8:291-295; Seale et al., Thrombosis and Haemostasis, 1997, 77(5):959-963). Tridegin isa polypeptide of 66 amino acids (sequence of SEQ ID NO: 14:KLLPCKEWHQGIPNPRCWCGADLECAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE) having different disulfide bond connectivities (cf. Böhm et al.,Journal of Medicinal Chemistry, 2014, 57:10355-10365). In the wild-type form, thetridegin may optionally form a triple disulfide-bridged polypeptide. Tridegin wasisolated from the saliva of the leech Haementeria ghilianii in 1997 and has sincebeen recognized as one of the most potent, naturally occurring FXIIIa inhibitors(Finney et al., Biochemical Journal, 1997, 324:797-805; Wallis et al., BloodCoagulation and Fibrinolysis, 1997, 8:291-295; Seale et al., Thrombosis andHaemostasis, 1997, 77(5):959-963). Compared to other FXIIIa inhibitors, trideginbears the technical advantage that it inhibits FXIIIa efficiently, while at the same timeaffecting other enzymes such as the transglutaminase TG2 to a considerably and desirable low extent. Tridegin has a high potency against FXIIIa (which may be IC50 = 10 nM) and is considered as being rather specific, as it has essentially no inhibitory effect against other coagulation factors (thrombin, FXa) and cysteine proteases (e.g.bromelain, papain) (Finney et al., Biochemical Journal, 1997, 324:797-805).Tridegin is considered having a 23-fold weaker inhibitory effect against thetransglutaminase TG2 compared to FXIIIa. Tridegin is considered as a specific reversible peptide inhibitor of factor XIIIa in the last step of the blood coagulation cascade.In view of the technical advantages, tridegin has been considered as an interestingpharmacological tool and potential drug for treating or preventing thrombotic events, including addressing certain cardiovascular diseases.However, native tridegin also bears non-negligible technical drawbacks. Access tolarger amounts and high purity of native tridegin from natural resources ishampered. Truncated polypeptides with a focus of the C-terminal parts partly corresponding totridegin have been described in US-A 2009 / 0226415 and Böhm et al., Journal ofMedicinal Chemistry, 2014, 57:10355-10365. Such truncated forms are, however,less active. The heterologous expression or synthetic provision of tridegin facessevere challenges. The tridegin having three disulfide bridges bears 15conformational isomers (Böhm et al., Journal of Medicinal Chemistry, 2014,57:10355-10365, Bäuml et al., Journal of Medicinal Chemistry, 2019, 62:3513-3523; Bäuml et al., European Journal of Medicinal Chemistry, 2020, 201:112474). Thus,the preparation of tridegin is severely complicated due to the multiple disulfide bondsand consequently the excessive yield loss associated with it.Additionally, US-A 2009 / 0226415 describes substitutions of amino acid moieties atdifferent positions, including up to four Cys, with small amino acid moieties Gly, Ala, Val and Ser. Furthermore, it has been tried to prepare tridegin derivatives having only two disulfide bonds (Bäuml et al., Journal of Medicinal Chemistry, 2019, 62:3513-3523; Schmitz et al., International Journal of Molecular Sciences, 2021, 22:880). This, however, bears the technical drawback that still undesirable complex synthesis steps are needed and structural isomers by the generation of different disulfide bonds and re-arrangements of disulfide bonds (“shuffling”) may occur. It has been tried to replace cysteine residues by serine residues to observe the impact of the disulfide bonds on the three-dimensional structure and inhibitoryactivity (Schmitz et al., International Journal of Molecular Sciences, 2021, 22:880).The synthesis of such a linear tridegin analogue was greatly simplified, the yield was enhanced and the loss of inhibitory activity was seen, but in an acceptable range (Böhm et al., Journal of Medicinal Chemistry, 2014, 57:10355-10365, Bäuml et al.,Journal of Medicinal Chemistry, 2019, 62:3513-3523), while - at the same time -specificity for FXIIIa was kept. Substitutions of amino acids with non-naturalmoieties, in particular such as ornithine, citrulline and D-amino acids, particularly inpositions X1, X6, X12, X16, X23, X34, X38, X40, X45, X53, X63, X65, have not been suggested or reported yet. As the number of substitutions on amino acid residues of a peptide increases, so does the complexity and predictability of both the synthesis to be performed and the resulting peptide. Therefore, possible substitution sites as well as substituents must be carefully analyzed, tested and selected in order to obtain a substituted peptide with the desired properties.Therefore, there was an unmet need for polypeptides that bear a sufficient inhibitoryeffect on FXIIIa that can be prepared more efficiently and have a desirably goodinhibitory effect on and specificity for FXIIIa as well as proteolytic stability.Surprisingly, it has been found that a polypeptide as claimed can also be very wellused as inhibitor of FXIIIa and as agent usable for treating and / or preventing athrombotic event in a patient or as an endoprosthesis in biomedical applications.A first aspect relates to a polypeptide comprising a polypeptide strand ofSEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38P X40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E,wherein amino acid moieties X19 and X25 are each independently selected from thegroup consisting of Ala and Cys(Me); wherein amino acid moieties X1, X6, X16, X38, X40, X45, X53, X63, and X65are each independently selected from the group consisting of Lys, D-Lys, Arg, D-Arg, Orn, and Cit; wherein amino acid moieties X5, X17, X31, and X37are each independently selectedfrom the group consisting of Ser, Ala, Cys(Me), an amino acid moiety forming alactam bond with another one of the amino acid moieties selected from the groupconsisting of X5, X17, X31, and X37, and Cys optionally forming a disulfide bond withanother Cys at an amino acid moiety selected from the group consisting of X5, X17, X31, and X37;wherein not more than two of X5, X17, X31, and X37 are Cys,wherein optionally one or more of the L-amino acid moieties may be replaced by therespective one or more D-amino acids, or a retro-inverso analogue of the sequence thereof, or a peptidomimetic analogue of the sequence thereof, or a pharmaceutical salt thereof. A preferred embodiment of the invention relates to a polypeptide comprising apolypeptide strand of SEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38P X40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E, differing in at least 6 positions from the sequence of natural tridegin (SEQ IDNO: 14),wherein amino acid moieties X19 and X25 are each independently selected from thegroup consisting of Ala and Cys(Me); wherein amino acid moieties X1, X6, X16, X38, X40, X45, X53, X63, and X65are each independently selected from the group consisting of Lys, D-Lys, Arg, D-Arg, Orn, and Cit; wherein amino acid moieties X5, X17, X31, and X37are each independently selected from the group consisting of Ser, Ala, Cys(Me), an amino acid moiety forming a lactam bond with another one of the amino acid moieties selected from the group consisting of X5, X17, X31, and X37, and Cys optionally forming a disulfide bond with another Cys at an amino acid moiety selected from the group consisting of X5, X17, X31, and X37; wherein not more than two of X5, X17X31, and X37are Cys, wherein optionally one or more of the L-amino acid moieties may be replaced by the respective one or more D-amino acids, or a retro-inverso analogue of the sequence thereof, or a peptidomimetic analogue of the sequence thereof, or a pharmaceutical salt thereof.A second aspect of the invention relates to a polypeptide comprising a polypeptidestrand of SEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38P X40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E,wherein amino acid moieties X19 and X25 are each independently selected from thegroup consisting of Ser, Ala and Cys(Me); wherein amino acid moieties X1, X6, X16, X38, X40, X45, X53, X63, and X65are each independently selected from the group consisting of Lys, D-Lys, Arg, D-Arg, Orn, and Cit; wherein amino acid moieties X5, X17, X31, and X37are each independently selectedfrom the group consisting of Ser, Ala, Cys(Me), an amino acid moiety forming alactam bond with another one of the amino acid moieties selected from the groupconsisting of X5, X17, X31, and X37, and Cys optionally forming a disulfide bond withanother Cys at an amino acid moiety selected from the group consisting of X5, X17,X31, and X37;wherein optionally one or more of the L-amino acid moieties may be replaced by therespective one or more D-amino acids, or a retro-inverso analogue of the sequence thereof,or a peptidomimetic analogue of the sequence thereof,or a pharmaceutical salt thereof. A preferred embodiment of the invention relates to a polypeptide comprising apolypeptide strand of SEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38P X40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E, differing in at least 6 positions from the sequence of natural tridegin(SEQ ID NO: 14),wherein amino acid moieties X19and X25are each independently selected from the group consisting of Ser, Ala and Cys(Me); wherein amino acid moieties X1, X6, X16, X38, X40, X45, X53, X63, and X65are each independently selected from the group consisting of Lys, D-Lys, Arg, D-Arg, Orn, and Cit; wherein amino acid moieties X5, X17, X31, and X37are each independently selected from the group consisting of Ser, Ala, Cys(Me), an amino acid moiety forming a lactam bond with another one of the amino acid moieties selected from the group consisting of X5, X17, X31, and X37, and Cys optionally forming a disulfide bond with another Cys at an amino acid moiety selected from the group consisting of X5, X17, X31, and X37; wherein optionally one or more of the L-amino acid moieties may be replaced by the respective one or more D-amino acids, or a retro-inverso analogue of the sequence thereof, or a peptidomimetic analogue of the sequence thereof, or a pharmaceutical salt thereof. It was found that such a polypeptide could be feasibly prepared and that such apolypeptide bears beneficial properties on inhibiting FXIIIa. The polypeptides of thepresent invention are comparably stable against proteolytic degradation while at the same time maintaining their inhibitory activity and enabling comparably easierproduction and purification protocols. These may additionally show superiorbehavior concerning physicochemical features and pharmacokinetics. The polypeptide of the present invention may serve as a specific pharmacologicalinhibitor of active FXIIIa with (essentially) no influence on thrombin generation. Itmay thereby act as a potent protectant against thrombotic predispositions and complementary reducing endothelial inflammation, henceforth unwanted bleedings.In the context of the present invention, the terms “polypeptide” and “peptide” maybe understood interchangeably in the broadest sense as a compound mainlycomposed of amino acid moieties, preferably mainly composed of proteinogenicamino acid moieties, consecutively conjugated with another via amide bonds.The person skilled in the art generally knows the proteinogenic amino acid moietiesand its abbreviations in three-letter code and one-letter code. In addition, thepolypeptide of the present invention may include one or more further non-proteinogenic amino acid moieties. For example, these may include one or morefurther amino acid moieties selected from the group consisting of citrulline (Cit),ornithine (Orn), and S-methylcysteine (Cys(Me)). Optionally, ornithine may be usedfor replacing lysine and citrulline may be used for arginine. This may increase proteolytic stability and may thus increase half-life and may even allow new routesof administrations, including e.g. transdermal or oral administration. S-methylcysteine may be used as a replacement of cysteine to prevent that therespective residue participates in a disulfide bond, while at the same time mimicking a blocked thiol function in cysteine. As used herein, the terms “amide bond” and “peptide bond” of the backbonestructure may be understood interchangeably as any -CO-NH-, -CO-NRx- group, ora pharmaceutically acceptable derivative thereof, wherein Rx and Ry are eachindependently from another any organic moiety preferably comprising not more than 20 carbon atoms, more preferably a moiety selected from the group consisting of anoptionally substituted C1-C6-alkyl, an optionally substituted C1-C6-alkylene-C3-C10- aryl, an optionally substituted C1-C6-alkylene-C3-C10-heteroaryl, an optionally substituted C1-C6-heteroalkyl, an optionally substituted C1-C6-heteroalkylene-C3-C10-aryl, and an optionally substituted C1-C6-heteroalkylene-C3-C10-heteroaryl, anoptionally substituted C1-C6-alkenyl, an optionally substituted C1-C6-alkenylene-C3- C10-aryl, an optionally substituted C1-C6-alkenylene-C3-C10-heteroaryl, an optionally substituted C1-C6-heteroalkenyl, an optionally substituted C1-C6-heteroalkenylene- C3-C10-aryl, and an optionally substituted C1-C6-heteroalkenylene-C3-C10- heteroaryl. As used in this context, a C1-C6-alkyl, a C1-C6-heteroalkyl, a C1-C6-alkylene, and a C1-C6-heteroalkylene may each be linear or cyclic, unbranched orbranched. In a preferred embodiment, most of the amino acid moieties, in particular all of the amino acid moieties, of the polypeptide of the present invention are alpha amino acids, typically having the structure –NH-CRxRy-CO-, wherein the moieties Rxand Ryare defined as above, in particular wherein Rxis hydrogen, i.e., having a structure NH-CHRy-CO-. As used herein, “hetero” may be understood in the broadest sense in that the respective moiety such as a side chain of an amino acid moiety contains one or more heteroatoms binding two or more carbon atoms, wherein the one or more heteroatoms may optionally be selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), selene (Se), a sulfate group, a sulfonate group, a phosphate group, a phosphonate group, a silicate group, a silane group, an ureate, an ester, a thioester, an amide, a guanidinium group, an imine, and a combination of two ormore thereof. It will be understood that such atoms or functional groups will be eachbound with the respective binding partners, including carbon atoms and hydrogen or other atoms, to fulfill its binding properties and that such atoms or functional groups may optionally also form a salt, in particular when binding or releasing one or more protons. As used herein, the term “substituted” may be understood in the broadest sense in that one or more hydrogen atoms are replaced by one or more heteroatoms or functional groups. For example, a hydrogen atom may be replaced by an atom or functional group selected from the group consisting of deuterium, fluorine, a hydroxyl group, a thiol group, a sulfate group, a sulfonate group, a phosphate group,a phosphonate group, a silicate group, a silane group, an ureate, a carboxylic group,and a combination of two or more thereof. As used in the context of the present invention, the term “pharmaceutically acceptable salt” may be understood in the broadest sense as generally understoodin the art. A pharmaceutically acceptable salt may be any salt that may beadministered to a patient without harming the patient. For example, a pharmaceutically acceptable cation may be selected from the group consisting of one or more cations of sodium, potassium, magnesium, calcium, ammonium, and (preferably positively charged) amino acids. For example, a pharmaceutically acceptable anion may be selected from the group consisting of one or more anions of chloride, sulfate, phosphate, acetic acid, and (preferably negatively charged) amino acids. The polypeptide of the present invention may also comprise one or more peptidomimetic moieties and may optionally be mainly or completely composed ofpeptidomimetic moieties and / or may optionally be a peptidomimetic structure as awhole. As used in the context of the present invention, the term “peptidomimetic” may be understood in the broadest sense as any mimic of a peptide that has similar properties like a peptide, but typically bears higher (biological) stability. In apreferred embodiment, a peptidomimetic in the sense of the present invention isselected from the group consisting of beta amino acid moieties, N-acetylated amino acid moieties (e.g., N-methylated amino acid moieties) and peptoids (i.e., poly-N- substituted glycinyl moieties). The polypeptide may be partly or completely composed of peptidomimetic moieties. In a preferred embodiment, if the polypeptideis a peptidomimetic, all amino acid moieties of the polypeptide are amino acidanalogues of one type (e.g. all are on beta amino acid moieties, all are N-acetylatedamino acid moieties or all are N-substituted glycinyl moieties). Likewise, if thepolypeptide is a D-peptide analogue, in a preferred embodiment, one or more aminoacid moieties of the polypeptide may be D-amino acid moieties.As used herein, the term “retro-inverso analogue” will be unambiguously understood by a person skilled in the art. A retro-inverso analogue may be understood as therespective sequence is reversed and / or D-amino acid moieties are used instead ofL-amino acid moieties in different combinations.The polypeptide may or may not comprise further chemical structures at one or both of its termini. In other words, the polypeptide may be an independent molecular structure (i.e., an unbound polypeptide) or a polypeptide conjugated to another molecular structure. As indicated herein, the polypeptide of the present invention may or may not form a bridging moiety. In one embodiment, the polypeptide comprises two intramolecularbridging moieties (e.g.one disulfide bond, and one lactam bond or two lactambonds). In one embodiment, the polypeptide comprises a single (one) intramolecular bridging moiety (e.g. one lactam bond, or one disulfide bond). In one embodiment,the polypeptide does not comprise an intramolecular bridging moiety, i.e. is a linearpolypeptide. In a particularly preferred embodiment, the polypeptide comprises a single (one) lactam bond. In preferred embodiments, at least two, in particular two to six, Cys moieties may besubstituted compared to native tridegin (SEQ ID NO: 14).In preferred embodiments, two or none of X5, X17, X31, and X37may be cysteine. Inone embodiment, X19 and X25 are both Ala. In another embodiment, X19 and X25 areboth Cys(Me). In another embodiment, X19 is Ala and X25 is Cys(Me). In anotherembodiment, X19 is Cys(Me) and X25 is Ala.In other preferred embodiments, X19and X25may be Ser, Ala or Cys(Me). In oneembodiment, X19 may be Ser and X25 may be Ser, Ala or Cys(Me). In anotherembodiment X25 may be Ser and X19 may be Ser, Ala or Cys(Me). In oneembodiment, X19and X25may both be Ser. In a preferred embodiment, at least two of X5, X17, X31, and X37are eachindependently selected from the group consisting of Cys(Me), Ser and Ala. In apreferred embodiment, X5, X17, X31, and X37are each independently selected from the group consisting of Cys(Me), Ser and Ala. In a preferred embodiment, X5, X17, X31, and X37are each independently selected from the group consisting of Cys(Me), Ser and Ala. In a preferred embodiment, X5, X17, X31, and X37are each Ser. In a preferred embodiment, X5, X17, X31, and X37are each Ala. In a preferred embodiment, X5, X17, X31, and X37are each Cys(Me). In a preferred embodiment, one of X5, X17, X31, and X37is Cys(Me) and the otherones are Ser or Ala. In a preferred embodiment, two of X5, X17, X31, and X37 areCys(Me) and the other ones are each independently from each other Ser or Ala. Ina preferred embodiment, three of X5, X17, X31, and X37are Cys(Me) and the remaining moiety is Ser or Ala. In a preferred embodiment, one of X5, X17, X31, and X37is Ala and the other onesare Ser or Cys(Me). In a preferred embodiment, two of X5, X17, X31, and X37 are Alaand the other ones are each independently from each other Ser or Cys(Me). In apreferred embodiment, three of X5, X17, X31, and X37are Ala and the remaining moiety is Ser or Cys(Me). In a preferred embodiment, one of X5, X17, X31, and X37is Ser and the other onesare Ala or Cys(Me). In a preferred embodiment, two of X5, X17, X31, and X37 areSerand the other ones are each independently from each other Alaor Cys(Me). In apreferred embodiment, three of X5, X17, X31, and X37are Ser and the remaining moiety is Ala or Cys(Me). In another preferred embodiment, at least two, or all of X5, X17, X31, and X37are pairwise selected from amino acid moieties able to from a lactam bond (either X5- X17, X5-X31, X5-X37, X17-X31, X17-X37or X31-X37).As used herein, a lactam bond may be understood in the broadest sense as anamide bond formed between two suitable amino acid side chains. Herein, a cyclicstructure is formed. As used herein, an “amide bond” may be broadly defined as laid out above. As used herein, an amino acid moiety forming a lactam bond with another one of the amino acid moieties selected from the group consisting of X5, X17, X31, and X37may be any amino acid moiety that comprises a carboxyl group or an amino group in its side chain. Typically, a carboxyl group of a side chain of one amino acid moietymay react with an amino group of the side chain of another amino acid moiety.In this context, an amino acid moiety having a carboxylic group (-COOH / -COO-) inits side chain may be any amino acid moiety having a carboxylic group in its sidechain. It may be a naturally occurring amino acid moiety or an amino acid moiety ofartificial origin. In a preferred embodiment, an amino acid moiety having a carboxylicgroup in its side chain may be selected from the group consisting of glutamic acid(Glu) and aspartic acid (Asp). In a preferred embodiment, an amino acid moiety having a carboxylic group in its side chain is Asp. In this context, an amino acid moiety having an amino group (-NH2 / -NH3+) in its side chain may be any amino acid moiety having an amino group in its side chain. It may be a naturally occurring amino acid moiety or an amino acid moiety of artificial origin. In a preferred embodiment, an amino acid moiety having an amino group in its sidechain may be selected from the group consisting of lysine (Lys), ornithine (Orn), and2,3-diaminopropionic acid (Dap). In a preferred embodiment, an amino acid moietyhaving an amino group in its side chain is Orn.In a preferred embodiment, one or two lactam bonds may be formed by a combination of amino acid moieties selected from the group consisting of Asp / Lys,Asp / Orn, Asp / Dap, Glu / Lys, Glu / Orn, and Glu / Dap in any orientation.A lactam bond may be prepared by any means. In a preferred embodiment, it is prepared by using orthogonal protecting groups at the side chains of an amino acid moiety having an amino group and / or an amino acid moiety having a carboxylic group. This allows the preparation of a specific lactam group. In a preferred embodiment, the N-terminus of polypeptide strand is:(i) acetylated;(ii) an unbound primary amino group;(iii) conjugated to a peptidic moiety, a non-peptidic moiety or a moiety comprisingpeptidic and non-peptidic structure elements, of a total molecular weight of not more than 500 Da,(iv) conjugated to a peptidic polymeric structure, a non-peptidic polymericstructure or a polymeric structure comprising peptidic and non-peptidic structure elements, of a total molecular weight of more than 500 Da,preferably of 500 to 100,000 Da;(v) conjugated to solid support, optionally via a peptidic linker structure, a non-peptidic linker structure or a linker structure comprising peptidic and non-peptidic structure elements, preferably wherein the total linker structure has amolecular weight of 100 to 10,000 Da; or(vi) conjugated to a fibrinolytic enzyme, preferably streptokinase, nattokinase, ortissue plasminogen activators (tPA), via a flexible peptide linker for constitution of a fusion protein that combines two functions – inhibition ofFXIIIa and fibrinolytic activity on the thrombus. In a preferred embodiment, the C-terminus of polypeptide strand is:(i) amidated,(ii) an unbound carboxylic group;(iii) conjugated to a peptidic moiety, a non-peptidic moiety or a moiety comprisingpeptidic and non-peptidic structure elements, of a total molecular weight of not more than 500 Da;(iv) conjugated to a peptidic polymeric structure, a non-peptidic polymericstructure or a polymeric structure comprising peptidic and non-peptidic structure elements, of a total molecular weight of more than 500 Da,preferably of 500 to 100,000 Da;(v) conjugated to solid support, optionally via a peptidic linker structure, a non-peptidic linker structure or a linker structure comprising peptidic and non- peptidic structure elements, preferably wherein the total linker structure has amolecular weight of 100 to 10,000 Da; or(vi) conjugated to a fibrinolytic enzyme, preferably streptokinase, nattokinase, ortissue plasminogen activators (tPA), via a flexible peptide linker for constitution of a fusion protein that combines two functions – inhibition ofFXIIIa and fibrinolytic activity on the thrombus. As used throughout the present invention, a peptidic moiety may be comprised of one or more amino acid residues, which may be natural amino acids, non-naturalamino acids or a combination thereof. It will be understood that an amino acidresidue is not necessarily be bound via the alpha amino group. For instance, a lysyl residue may also be bound via its epsilon amino group. As used throughout the present invention, a non-peptidic moiety may be comprised of any chemical structure that is not an amino acid. For example, this may be acarboxylic acid, preferably of 1 to 20 carbon atoms in lengths, which may optionallybe saturated or unsaturated, and may optionally comprise one or more further functional groups in addition to a carboxylic group. For example, this may be anamino group-containing moiety, preferably of 1 to 20 carbon atoms in lengths, whichmay optionally be saturated or unsaturated, and may optionally comprise one ormore further functional groups in addition to an amino group. When conjugated to ahydrophobic structure, the polypeptide of the present invention may also beattached to or embedded in a micelle or liposome or microsome or nanoparticles ormicroparticles, SFD, HME (typically non-covalently). A non-peptidic moiety mayoptionally also be a dye (e.g., a fluorescence dye).Optionally, the polypeptide of the present invention may be conjugated to anotherchemical moiety such as, e.g., a cell-penetrating peptide (e.g. polyarginine) for improving intracellular uptake and / or improving half-life and / or the plasma protein binding. As used throughout the present invention, a peptidic polymeric structure may be any polypeptide structure. In one embodiment, a peptidic polymeric structure is a polypeptide of the present invention. In another embodiment, a peptidic polymeric structure is another polypeptide. In a preferred embodiment, a peptidic polymeric structure is a serum protein, in particular serum albumin. As used throughout the present invention, a non-peptidic polymeric structure may be any structure that is not a polypeptide. In a preferred embodiment, a non-peptidic polymeric structure is polyethylene glycol (PEG).As used throughout the present invention, a polymeric support may be any solidstructure. In a preferred embodiment, a solid support is an endoprosthesis, in particular such as described below. As used throughout the present invention, a linker structure is any bivalent structure that can link the polypeptide of the present invention with a biocompatible polymer. For instance, a linker structure may comprise polyethylene glycol (PEG). Two or none of the amino acid moieties X5, X17, X31, and X37may be a cysteine residue (Cys), which may optionally be or not be involved in a disulfide bond, in particular an intramolecular disulfide bond, i.e. forming a disulfide bond betweenamino acid moieties X5 and X37, X5 and X31, X17 and X37 or X17 and X31. In a preferredembodiment, a disulfide bond is formed between amino acid moieties X5 and X37. Inanother preferred embodiment, a disulfide bond is formed between amino acidmoieties X17and X31. In a preferred embodiment, amino acid moieties X5, X17, X31, and X37are each not Cys.In some embodiments amino acid moieties X5, X17, X31, and X37 may each be Cys.As indicated above, any of the amino acid moieties of the polypeptide of the present invention may optionally be replaced by its respective D-amino acid moiety. In one embodiment, none of the amino acid moieties of the polypeptide is replaced by a D-amino acid moiety. In another embodiment, one or more amino acid moieties of the polypeptide are replaced by the respective D-amino acid moieties. In a preferred embodiment, one or more amino acid moieties of the N-terminal part of the polypeptide ranging fromamino acid moieties at positions 1 to 37 are replaced by the respective D-aminoacid moieties. In a preferred embodiment, at least one of the moieties selected from the group consisting of Lys6, Ile12, Leu23or Ile34is replaced by the respective D-amino acid residue. In a preferred embodiment, at least two of the moieties selected from the group consisting of Lys6, Ile12, Leu23or Ile34are each replaced by the respective D-amino acid residues. In a preferred embodiment, at least three of the moietiesselected from the group consisting of Lys6, Ile12, Leu23or Ile34are each replaced by the respective D-amino acid residues. In a preferred embodiment, the moieties selected from the group consisting of Ile12, Leu23or Ile34are each replaced by the respective D-amino acid residues. In a preferred embodiment, Lys6is replaced by the respective D-amino acid residue. In a preferred embodiment, Ile12is replaced by the respective D-amino acid residue. In a preferred embodiment, Leu23is replaced by the respective D-amino acidresidue. In a preferred embodiment, Ile34 is replaced by the respective D-amino acidresidue.In a preferred embodiment, at Lys6 and Ile12 are replaced by the respective D-aminoacid residue. In a preferred embodiment, at Lys6 and Leu23 are replaced by therespective D-amino acid residue. In a preferred embodiment, at Lys6 and Ile34 arereplaced by the respective D-amino acid residue. In a preferred embodiment, Ile12and Leu23are each replaced by the respective D-amino acid residue. In a preferred embodiment, Ile12 and Ile34 are each replaced bythe respective D-amino acid residue. In a preferred embodiment, Ile12 and Ile34 areeach replaced by the respective D-amino acid residue. In a preferred embodiment,Leu23and Ile34are each replaced by the respective D-amino acid residue.In a preferred embodiment, Lys6, Ile12 and Leu23 are each replaced by the respectiveD-amino acid residue. In a preferred embodiment, Ile12, Leu23 and Ile34 are eachreplaced by the respective D-amino acid residue. In a preferred embodiment, Lys6,Leu23 and Ile34 are each replaced by the respective D-amino acid residue. In apreferred embodiment, Lys6, Ile12 and Ile34 are each replaced by the respective D-amino acid residue.In a preferred embodiment, Lys6, Ile12, Leu23 and Ile34 are each replaced by therespective D-amino acid residue.Optionally, one or more lysine moieties of the original tridegin sequence (SEQ IDNO: 14) may be replaced by ornithine (Orn). Optionally, one or more argininemoieties of the original tridegin sequence (SEQ ID NO: 14) may be replaced bycitrulline (Cit).In a preferred embodiment, one or more amino acid moiety positions X1, X6, X45 andX63are each independently from each other selected from the group consisting of D-Lys and Orn and / or one or more amino acid moiety positions X16, X38, X40, X53, X65are each independently from each other selected from the group consisting of D-Arg and Cit.In a preferred embodiment, amino acid moiety positions X1, X6, X45 and X63 are eachindependently from each other selected from the group consisting of D-Lys and Ornand amino acid moiety positions X16, X38, X40, X53, X65 are each independently fromeach other selected from the group consisting of D-Arg and Cit.In another preferred embodiment, at least one of amino acid moiety positions X1,X6, X45and X63is Orn or D-Lys and / or at least one of amino acid moiety positionsX16, X38, X40, X53, X65 is Cit or D-Arg, in particular wherein at least one of amino acidmoiety positions X1, X6, X45and X63is Orn and / or at least one of amino acid moiety positions X16, X38, X40, X53, X65is Cit. In a preferred embodiment, one, two, three, or all of amino acid moiety positions X1, X6, X45and X63are each Orn.In a preferred embodiment, amino acid moiety position X1 is Orn. In a preferredembodiment, amino acid moiety position X6 is Orn. In a preferred embodiment,amino acid moiety position X45 is Orn. In a preferred embodiment, amino acid moietyposition X63 is Orn.In a preferred embodiment, amino acid moiety positions X1 and X6 are each Orn. Ina preferred embodiment, amino acid moiety positions X1 and X45 are each Orn. In apreferred embodiment, amino acid moiety positions X1 and X63 are each Orn. In apreferred embodiment, amino acid moiety positions X6 and X45 are each Orn. In apreferred embodiment, amino acid moiety positions X6 and X63 are each Orn. In apreferred embodiment, amino acid moiety positions X45 and X63 are each Orn.In a preferred embodiment, amino acid moiety positions X1, X6 and X45 are eachOrn. In a preferred embodiment, amino acid moiety positions X6, X45 and X63 areeach Orn. In a preferred embodiment, amino acid moiety positions X1, X6 and X63are each Orn. In a preferred embodiment, amino acid moiety positions X1, X45 andX63 are each Orn.In a preferred embodiment, amino acid moiety positions X1, X6, X45 and X63 are eachOrn. In a preferred embodiment, one, two, three, four or all of amino acid moiety positions X16, X38, X40, X53, X65are each Cit.In a preferred embodiment, amino acid moiety position X16 is Cit. In a preferredembodiment, amino acid moiety position X38 is Cit. In a preferred embodiment,amino acid moiety position X40 is Cit. In a preferred embodiment, amino acid moietyposition X53 is Cit. In a preferred embodiment, amino acid moiety position X65 is Cit.In a preferred embodiment, amino acid moiety positions X16 and X38 are each Cit. Ina preferred embodiment, amino acid moiety positions X16 and X40 are each Cit. In apreferred embodiment, amino acid moiety positions X16 and X53 are each Cit. In apreferred embodiment, amino acid moiety positions X16 and X65 are each Cit. In apreferred embodiment, amino acid moiety positions X38 and X40 are each Cit. In apreferred embodiment, amino acid moiety positions X38 and X53 are each Cit. In apreferred embodiment, amino acid moiety positions X38 and X65 are each Cit. In apreferred embodiment, amino acid moiety positions X40 and X53 are each Cit. In apreferred embodiment, amino acid moiety positions X40 and X65 are each Cit. In apreferred embodiment, amino acid moiety positions X53 and X65 are each Cit.In a preferred embodiment, three of amino acid moiety positions X16, X38, X40, X53,X65are each Cit. In a preferred embodiment, four of amino acid moiety positions X16, X38, X40, X53, X65are each Cit. In a preferred embodiment, amino acid moietypositions X16, X38, X40, X53, X65 are each Cit.In a preferred embodiment, one or more amino acid moiety positions X1, X6, X45and X63are each Orn and / or one or more amino acid moiety positions X16, X38, X40, X53, X65are each Cit.In a preferred embodiment, amino acid moiety positions X1, X6, X45 and X63 are eachOrn and amino acid moiety positions X16, X38, X40, X53, X65 are each Cit.In a preferred embodiment, the polypeptide comprises at least one non-proteinogenic amino acid moiety, preferably selected from the group consisting of aD-amino acid moiety, Cit and Orn. In a preferred embodiment, the polypeptidecomprises at least two non-proteinogenic amino acid moieties, preferably eachindependently selected from the group consisting of a D-amino acid moiety, Cit andOrn. In a preferred embodiment, the polypeptide comprises at least three non-proteinogenic amino acid moieties, preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn. In a preferred embodiment,the polypeptide comprises at least four non-proteinogenic amino acid moieties,preferably each independently selected from the group consisting of a D-amino acidmoiety, Cit and Orn. In a preferred embodiment, the polypeptide comprises at leastfive non-proteinogenic amino acid moieties, preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn. In a preferredembodiment, the polypeptide comprises at least six non-proteinogenic amino acidmoieties, preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn. In a preferred embodiment, the polypeptidecomprises at least seven non-proteinogenic amino acid moieties, preferably eachindependently selected from the group consisting of a D-amino acid moiety, Cit andOrn. In a preferred embodiment, the polypeptide comprises at least eight non-proteinogenic amino acid moieties, preferably each independently selected from thegroup consisting of a D-amino acid moiety, Cit and Orn. In a preferred embodiment,the polypeptide comprises at least nine non-proteinogenic amino acid moieties,preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn.In a preferred embodiment, the polypeptide comprises at least one non-proteinogenic amino acid moiety within the N-terminal amino acid moieties 1 to 37, preferably selected from the group consisting of a D-amino acid moiety, Cit and Orn.In a preferred embodiment, the polypeptide comprises at least two non-proteinogenic amino acid moieties within the N-terminal amino acid moieties, preferably each independently selected from the group consisting of a D-amino acidmoiety, Cit and Orn. In a preferred embodiment, the polypeptide comprises at least three non-proteinogenic amino acid moieties within the N-terminal amino acid moieties, preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn. In a preferred embodiment, the polypeptidecomprises at least four non-proteinogenic amino acid moieties within the N-terminalamino acid moieties, preferably each independently selected from the groupconsisting of a D-amino acid moiety, Cit and Orn. In a preferred embodiment, thepolypeptide comprises at least five non-proteinogenic amino acid moieties within theN-terminal amino acid moieties, preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn. In a preferred embodiment,the polypeptide comprises at least six non-proteinogenic amino acid moieties withinthe N-terminal amino acid moieties, preferably each independently selected from the group consisting of a D-amino acid moiety, Cit and Orn.In a preferred embodiment, the polypeptide comprises at least one non-proteinogenic amino acid moiety, selected from the group consisting of one or morerespective D-amino acid moieties at one or more amino acid moiety positions Lys6,Ile12, Leu23 and Ile34; Orn at one or more amino acid moiety positions X1, X6, X45 andX63; and Cit at one or more amino acid moiety positions X16, X38, X40, X53, X65.In a preferred embodiment, the polypeptide comprises at least two, at least three,at least four, at least five, at least six, at least eight, or at least nine non-proteinogenic amino acid moiety, selected from the group consisting ofone or more respective D-amino acid moieties at one or more amino acid moietypositions Lys6, Ile12, Leu23 and Ile34;Orn at one or more amino acid moiety positions X1, X6, X45and X63; and Cit at one or more amino acid moiety positions X16, X38, X40, X53, X65. In a preferred embodiment, the polypeptide comprises (or consists of) a polypeptidestrand of SEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E, wherein amino acid moieties X1, X6, X45and X63are each independently selectedfrom the group consisting of Lys, D-Lys, and Orn;wherein amino acid moieties X16, X38, X40, X53, X65are each independently selectedfrom the group consisting of Arg, D-Arg, and Cit; wherein amino acid moieties X5, X17, X31, and X37are each independently selectedfrom the group consisting of Ser, Ala, Cys(Me), an amino acid moiety forming alactam bond with another one of the amino acid moieties selected from the groupconsisting of X5, X17, X31, and X37, and Cys optionally forming a disulfide bond withanother Cys at an amino acid moiety selected from the group consisting of X5, X17, X31, and X37;wherein not more than two of X5, X17, X31, and X37 are Cys;wherein amino acid moieties X19and X25are each independently selected from the group consisting of Cys(Me), Ser, and Ala,or a pharmaceutically acceptable salt of the sequence SEQ ID NO: 1,wherein optionally one or more of the L-amino acid moieties may be replaced by therespective one or more D-amino acids,or a retro-inverso analogue of the sequence of SEQ ID NO: 1,or a peptidomimetic analogue of the sequence of SEQ ID NO: 1.In a preferred embodiment, the polypeptide comprises (or consists of) a polypeptide strand selected from the group consisting of KLLPCkEWHQGiPNPRCWSGADlESAQDQYCAFiPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 2), wherein k6 is a D-lysyl moiety, l23 is a D-leucineand i12 and i34 are D-isoleucine moieties,X1LLPSX6EWHQGIPNPX16SWSGADLESAQDQYSAFIPQSX38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E (SEQ ID NO: 3),wherein X1, X6, X45and X63are each Orn; and wherein X16, X38, X40, X53, X65are each Cit, KLLPAKEWHQGIPNPRAWSGADLEAAQDQYAAFIPQARPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 4), andX1LLPAX6EWHQGIPNPX16AWSGADLEAAQDQYAAFIPQAX38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E (SEQ ID NO: 5),wherein X1, X6, X45and X63are each Orn; and wherein X16, X38, X40, X53, X65are each Cit,wherein optionally one or more of the L-amino acid moieties may be replaced by therespective one or more D-amino acids, or a retro-inverso analogue of any one of the sequences, or a peptidomimetic analogue of any one of the sequences, or a pharmaceutically acceptable salt of any one of the sequences. In a preferred embodiment, each polypeptide comprising a polypeptide strand ofany one of SEQ ID NOs: 1 to 5 is acetylated at the N-terminus (i.e., the N-terminalalpha nitrogen, Ac-NH-) and amidated at the C-terminus (i.e., the C-terminalcarboxyl group, -CO-NH2).In one embodiment, one or more cysteine moieties may optionally be eachsubstituted by Cys(Me). In one embodiment, each cysteine moiety may optionallybe substituted by Cys(Me). In one embodiment, one or more serine moieties may optionally be each substituted by Cys(Me), in particular of one or more serine residues that replace cysteineresidues e.g., in SEQ ID NO: 3). In one embodiment, each serine moiety mayoptionally be substituted by Cys(Me), in particular of one or more serine residuesthat replace cysteine residues e.g., in SEQ ID NO: 3).In one embodiment, one or more alanine moieties may optionally be eachsubstituted by Cys(Me), in particular of one or more alanine moieties that replacecysteine residues e.g., in SEQ ID NOs: 4 and / or 5). In one embodiment, eachalanine moiety may optionally be substituted by Cys(Me), in particular of one ormore alanine residues that replace cysteine residues e.g., in SEQ ID NOs: 4 and / or5). As indicated above, the polypeptide may or may not comprise a bridging moiety. Inother words, the polypeptide may be cyclic (i.e., with one or two intramolecularbridges) or may be linear (i.e., without an intramolecular bridge). As noted above, insome preferred embodiments, the polypeptide may optionally comprise one or two disulfide bonds.In a preferred embodiment, the polypeptide does not comprise a disulfide bond. Thismay help to avoid disulfide shuffling and / or disulfide modification. This may reducethe number of structural isomers. In this context, it may be noted that a lactam bondmay be optionally used instead that may avoid shuffling. In a preferred embodiment, the polypeptide does not comprise an intramolecularbridge. In this case, it is a linear polypeptide. In another preferred embodiment, thepolypeptide comprises one or two lactam bonds (e.g., between one or more Asp orGlu moieties and an equivalent number of Lys or Orn or Dap moieties). Such alactam bond may optionally replace a disulfide bond. It may optionally be at thecorresponding positions of the sequence.In a preferred embodiment a lactam bond is formed between amino acid moietiesX5 and X37 and / or a lactam bond is formed between amino acid moieties X17 andX31 or between amino acid moieties X5 and X17 and between amino acid moietiesX31 and X37, or between amino acid moieties X5 and X31 and between amino acidmoieties X17 and X37.In another preferred embodiment, a lactam bond may be formed between aminoacid moieties X5 and X37. In a preferred embodiment, X5 is Asp or Glu and X37 is Lysor Orn or Dap and a lactam bond is formed between these groups. In anotherpreferred embodiment, X5 is Lys or Orn or Dap and X37 is Asp or Glu and a lactambond is formed between these groups. In a particularly preferred embodiment, X5isAsp and X37 is Orn and a lactam bond is formed between these groups. In anotherparticularly preferred embodiment, X5 is Orn and X37 is Asp and a lactam bond isformed between these groups.In another preferred embodiment, a lactam bond may be formed between aminoacid moieties X17 and X31. In another preferred embodiment, X17 is Asp or Glu andX31 is Lys, Orn or Dap and a lactam bond is formed between these groups. Inanother preferred embodiment, X17 is Lys, Orn or Dap and X31 is Asp or Glu and alactam bond is formed between these groups. In another preferred embodiment, X17is Asp and X31 is Orn and a lactam bond is formed between these groups. In anotherpreferred embodiment, X17 is Orn and X31 is Asp and a lactam bond is formedbetween these groups.In one embodiment, a lactam bond may be formed between amino acid moieties X5and X37 and a further lactam bond may be formed between amino acid moieties X17and X31. In a more preferred embodiment, a single lactam bond may be formedbetween amino acid moieties X5 and X37 or between amino acid moieties X17 andX31.In another preferred embodiment, the polypeptide of the present invention is part ofa recombinant fusion protein comprising:(i) the inventive polypeptide, i.e. comprising a tridegin-derived domain thatspecifically inhibits FXIIIa;(ii) a fibrinolytic enzyme, preferably streptokinase, nattokinase, or tPAs with afibrinolytic effector domain; and(iii) a flexible peptide linker connecting the polypeptide and the fibrinolyticenzyme, preferably comprising a thrombin-cleavable amino acid sequence, in particular comprising the sequence VSQTSKLTRAETVFPDV (SEQ ID NO:12).The thrombin-cleavable linker, also referred to as thrombin-sensitive peptide linker,contains a thrombin-cleavable site and allows for selective proteolytic cleavagewithin the thrombus environment, where thrombin is abundantly active. Cleavage bythrombin occurs between the amino acids at positions 9 (Arg) and 10 (Ala) in thesequence of the linker. Upon cleavage, the fibrinolytic enzyme is locally released,activating fibrinolysis in situ while the tridegin domain continues to inhibit FXIIIa-mediated fibrin crosslinking. This fusion construct thus enables spatially confined, synergistic inhibition of thrombus stabilization and promotion of clot dissolution withreduced systemic exposure. In other words, the recombinant fusion protein of theinvention allows selective inhibition of FXIIIa within a thrombus, while enabling thrombin-triggered release of the fibrinolytic enzyme to induce localized fibrinolysis, thereby achieving targeted dual-action thrombolytic therapy.The polypeptide of the present invention may be obtained by any means known forthis purpose in the art. In a preferred embodiment, the polypeptide of the presentinvention is obtained by solid phase peptide synthesis (SPPS) such as of Fmoc- orBoc-based SPPS. Alternatively, the polypeptide of the present invention may also be obtained by liquid phase peptide synthesis (LPPS) or, in the case of consisting of L-amino acid moieties, by means of biotechnology means such as heterologous expression in a genetically modified organism excluding human bodies such as, e.g., bacteria (e.g., E. coli), fungi (e.g., yeast), mammalian cells or mammalians excluding humans, insect cells or insects, plant cells or plants, etc. In some hostcells, one or more posttranslational modifications may occur. Accordingly, geneticmanipulation of a host organism with the sequence comprising the polypeptide of the present invention may be used.In SPPS (or also LPPS), the synthesis is typically based on the stepwise couplingof amino acid moieties bearing protected side chains (orthogonal protecting groups). Typically, during synthesis, the peptide strand grows from the C-terminus to the N- terminus. However, there are alternative methods wherein the peptide strand grows from the N-terminus to the C-terminus. Nowadays, the most common methods arebased on at least two or more different types of protecting groups that are cleavableunder at least two or more different conditions, such as, e.g., the fluorenyl-9-methoxycarbonyl / tert-butyl- (Fmoc / tBu) protecting group scheme (SheppardTactics) or the tert-butoxycarbonyl / benzyl- (Boc / Bzl) protecting group scheme (Merrifield Tactics). A protecting group may be any protecting group known in the art such as, e.g., an amino-protecting group of the urethane type (e.g., benzyloxycarbonyl (Z), 4-methoxybenzyloxycarbonyl (Z(OMe)), 2- nitrobenzyloxycarbonyl (Z(2-NO2)), 4-nitrobenzyloxycarbonyl (Z(NO2)), chlorobenzyloxycarbonyl (Z(Cl), Z(2-Cl), Z(3-Cl), Z(2,4-Cl)), 3,5- dimethoxybenzyloxycarbonyl (Z(3,5-OMe), alpha,alpha-dimethyl-3,5- dimethoxybenzyloxycarbonyl (Ddz), 6-nitroveratryloxycarbonyl (Nvoc), 4- (phenyldiazenyl)-benzyloxycarbonyl (Pz), 2-(biphenyl-4-yl)-2-propoxycarbonyl (Bpoc), isonicotinyloxycarbonyl (iNoc), tert-butoxycarbonyl (Boc), 2-cyano-tert- butoxycarbonyl (Cyoc), 2,2,2-trichloro-tert-butoxycarbonyl (Tcboc), adamantyl-1- oxycarbonyl (Adoc), 1-(1-adamantyl)-1-methoxycarbonyl (Adpoc), fluorenyl-9- methoxycarbonyl (Fmoc), (2-nirofluoren-9-yl)methoxycarbonyl (Fmoc(NO2)), 2-(4- toluenesulfonyl)-ethoxycarbonyl (Tsoc), methylsulfonylethoxycarbonyl (Msc), 2-(4- nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-(tert-butylsulfonyl)-2- propenyloxycarbonyl (Bspoc), 1,1-dioxobenzo[b]-thien-2-ylmethoxycarbonyl (Bsmoc), 2- / methylsulfonyl)-3-phenyl-2-propenyloxycarbonyl (Mspoc), allyloxycarbonyl (Aloc), 2-(trimethylsilyl)-ethoxycarbonyl (Teoc), triisopropylsilylethoxycarbonyl (Tipseoc), piperidinyloxycarbonyl (Pipoc), cyclopententyloxycarbonyl (Poc)), a carboxy-protecting group of the ester type (e.g., methyl (Me), ethyl (Et), benzyl (Bzl), 4-nitrobenzyl (Nbz), 4-methoxybenzyl (Mob), 2,4-di methoxybenzyl (2,4-Dmb), o-chlorotrityl (Trt(2-Cl), pyrimidyl-4-methyl(4-picolyl (Pic), phenacyl (Pac), 4-methoxyphenacyl (Pac(OMe), diphenylmethyl(Dpm), tert-butyl (tBu), cyclohexyl (Cy), 1-adamantyl (1-Ada), 2-adamantyl (2-Ada), dicyclopropylmethyl (Dcpm), 9-phenylfluoren-9-yl (Pf), 9-fluorenylmethyl (Fm), 2- trimethylsilylethyl (TMSE), 2-phenyl-trimethyl-silyl (PTMSE), allay (Al), 4-{N-[1-(4,4- dimethyl-2,6-dioxocyclohexanylidene)-3-methylbutyl]-amino}benzyl (Dmab)), a thiol-protecting group (e.g., benzyl (Bzl), 4-methylbenzyl (Bzl(4-Me)), 4- methoxybenzyl (Mob), 2,4,6-trimethoxybenzyl (Tmb), diphenylmethyl (Dpm), trityl (Trt), tert-butyl (tBu), acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), 9- fluorophenylmethyl (Fm), tert-butylsulfanyl (StBu), 3-nitro-2-pyridylsulfanyl (Npys), 9H-xanthen-9-yl (Xan)), an imidazole protecting group (e.g., benzyl (Bzl), 2,4-dinitrophenyl (Dnp), benzyloxycarbonyl (Bom), adamantly-1-oxycarbonyl (Adoc),triphenylmethyl (Trt), diphenylmethyl (Dpm), pyridyldiphenylmethyl (Pdpm), 4- toluenesulfonyl (Tosyl, Tos), 4-methoxybenzenesulfonyl (Mbs), tert-butoxymethyl (Bum), allyl (Al), allyloxymethyl (Alom)), an a hydroxyl-protecting group (e.g., benzyl (Bzl), 2,6-dichlorobenzyl (Dcb), diphenylmethyl (Dbm), cyclohexyl (Cy), 2- bromobenzyloxycarbonyl (Z(2-Br)), tert-butyl (tBu), 1-benzyloxycarbonyl-amino- 2,2,2-trifluoroethyl (Zte), methylthiomethyl (Mtm), allyl (Al), or a guanidinium protecting group (e.g., 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), tert-butoxycarbonyl (Boc), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc)). It will be known that in SPPS, the peptide is typically bound to a solid support during synthesis. In the context of peptide synthesis, a solid support may be understood interchangeably in the broadest sense as any solid matrix known for peptidesynthesis in the art. Typically, the solid support used during peptide synthesis is apolymeric bead. The solid support may be, but may not be limited to, chloromethylresin (Merrifield resin), 4-benzyloxybenzyl alcohol resin (Wang resin), (2,4- dimethoxy)benzhydrylamine resin (Rink amide resin), 2,4-dialkoxybenzyl resin (super acid-sensitive resin, SASRIN®), 2-chlorotrityl resin, alpha-chlorotritylchloride resin (Barlos resin), benzhydrylamine resin (BHA resin), chloromethyl resin, hydroxymethylbenzoic acid resin (HMBA resin), 4-hydroxymethyl-3-methoxyphenoxybutyric acid resin (HMPB resin), hydroxycrotonoyl aminomethylresin (HYCRAM resin), MBHA resin, oxime resin, Sieber resin, Ramage-amide resinand / or a resin with special cleavable linkers (e.g., photolabile linkers or safety-catch linkers). Alternatively or additionally, the polypeptide may be also provided by conjugating two or more peptide strand(s) with another by any conjugation method known in the art such as, e.g., Native Chemical Ligation (NCL), Click Chemistry, maleimide-thiol conjugation, enzymatic conjugation, biochemical protein ligation and / or soluble handling conjugation. Additionally, the polypeptide may be optionally purified by any means known in theart, such as, e.g., one or more chromatographic methods (e.g. high-performanceliquid chromatography (HPLC), preferably reversed phase HPLC (RP-HPLC), ultra- high-performance liquid chromatography (UPLC or UHPLC), fast protein liquidchromatography (FPLC), etc.), one or more filtration methods (e.g., one or morediafiltration methods, one or more dead-end filtration methods, one or more cross- flow filtration methods), one or more electrophoretic methods, one or more precipitation-based methods, one or more dialysis methods, one or more other concentration methods, one or more precipitation steps (e.g., salting in, salting out,contacting with an anti-solvent, etc.), one or more crystallization steps, one or morefreeze-drying steps (also: lyophilization), or a combination of two or more thereof. Additionally, the polypeptide may optionally be oxidized to form one intramolecular disulfide bond (also: cystine bond, including the structural pattern -S-S-). Theoptional formation of a disulfide bond may depend on the primary structure of thepolypeptide. It may also depend of the secondary structure of the polypeptide. Optionally, oxidation may be accomplished by storage of the polypeptide in an oxygen-containing environment (e.g., in a solution or at dry state), by purging oxygen in a solution containing the polypeptide or pharmaceutically acceptable salt thereof, and / or by adding one or more oxidizing agents to a solution containing the polypeptide or pharmaceutically acceptable salt thereof. When expressed in an organism, i.e., prepared by biotechnological means, it will be understood that a polypeptide in the sense of the present invention may or may not be subjected to one or more posttranslational modifications. The one or more optional posttranslational modifications typically depend on the organism in which the polypeptide of interest is expressed (in case of being prepared bybiotechnological means). The termini of the polypeptide may optionally be cappedby any means known in the art, such as, e.g., amidation, acetylation, methylation, and / or acylation. Posttranslational modifications are well-known in the art and may be but may not be limited to lipidation, phosphorylation, sulfatation, glycosylation, truncation, oxidation, reduction, decarboxylation, acetylation, amidation, deamidation, disulfide bond formation, hydroxylation, amino acid addition, cofactor addition (e.g., biotinylation, heme addition, eicosanoid addition, steroid addition) and complexation of metal ions, non-metal ions, peptides or small molecules and addition of iron-sulphide clusters. Moreover, optionally, co-factors, in particular cyclic guanidinium monophosphate (cGMP), but optionally also such as, e.g., ATP, ADP, NAD+, NADH+H+, NADP+, NADPH+H+, metal ions, anions, lipids, etc. may be bound to the polypeptide, irrespective on the biological influence of these co- factors. It will be understood that such polypeptide may also contain one or more non-natural amino acid moiety / moieties and / or one or more posttranscriptional modifications. The polypeptide of the present invention may be stored at any condition suitable for such purpose. For example, it may be dried or freeze-dried. For example, it may be stored in a temperature range of -100 to 30°C, or -80 to 30°C, or -25 to 25°C, or -25 to 0°C, or 0 to 25°C, or 2 to 22°C, or 5 to 15°C, or 10 to 25°C, or 15 to 25°C, or 18 to 22°C. For example, it may be stored at ambient temperature (e.g., 15 to 30°C, preferably 18 to 25°C), in a fridge (e.g., 2 to 20°C), a freezer (e.g., -25 to -5°C), a deep freezer (e.g., -80 to -60°C), or in a liquid gas (e.g., -150 to -90°C). As indicated above, in some embodiments, it is beneficial when the polypeptide of the present invention is pharmaceutically administrable to a patient. The polypeptide of the present invention may be administered to a patient in any form. Accordingly, in a preferred embodiment, the polypeptide of the present invention forms part of a pharmaceutical composition. Thus, a further aspect of the present invention is related to a pharmaceutical composition comprising:(A) the polypeptide of the present invention; and(B) at least one pharmaceutically acceptable carrier.As used herein, the terms “pharmaceutical composition” and “pharmaceuticalformulation” may be understood interchangeably as any composition that may beused in a pharmaceutical context such as for treating or preventing a patient. As used herein, the terms “pharmaceutically acceptable carrier”, “pharmaceutically acceptable excipient”, “carrier” and “excipient” may be understood interchangeably in the broadest sense as any substance that may support the pharmacological acceptance of the polypeptide of the present invention. Such pharmaceutical composition may be ready to use and may preferably be a liquid formulation, in particular an injection portion. The storage form may also be liquid, but may also be a dried form (e.g. a powder such as a powder comprising dried or freeze-dried polypeptide of the present invention) or may be a paste or syrup or the like. Optionally, a dried form, paste or syrup may be dissolved or emulsified prior to being administered to the patient. A pharmaceutically acceptable carrier may exemplarily be selected from the list consisting of an aqueous buffer, saline, water, dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or combinations of two or more thereof. Furthermore, the pharmaceutically acceptable carrier may optionally contain one or more detergent(s), one or more foaming agent(s) (e.g., sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS)), one or more coloring agent(s) (e.g., food coloring), one or more vitamin(s), one or more salt(s) (e.g., sodium, potassium, calcium, zinc salts), one or more humectant(s) (e.g., sorbitol, glycerol, mannitol, propylenglycol,polydextrose), one or more enzyme(s), one or more preserving agent(s) (e.g.,benzoic acid, methylparaben, one or more antioxidant(s), one or more herbal and plant extract(s), one or more stabilizing agent(s), one or more chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), and / or one or more uptake mediator(s) (e.g., polyethylene imine (PEI), a cell-penetrating peptide (CPP), a protein transduction domain (PTD), an antimicrobial peptide, etc.). The present invention also relates to a dosage unit of the pharmaceutical composition usable in the context of the treatment or prevention of the present invention. Exemplarily, the present invention may refer to a single dose container or to a multiple dosage form. Preferably, a pharmaceutical composition is prepared for final administration toenable routes of administration, which circumvent the first pass effect. Morepreferably, the pharmaceutical composition is prepared to be suitable for administration by injection into the patient (e.g., suitable for administration routes selected from the group consisting of intravenous (i.v.), intraarterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), and subcutaneous (s.c.) injection).Alternatively or additionally, the pharmaceutical composition may also be suitablefor other routes of administration such as, e.g., nasal or transdermal administration. The polypeptide of the present invention may optionally also be included in or attached to a liposome and / or a micelle. The polypeptide and the pharmaceutical composition may be used for any purpose. As indicated above, the polypeptide may be used to treat and / or prevent a thrombotic event. Accordingly, a further aspect of the present invention relates to the polypeptide of the present invention or a pharmaceutical composition of the present invention foruse in a method for treating or preventing a thrombotic event in a patient.It will be understood that the definitions and preferred embodiments laid out in thecontext of the polypeptide and the pharmaceutical composition as laid out abovemutatis mutandis apply to the pharmaceutical use thereof.In other words, the present invention relates to a method for treating or preventinga thrombotic event in a patient, wherein the patient is administered with a sufficientamount of the polypeptide of the present invention or a pharmaceutical compositionof the present invention. As used in the context of the present invention, the term “patient” may be understood in the broadest sense as any living being, which is preferably an animal, morepreferably a mammal including human, in particular a human being. A non-humanmammal as used herein may be any non-human mammal, preferably a domestic or agriculturally used animal such as, e.g., a horse, a dog, a cat, a bovine, a goat, a sheep, a donkey, or a camel. The polypeptide of the present invention or a pharmaceutical composition of the present invention may be administered to the patient by any means. In a preferredembodiment, it is administered to the patient by a route of administration, whichcircumvents the first pass effect. In a preferred embodiment, the polypeptide or a pharmaceutical composition is administered to the patient by injection, in particular injection selected from the group consisting of intravenous (i.v.), intraarterial (i.a.),intraperitoneal (i.p.), intramuscular (i.m.), and subcutaneous (s.c.) injection. In analternative preferred embodiment, the polypeptide or a pharmaceutical compositionis administered by nasal or transdermal administration. Alternatively, also otherroutes of administration such as, e.g., transdermal or oral administration may be used.The term “thrombotic event” may be understood in the broadest sense as generallyunderstood in the art. Typically, a thrombotic event includes the formation of one or more blood clots. The thrombotic event may be a vascular thrombotic event (VTE). In a preferred embodiment, the patient is at risk of developing or is suffering from thrombosis or embolization of the large blood vessels. In another preferred embodiment, the patient is at risk of developing or is suffering from microthrombi. In another preferred embodiment, the patient is at risk of developing or is sufferingfrom thrombosis or embolization of the large blood vessels and microthrombi. Asused herein, large blood vessels may have an inner diameter of the lumen of at least2 mm and microthrombi are thrombi occurring in blood vessels having an innerdiameter of the lumen of below 2 mm.In a preferred embodiment, the patient is suffering from or is at risk of developing ora pathological state associated with a thrombotic event selected from the groupconsisting of stenosis of one or more veins, venules, arteria, arterioles, and / orcapillaries. A stenosis of one or more veins and / or venules may be any type of at least partlyocclusion of one or more veins and / or venules hampering blood flow in one or moreveins and / or venules. In a preferred embodiment, stenosis of veins and / or venulesis selected from the group consisting of deep vein thrombosis, pelvic vein thrombosis, and retinal vein occlusion.A stenosis of one or more arteries and / or arterioles may be any type of at least partlyocclusion of one or more arteries and / or arterioles. In a preferred embodiment,stenosis of one or more arteries and / or arterioles may be atherosclerosis or stenosisof arteries. A stenosis of one or more capillaries may be any type of at least partly occlusion of one or more capillaries. In a preferred embodiment, stenosis of capillaries may be an angiopathy coincidence with thrombotic events in capillary flow path, in particular diabetic angiopathy. In a preferred embodiment, the patient is suffering from one or more ischemic regions that lead to or are at risk of leading to necrosis of at least a part of a tissuewithout administration of the polypeptide or pharmaceutical salt thereof of thepresent invention to said patient. In a preferred embodiment, the patient is suffering from or is at risk of developing or a pathological state associated with a thrombotic event selected from the group consisting of stroke, myocardial infarction, embolism, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), thrombophlebitis,sepsis, or a combination of two or more thereof.Embolism may be embolism of any tissue. In a preferred embodiment, embolism is pulmonary embolism. Infarction may be infarction of any organ. In a preferred embodiment, infarction is myocardial infarction. The administration of the polypeptide of the present invention may enhance reperfusion and reduce the recurrence of one or more thrombotic events in post- operative settings. Optionally, the administration of the polypeptide of the presentinvention may reduce FXIII-mediated, optionally local, inflammation such as, e.g., incase of arthropathies. In one embodiment, locally reversing thrombotic events may be destined by FXIIIa mediated reactions such as alpha2-macroglobulin placement within the growing thrombotic event (e.g., blood clot), reduced platelet adhesion viareduced alpha2-antiplasmin incorporation. Children with severe thrombotic events(e.g., blood clots) may benefit from shorter treatments (tailored treatments for youngchildren). As this peptide inhibitor may have a shorter half-life than many syntheticagents, it may significantly reduce recurrent thrombotic event risk as well as(subsequent) bleeding risk. The half-life may be adapted by routes of administration as well as structural modifications. In a preferred embodiment, the patient is subjected to a surgery. Surgery may be any type of surgery. In a preferred embodiment, surgery is bypass surgery. The polypeptide may be administered to the patient prior to a surgery, may be administered to the patient subsequent to receiving a surgery, or may be administered to the patient prior to and subsequent to receiving a surgery. In a preferred embodiment, the patient is receiving an endoprosthesis. Anendoprosthesis may be any type of an endoprosthesis. In a preferred embodiment,an endoprosthesis is selected from the group consisting of a blood vesselendoprosthesis (e.g., a stent, artificial vessel, etc.), an artificial joint (e.g., an artificialhip joint, an artificial knee joint, etc.), a cardiac pacemaker, an artificial bowel outlet, and a cosmetic implant.The target FXIIIa may be associated with the regulation of Insulin resistance andadipogenesis. Stent failures among T2D patients often involve endothelialdysfunction, and platelet hyperactivity, which in turn is again expected to raise localFXIIIa. Administration of FXIIIa inhibitor to the stent (coated with the polypeptide)may improve biofunctionalization by improving stent endothelium interactions.In a preferred embodiment, the patient is receiving an organ transplant and / or atissue transplant. As used herein, an organ transplant may be understood in thebroadest sense. For instance, an organ transplant may be a transplant of an inner organ (e.g., a kidney, a liver, a heart, a stomach, a part of a gut, or a blood vessel, etc.). As used herein, a tissue transplant may be understood in the broadest sense. For instance, a tissue transplant may be a transplant of tissue of an inner organ (e.g., of liver, of lung, of gut), or retina, or of skin. As noted above, the polypeptide and the pharmaceutical composition may be used for any purpose. The polypeptide may optionally be used for a coating such as, e.g., for an endoprosthesis. Accordingly, a yet further aspect of the present invention relates to anendoprosthesis or an enzyme-linked immunosorbent assay (ELISA) plate coatedwith the polypeptide or pharmaceutically acceptable salt thereof. It will be understood that the definitions and preferred embodiments laid out in the context of the polypeptide or pharmaceutically acceptable salt thereof, thepharmaceutical composition and use thereof as laid out above mutatis mutandisapply to the endoprosthesis coated with the polypeptide or pharmaceutically acceptable salt thereof. As used herein, the term “endoprosthesis” may be understood in the broadest sense as any artificial implant that remains in the patient’s body. Typically, an endoprosthesis is of a solid material and may thus be considered as a solid support of the polypeptide or pharmaceutically acceptable salt thereof. The endoprosthesismay be any endoprosthesis. In a preferred embodiment, the endoprosthesis isselected from the group consisting of a blood vessel endoprosthesis (e.g., a stent,artificial vessel, etc.), an artificial joint (e.g., an artificial hip joint, an artificial kneejoint, etc.), a cardiac pacemaker, an artificial bowel outlet, and a cosmetic implant(e.g., a breast implant, a chin implant, a buttock implant, etc.). Coating may be performed by any means. In a preferred embodiment, coating is a covalent binding of the polypeptide to the solid endoprosthesis. Such covalent binding may be directly or may be via a linker structure. A linker structure may be any linker structure. In a preferred embodiment, a linker structure is a peptidic linker structure, a non-peptidic linker structure or a linker structure comprising peptidic and non-peptidic structure elements, preferably wherein the total linker structure has amolecular weight of 100 to 10,000 Da. For instance, a linker may have a molecularweight of 100 to 1,000 Da, or 200 to 2,000 Da, or 500 to 5,000 Da, or 1,000 to10,000 Da.Alternatively or additionally, coating may involve complex formation between the polypeptide and the solid endoprosthesis. This may be achieved by any type of complex formation such as, e.g., by a chelate complex (e.g., a histidine tag bound to a nickel ion). A complex may also be formed with a linker structure as described before. In another preferred embodiment, coating is a non-covalent coating. This may beperformed by any means such as, e.g., by soaking and / or spraying of theendoprosthesis with a solution of the polypeptide or pharmaceutically acceptable salt thereof. It will be understood that the polypeptide may also be used for any non-therapeutic purpose. In one aspect, the polypeptide is used in assay development such as, e.g. in anenzyme-linked immunosorbent assay (ELISA) involving FXIIIa or an enzyme kinetictest based on FXIIIa. Herein, the polypeptide may specifically bind to FXIIIa andmay replace antibodies. The polypeptide may optionally be labelled (e.g., fluorescently, by an enzyme, by colloidal gold, by a binding group, etc.). This mayenable fluorescence-based localization / visualization. Also, cellular assays may bepossible that involve binding of FXIIIa. The functionality and biochemical processes of formation of thrombotic events may be further revealed. The polypeptide may alsoserve as a pharmacological tool for in vitro and ex vivo testing and chromogenic aswell as fluorogenic FXIIIa activity assays. The following examples and figures are intended to provide illustrative embodiments of the present invention. These examples are not intended to provide any limitation on the scope of the invented subject-matter. Brief description of the FiguresFigure 1 shows an overview of tridegin derivatives. These may be trideginderivatives having two disulfide bonds between Cys5 / Cys17and Cys31 / Cys37(Fig. 1A, also designated as “isomer A” herein) and tridegin derivatives having twoother disulfide bonds between Cys5 / Cys37 and Cys5 / Cys17 (Fig. 1A, also designatedas “isomer B” herein) and tridegin derivatives having two other disulfide bondsbetween (Fig. 1A, also designated as “isomer C” herein). Furthermore, an NMR-based structural characterization of the double-bridged isomer B (Fig. 1B) and asuperimposition of the NMR-based structural characterization with the previouslyused exclusively computer-based model of isomer B (Fig. 1C) are depicted.Figure 2 shows a functional in vitro analysis of tridegin derivatives. (A) Curve forfluorogenic enzyme activity assay of polypeptide isomer B (n=3). (B) IC50 valuesderived from the fluorogenic enzyme activity assay for the investigated polypeptidesisomer B (SEQ ID NO: 6) (1), SEQ ID NO: 2 (2), All-Ser (SEQ ID NO: 7) (3), SEQID NO: 3 (4), SEQ ID NO: 4 (5), SEQ ID NO: 5 (6), SEQ ID NO: 8 (7), SEQ ID NO:9 (8), SEQ ID NO: 10 (9), and SEQ ID NO: 11 (10). (C) Relative inhibitory effects at 1.1 µM polypeptide concentration obtained by the Berichrom®assay. Inhibitoryeffects were normalized to 100% for polypeptide isomer B (SEQ ID NO: 6). (D)FXIIIa inhibition upon addition of several polypeptides isomer B (SEQ ID NO: 6) (1),SEQ ID NO: 2 (2), and All-Ser (SEQ ID NO: 7) (3) at varying concentrations in theBerichrom®assay (n = 4). Activity is correlated to a standard human plasma with a reference interval of 70 to 140%. Consequently, data points below 15% wereconsidered inactive. (E-F) Whole blood clot contraction assay of polypeptide isomerB (SEQ ID NO: 6) and polypeptide All-Ser (SEQ ID NO: 7) performed in humanwhole blood (n = 1) (E) and polypeptide All-Ser (SEQ ID NO: 7) performed in whole blood from mice (n = 1) (F). x represents inhibition by irreversible FXIIIa inhibitor T101 (Zedira GmbH).Figure 3 shows an in vivo analysis of the effect of polypeptide isomer B (SEQ IDNO: 6) on FXIIIa activity in mice.Figure 4 shows the fusion-molecule encoding sequence inserted into theexpression vector pETDuet-1 (top). Below the map of an expression vectorpETDuet-1 containing DsbC (left) and the specifications of the fusion moleculelinking streptokinase and tridegin (SEQ ID NO: 14) with a thrombin-cleavable linkersequence (bottom right) are shown.Figure 5 shows the expression analysis and functional testing of a recombinantfusion molecule consisting of streptokinase, a thrombin-cleavable linker, andtridegin (SEQ ID NO: 14). (A) Coomassie-stained SDS-PAGE analysis of whole celllysates of two clones (#1, #2), that express the recombinant His-tagged fusionmolecule (lanes 3-8 that show cell pellets, cleared cell lysate and conditionedmedium for each clone, respectively). Arrows in lines 4 and 7 indicate the expectedband for the recombinant construct (~70 kDa). The arrow in line 2 representsrecombinantly expressed streptokinase (47 kDa). (B) Western Blot using anti-His-antibodies confirm the expression of the recombinantly expressed proteins in the clones (arrows). (C) Functional analysis of the crude lysate of both clones applyingthe Berichrom® assay shows an inhibition of FXIIIa activity in comparison to normalpooled human plasma (NPP). ExamplesPreparation of polypeptides (tridegin derivatives)Different tridegin derivatives were prepared by standard solid phase peptidesynthesis (SPPS). Experimental sectionMaterials. All solvents, reagents, and buffer chemicals were of analytical grade ifnot stated otherwise. Further details about the chemicals are given below.Synthesis of peptides and purification. General procedure for SPPS. Peptidesynthesis of all peptides was carried out by automated solid phase peptide synthesis (SPPS) using a standard Fmoc (N-(9-fluorenyl)methoxy-carbonyl) protocol on an EPS 221 synthesizer (Intavis Bioanalytical Instruments AG, Cologne, Germany). A Rink amide MBHA resin (0.56 mmol / g) was utilized. The coupling reagent 2-(1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and the base N-methylmorpholine were applied to perform coupling of the Fmoc-amino acid derivatives. Fmoc deprotection was carried out with 20% piperidine in dimethylformamide. Introduction of non-proteinogenic amino acids ornithine, citrulline and the D-amino acid derivatives of Lys, Ile and Leu was achieved by using Fmoc-L-Orn(Boc)-OH, Fmoc-L-Cit-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ile-OH, and Fmoc-D-Leu-OH as derivatives. For the selective formation of disulfide bridgesFmoc-L-Cys(Trt)-OH and Fmoc-L-Cys(Acm)-OH at the respective positions havebeen applied as outlined earlier. Peptide cleavage and side-chain deprotection (except for acetamidomethylated cysteine) were accomplished by addition of a cocktail of 1.0 ml reagent K (75 mgphenol, 25 µl 1,2-ethandithiol, 50 µl thioanisol, 50 µl water in 0.95 ml TFA) per 100mg resin on ice and subsequent stirring for 3 h at room temperature. The cleavage mixture was filtered and precipitated in ice-cold diethyl ether. Peptide pellets were washed for three times with diethyl ether and re-dissolved in 80% tert-butanol for freeze-drying.Oxidation of linear precursor peptides. Crude linear precursor peptides containingcysteines, i.e., peptides 1 and 2 were oxidized as described earlier with slightmodifications as follows. Peptides (0.025 – 0.050 mM, 1 equiv.) in 50% acetic acidwere mixed with 1.1 equiv. iodine (0.1 M in methanol) under argon for 1 h to complete the first oxidation. Subsequently, 8.9 equiv. iodine (0.1 M in methanol) were added to the same reaction mixture and left stirring for 4 h. The solution was diluted with the same volume of water and iodine was extracted 3 times with ethyl acetate. The aqueous phase was freeze-dried and stored at –20 °C.General procedure for purification of peptides. Crude (oxidized) peptides werepurified by semi-preparative RP-HPLC using a Shimdazu LC-8A system (Duisburg, Germany) or a JASCO PV-987 instrument (Gross-Umstadt, Germany) equippedwith a Knauer Eurospher 100 column (C18, 250 ^ 32 mm, 5.0 µm particle size, 100Å pore size). The separation was performed in a gradient elution system of eluent A (0.1% TFA in water) and eluent B (acetonitrile: / water 9 / 1, 0.1 % TFA). By continuously increasing the concentration of eluent B by 50 % within 120 min at a flow rate of 10 ml / min elution of the peptides was achieved and detected at 220 nm. The applied gradients for each peptide are described in Table 1 below. Collected fractions were combined, evaporated from acetonitrile, freeze-dried, and stored at –20 °C. Peptide purity was confirmed by analytical HPLC on a ShimadzuLC-20AD system equipped with a Vydac 218TP column (C18, 250 ^ 4.6 mm, 5.0µm particle size, 300 Å pore size). Gradient elution was carried out using acetonitrile containing 0.1 % TFA (eluent B) and 0.1 % TFA in water (eluent A) at a flow rate of 1.0 mL / min. The detection of the peptides was at λ = 220 nm. Retention times and methods are given in Table 1 below. All peptides were obtained in purities >95 %.Peptides analytical characterization. Analytical HPLC (as aforementioned, orperformed on a Shimadzu Prominence-i LC-2030 system equipped with a Vydac208TP column (C8, 250 ^ 4.6 mm, 5.0 µm particle size, 300 Å pore size) in gradientelution mode using acetonitrile containing 0.1 % TFA (eluent B) and 0.1 % TFA in water (eluent A) at a flow rate of 1.0 mL / min), mass spectrometry, and thin-layer chromatography (TLC) were applied to verify peptide identities. Peptide molar masses were analyzed by matrix-assisted laser desorption-ionization / time-of-flight (matrix-assisted laser desorption / ionization (MALDI), MALDI-TOF) on an ultrafleXtreme instrument. Peptide contents were determined by RP-HPLC usingthe above-mentioned LC-20AD system based on peak area comparison ofpreviously determined peptide samples.The tridegin derivative isomer B (SEQ ID NO: 6) containing disulfide bridges C5-C37and C17-C31was selected as a starting point for further analysis and optimization toevaluate its applicability for future drug development. In one tridegin derivative, allcysteine moieties were replaced by serine moieties (All-Ser, SEQ ID NO: 7). These two tridegin derivatives are known in the art and were herein used as control peptides. In another tridegin derivative, all cysteine moieties were replaced byalanine moieties (SEQ ID NO: 4). Replacements of amino acids in positions 19 and25 were analyzed to identify feasibility and cost savings for the synthesis of suchanalogues (SEQ ID NO: 8) and to investigate the impact of the polarity of theseamino acids using alternatives with similarity to a disulfide bridge between C19andC25, i.e. S-methyl-cysteine (SEQ ID NO: 11). At the same time, introduction of non-proteinogenic amino acids, such as D-amino acids, or replacements of amino acids by non-proteinogenic amino acids, such as ornithine for lysine and citrulline for arginine, were tested to reduce accessibility of these peptides for peptidases and proteases in the plasma or organism, respectively, depending on the route of administration. As a consequence, further modifications of polypeptides All-Ser(SEQ ID NO: 7) or SEQ ID NO: 4 by substituting all lysine moieties and argininemoieties for ornithine moieties and citrulline moieties, respectively, have beencarried out and yielded polypeptides SEQ ID NO: 3 and SEQ ID NO: 5. In anotherderivative of SEQ ID NO: 7, K1and L34were replaced for the corresponding D-aminoacids (SEQ ID NO: 9, SEQ ID NO: 10). Indeed, synthesis yielded for the linearpeptides without disulfide bridges significantly increased since oxidation of cysteines as an additional synthesis step was not required anymore. The aforementioned peptides are as follows (wherein the experimentally usedpolypeptides amidated at the C-terminus (i.e., the C-terminal carboxyl group, -CO-NH2):Polypeptide No. 1, Polypeptide isomer B, used herein as control:KLLPCKEWHQGIPNPRCWSGADLESAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 6),wherein preferably disulfide bonds form between cysteine residues Cys5and Cys37as well as between Cys17and Cys31. Polypeptide No.2: KLLPCkEWHQGiPNPRCWSGADlESAQDQYCAFiPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 2),wherein k6 is a D-lysyl moiety, l23 is a D-leucine and i12 and i34 is a D-isoleucinemoiety, and wherein preferably disulfide bonds form between cysteine residues Cys5and Cys37as well as between Cys17and Cys31.Polypeptide No. 3, Polypeptide All-Ser, used herein as control:KLLPSKEWHQGIPNPRSWSGADLESAQDQYSAFIPQSRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 7).Polypeptide No.4: OrnLLPSOrnEWHQGIPNPCitSWSGADLESAQDQYSAFIPQSCitPCitSELIOrnPM DDIYQCitPVEFPNLPLOrnPCitE, which can also be depicted as: X1LLPSX6EWHQGIPNPX16SWSGADLESAQDQYSAFIPQSX38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E (SEQ ID NO: 3),wherein X1, X6, X45and X63are each Orn; and wherein X16, X38, X40, X53, X65are each Cit. Polypeptide No.5: KLLPAKEWHQGIPNPRAWAGADLEAAQDQYAAFIPQARPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 4).Polypeptide No.6: OrnLLPAOrnEWHQGIPNPCitAWAGADLEAAQDQYAAFIPQACitPCitSELIOrnPM DDIYQCitPVEFPNLPLOrnPCitE, which can also be depicted as X1LLPAX6EWHQGIPNPX16AWAGADLEAAQDQYAAFIPQAX38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E (SEQ ID NO: 5),wherein X1, X6, X45and X63are each Orn; and wherein X16, X38, X40, X53, X65are each Cit. Polypeptide No.7: KLLPSKEWHQGIPNPRSWAGADLEAAQDQYSAFIPQSRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 8).Polypeptide No.8: kLLPSKEWHQGIPNPRSWAGADLEAAQDQYSAFIPQSRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 9),wherein k1is a D-lysyl moiety. Polypeptide No.9: KLLPSKEWHQGIPNPRSWAGADlEAAQDQYSAFIPQSRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 10),wherein l23is a D-leucine moiety. Polypeptide No.10: KLLPSKEWHQGIPNPRSWX19GADLEX25AQDQYSAFIPQSRPRSELIKPMDDIYQRPVEFPNLPLKPRE (SEQ ID NO: 11),wherein X19 and X25 are each Cys(Me).As experimentally used, the polypeptides were synthesized as C-terminal amides.Changes in cysteine positions compared to isomer B (SEQ ID NO: 6) are underlinedabove. Changes for other amino acids are given in italics in the respective sequences, e.g. k = D-Lys, i = D-Ile, etc. Initially, a careful analysis of potential cleavage sites for peptidases and proteases was performed applying primarily the MEROPS database and yielded 51 potential cleavage sites for proteases and peptidases. This is depicted for polypeptide 2(SEQ ID NO: 2), wherein, in the first line, the cleavage sites are depicted in boldletters, in the second line the solvent-accessible surface area is depicted in bold letters, in the third row, the accessible cleavage sites are depicted in bold letters,the fourth row the interaction sites (no change allowed) are depicted in bold letters,and in the last line, the accessible cleavage sites without interaction are depicted inbold letters (in SEQ ID NO: 6):KLLPCKEWHQGIPNPRCWSGADLESAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE KLLPCKEWHQGIPNPRCWSGADLESAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE KLLPCKEWHQGIPNPRCWSGADLESAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE KLLPCKEWHQGIPNPRCWSGADLESAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE KLLPCKEWHQGIPNPRCWSGADLESAQDQYCAFIPQCRPRSELIKPMDDIYQRPVEFPNLPLKPRE In parallel, the solvent-accessible surface area of polypeptide isomer B (SEQ IDNO: 6) was examined considering the earlier reported simulations of this peptide.This allowed narrowing down the number of accessible cleavage sites to a total of27 amino acids. Exclusion of relevant interaction sites with FXIIIa was implemented as a third criterion to decide for potential amino acids for replacement by e.g. D-amino acids in polypeptide isomer B (SEQ ID NO: 6) without affecting its inhibitorypotential and reduced the number of relevant positions to 15. Considering that the main impact on the inhibitory potential of tridegin is inferred by the C-terminal (amino acids 38-66), further exchanges in this region in the beginning have been excluded, which enabled to limit our selection to six potential amino acid positions for replacement. In combination with computational studies, revealing which amino acids are of interest for the interaction of the N-terminal fragment with FXIIIa, we decided first to substitute the amino acid moieties Lys6, Ile12, Leu23and Ile34by their corresponding D-amino acid. Mutations of Glu7and Glu24were avoided as glutamic acid cleavage sites appear with only very minor frequency in a physiological context. This led us to the design of polypeptide 2 (SEQ ID NO: 2). Analysis of the formed disulfide connectivities by the applied selective protecting group strategy was performed as earlier and confirmed the expected bonds between Cys5-Cys37and Cys17-Cys31.Table 1. Analytical characterization of the peptides.PolypeptidetR (min) [M+H]+ (calc.)a [M+H]+ (meas.)a Rf1 Rf2No.* 119.1b 7741.8 7742.3 0.25d 0.20e2 19.7b 7741.8 7741.6 0.31d 0.25e3 Analytical studies were performed before4 20.0b 7631.0 7630.8 0.64f 0.15e5 20.3b 7586.0 7586.0 0.94g 0.04e6 21.8b 7535.0 7535.2 0.64f 0.11e7 18.6b / 38.5c 7649.0 7649.6 - -8 18.7b / 38.6c 7649.0 7649.8 - -9 18.1b / 38.0c 7649.0 7649.9 - -10 19.0b / 38.9c 7740.9 7741.5 - -*as indicated above.amonoisotopic mass.b20-60% in 40 min measured on a Shimadzu LC-20A system equipped with a C18Vydac 218TP column.c0-60% in 60 min measured on a Shimadzu Prominence-I LC20-30 systemequipped with a C8 Vydac 208TP column.dtert-butanol / acetic acid / ethyl acetate / water (1 / 1 / 1 / 1).epyridine / ethyl acetate / acetic acid / water (5 / 5 / 3 / 1).facetonitrile / water (1 / 1).gacetonitrile / water / 25% ammonia (25 / 27 / 10).Recombinant expression. For the design of a fusion molecule consisting of a His-tag, streptokinase (Uniprot: Q7X0Y3) from Streptococcus pyogenes, a thrombin-cleavable linker (VSQTSKLTRAETVFPDV, SEQ ID NO: 12), and tridegin (SEQ IDNO: 14), the relevant genes were cloned and fused into the pETDuet-1 vector(Figure 4) and transformed into SHuffle® T7 Competent E. coli cells. This isintended to co-express IPTG induced DsbC chaperone and the fusion molecule viatheir respective T7 promoters within this bicistronic plasmid. The transformed cellswere selected on LB agar plates containing Ampicillin and Spectinomycin. SHuffle®T7 E. coli cells transformed with the recombinant plasmid were grown in LB mediumsupplemented with 100 μg / mL of ampicillin and 60 μg / mL of spectinomycin. Expression of the fusion protein was induced by adding 0.4 mM IPTG to an exponential growth culture and further incubated for 4 hours at 30 °C with shaking. Cultured cells were harvested by centrifugation at 5000 g for 10 min at 4 °C. The harvested cells containing the fusion protein were resuspended in an ice-cold lysisbuffer (50 mM Tris, 1 mM EDTA, pH 8.0, 10 mM imidazole, Protease inhibitor tablet,Lysozyme) and completely lysed by ultrasonication. The soluble and insoluble formsof the extracted protein were separated by centrifugation at 13,500 g for 20 min at4 °C. Cell pellets, and supernatants were also subjected to SDS-PAGE and WesternBlot (Figure 5A, B).Sequence of recombinant fusion molecule (SEQ ID NO: 13):MHHHHHHENL YFQSMKNYLS FGMFALLFAL TFGTVKPVQA IAGYEWLLDR PSVNNSQLVV SMAGIVEGTD KKVFINFFEI DLTSQPAHGG KTEQGLSPKS KPFATDNGAM PHKLEKADLL KAIQERLIAN VHSNDGYFEV IDFASDATIT DRDDNIYFAN QDGSVTLPTQ PIQQFLLRGH VRVRPYKEKP IQTPAKSVDI RYTVQFTPLN PDDDFKPVLK DTKLLKTLAI GDTITSQELL AQAQSILNES HPDYTIYERD SSIVTHDNDI FRTILPTDQE FTYHVKNREQ AYQNDNKTGL KKETKNTDLI SEKYYILKKG EKPYDPFDRS HLKLFTIKYV DVDTKDLLKS EQLLTASERN LDFRDLYDPR DKAKLLYNNL DAFDIMDYTL TGKVEDNHDK NNRVVTVYMG KRPKGAKGSY HLAYDKDLYT EEEREVYSYL RDTGTPIPDN PNDKVSQTSK LTRAETVFPD VKLLPCKEWH QGIPNPRCWC GADLECAQDQ YCAFIPQCRP RSELIKPMDD IYQRPVEFPN FPLKPREFunctional studies. Enzyme activity assay. Enzyme activity was measured usinga fluorogenic enzyme activity assay. The measurements were carried out with the substrate H−Tyr(3-NO2)−Glu(NH−(CH2)4−NH−Abz)−Val−Lys−Val−Ile−NH2asdescribed previously. IC50 plots were fitted in GraphPad Prism 9.3 (SynergySoftware, Reading, PA, USA) by nonlinear regression using the equation y = vmax / (1 + (x / x0)s) with y = response, vmax= maximum velocity, x = inhibitor concentration, x0= relative IC50, and s = slope factor. Results were subsequently illustrated in GraphPad Prism 9.3. IC50values are shown as the means ± standard deviation. Berichrome®assay for FXIIIa activity. For the assay, an inhibitor stock solution(11 mM in H2O) was diluted with water to 5500, 1100, 550, and 110 μM. Eachconcentration was diluted 1:10 with normal pooled plasma (NPP). 15 μl of each dilution was added to a mixture of 75 μl activator reagent and 75 µl detection reagent (Siemens, Berichrom®Assay Kit). As a positive control, human plasma was mixed 10:1 with H2O and measured analogously. As negative control, FXIII-deficient plasma was used instead of human plasma. For detection, the released ammonia was used, which was converted into glutamate in a glutamate dehydrogenase side reaction. The decrease in NADH was determined photometrically at 340 nm. The measurements were carried out in triplicates on two different days. Data were evaluated applying GraphPad Prism 9.3.Whole blood and plasma preparation. Phlebotomy was performed on consentinghealthy donors in accordance with the Declaration of Helsinki and the University ofNorth Carolina Institutional Review Board. Blood was collected by venipuncture using a 21G butterfly needle (Becton, Dickenson and Company, Franklin Lakes, NJ) into 0.105 M sodium citrate, pH 5.5 (10% v / v, final concentration). Normal pooled plasma (NPP) was obtained from four healthy donors. For each donor, the first 5 ml were discarded. Platelet-poor plasma was prepared by sequential centrifugation (150 g for 25 min, then 20000 g for 20 min), pooled, aliquoted, flash frozen withliquid nitrogen, and stored at −80 °C.Whole blood clot contraction assay. Clotting was triggered in recalcified (10 mM, final concentration) whole blood via the addition of a tissue factor (Innovin diluted1:12000, 1 pM, final concentration). Final reaction volumes were 200 μl (85% wholeblood; 10% tridegin variant, T101, or HBS; 2.5% Innovin; and 2.5% calcium chloride). Clot contraction proceeded at 37 °C for 120 min in siliconized multiwell plates. Contracted clots were removed and weighed. Analysis of clot weight was performed in GraphPad v 7.02 (Synergy Software, Reading, PA, USA) using the equation y = bottom + (top − bottom) / (1 + (x / IC50)) with y = response and x = inhibitor concentration. IC50values are shown as the means ± standard deviation. Statistical analysis was performed using an unpaired t-test (Holm−Sidak method) by means of GraphPad v 7.04 (Synergy Software, Reading, PA, USA). P values less than 0.01 were considered statistically significant.Pharmacokinetics. Aqueous solubility. Peptides were dissolved in 0.07 mMphosphate buffer (pH 7.4, 6.2 or 5.0) at 100 µM and incubated for 18 h at 25 °C. After incubation samples were centrifuged at 14000 rpm for 15 min and supernatants were again centrifuged for 15 min. No pellet formation was observed at any step for the investigated peptides. Supernatants were finally analyzed by analytical HPLC as described above applying a gradient of 20%-60% eluent B in 40 min. Peak areas were quantified and compared to a standard curve of polypeptideTS2 that was prepared in advance.Measurement of LogD7.4. LogD7.4 was measured by adapting the shake flaskprocedure from OECD19 at room temperature in n-octanol (> 99 %) and 20 mM phosphate-buffer pH 7.4. The peptides (100 µg / ml) were dissolved in a solution with equal amounts of n-octanol and phosphate buffer, shaken for 1 h and measured for the amount of peptide in each phase with RP-HPLC. The LogD7.4values were estimated using the relation log([peptide]octanol / [peptide]buffer).Plasma protein binding. For the PPB studies, plasma (total volume 200 µl) wasspiked with the peptides (10 µM) and incubated for 5 min for PPB equilibrium. Thepeptide containing plasma probes were transferred into 500 µl 30 kDa MWCO filterdevices (Amicon Ultra, Merck, Darmstadt) and filled up with 300 µl PBS buffer. Thedevices were centrifuged at 14,000 rcf for 15 min and subsequently washed with500 µl of PBS buffer. This procedure was repeated once. After the elution of thefilter concentrate, the slurry was exactly reconstituted with PBS buffer to a final volume of 200 µl. The PPB samples as well as the reference (10 µM of the peptidein 200 µl PBS) were diluted 1:100 with 0.2 % formic acid and finally measured viaLC-MS.Plasma stability. Compounds were incubated for 3 h in 1 ml human plasma at roomtemperature (10 µM and 100 µM). Reaction samples were taken at defined times and measured by selected reaction monitoring.Human liver microsomal stability. Compounds were incubated for 1 h at roomtemperature at a concentration of 10 µM. Reaction samples were taken at defined times and measured by selected reaction monitoring.Preparation of cell cultures. Human hepatocellular carcinoma (HepG2) cells werecultured in Dulbecco´s modified Eagle´s medium (DMEM, Gibco, Austria) supplemented with 10% fetal bovine serum (FBS; Gibco, Austria), penicillin(100 U / ml) and streptomycin (0.1 mg / ml) solution (Gibco, USA). All cells werecultured under a humidified CO2(5.0 %) atmosphere at 37°C and passaged by trypsinization when reached approximately 80% confluence. For experiments, cells in exponential phase of growth (at a density of 5,000 cells / well) were seeded into 96-well flat-bottom plates after treatment with trypsin-EDTA (Greiner, Germany) solution at a final volume of 100 µl / well. Cells were incubated overnight before treatment with test substances. Cell viability assay. The cytotoxicity of the above-references six polypeptides(1.0 mM stock solutions in PBS7.4) was evaluated in HepG2 cells by a colorimetricassay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).Cells were seeded in 96-well culture plates with a density of 2.5 ^ 104 cells per well.Following adherence, the cells were treated with the polypeptides at different concentrations (0.1, 1.0, 5.0, 10, 25, 50 and 100 µM) and further incubated for 24 h at 37°C. After incubation period, MTT solution (0.5 mg / ml) was added and cells were incubated for further 90 min. Then, the medium was removed and the plates were placed in a plate shaker at room temperature until complete dissolution of purple formazans. The quantification of formazans produced from the biological sample after reduction of MTT was monitored using a microplate ELISA reader VarioscanTM LUX (Thermo Fisher Scientific Inc., USA) at a wavelength of 540 nm with a reference wavelength of 630 nm. The cytotoxicity of the test peptides determined by MTT assay was expressed as percentage cell viability according to the following equation: %Cell viability = (Asample – Ablank) / (Acontrol – Ablank) ^ 100In this equation, Asample, Ablankand Acontrolare the measured absorption of the respective test sample, blank solution, and control sample. The results were expressed as the mean % of the untreated controls ± SD from three independent experiments (n = 3).Computational Studies. MD simulations. Molecular dynamics (MD) simulations inthis study were carried out using the Gromacs 2018 package and the YASARA platform. In general, NMR studies may be conducted as described in the art (Schmitz et al., International Journal of Molecular Sciences, 2021, 22:880). The first model of the 100-member NMR ensemble of the full-length model of polypeptide TS2 was used as the starting structure of the 300-ns long simulation. The polypeptide was placed in the center of a cubic simulation cell, with the edges of the cube separated by at least 12 Å from all atoms of the peptide. The polypeptide wassolvated using the TIP3P water model with additional Na+ and Cl– counter ionsadded to achieve a physiological salt concentration of 0.9%, while maintaining azero net charge on the entire system. The solvated system was subjected to 5000steps of steepest-descents energy minimization with the Amber-ff14sb force field, which was used to describe atomic motions for all parts of the simulation. The energy-minimized system was first subjected to a temperature equilibration in the NVT (constant number of atoms, volume, temperature) ensemble for 2 ns, with temperature maintained by the velocity-rescaled variant of the Berendsenthermostat at 300 K. Subsequently a 2 ns simulation to equilibrate the pressure ofthe system at 1 atm was conducted in the NPT (constant number of atoms, pressure, temperature) ensemble aided by the Parrinello–Rahman barostat. During both the temperature and pressure equilibration runs, all heavy atoms were position- restrained by the LINCS algorithm. The production run was conducted for 300 ns using a 2 fs timestep. Long-range interactions were cutoff at 10 Å, and theelectrostatics were described by a particle-mesh (Ewald method). Periodicboundary conditions were employed in the simulations, the effects of which were adjusted prior to conducting analyses on the trajectory. Trajectory analysis, namely the backbone root mean square deviation (RMSD) computation was conducted inVMD 1.9.3., which was also used in the creation of molecular graphics. Snapshotswere written at a 100 ps interval to disc, resulting in a total of 3000 snapshots collected for analysis, from this simulation.Molecular docking studies. The full-length model of polypeptide isomer B (SEQ IDNO: 6)2 was used for docking onto the modified and simulation-equilibrated crystalstructure of FXIIIa originally derived from the source PDB ID: 4KTY. The PDBstructure used as a receptor for docking was a simulation-averaged structure of the non-proteolytically activated FXIIIa, which had been modified, i.e., missingregions / loops filled, all heteroatoms / water molecules removed, and subjected toclassical molecular dynamic simulation. The details of this simulation have beenreported earlier. The full-length model obtained as the final snapshot of the 300 nsMD simulation was used as the ligand for the docking simulation. Docking was performed on the Hdock server (http: / / hdock.phys.hust.edu.cn / ) in a blind fashionand under the default conditions of the server. The Hdock server is based on ahybrid algorithm of template-based modeling and ab initio free docking and is currently ranked favorably amongst the topmost automated docking servers in therecent CASP (critical assessment of protein structure prediction) competitions. Only the top-ten docking poses generated by the server were closely inspected. The docking pose that strongly agreed with the experimental data was subjected to a docking refinement protocol on the Haddock 2.2 webserver (https: / / milou.science.uu.nl / services / HADDOCK2.2 / haddockserver- refinement.html) that allows for flexible refinement in explicit solvent of the dockedcomplex. The model with the best Haddock score from the output clusters generatedout of the refinement protocol was finally inspected in terms of interatomic interactions on the protein interaction calculator webserver(http: / / pic.mbu.iisc.ernet.in / ). Similarly docking was also performed with the mutantstructures (polypeptides isomer B (SEQ ID NO: 6) and 2 (SEQ ID NO: 2)) of thewell-defined N-terminal fragment as the ligand and the above-described FXIIIa structure as the receptor. Functional studies of tridegin analogsAs earlier reported, polypeptides 3 (SEQ ID NO: 7) and 5 (SEQ ID NO: 4) wereused as controls and for reasons of comparison. In previous studies, inhibition ofthe target protein FXIIIa by tridegin (SEQ ID NO: 14) was extensively investigatedby a fluorogenic enzyme activity assay: This assay is based on the isopeptidase activity of FXIIIa and releases a fluorophoreupon isopeptide bond cleavage within the substrate used. The cleavage indicatesfull functional enzyme activity, which is changed in the presence of activity-modulating compounds and therefore can be determined accordingly.The impact of the peptides investigated, herein on FXIIIa activity, was monitoredusing this fluorometric assay and the substrate H–Tyr(3-NO2)–Glu(Dab-2-Abz)–Val–Lys–Val–Ile–NH2. Linear progress curves for the above-mentionedpolypeptides were recorded and indicated stable inhibition of FXIIIa as describedearlier. The Cys19–Cys25-deficient tridegin derivatives (e.g., polypeptide isomer B(SEQ ID NO: 6)) displayed significant inhibition of FXIIIa with an IC50 value of 1.02± 0.06 µM (n = 3) in a comparable way as was already described (Figs.3A, 3B).Interestingly, the polypeptides 3 (SEQ ID NO: 7), 5 (SEQ ID NO: 4), 7 (SEQ ID NO:8), and 10 (SEQ ID NO: 11), all of them lacking cysteines, revealed slightly reducedIC50 values of 1.51 ± 0.11 µM (n = 3), 1.53 ± 0.11 µM (n = 3), 1.66 ± 0.09 µM (n =3), and 1.76 ± 0.10 µM (n = 3), respectively. Linear polypeptides 8 (SEQ ID NO: 9,2.06 ± 0.09 µM, n = 3) and 9 (SEQ ID NO: 10, 1.33 ± 0.08 µM, n = 3) with one D-amino acid provided results in a similar range. Polypeptide 2 (SEQ ID NO: 2)containing even four D-amino acids also maintained a significant inhibitory potentialtowards FXIIIa with an IC50of 1.93 ± 0.11 µM (n = 3). Although several amino acidshave been exchanged in polypeptides 3 (SEQ ID NO: 7) and 5 (SEQ ID NO: 4),there was still some inhibitory potency maintained for both peptides (4.76 ± 0.37 µM and 5.02 ± 0.34 µM, respectively). Thus, all peptides investigated in this study, some representing proteolytically stabilized tridegin derivatives, were still potential reversibly binding inhibitors and none lost its activity completely despite the extensive number of amino acid changes or modifications that had been introduced in the sequence. For validation of the results of the fluorogenic assay, the Berichrom®assay for determining the FXIIIa activity was applied for the most potent polypeptides isomerB (SEQ ID NO: 6), 2 (SEQ ID NO: 2), and 3 (SEQ ID NO: 7) (IC50 < 2 µM). Thepolypeptides 5-10 (SEQ ID NOs: 4 and 8-11) were excluded due to the closeprinciple of both assays (i.e. measurement of factor XIIIa activity and its inhibition)and its foreseeable effect. Due to the same basis of both assay systems (i.e.measurement of activity of factor XIIIa and its inhibition) no different result isexpected to be determined. In this assay, ammonia is released from a reaction inwhich FXIIIa links a peptide substrate to a glycine ethyl ester in normal pooled plasma (NPP). Ammonia is converted into glutamate by glutamate dehydrogenase upon consumption of NADH that results in a reduction in absorbance. The decrease observed for untreated NPP was measured relative to NPP treated with different inhibitor concentrations and the effect of the tridegin analogs was normalized to theeffect of lead polypeptide isomer B (SEQ ID NO: 6) as described earlier (Fig. 2C).Polypeptides 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 7) showed a lower level ofinhibition (81.2 ± 0.3%, n = 4 and 51.4 ± 0.2%, n = 4, respectively) than polypeptideisomer B (SEQ ID NO: 6), which is in agreement with the activity reduction that wasobserved in the fluorogenic enzyme assay. However, only approx. 20% FXIIIainhibition was achieved at a concentration of 1 µM for polypeptide isomer B (SEQID NO: 6) in NPP (Fig. 2D). For complete inactivation higher compound concentrations were required (>100 µM). A continuous reduction was observed at increasing inhibitor concentrations (1-100 µM) for all three compounds investigatedwhile ratios between polypeptides isomer B (SEQ ID NO: 6), 2 (SEQ ID NO: 2), and3 (SEQ ID NO: 7) remained unaffected (Fig. 2D). A recombinant fusion proteinconsisting of a His-tag, streptokinase from Streptococcus pyrogenes, a thrombin- cleavable linker, and tridegin (SEQ ID NO: 14) was included in the testing as well. Cell pellets that contained the protein were analyzed and exhibited a clear inhibitoryeffect on FXIIIa activity (Fig. 5C).Additionally, inhibition of FXIIIa was determined in human and mouse whole blood by measuring the clot size in a whole blood contraction assay as performed earlier. Red blood cell retention (promoted by FXIIIa) is inhibited by polypeptides isomer B(SEQ ID NO: 6) and 3 (SEQ ID NO: 7) in equipotent amounts in human blood withan IC50between 1-5 µM and, therefore, whole blood clot size and weight are reduced (Fig.2E). At the same time a slightly more potent inhibition of the clot weightformation was observed when polypeptide isomer B (SEQ ID NO: 6) and 3 (SEQ IDNO: 7) were added to whole blood from mice (IC50: 2.2 ± 0.8 µM with n = 3 and 2µM with n = 1, respectively). Thus, the effects observed for the linear polypeptide 3(SEQ ID NO: 7) and oxidized tridegin with two disulfide bridges (Cys5–Cys37, Cys17–Cys31) of isomer B (SEQ ID NO: 6) are in line with the aforementioned findings ofthe fluorogenic assay. The slight drop in activity of the linear polypeptides may not be reflected to the same extent by the whole blood assays as compared to theisolated in vitro FXIIIa assays. This is probably due to the more complexenvironment that may compensate these slight differences. In order to figure out which impact the environment in the blood may have on the inhibitory effect, i.e., how pharmacokinetic parameters such as plasma protein binding or metabolization by liver microsomes may be affected, ADME-T studies have been conducted. Pharmacokinetics of tridegin derivativesThe above-referenced six polypeptides were subjected to pharmacokinetic studiesto validate their early-stage potential as future drugs as follows in Table 2.Table 2. In vitro analysis for ADME-T properties of polypeptides isomer B (SEQ IDNO: 6) and 3 (SEQ ID NO: 7) are shown as representatives for all peptidesinvestigated in this study. Poly Solubility Log Plasma Plasma Hepatic Cytotoxi pepti (mg / ml)aD7.4stability (3 h) protein microsome city de binding (%) stability (1 h) (LC50, µM)b1> 100 << 0 stable > 99 stable > 1003 > 100 << 0 stable > 99 stable > 100ameasured in 0.1 M phosphate buffer, pH7.4.btested in an MTT-based assay on HepG2 cells.Accordingly, proper assessment of parameters such as aqueous solubility,hydrophobicity, stability, and toxicity are essential key criteria. All polypeptides showed solubility in aqueous media (phosphate buffers at pH 7.4, 6.2 and 5.0) above 100 µM combined with a high hydrophilicity (Log D7.4<< 0). Stability upon application and incubation in plasma or with hepatic microsomes wassuitable, i.e., no degradation or modification was observed in the studied time range.Also, no compound showed cytotoxic effects towards HepG2 cells below 100 µM (LC50> 100 µM), whereas we observed plasma protein binding to be > 99 %(Table 2).Behavior of all analogs of tridegin was comparable in our pharmacokinetic studiesand for representation polypeptides isomer B (SEQ ID NO: 6) and 3 (SEQ ID NO:7) have been shown in detail in Table 2.In addition to the aforementioned in vitro pharmacokinetics, we also conducted invivo stability studies for polypeptide isomer B (SEQ ID NO: 6) in mice. After retro-orbital injection of the compound (20 µM) blood of the treated mice was drawn from the inferior vena cava at defined times and clotting properties were evaluated bymeasuring the clot mass that was formed in the whole blood contraction assay.While up to 2 min a clear inhibition of FXIIIa could be detected, at 15 min no furthereffect on the clot mass was observed (Figure 3). Consequently, these results alignwith the observation of high plasma protein binding that most likely leads to fastelimination of the polypeptide from the mice.

Claims

Rheinische Friedrich-Wilhelms-Universität Bonn 23 July 2025P74921EP JA / ZIT / staClaims1. A polypeptide comprising a polypeptide strand of SEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E, differing in at least 6 positions from the sequence of natural tridegin(SEQ ID NO: 14),wherein amino acid moieties X19 and X25 are each independently selectedfrom the group consisting of Ala and Cys(Me); wherein amino acid moieties X1, X6, X16, X38, X40, X45, X53, X63, and X65are each independently selected from the group consisting of Lys, D-Lys, Arg,D-Arg, Orn, and Cit;wherein amino acid moieties X5, X17, X31, and X37are each independently selected from the group consisting of Ser, Ala, Cys(Me), an amino acidmoiety forming a lactam bond with another one of the amino acid moieties selected from the group consisting of X5, X17, X31, and X37, and Cys optionally forming a disulfide bond with another Cys at an amino acid moiety selected from the group consisting of X5, X17, X31, and X37;wherein not more than two of X5, X17X31, and X37 are Cys,wherein optionally one or more of the L-amino acid moieties may be replacedby the respective one or more D-amino acids, or a retro-inverso analogue of the sequence thereof,or a peptidomimetic analogue of the sequence thereof, or a pharmaceutical salt thereof.

2. The polypeptide of claim 1, wherein at least two of X5, X17, X31, and X37 areeach independently selected from the group consisting of Cys(Me), Ser andAla, preferably wherein all of X5, X17, X31, and X37are each independently selected from the group consisting of Cys(Me), Ser and Ala.

3. The polypeptide of any one of claims 1 or 2, wherein the N-terminus ofpolypeptide strand is:(i) acetylated;(ii) an unbound primary amino group;(iii) conjugated to a peptidic moiety, a non-peptidic moiety or a moietycomprising peptidic and non-peptidic structure elements, of a total molecular weight of not more than 500 Da,(iv) conjugated to a peptidic polymeric structure, a non-peptidic polymericstructure or a polymeric structure comprising peptidic and non-peptidic structure elements, of a total molecular weight of more than 500 Da,preferably of 500 to 100,000 Da;(v) conjugated to solid support, optionally via a peptidic linker structure, anon-peptidic linker structure or a linker structure comprising peptidic and non-peptidic structure elements, preferably wherein the total linker structure has a molecular weight of 100 to 10,000 Da; or(vi) conjugated to a fibrinolytic enzyme, preferably streptokinase,nattokinase, or tissue plasminogen activators (tPA), via a flexible peptide linker for constitution of a fusion protein.

4. The polypeptide of any one of claims 1 to 3, wherein the C-terminus ofpolypeptide strand is: (i) amidated,(ii) an unbound carboxylic group;(iii) conjugated to a peptidic moiety, a non-peptidic moiety or a moietycomprising peptidic and non-peptidic structure elements, of a total molecular weight of not more than 500 Da;(iv) conjugated to a peptidic polymeric structure, a non-peptidic polymericstructure or a polymeric structure comprising peptidic and non-peptidic structure elements, of a total molecular weight of more than 500 Da,preferably of 500 to 100,000 Da; or(v) conjugated to solid support, optionally via a peptidic linker structure, anon-peptidic linker structure or a linker structure comprising peptidic and non-peptidic structure elements, preferably wherein the total linker structure has a molecular weight of 100 to 10,000 Da(vi) conjugated to a fibrinolytic enzyme, preferably streptokinase,nattokinase, or tissue plasminogen activators (tPA), via a flexible peptide linker for constitution of a fusion protein.

5. The polypeptide of any one of claims 1 to 4, wherein amino acid moieties X5,X17, X31, and X37are each not Cys.

6. The polypeptide of claim 5, wherein a lactam bond is formed between aminoacid moieties X5 and X37 and / or a lactam bond is formed between amino acidmoieties X17 and X31, or between amino acid moieties X5 and X17 and betweenamino acid moieties X31 and X37, or between amino acid moieties X5 and X31and between amino acid moieties X17 and X37.

7. The polypeptide of any one of claims 1 to 6, wherein at least one of themoieties selected from the group consisting of Lys6, Ile12, Leu23or Ile34is replaced by the respective D-amino acid residue, in particular wherein the moieties selected from the group consisting of Lys6, Ile12, Leu23or Ile34are each replaced by the respective D-amino acid residues.

8. The polypeptide of any one of claims 1 to 7, wherein one or more amino acidmoiety positions X1, X6, X45and X63are each independently from each other selected from the group consisting of D-Lys and Orn and / or one or more amino acid moiety positions X16, X38, X40, X53, X65are each independently from each other selected from the group consisting of D-Arg and Cit,in particular wherein amino acid moiety positions X1, X6, X45and X63are each independently from each other selected from the group consisting of D-Lys and Orn and amino acid moiety positions X16, X38, X40, X53, X65 are eachindependently from each other selected from the group consisting of D-Arg and Cit.

9. The polypeptide of any one of claims 1 to 8, wherein the polypeptidecomprises (or consists of) a polypeptide strand selected from the group consisting of KLLPAKEWHQGIPNPRAWAGADLEAAQDQYAAFIPQARPRSELIKPMDDI YQRPVEFPNLPLKPRE (SEQ ID NO: 4), andX1LLPAX6EWHQGIPNPX16AWAGADLEAAQDQYAAFIPQAX38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E (SEQ ID NO: 5),wherein X1, X6, X45and X63are each Orn; and wherein X16, X38, X40, X53, X65are each Cit,wherein optionally one or more of the L-amino acid moieties may be replacedby the respective one or more D-amino acids, or a retro-inverso analogue of any one of the sequences, or a peptidomimetic analogue of any one of the sequences,or a pharmaceutically acceptable salt of any one of the sequences.

10. A polypeptide comprising a polypeptide strand of SEQ ID NO: 1:X1LLPX5X6EWHQGIPNPX16X17WX19GADLEX25AQDQYX31AFIPQX37X38P X40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E, wherein amino acid moieties X19 and X25 are each independently selectedfrom the group consisting of Ser, Ala and Cys(Me); wherein amino acid moieties X1, X6, X16, X38, X40, X45, X53, X63, and X65are each independently selected from the group consisting of Lys, D-Lys, Arg, D- Arg, Orn, and Cit;wherein amino acid moieties X5, X17, X31, and X37are each independently selected from the group consisting of Ser, Ala, Cys(Me), an amino acidmoiety forming a lactam bond with another one of the amino acid moieties selected from the group consisting of X5, X17, X31, and X37, and Cys optionallyforming a disulfide bond with another Cys at an amino acid moiety selected from the group consisting of X5, X17, X31, and X37;wherein optionally one or more of the L-amino acid moieties may be replacedby the respective one or more D-amino acids, or a retro-inverso analogue of the sequence thereof, or a peptidomimetic analogue of the sequence thereof, or a pharmaceutical salt thereof.

11. The polypeptide of claim 10 comprising a polypeptide strand ofSEQ ID NO: 1,differing in at least 6 positions from the sequence of natural tridegin (SEQ ID NO: 14).

12. The polypeptide of claim 10 or 11 comprising a polypeptide strand ofSEQ ID NO: 1:wherein amino acid moieties X1, X6, X45and X63are each independently selected from the group consisting of Lys, D-Lys, and Orn;wherein amino acid moieties X16, X38, X40, X53, X65are each independently selected from the group consisting of Arg, D-Arg, and Cit; wherein optionally one or more of the L-amino acid moieties may be replaced by the respective one or more D-amino acids.

13. The polypeptide of any of claims 10 to 12, wherein not more than two of X5,X17, X31, and X37are Cys.

14. The polypeptide of any one of claims 10 to 12, wherein the polypeptidecomprises (or consists of) a polypeptide strand selected from the group consisting of KLLPCkEWHQGiPNPRCWSGADlESAQDQYCAFiPQCRPRSELIKPMDDIY QRPVEFPNLPLKPRE (SEQ ID NO: 2), wherein k6 is a D-lysyl moiety, l23 isa D-leucine and i12 and i34 are a D-isoleucine moiety, andX1LLPSX6EWHQGIPNPX16SWSGADLESAQDQYSAFIPQSX38PX40SELIX45PMDDIYQX53PVEFPNLPLX63PX65E (SEQ ID NO: 3),wherein X1, X6, X45and X63are each Orn; and wherein X16, X38, X40, X53, X65are each Cit, wherein optionally one or more of the L-amino acid moieties may be replacedby the respective one or more D-amino acids, or a retro-inverso analogue of any one of the sequences, or a peptidomimetic analogue of any one of the sequences, or a pharmaceutically acceptable salt of any one of the sequences.

15. The polypeptide of any one of claims 1 to 14, wherein the polypeptide doesnot comprise a disulfide bond, in particular does not comprise an internal bridge or comprises one, two or three lactam bonds between one or moreAsp residues and an equivalent number of Lys, Orn or Dap moieties.

16. A pharmaceutical composition comprising:(A) the polypeptide of any one of claims 1 to 15; and(B) at least one pharmaceutically acceptable carrier.

17. The polypeptide of any one of claims 1 to 15 or a pharmaceutical compositionof claim 16 for use in a method for treating or preventing a thrombotic eventin a patient.

18. The polypeptide for use of claim 17, wherein the patient is:(a) at risk of developing or is suffering from a pathological state associatedwith a thrombotic event selected from the group consisting of stenosis of one or more veins, venules, arteria, arterioles, and / or capillaries, (b) at risk of developing or is suffering from a pathological state associatedwith a thrombotic event selected from the group consisting of stroke, infarction, embolism, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), thrombophlebitis, sepsis, or a combination of two or more thereof; and / or(c) subjected to a surgery, in particular is receiving an endoprosthesisand / or is receiving an organ transplant and / or a tissue transplant.

19. A recombinant fusion protein comprising:(a) the polypeptide of any one of claims 1 to 15;(b) a fibrinolytic enzyme, preferably streptokinase, nattokinase or tPAs;and (c) a thrombin-cleavable flexible linker connecting the polypeptide of (a)and the fibrinolytic enzyme of (b)20. The recombinant fusion protein of claim 19, wherein the thrombin-cleavablelinker comprises the sequence VSQTSKLTRAETVFPDV (SEQ ID No: 12).

21. The recombinant fusion protein of claim 19 or 20 for use in a method fortreating or preventing a thrombotic condition in a patient comprising administering the recombinant fusion protein, whereby the tridegin domain inhibits FXIIIa and the fibrinolytic enzyme is released locally upon thrombin cleavage to promote targeted fibrinolysis.

22. An endoprosthesis or an enzyme-linked immunosorbent assay (ELISA) platecoated with the polypeptide of any one of claims 1 to 15.

23. The endoprosthesis of claim 22, wherein the endoprosthesis is selected fromthe group consisting of a blood vessel endoprosthesis, an artificial joint, a cardiac pacemaker, an artificial bowel outlet, and a cosmetic implant.

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