Improved, highly potent and specific human Kunitz inhibitors of the fibrinolytic enzyme plasmin.

Abstract

Disclosed are compositions and methods relating to novel plasmin inhibitory polypeptides that are structural variants of the human TFPI-2 Kunitz-type proteinase first inhibitor domain (KD1). The polypeptides are potent plasmin inhibitors, have antifibrinolytic activity, and / or have reduced anticoagulant activity relative to wild-type TFPI-2 KD1. The plasmin inhibitory polypeptides are useful as anticancer agents, antifibrinolytic agents, protease inhibitors, and in other contexts.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of co-pending U.S. Provisional Patent Application No. 63 / 076,516, filed September 10, 2020, and co-pending U.S. Provisional Patent Application No. 63 / 112,840, filed November 12, 2020, which applications are incorporated by reference herein in their entireties.

[0002] STATEMENT AS TO RIGHTS TO INVETIONS MADE UNDER GOVERNMENT SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant No. HL141850 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Technical Field The field of the invention relates to polypeptide agents that can inhibit the action of plasmin. [Background technology]

[0004] 2. Background of the Invention Fibrinolysis is a physiological process that regulates the extent of clot formation and its excessive growth under normal physiological circumstances. However, after trauma or surgery, ischemia and reperfusion, blood comes into contact with large non-endothelial surfaces (e.g., cardiopulmonary bypass (CPB) circuits) resulting in excessive fibrinolysis. This excessive fibrinolysis contributes to coagulopathy, bleeding, and inflammatory responses. Under these circumstances, antifibrinolytic agents that inhibit plasmin, a key mediator of this process, are used by medical professionals to reduce bleeding, allogeneic blood administration, and adverse clinical outcomes.

[0005] The antifibrinolytic agents used are aprotinin, tranexamic acid (TXA) and epsilon-aminocaproic acid (EACA). Aprotinin was removed from the market in 2008 due to its adverse effects in patients, mainly due to renal dysfunction and anaphylaxis (1). Significantly, aprotinin has been reintroduced with restricted use in Europe and Canada because of its risk-benefit profile (2). However, the ban on aprotinin has not been lifted in the United States. The currently used lysine analogs, TXA and EACA, are not as effective as aprotinin and also cause seizures and renal dysfunction (3,4). A recent paper from the Montreal Cardiac Surgery Group (5) showed that transfusions of more than 4 units of red blood cells occurred in nearly one-quarter of all patients undergoing cardiac surgery at their institution between 2012 and 2015, despite nearly 100% use of TXA. From this, the authors concluded that there remains a need for efficient blood-sparing agents. The desired products must demonstrate a protease inhibition profile with respect to fibrinolysis and inflammation that has the best balance of benefit to risk (6).

[0006] Recently, two products that inhibit plasmin, namely, Ecallantide and MDCO-2010, have completed cardiac bypass surgery trials up to Phase II clinical trials. However, each product failed to provide a satisfactory efficacy and safety profile in Phase III cardiac bypass surgery trials, and both trials were terminated prematurely (7,8). This may be related to the fact that Ecallantide and MDCO-2010 are potent inhibitors of kallikrein. In addition, MDCO-2010 also inhibits the coagulation protease factor Xa. Another product, Textilinin-1, a snake venom-derived Kunitz-type serine protease inhibitor, is in early development (9). In this case, the Kunitz domain is derived from snake venom, as compared to the aprotinin Kunitz domain, which is derived from bovine lung. Thus, similar to aprotinin, the snake venom Kunitz domain is predicted to produce an anaphylactic response in humans. The problems with aprotinin and the ongoing efforts of those skilled in the art to identify antifibrinolytic agents more suitable for in vivo use indicate that this recognized problem has existed in the art for a long time without resolution and that this need is a persistent one recognized by those skilled in the art.

[0007] One group of agents that have been explored for use as antifibrinolytic agents include variants of human Kunitz-type inhibitor polypeptides (see, e.g., U.S. Patent No. 8,993,719 and U.S. Patent Publication 20080026998). However, because these human Kunitz-type inhibitor polypeptides can interact with a variety of polypeptides in vivo, including plasmin and serine proteases (e.g., kallikrein), the complex pharmacokinetics of such polypeptides poses challenges in finding human Kunitz-type inhibitor variant polypeptides that have a constellation of amino acid residues that provide such variant polypeptides with optimized functional properties for use as therapeutic agents in vivo. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Pat. No. 8,993,719 [Patent Document 2] US Patent Application Publication No. 2008 / 0026998 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention As disclosed herein, new non-naturally occurring polypeptide variants of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) have been created and discovered to have highly desirable pharmacokinetic profiles. For example, the disclosed polypeptides have pharmacokinetic profiles that include the ability to inhibit the activity of plasmin better than the previously utilized but problematic antifibrinolytic agent aprotinin. Furthermore, in addition to the plasmin inhibitory activity of the new polypeptide variants being superior to that observed with aprotinin, the polypeptide variants disclosed herein further avoid certain adverse side effects observed with aprotinin and related molecules. In one illustration of this, the polypeptide variants disclosed herein are observed to exhibit minimal inhibitory activity against other coagulation serine proteases (e.g., kallikrein).

[0010] The 60-residue polypeptide variant disclosed herein comprises a unique grouping of amino acid residues that includes a C-terminal structure that includes a lysine residue. Without being bound by any particular theory or mechanism of action, this C-terminal structure appears to facilitate binding of the 60-residue polypeptide variant to plasmin or plasminogen via its kringle domain in a manner that inhibits plasminogen binding to fibrin clots. The polypeptide variant disclosed herein also comprises three amino acid mutations, including a lysine amino acid substitution at position 15 ("KD1"). Y11T / R15K / L17R -K T "). Surprisingly, the Y11T / R15K / L17R triple mutant is observed to be 4-5 times more potent in inhibiting plasmin when compared to a 60 residue polypeptide variant having only the double mutation Y11T / L17R. Without being bound by a particular theory or mechanism of action, the triple mutant having a lysine amino acid substitution at position 15 appears to function by promoting the interaction of the variant polypeptide with residues Asp189 and Ser190 in plasmin. Unexpectedly, the 60 residue Y11T / R15K / L17R triple mutant polypeptide further exhibits at least 10 times weaker inhibition of kallikrein, factor XIa and factor VIIa / tissue factor when compared to the corresponding 60 residue polypeptide variant having only the double mutation Y11T / L17R. Thus, the 60-residue variant polypeptides disclosed herein exhibit highly desirable pharmacokinetic / material profiles, including, for example, the ability to potently inhibit plasmin while simultaneously avoiding certain side effects associated with similar inhibitory molecules in the art.

[0011] The invention disclosed herein has many embodiments. The embodiments of the present invention include, for example, a polypeptide comprising the sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCE GNANNFYTWEACDDACWRIEK (SEQ ID NO: 1) and / or a composition comprising an isolated non-naturally occurring polypeptide (SEQ ID NO: 1). Typically, such compositions also include additional agents, such as pharma- ceutically acceptable carriers (e.g., preservatives, tonicity adjusters, detergents, hydrogels, viscosity adjusters, pH adjusters, etc.). Such embodiments include, for example, pharmaceutical compositions comprising pharma-ceutically acceptable excipients selected for use in intravenous injection or infusion.

[0012] Another embodiment of the present invention is a composition comprising a polynucleotide encoding the polypeptide sequence: NAEICLLPLDTGPCKARLLR YYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1). In a working embodiment of the invention disclosed herein, the polynucleotide comprises the sequence: AACGCGGAGATCTGTCTGCCCCTAGACACCGGACCCTGCAAAGCCAGACTTCTCCGTTACTACTACGACAGGTACACGCAGAGCTGCCGCCAGTTCCTGTACGGGGGCTGCGAGGGCAACGCCAACAATTTCTACACCTGGGAGGCTTGCGACGATGCTTGCTGGAGGATAGAAAA (SEQ ID NO: 2). Those skilled in the art will understand that this specific sequence includes the codons used to generate the Kunitz domain 1 inhibitor polypeptides disclosed herein in humans, but that the polynucleotide sequences encoding these polypeptides may vary depending on the system used to express the polypeptide (i.e., different codons may be used in bacteria, yeast, and insect cells). Typically, such polynucleotides are placed in a vector that contains one or more regulatory sequences for expressing the polypeptide in a cell. Embodiments of the invention also include cells (e.g., bacterial, yeast, insect or mammalian cells) that contain such vectors.

[0013] As discussed below, embodiments of the present invention also include methods of using the polypeptides disclosed herein. Such embodiments of the present invention include, for example, a method of inhibiting at least one activity of plasmin, the method comprising contacting plasmin with an effective amount of a polypeptide disclosed herein. Related embodiments of the present invention include a method of inhibiting fibrinolysis in a patient, the method comprising administering to the patient an amount of a polypeptide disclosed herein sufficient to inhibit fibrinolysis, such that fibrinolysis is inhibited. Other illustrative embodiments of the present invention include a method for treating a subject in need of inhibition of plasmin activity, the method comprising administering to the subject an effective amount of a polypeptide disclosed herein. Other illustrative embodiments of the present invention include a method for treating a subject in need of surgery, the method comprising administering to the subject an effective amount of a polypeptide disclosed herein before, during, and / or after surgery. Other illustrative embodiments of the present invention include a method for treating a subject suffering from cancer or a precancerous condition, the method comprising administering to the subject an effective amount of a polypeptide disclosed herein. Other illustrative embodiments of the present invention include a method of treating a subject for a condition treatable by aprotinin, the method comprising administering to the subject an effective amount of a polypeptide disclosed herein.

[0014] One illustrative embodiment of the present invention is a method for inhibiting bleeding in a subject, comprising administering to the subject an effective amount of a polypeptide disclosed herein. In certain embodiments of the present invention, the bleeding results from surgery (e.g., heart surgery, or organ transplant surgery such as liver transplant) or traumatic injury (e.g., traumatic brain injury, gunshot wound, accident, etc.). Some embodiments of the present invention include a patch or dressing or the like in which a polypeptide as set forth in SEQ ID NO:1 is disposed. In this context, another embodiment of the present invention is a method for inhibiting at least one activity of plasmin in a subject by contacting a bleeding tissue in the subject with such a patch or the like in which a polypeptide as set forth in SEQ ID NO:1 is disposed so that these polypeptides can inhibit at least one activity of plasmin in the subject.

[0015] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain embodiments of the present invention, are given by way of illustration and not by way of limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. [Brief description of the drawings]

[0016] [Figure 1] Figure 1. Data from SDS-PAGE gel electrophoresis of purified KD1Y11T / R15K / L17R-KT. Lane 1, protein marker; lane 2, reduced KD1Y11T / R15K / L17R-KT; lane 3, non-reduced KD1Y11T / R15K / L17R-KT.

[0017] [Diagram 2] Figure 2. Data from the equilibrium inhibition constants (Ki) of KD1Y11T / R15KL17R-KT with plasmin, FXIa, kallikrein (KLK), FVIIa / sTF and FXa. [ka] Abbreviations: Pm, plasmin; BPTI, bovine pancreatic trypsin inhibitor (aprotinin, trasylol); KD1TM, KD1 Y11T / R15KL17R -K T ;KLK, kallikrein; FXIa, factor XIa, FVIIa / sTF, factor VIIa / soluble tissue factor; FXa, factor Xa.

[0018] [Diagram 3] 3A and 3B. Data examining the effect of KD1Y11T / R1 5KL17R-KT (A) or BPTI (B) on fibrinolysis in human plasma. Thrombin (IIa) was added to human plasma to initiate clot formation, which is associated with an increase in OD405 (black; open circles; IIa, zero tPA). Addition of 3 μM KD1Y11T / R1 5KL17R-KT or BPTI does not affect clot formation (brown curve; IIa, 3 μM KD1TM (panel A) or BPTI (panel B)). Addition of tPA converts plasminogen to plasmin, which completely dissolves the fibrin clot within approximately 12 minutes, as indicated by an initial increase, followed by a decrease, in OD405 (black; filled circles; IIa, tPA). Addition of KD1Y11T / R15K / L17R-KT or BPTI inhibits fibrinolysis in a dose-dependent manner - KD1Y11T / R15K / L17R-KT (A) or BPTI (B) at the following concentrations: 0.5 μM (blue), 1 μM (red), 2 μM (green), 3 μM (magenta). Abbreviations: tPA, tissue plasminogen activator; KD1TM, KD1Y11T / R15K / L17R-KT; BPTI, bovine pancreatic trypsin inhibitor (aprotinin / trasylol).

[0019] [Figure 4]Figure 4. Data from HUVEC viability studies. HUVECs were either untreated or treated with the antifibrinolytic agents: KD1TM, BPTI, EACA, or TXA at ​​the indicated concentrations for 24 hours. Cells were incubated with resazurin, a cell-permeable, non-toxic, and weakly fluorescent blue indicator dye (alamarBlue viability assay). Fluorescence intensity is proportional to relative cell number. Error bars represent standard error of the mean (SEM). The viability of cells treated with each antifibrinolytic agent was found to be not significantly different from untreated cells (p>0.05). Abbreviations: KD1TM, KD1Y11T / R15K / L17R-KT; BPTI, bovine pancreatic trypsin inhibitor; EACA, epsilon aminocaproic acid; TXA, tranexamic acid.

[0020] [Diagram 5] Figure 5. Data from fibroblast viability studies. Fibroblasts were either untreated or treated with the following antifibrinolytic agents at the indicated concentrations for 24 hours: KD1TM, BPTI, EACA, or TXA. Cells were incubated with resazurin, a cell-permeable, non-toxic, weakly fluorescent blue indicator dye (alamarBlue viability assay). Fluorescence intensity is proportional to relative cell number. Error bars represent standard error of the mean (SEM). The viability of cells treated with each antifibrinolytic agent was found to be not significantly different from untreated cells (p>0.05). Abbreviations: same as Figure 4 above.

[0021] [Figure 6A]Figures 6A and 6B. Data from apoptosis studies of HUVEC (A) and human fibroblasts (B). HUVEC or fibroblasts were either untreated or treated with the following antifibrinolytic agents for 24 hours: 30 μM KD1TM, 30 μM BPTI, 60 mM EACA, 30 mM TXA or 0.05 μM taxol (positive control). Luminescence (shown as relative light units (RLU)) is proportional to caspase-3 / 7 activity. Error bars represent standard error of the mean (SEM). Abbreviations: KD1TM, KD1Y11T / R15K / L17R-KT; BPTI, bovine pancreatic trypsin inhibitor, EACA, epsilon aminocaproic acid, TXA, tranexamic acid; HUVEC, human umbilical vein endothelial cells. [Figure 6B] Figures 6A and 6B. Data from apoptosis studies of HUVEC (A) and human fibroblasts (B). HUVEC or fibroblasts were either untreated or treated with the following antifibrinolytic agents for 24 hours: 30 μM KD1TM, 30 μM BPTI, 60 mM EACA, 30 mM TXA or 0.05 μM taxol (positive control). Luminescence (shown as relative light units (RLU)) is proportional to caspase-3 / 7 activity. Error bars represent standard error of the mean (SEM). Abbreviations: KD1TM, KD1Y11T / R15K / L17R-KT; BPTI, bovine pancreatic trypsin inhibitor, EACA, epsilon aminocaproic acid, TXA, tranexamic acid; HUVEC, human umbilical vein endothelial cells.

[0022] [Figure 7]Figure 7. Data from HUVEC cytotoxicity studies. HUVEC were either untreated or treated for 24 hours with the following antifibrinolytic agents: 30 μM KD1™, 30 μM BPTI, 60 mM EACA, 30 mM TXA or 0.05 μM Taxol as a positive control (top panel). FITC: CellTox green dye binds to DNA when membrane integrity is compromised. Fluorescence signal (quantified in bar graph, bottom panel) is proportional to cytotoxicity. DAPI: nuclear stain binds to all nuclei. Abbreviations: FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cells; KD1TM, KD1Y11T / R15K / L17R-KT; BPTI, bovine pancreatic trypsin inhibitor, EACA, epsilon aminocaproic acid, TXA, tranexamic acid.

[0023] [Figure 8] FIG. 8. Data from fibroblast cytotoxicity studies. Fibroblasts were either untreated or treated with the following antifibrinolytic agents: 30 μM KD1™, 30 μM BPTI, 60 mM EACA, 30 mM TXA or 0.05 μM taxol as a positive control and lysis buffer for 24 h (upper panel). FITC: CellTox green dye binds to DNA when membrane integrity is compromised. Fluorescence signal (quantified in bar graph, lower panel) is proportional to cytotoxicity. DAPI: nuclear stain, binds to all nuclei. Abbreviations: FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; KD1™, KD1Y11T / R15K / L17R-KT; BPTI, bovine pancreatic trypsin inhibitor; EACA, epsilon aminocaproic acid; TXA, tranexamic acid.

[0024] [Figure 9A]9A-9B. Modeled complexes of Kunitz domain 1 (KD1) of human tissue factor pathway inhibitor. FIG. 9A shows a modeled complex of KD1Y11T / R15K / L17R-KT interaction with plasmin. (A) Modeled interaction of KD1Y11T / R15K / L17R-KT with the plasmin protease domain. Shown are the electrostatic surface of the plasmin protease domain and a cartoon representation of KD1Y11T / R15K / L17R-KT (light green). The P1 (Lys15), P5 (Thr11) and P2' (Arg17) residues of the KD1Y11T / R15K / L17R-KT interaction with plasmin are shown in stick representation. For the electrostatic surface, blue represents positive charge, red represents negative charge and white represents neutral charge. (B) Modeled interactions between KD1Y11T / R15K / L17R-KT and the plasmin kringle domain. The electrostatic surface of plasminogen kringle domain 1 and a cartoon representation of KD1Y11T / R15K / L17R-KT (light green) are shown. Residues that form hydrogen bonds and salt bridges (shown as dashed lines) between the kringle domain and KD1Y11T / R15K / L17R-KT are shown in stick representation. Carbon atoms are shown in green for the kringle domain and light green for KD1Y11T / R15K / L17R-KT. As in (A), oxygen atoms are shown in red and nitrogen atoms are shown in blue. KD1Y11T / R15K / L17R-KT residues are shown with suffix I. For the electrostatic surface, blue represents positive charge, red represents negative charge, and white represents neutral charge. Figure 9B shows a modeled complex of KD1-Y11T / L17R-KT interaction with plasmin. (A) Modeled interaction of KD1-Y11T / L17R-KT with the plasmin protease domain. Shown is the electrostatic surface of the plasmin protease domain and a cartoon representation of KD1-Y11T / L17R-KT (yellow). The P1 (Arg15), P5 (Thr11), and P2' (Arg17) residues of the KD1-Y11T / L17R-KT interaction with plasmin are shown in stick representation. For the electrostatic surface, blue represents positive charge, red represents negative charge, and white represents neutral charge. (B) Modeled interaction of KD1-Y11T / L17R-KT with the plasmin kringle domain.The electrostatic surface of plasminogen Kringle domain 1 and a cartoon representation of KD1-Y11T / L17R-KT (yellow) are shown. Residues that form hydrogen bonds and salt bridges (shown as dashed lines) between the Kringle domain and KD1-Y11T / L17R-KT are shown in stick representation. Carbon atoms are shown in green for the Kringle domain and in yellow for KD1-Y11T / L17R-KT. As in (A), oxygen atoms are shown in red and nitrogen atoms are shown in blue. The KD1-Y11T / L17R-KT residues are shown with the suffix I. In the electrostatic surface, blue represents a positive charge, red represents a negative charge, and white represents a neutral charge. [Figure 9B]9A-9B. Modeled complexes of Kunitz domain 1 (KD1) of human tissue factor pathway inhibitor. FIG. 9A shows a modeled complex of KD1Y11T / R15K / L17R-KT interaction with plasmin. (A) Modeled interaction of KD1Y11T / R15K / L17R-KT with the plasmin protease domain. Shown are the electrostatic surface of the plasmin protease domain and a cartoon representation of KD1Y11T / R15K / L17R-KT (light green). The P1 (Lys15), P5 (Thr11) and P2' (Arg17) residues of the KD1Y11T / R15K / L17R-KT interaction with plasmin are shown in stick representation. For the electrostatic surface, blue represents positive charge, red represents negative charge and white represents neutral charge. (B) Modeled interactions between KD1Y11T / R15K / L17R-KT and the plasmin kringle domain. The electrostatic surface of plasminogen kringle domain 1 and a cartoon representation of KD1Y11T / R15K / L17R-KT (light green) are shown. Residues that form hydrogen bonds and salt bridges (shown as dashed lines) between the kringle domain and KD1Y11T / R15K / L17R-KT are shown in stick representation. Carbon atoms are shown in green for the kringle domain and light green for KD1Y11T / R15K / L17R-KT. As in (A), oxygen atoms are shown in red and nitrogen atoms are shown in blue. KD1Y11T / R15K / L17R-KT residues are shown with suffix I. For the electrostatic surface, blue represents positive charge, red represents negative charge, and white represents neutral charge. Figure 9B shows a modeled complex of KD1-Y11T / L17R-KT interaction with plasmin. (A) Modeled interaction of KD1-Y11T / L17R-KT with the plasmin protease domain. Shown is the electrostatic surface of the plasmin protease domain and a cartoon representation of KD1-Y11T / L17R-KT (yellow). The P1 (Arg15), P5 (Thr11), and P2' (Arg17) residues of the KD1-Y11T / L17R-KT interaction with plasmin are shown in stick representation. For the electrostatic surface, blue represents positive charge, red represents negative charge, and white represents neutral charge. (B) Modeled interaction of KD1-Y11T / L17R-KT with the plasmin kringle domain.The electrostatic surface of plasminogen Kringle domain 1 and a cartoon representation of KD1-Y11T / L17R-KT (yellow) are shown. Residues that form hydrogen bonds and salt bridges (shown as dashed lines) between the Kringle domain and KD1-Y11T / L17R-KT are shown in stick representation. Carbon atoms are shown in green for the Kringle domain and in yellow for KD1-Y11T / L17R-KT. As in (A), oxygen atoms are shown in red and nitrogen atoms are shown in blue. The KD1-Y11T / L17R-KT residues are shown with the suffix I. In the electrostatic surface, blue represents a positive charge, red represents a negative charge, and white represents a neutral charge.

[0025] [Figure 10] Figure 10. Sequence of the KD1 polypeptide. Sequence of the human TFPI-2 KD1 single mutant (KD1-L17R-KT) expressed in E. coli (SEQ ID NO: 4) and the double mutant (KD1-Y11T / L17R-KT) expressed in E. coli (SEQ ID NO: 5) and Pichia pastoris (SEQ ID NO: 6). The down arrow indicates the enterokinase cleavage site introduced to remove the His tag. The mutated residues, Tyr11Thr and Leu17Arg, are also marked. Residue 1 is numbered according to the BPTI-Kunitz domain numbering and corresponds to amino acid 10 in the TFPI-2 Kunitz domain 1 sequence.

[0026] [Figure 11]FIG. 11. Data from SDS-PAGE analysis of TFPI-2 KD1 single and double mutants. Lane 1, molecular weight (MW) markers; lane 2, reduced E. coli KD1-L17R-KT; lane 3, reduced E. coli KD1-Y11T / L17R-KT; lane 4, reduced P. pastoris KD1-Y11T / L17R-KT; lane 5, non-reduced E. coli KD1-L17R-KT; lane 6, non-reduced E. coli KD1-Y11T / L17R-KT; lane 7, non-reduced P. pastoris KD1-Y11T / L17R-KT. 5 μg of protein was loaded in each lane. KD1SM, KD1-L17R-KT; and KD1DM, KD1-Y11T / L17R-KT.

[0027] [Figure 12] Figures 12A and 12B. 12(A) Data from the study of E. coli expressed KD1-WT (IIa cleavage site, 32), E. coli expressed KD1-L17R-KT, KD1-Y11T / L17R-KT with enterokinase cleavage site, P. pastoris expressed KD1-Y11T / L17R-KT and determination of the equilibrium dissociation constant (Ki) between aprotinin and plasmin. Enzyme activity is expressed as percent fractional activity (inhibited / uninhibited) at increasing inhibitor concentrations. Inhibition constants (Ki) were determined using Equations 1 and 2 as outlined in the experimental section. Data represent the average of three experiments. The concentration of plasmin used was 3 nM. 12(B) Inhibition profile of KD1-Y11T / L17R-KT with FVIIa / sTF, pKLK and FXIa. The concentration of FVIIa / sTF was 20 nM, whereas pKLK and FXIa were each 1 nM. In triplicate experiments performed, no inhibition was observed up to a concentration of 3 μM of KD1-Y11T / L17R-KT.

[0028] [Figure 13]13A and 13B. Data from the study of the interaction of KD1-Y11T / L17R-KT with DIP-δ plasmin and tPA as measured by SPR. (A) DIP-δ plasmin binding to KD1-Y11T / L17R-KT. DIP-δ plasmin was coupled to a CM5 chip by the amine coupling method, achieving a fixed level of 734 response units (RU) for bound protein. Five concentrations of KD1-Y11T / L17R-KT (0.1 μM, 0.3 μM, 0.5 μM, 0.75 μM and 1 μM) were used with an association time of 6 minutes and a dissociation time of 10 minutes (flow rate 10 μL / min). Details are provided in the experimental section. (B) tPA binding to KD1-Y11T / L17R-KT. tPA was coupled to a CM5 chip to achieve a fixed level of 1182 RU for bound protein. Five concentrations of KD1-Y11T / L17R-KT (0.1 μM, 0.3 μM, 0.75 μM, 1 μM and 2 μM) were used. The association and dissociation protocols of the analytes were the same as in panel (A). Experiments in panels A and B were performed in duplicate. Each data set was then used to calculate the k, k and K values, resulting in the mean ± SD values ​​provided in the text.

[0029] [Figure 14] 14A-14C. Data from a study of the effects of KD1-L17R-KT, KD1-Y11T / L17R-KT and aprotinin on fibrinolysis in human NPP. [ka]

[0030] [Figure 15]Figures 15A-15D. Data from a study comparing the fibrinolysis midpoint for KD1-L17R-KT, KD1-Y11T / L17R-KT and aprotinin at various concentrations used in a plasma clot lysis assay. Bar graphs are presented showing the time (min) to reach the fibrinolysis midpoint for the indicated concentrations of KD1-L17R-KT, KD1-Y11T / L17R-KT and aprotinin. The concentration of each inhibitor used is indicated for each panel. All experiments were performed in triplicate and mean ± SD values ​​are shown. Note: * without bar represents significant difference from all other agents listed. * indicates p<0.05.

[0031] [Figure 16] 16A-16G. Data from thromboelastograms illustrating dose-response analysis of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT, aprotinin and EACA. All experiments included citrated whole blood (300 μl), 1.5 μM plasmin and 10 mM CaCl2. Antifibrinolytic agents were added to the blood first, to which 1.5 μM plasmin and 10 mM CaCl2 were then added. Clot formation and lysis were monitored for 180 min. Control experiments were performed without any antifibrinolytic agent, and in the presence or absence of plasmin. (A) Plasmin effect on clot formation and fibrinolysis. Citrated whole blood (300 μl) was added with various concentrations of plasmin (0-3 μM) and 10 mM CaCl2. Clot formation and lysis were monitored for 180 min. Effects of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT and aprotinin at 1 μM (B), 2 μM (C), 3 μM (D), 4 μM (E), 5 μM (F) and 7.5 μM (G) on clot formation and fibrinolysis using 1.5 μM plasmin. (G) Effects of 200 μM to 3000 μM EACA on clot formation and fibrinolysis using 1.5 μM plasmin. Pm, plasmin; NHB, normal human blood.

[0032] [Figure 17]17A-17F. Data from a study of the comparison of maximum amplitude (MA) from TEG experiments for various concentrations of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT, aprotinin and EACA. Bar graphs represent MA achieved with various concentrations of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT, aprotinin and EACA-A, 1 μM; B, 2 μM, C, 3 μM; D, 5 μM D, E, 7.5 μM and F, EACA. All experiments were performed in duplicate and mean ± SD values ​​are represented. Note: * without bar represents significant difference from all other agents listed. * indicates p<0.05.

[0033] [Figure 18] 18A-18F. Data from a study of the comparison of shear modulus strength (G, TEG experiments) for various concentrations of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT, aprotinin and EACA. Bar graphs represent "G" achieved with various concentrations of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT, aprotinin and EACA-A, 1 μM; B, 2 μM, C, 3 μM; D, 5 μM D, E, 7.5 μM and F, EACA. All experiments were performed in duplicate and represent the mean ± SD values. Note: * without bar represents significant difference from all other agents listed. * indicates p<0.05.

[0034] [Figure 19] Figures 19A-19D. Data from a study comparing LY60% (TEG experiments) for various concentrations of KD1-WT, KD1-L17R-KT, KD1-Y11T / L17R-KT and aprotinin. Bar graphs showing percent lysis at 60 min are shown. All experiments were performed in duplicate and represent mean ± SD values. Note: * without bar represents significant difference from all other agents listed. * indicates p<0.05.

[0035] [Figure 20]Figure 20. Thromboelastograms illustrating dose-response analysis of KD1TM. All experiments included citrated whole blood (300 μL), 0.15 μM thrombin and 10 mM CaCl2. KD1TM was added to the blood first, which was then supplemented with 0.15 μM thrombin, 2 nM tPA and 10 mM CaCl2. Clot formation and lysis were monitored for 180 min. Control experiments were performed without KD1TM and in the presence or absence of 2 nM tPA (curve 1 and curve 2). KD1TM effect on clot formation and fibrinolysis with 0.15 μM α-thrombin and 2 nM tPA (curve 3, 2 μM and curve 4, 4 μM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Invention In the description of the embodiments, reference may be made to the accompanying drawings, which form a part hereof and which show by way of illustration specific embodiments in which the invention may be practiced. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. Unless otherwise defined, all terms of technology, notation and other scientific terms or terminology used herein are intended to have the meanings that are commonly understood by those skilled in the art to which the invention belongs. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference beyond that which is commonly understood in the art. Many of the aspects of the techniques and procedures described or referred to herein are well understood and commonly used by those skilled in the art. The following text discusses various embodiments of the invention.

[0037] Antifibrinolytic agents such as the polypeptides of the present disclosure are useful in reducing bleeding and transfusions during major surgical procedures and trauma (e.g., cardiac surgery, orthopedic surgery, liver surgery, neurosurgery, and obstetrics). As disclosed herein, a new polypeptide variant of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) having the following sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1) has been generated and discovered to have a highly desirable pharmacokinetic profile for in vivo use. This 60-residue polypeptide variant disclosed herein contains a unique group of amino acid residues that includes a C-terminal structure that includes a lysine residue. The polypeptide variant disclosed herein further includes a group of three amino acid mutations that include a lysine amino acid substitution at position 15 ("KD1"). Y11T / R15K / L17R "). Surprisingly, this Y11T / R15K / L17R triple mutant is observed to be 4-5 times more potent in inhibiting plasmin when compared to a 60 residue polypeptide variant having only the double mutation Y11T / L17R. Surprisingly, this 60 residue Y11T / R15K / L17R triple mutant polypeptide further exhibits at least 10 times weaker inhibition of kallikrein, factor XIa and factor VIIa / tissue factor when compared to a comparable 60 residue polypeptide variant having only the double mutation Y11T / L17R. Thus, the 60 residue variant polypeptides disclosed herein exhibit a highly desirable pharmacokinetic / material profile, including, for example, the ability to strongly inhibit plasmin while simultaneously avoiding certain side effects associated with similar inhibitory molecules in the art (e.g., aprotinin). Thus, the 60 residue variant polypeptides disclosed herein fulfill a long-standing need that has been recognized, persists and has not been addressed by others.

[0038] The invention disclosed herein has many embodiments. An embodiment of the present invention includes, for example, a composition comprising a polypeptide comprising (or consisting essentially of) the following sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1). Typically, such a composition also comprises additional agents, such as pharma- ceutically acceptable carriers (e.g., preservatives, tonicity adjusters, detergents, hydrogels, viscosity adjusters, pH adjusters, etc.). Such an embodiment of the present invention includes, for example, a 60 amino acid protein sequence represented by: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1); and a pharma-ceutically acceptable excipient suitable for intravenous injection or infusion. Such pharma- ceutically acceptable excipients are well known in the art, and a thorough discussion of pharma- ceutically acceptable carriers, diluents, and other excipients is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ latest edition).

[0039] Moreover, the polypeptide variants of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) disclosed herein further exhibit desirable and unexpected stability profiles. For example, in certain embodiments of the present invention, the 60-residue polypeptide variants disclosed herein are placed in a composition, where the plasmin inhibition constant (Ki) of the polypeptide changes by less than 10% (or less than 5%) when the polypeptide composition is incubated in Tris-buffered saline (TBS) containing 0.1 mg / mL bovine serum albumin (BSA) and 2 mM calcium for at least 2 days, 4 days, or 1 week at 37°C. See, for example, Table 7 in Example 2 below.

[0040] An embodiment of the present invention includes the polypeptide composition disclosed herein further disposed within a substrate, such as a patch or pressure bandage material. As known in the art, such a patch or pressure bandage material can be used to provide hemostasis for bleeding wounds. The substrate, in the context of the present invention, is understood to include any type of medical pressure bandage, patch, sponge, pad, swab, bandage, etc., as conventionally used in the medical field for wound treatment. The substrate can be made of cotton and / or cellulose-based materials (viscose or rayon), for example, in the form of absorbent woven or nonwoven textile products. In this context, one such embodiment of the present invention is a patch, sponge, pad, swab, bandage, etc., in which the 60 amino acid polypeptide disclosed herein (as shown in SEQ ID NO: 1) is disposed within the matrix of the patch, sponge, pad, swab, bandage, etc. In a representative embodiment, the patch or the like is designed to be placed at an in vivo location (e.g., at the site of an injury) and the 60 amino acid polypeptide is placed within the patch such that the polypeptide can diffuse away from the matrix of the patch or the like and into the tissue surrounding the site where the patch is placed. Illustrative patches and similar materials and methods that can be adapted for use with such embodiments of the invention are disclosed, for example, in U.S. Patent Publication Nos. 20020049471, 20110071498 and 20210038758, the contents of which are incorporated by reference.

[0041] Another embodiment of the present invention is a composition comprising a polynucleotide encoding the above polypeptide sequence: NAEICLLPLDTGPCKARLLR YYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1). In a working embodiment of the invention disclosed herein, the polynucleotide comprises the sequence: AACGCGGAGATCTGTCTGCCCCTAGACACCGGACCCTGCAAAGCCAGACTTCTCCGTTACTACTACGACAGGTACACGCAGAGCTGCCGCCAGTTCCTGTACGGGGGCTGCGAGGGCAACGCCAACAATTTCTACACCTGGGAGGCTTGCGACGATGCTTGCTGGAGGATAGAAAA (SEQ ID NO: 2). Those skilled in the art will understand that while this specific sequence contains the codons used to generate the Kunitz domain 1 inhibitor polypeptides disclosed herein in humans, the polynucleotide sequences encoding these polypeptides may vary depending on the system used to express the polypeptide (i.e., different codons may be used in bacteria, yeast and insect cells). Typically, such polynucleotides are placed into a vector that contains one or more regulatory sequences for expressing the polypeptide in a cell. Embodiments of the invention also include cells (e.g., bacterial, yeast, insect or mammalian cells) that contain such vectors.

[0042] The embodiments of the present invention also include methods of using the polypeptides disclosed herein. Such embodiments of the present invention include, for example, a method of inhibiting fibrinolysis in a patient, comprising administering to the patient an amount of a polypeptide disclosed herein sufficient to inhibit fibrinolysis such that fibrinolysis is inhibited. A related embodiment of the present invention includes a method of inhibiting at least one activity of plasmin, comprising contacting plasmin with an effective amount of a polypeptide disclosed herein. Another illustrative embodiment of the present invention includes a method of treating a subject for a condition treatable by aprotinin, comprising administering to the subject an effective amount of a polypeptide disclosed herein. Another illustrative embodiment of the present invention includes a method for treating a subject in need of inhibition of plasmin activity, comprising administering to the subject an effective amount of a polypeptide disclosed herein. Another illustrative embodiment of the present invention includes a method for treating a subject in need of surgery, comprising administering to the subject an effective amount of a polypeptide disclosed herein before, during, and / or after surgery. Other illustrative embodiments of the present invention include a method for treating a subject suffering from cancer or a precancerous condition, comprising administering to the subject an effective amount of a polypeptide disclosed herein. One such illustrative embodiment of the present invention includes a method for treating a subject suffering from cancer metastasis, comprising administering to the subject an effective amount of a polypeptide disclosed herein.

[0043] Related embodiments of the present invention include a method for inhibiting bleeding in a subject, comprising administering to a subject an effective amount of a polypeptide disclosed herein. In certain embodiments of the present invention, the bleeding occurs from a laceration (e.g., a liver laceration), surgery (e.g., an organ such as a liver transplant), or a traumatic injury (e.g., a traumatic brain injury, a gunshot wound, an accident, etc.). In certain of these methods of the present invention, the polypeptide is delivered to an in vivo location in a patch in which the 60 amino acid polypeptide is disposed. In this context, another embodiment of the present invention is a method for inhibiting bleeding in a subject, comprising contacting the patch with a bleeding tissue such that the 60 amino acid polypeptide can diffuse away from the patch matrix and into an in vivo environment (e.g., the bleeding tissue). For example, a method for treating a patient suffering from excessive bleeding is provided herein, comprising administering to the patient a therapeutically effective amount of a polypeptide of the present disclosure. In other embodiments, provided herein are methods of treating a patient suffering from, for example, stroke (e.g., acute stroke), cerebral ischemia caused by stroke, hematoma, edema, hypoxic / anoxic brain injury, or traumatic brain injury, the method comprising administering to the patient a therapeutically effective amount of a polypeptide of the present disclosure.

[0044] One illustrative embodiment of the present invention is a method of treating a patient undergoing cardiac surgery and in need of reduced blood loss, comprising administering a therapeutically effective amount of a 60 amino acid protein sequence represented by the following: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK SEQ ID NO: 1) before, during, and / or after surgery, a pharmacologic composition comprising a 60 amino acid protein sequence; and a pharmacologic excipient suitable for intravenous injection or infusion. In a particular embodiment of the present invention, the cardiac surgery is cardiopulmonary bypass surgery. Another embodiment of the present invention is a method of treating a patient in traumatic hemorrhagic shock, comprising administering a therapeutically effective amount of a pharmacologic composition comprising ...pharmacologic excipient suitable for intravenous injection or infusion. Furthermore, due to the strong plasmin inhibitory properties (and lack of anticoagulant properties) of rHuKD1-TM, the composition can be used to prophylactically treat hemophilia in a manner similar to how aprotinin has been used in the past to treat this condition. Moreover, the polypeptides disclosed herein have further advantages over aprotinin. In one example, aprotinin cannot be used long-term in such treatment regimens due to the anaphylactic reactions observed to occur with the use of aprotinin (which is of bovine origin).

[0045]

[0023] Embodiments of the invention include methods for administering the 60 amino acid polypeptide variant of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) disclosed herein. For example, embodiments of the invention encompass administering a dose of the 60 amino acid polypeptide variant in a therapeutic method (e.g., in a method of inhibiting at least one activity of plasmin in a patient) ranging from about 1 μg of the polypeptide variant per gram of patient body weight to about 10 μg of the polypeptide variant for each gram of the polypeptide variant for each gram of patient body weight (e.g., about 2 to about 8 μg of the polypeptide variant for each gram of the polypeptide variant for each gram of patient body weight, about 4 μg of the polypeptide variant for each gram of the polypeptide variant for each gram of patient body weight, etc.). Additionally, embodiments of the invention include dosing / administration methods specifically designed to treat acute and / or chronic medical conditions. For example, in certain embodiments of the invention, the 60 amino acid polypeptide variant is used in selected methods that administer the polypeptide variant shortly after surgery / injury (e.g., less than 48 hours, less than 24 hours, less than 12 hours, or less than 4 hours after surgery / injury). In some embodiments of the invention, the 60 amino acid polypeptide variant is used in selected methods that administer the polypeptide variant after an extended period of time after surgery / injury (e.g., at least 2 days, 4 days, 7 days, 14 days, or 21 days after surgery / injury).

[0046] Particular aspects and embodiments of the above polypeptides and related methods are disclosed in the Examples below. Further aspects and embodiments of the invention are discussed in the following sections.

[0047] Aspects and embodiments of the present invention In the context of the invention disclosed herein, the terms "polypeptide", "protein" and "peptide" as well as "glycoprotein" are used interchangeably and refer to a polymer of amino acids not limited to any particular length. The terms do not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation, formylation, and the addition or deletion of signal sequences. The terms "polypeptide" or "protein" refer to one or more chains of amino acids, where each chain comprises amino acids covalently linked by peptide bonds, and the polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked together by peptide bonds, and may include naturally occurring proteins, i.e., proteins that occur in nature and specifically are produced by non-recombinant cells, or genetically engineered or recombinant cells, and may include molecules that have the amino acid sequence of a naturally occurring protein, or molecules that have deletions, additions and / or substitutions of one or more amino acids of the naturally occurring sequence. Thus, a "polypeptide" or "protein" may comprise one (referred to as a "monomer") or multiple (referred to as a "multimer") amino acid chains. The terms "peptide," "polypeptide," and "protein" specifically encompass immunomodulatory polypeptides of the disclosure, or sequences having one or more amino acid deletions, additions, and / or substitutions of an immunomodulatory polypeptide.

[0048] The term "isolated" means that a material is removed from its original environment (e.g., the natural environment when the material is naturally occurring). For example, a naturally occurring polypeptide or nucleic acid present in a living animal is not isolated, but the same polypeptide or nucleic acid is isolated if it is separated from some or all of the materials that are present at the same time in the natural system. Such a nucleic acid may be part of a vector, and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), and is still isolated in that such a vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" refers to a segment of DNA involved in the production of a polypeptide chain; it includes the regions "leader and trailer" that precede and follow the coding region, as well as the intervening sequences (introns) between each coding segment (exon).

[0049] The terms "isolated protein" and "isolated polypeptide" as referred to herein mean that the subject protein or polypeptide is (1) free from at least some other proteins or polypeptides with which it would typically be found in nature; (2) essentially free from other proteins or polypeptides from the same source, e.g., from the same species; (3) expressed by cells from a different species; (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) not associated (by covalent or non-covalent interactions) with portions of proteins or polypeptides with which it may be naturally associated; (6) not operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated; or (7) not naturally occurring. Such isolated proteins or polypeptides may be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin by any of the many well-known chemical reactions for artificial peptide and protein synthesis, or any combination thereof. In certain embodiments, the isolated protein or polypeptide is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or other).

[0050] Certain embodiments relate to nucleic acid molecules encoding the plasmin inhibitory polypeptides described herein. Methods for the production of desired nucleic acids and / or polypeptides are well known in the art. For example, nucleic acids and / or polypeptides can be isolated from cells or synthesized de novo by chemical synthesis. Such nucleic acids or polypeptides can be introduced into vectors and transformed into host cells. Host cells can be cultured in standard nutrient media with necessary supplements or additives for inducing promoters, selecting transformants, or amplifying appropriate sequences.

[0051] Furthermore, a coding polynucleotide or polypeptide variant of a plasmin inhibitory polypeptide may contain one or more nucleotide or amino acid substitutions, additions, deletions, and / or insertions, respectively, relative to the native (e.g., wild-type or predominant or naturally occurring allelic form). In some embodiments, the variants include molecules in which an N-terminal L-amino acid is replaced with a D-amino acid, and in certain other embodiments, one or more other amino acids (e.g., not located at the N-terminus) may additionally or alternatively be replaced with a D-amino acid. In certain embodiments, the variants include molecules in which an N-terminal alpha amino acid is replaced with a beta or gamma amino acid. The variants preferably exhibit at least about 75%, 78%, 80%, 85%, 87%, 88% or 89% identity, and preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, with a portion of a plasmin inhibitory polypeptide sequence or a polynucleotide sequence encoding such a polypeptide. Percent identity can be easily determined by comparing the sequence of the polypeptide or polynucleotide variant with the corresponding portion of the full-length polynucleotide or polypeptide. Some techniques for sequence comparison include using computer algorithms well known to those skilled in the art, such as Align or BLAST algorithms (Altschul, J. Mol. Biol. 219:555-565, 1991; Henikoff and Henikoff, PNAS USA 89:10915-10919, 1992). Default parameters can be used.

[0052] Additionally, the plasmin inhibitory polypeptide variants disclosed herein may be linked to additional molecules or agents (e.g., imaging agents, particles, polymers, or other agents (e.g., antibodies, peptides, etc.) including those that facilitate polypeptide delivery to a particular in vivo location. Such embodiments of the invention include polypeptide variants of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) comprising (or consisting essentially of) the sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1) linked to additional molecules or agents. In one example thereof, embodiments of the invention may be modified in this manner to facilitate polypeptide delivery to the CNS (see, e.g., Behzad et al., (2019), Expert Opinion on Drug Delivery, 16:6, 583-605; Salameh et al., Adv Pharmacol. 2014;71:277-99; and U.S. Patent Publication Nos. 20200230218, 20060189515, 20150174267, and 20160213760, the contents of which are incorporated by reference.

[0053] The term "operably linked" means that the components to which the term is applied are in a relationship that permits those components to carry out their essential functions under appropriate conditions. For example, a transcription control sequence "operably linked" to a protein coding sequence is ligated to the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0054] The term "control sequence" as used herein refers to polynucleotide sequences that can affect the expression, processing or subcellular localization of coding sequences to which they are ligated or operably linked. The nature of such control sequences may depend on the host organism. In certain embodiments, transcription control sequences for prokaryotes may include promoters, ribosomal binding sites, and transcription termination sequences. In other specific embodiments, transcription control sequences for eukaryotes may include promoters containing one or more recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences, and polyadenylation sequences. In certain embodiments, "control sequences" may include leader sequences and / or fusion partner sequences.

[0055] The term "polynucleotide" as referred to herein means a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides constituting the polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide. Such modifications can include base modifications (e.g., bromouridine), ribose modifications (e.g., arabinoside and 2',3'-dideoxyribose) and internucleotide bond modifications (e.g., phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoroamidate). The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0056] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups, etc. The term "oligonucleotide linkage" includes oligonucleotide linkages (e.g., phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoraniladates, phosphoramidates, etc.). For example, see LaPlanche et al., 1986, Nucl. Acids Res., 14:9081;Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209;Zon et al., 1991, Anti-Cancer Drug Design, 6:539;Zon et al., 1991, Oligonucleotides And Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed.), Oxford University Press, Oxford England;Stec et al., U.S. Patent No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543 (the disclosures of which are hereby incorporated by reference for any purpose).Oligonucleotides can contain detectable labels to allow detection of the oligonucleotide or its hybridization.

[0057] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present).

[0058] As will be appreciated by those skilled in the art, polynucleotides may include genomic sequences, extragenomic, plasmid-encoded sequences, and smaller engineered gene segments, which may express or be adapted to express proteins, polypeptides, peptides, etc. Such segments may, of course, be isolated or synthetically modified by those skilled in the art.

[0059] As will also be appreciated by those skilled in the art, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules (which contain introns and correspond in a one-to-one manner to DNA molecules) and mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may, but need not, be present within a polynucleotide in accordance with the present disclosure, and polynucleotides may, but need not, be linked to other molecules and / or supporting materials. Polynucleotides may include naturally occurring sequences or may include sequences that code for variants or derivatives of such sequences.

[0060] Thus, in accordance with these and related embodiments, the disclosure also provides polynucleotides that encode the plasmin inhibiting polypeptides described herein. In certain embodiments, polynucleotides that include some or all of the polynucleotide sequences that encode the plasmin inhibiting polypeptides as described herein, and complements of such polynucleotides, are provided.

[0061] In other related embodiments, polynucleotide variants may have substantial identity with the polynucleotide sequence encoding the plasmin inhibitory polypeptide described herein.For example, the polynucleotide may be a polynucleotide that comprises at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, compared to a reference polynucleotide sequence (e.g., a sequence encoding a plasmin inhibitory polypeptide having an amino acid sequence disclosed herein) using the methods described herein (e.g., BLAST analysis using standard parameters as described below).Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of the protein encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame arrangement.

[0062] Typically, the polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions, preferably such that the binding affinity for plasmin of the plasmin inhibiting polypeptide encoded by said variant polynucleotide is not substantially reduced relative to the binding affinity of a plasmin inhibiting polypeptide having an amino acid sequence specifically set out herein.

[0063] According to certain related embodiments, there is provided a recombinant host cell comprising one or more constructs as described herein; a nucleic acid encoding a plasmin inhibitory polypeptide or variant thereof; and a method of producing the encoded product, said method comprising expression from the encoding nucleic acid for said product. Expression can conveniently be achieved by culturing a recombinant host cell comprising said nucleic acid under suitable conditions. After production by expression, the plasmin inhibitory polypeptide can be isolated and / or purified using any suitable technique and then used as desired.

[0064] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common and preferred bacterial host is E. coli.

[0065] Expression of peptides in prokaryotic cells such as E. coli is well established in the art. For a review, see, for example, Pluckthun, A. Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for producing recombinant polypeptides. See recent reviews, for example, references (1993) Curr. Opinion Biotech. 4:573-576; Trill et al. (1995) Curr. Opinion Biotech 6:553-560. Expression of peptides in yeast, such as Pichia pastoris, is also well known in the art (see, for example, U.S. Patent Publication Nos. 20180142038, 20190241645 and 20190119692).

[0066] A suitable vector can be selected or constructed, including suitable regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. The vector can be a plasmid, a virus, such as a phage, or a phagemid as appropriate. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, many known techniques and protocols for preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and manipulating nucleic acids in gene expression, and analyzing proteins are described in detail in Current Protocols in Molecular Biology, 2nd Edition, Ausubel et al., eds., John Wiley & Sons, 1992, or later latest editions.

[0067] The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the immunomodulatory polypeptides described herein has been introduced or can be introduced and which further expresses or can express a selected gene of interest (e.g., a gene encoding any of the plasmin inhibitory polypeptides described herein). The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent, so long as the selected gene is present. Thus, methods involving the introduction of such nucleic acids into a host cell are also contemplated. The introduction may use any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia), or for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. The introduction can be followed by causing or allowing expression from the nucleic acid, for example, by culturing a host cell under conditions for expression of the gene. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome, according to standard techniques.

[0068] The present invention also provides, in certain embodiments, methods that include using constructs as described above in an expression system to express a particular polypeptide (e.g., a plasmin inhibitory polypeptide as described herein). The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction" also refers to the attainment and transfer of eukaryotic cell sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when the exogenous DNA has been introduced into the cell membrane. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories; Davis et al., 1986, Basic Methods In Molecular Biology, Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0069] The term "transformation" as used herein refers to a change in the genetic characteristics of a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it has been genetically modified from its native state. After transfection or transduction, the transforming DNA may recombine with the cell's DNA by physically integrating into the cell's chromosome, may be maintained transiently as an episomal element without replication, or may replicate independently as a plasmid. A cell is considered stably transformed when the DNA replicates with cell division. The term "naturally occurring" or "native" when used in reference to biological material such as nucleic acid molecules, polypeptides, host cells, etc., refers to material that is found in nature and has not been manipulated by humans. Similarly, "non-naturally occurring" or "non-native" as used herein refers to material that is not found in nature or that has been structurally modified or synthesized by humans.

[0070] The present invention also relates in certain embodiments to pharmaceutical compositions comprising the plasmin inhibiting polypeptide disclosed herein. In one embodiment, the pharmaceutical composition comprises the plasmin inhibiting polypeptide in a pharma- ceutically acceptable excipient, carrier or diluent and in an amount effective to inhibit at least one activity of plasmin when administered to an animal, preferably a mammal, most preferably a human. In another embodiment, the pharmaceutical composition comprises the plasmin inhibiting polypeptide in a pharma- ceutically acceptable excipient, carrier or diluent and in an amount effective to treat a subject in need of inhibition of plasmin activity, for example, in a method comprising administering to the subject an effective amount of the plasmin inhibiting polypeptide disclosed herein.

[0071] Examples of diseases, disorders, and treatments involving the need for inhibition of plasmin include, but are not limited to, surgery, traumatic injury (e.g., traumatic brain injury), and other conditions and situations (e.g., tumor formation, angiogenesis, bone remodeling, hemophilia, coronary artery bypass graft surgery (CABG), etc.). Use of pharmaceutical compositions comprising the plasmin-inhibiting polypeptides described herein to control bleeding in other contexts is also contemplated, for example, as antifibrinolytic compositions and as antidotes for plasmin overdose, or overdose of tPA, or other hematologically active substances that may directly or indirectly promote the activity of plasmin or other related proteases.

[0072] Administration of the plasmin inhibiting polypeptide in pure form or in a suitable pharmaceutical composition may be via any of the accepted modes of administration of drugs to serve similar utilities. The pharmaceutical composition may be prepared by combining the plasmin inhibiting polypeptide with a suitable pharma- ceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gas form (e.g., tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, gels, microspheres, and aerosols). Representative routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, intranasal, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, subcutaneous, intramuscular, rectal, buccal, intranasal, liposomal, via inhalation, intraocular, via catheter (e.g., as in angioplasty), via topical delivery, subcutaneous, intraadipose, intraarticular, or intrathecal. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions are formulated to allow the active ingredient contained therein to be bioavailable upon administration of the composition to a patient. The composition administered to a subject or patient takes the form of one or more dosage units, where, for example, a tablet may be a single dosage unit and a container of the compound of the present invention in the form of an aerosol may hold multiple dosage units. The actual method of preparing such dosage forms is known or apparent to those skilled in the art; see, for example, The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will in any event contain a therapeutically effective amount of plasmin inhibiting polypeptide for the treatment of the disease or condition of interest according to the present teachings.

[0073] The pharmaceutical compositions useful herein also include pharma- ceutically acceptable carriers (including any suitable diluent or excipient), including any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol, ethanol, and the like. A thorough discussion of pharma-ceutically acceptable carriers, diluents, and other excipients is provided in Remington's Pharmaceutical Sciences (latest edition, Mack Pub. Co., NJ).

[0074] The pharmaceutical composition can be in the form of liquid, for example, elixir, syrup, solution, emulsion or suspension.The liquid can be for oral administration or for delivery by injection, as two examples.When intended for oral administration, the preferred composition contains one or more of the following in addition to the compound of the present invention: sweetener, preservative, dye / colorant and flavor enhancer.In the composition intended to be administered by injection, one or more of the following can be included: surfactant, preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer and isotonic agent.

[0075] The liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may contain one or more of the following adjuvants: a sterile diluent (e.g., water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils (e.g., synthetic mono- or diglycerides that can act as solvents or suspending media), polyethylene glycol, glycerin, propylene glycol or other solvents); antibacterial agents (e.g., benzyl alcohol or methylparabens); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid); buffers (e.g., acetates, citrates or phosphates) and agents for adjusting isotonicity (e.g., sodium chloride or dextrose). Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0076] Liquid pharmaceutical compositions intended for either parenteral or other administration should contain an amount of plasmin inhibiting polypeptide such that a suitable dosage will be obtained. Typically, this amount will be at least 0.01% of the plasmin inhibiting polypeptide in the composition. This amount may be varied to be between 0.1 and about 70% by weight of the composition. Certain illustrative pharmaceutical compositions and preparations according to the invention are prepared so that a parenteral dosage unit contains between 0.01 and 10% by weight of the plasmin inhibiting polypeptide.

[0077] The plasmin inhibiting polypeptide is administered in a therapeutically effective amount, which varies depending on a variety of factors, including the activity of the specific polypeptide; the metabolic stability and duration of action of the plasmin inhibiting polypeptide; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated. In general, the therapeutically effective daily dose is about 1 mg / Kg (i.e., 70 mg) to about 10 mg / Kg (i.e., 7.0 g) (for a 70 kg mammal); preferably, the therapeutically effective dose is about 2 mg / Kg to about 8 mg / Kg (for a 70 kg mammal); more preferably, the therapeutically effective dose is about 4 mg / Kg.

[0078] The ranges of effective doses provided herein are not intended to be limiting but rather represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as can be understood and determined by one of ordinary skill in the relevant art (see, e.g., Berkowet et al., eds., The Merck Manual, 16th ed., Merck and Co., Rahway, NJ, 1992; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., Pergamon Press, Inc., Elmsford, NY, (2001); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd ed., ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci et al., eds., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992).

[0079] The total dose required for each treatment may be administered in multiple doses or in a single dose over the course of a day, or a week or month, if desired. Generally, treatment is started with smaller doses that are less than the optimal dose of the plasmin inhibitory polypeptide. The dose is then increased in small increments until the optimal effect under the circumstances is reached. The plasmin inhibitory polypeptide may be administered alone or in conjunction with other diagnostic agents and / or other medications for the condition or for other symptoms of the condition. The recipient of administration of the plasmin inhibitory polypeptide may be any vertebrate (e.g., mammal). Among mammals, preferred recipients are mammals of the following orders: Primates (including humans, apes and monkeys), Artiodactyla (including horses, goats, cows, sheep, pigs), Rodentia (including mice, rats, rabbits and hamsters), and Carnivora (including cats and dogs). Among birds, preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipient is a human.

[0080] The pharmaceutical composition can be formulated to be placed in a matrix such as a patch. This can be placed in vivo so that the plasmin inhibiting polypeptide is then released from the matrix / patch and into the in vivo environment. Such compositions can include, for example, a backing, an active compound reservoir, a control membrane, a liner and a contact adhesive. Transdermal patches can be used to provide continuous pulsatile or on-demand delivery of the plasmin inhibiting polypeptide of the present invention as desired. Exemplary patch materials and methods that can be adapted for use with such embodiments of the present invention are disclosed, for example, in U.S. Patent Publication Nos. 20020049471, 20110071498 and 20210038758.

[0081] The plasmin inhibitory polypeptide can be formulated to provide rapid, sustained or delayed release of active ingredient after administration to patient by using procedures known in the art.Controlled release drug delivery systems include osmotic pump systems and dissolutional systems, including polymer-coated reservoirs or drug-polymer matrix formulations.Examples of controlled release systems are shown in U.S. Patent Nos. 3,845,770 and 4,326,525, and PJ Kuzma et al., Regional Anesthesia 22 (6):543-551 (1997), all of which are incorporated herein by reference.

[0082] The most suitable route will depend on the nature and severity of the condition being treated. Those skilled in the art will also be familiar with determining the method of administration (e.g., oral, intravenous, inhalation, subcutaneous, rectal, etc.), dosage form, appropriate pharmaceutical excipients, and other considerations related to delivery of the plasmin inhibiting polypeptide to a subject in need of inhibition of plasmin activity.

[0083] According to various contemplated embodiments, a subject in need of inhibition of plasmin activity may have or be suspected of having cancer (e.g., solid tumors such as lung, breast, prostate, or colon cancer, or another cancer), hemophilia, rheumatoid arthritis, or systemic inflammatory response syndrome (SIRS), or the subject may need to undergo or have undergone angiogenesis, bone remodeling, or coronary artery bypass graft surgery (CABG). The subject may be undergoing surgery or may have recently undergone surgery (e.g., within 1, 2, 4, 6, 8, 10, 12, or 24 hours, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days) (e.g., cardiovascular surgery, oncology surgery, genitourinary surgery, plastic surgery, thoracic surgery, plastic surgery, trauma surgery, abdominal surgery, transplant surgery, neurosurgery, or ear, nose, and throat surgery).

[0084] Those of skill in the relevant art are familiar with any number of diagnostic, surgical and other clinical criteria that may be appropriate for administration of the pharmaceutical compositions described herein. For example, Humar et al., Atlas of Organ Transplantation, 2006, Springer;Kuo et al., Comprehensive Atlas of Transplantation, 2004 Lippincott, Williams & Wilkins;Gruessner et al., Living Donor Organ Transplantation, 2007 McGraw-Hill Professional;Antin et al., Manual of Stem Cell and Bone Marrow Transplantation, 2009 Cambridge University Press;Wingard et al. (Ed.), Hematopoietic Stem Cell Transplantation:A Handbook for Clinicians, 2009 American Association of Blood Banks;Sabiston, Textbook of Surgery, 2012 Saunders & Co.;Mulholland, Greenfield's Surgery, 2010 Lippincott, Williams & Wilkins;Schwartz's Principles of Surgery, 2009 McGraw-Hill;Lawrence, Essentials of General Surgery See 2012 Lippincott, Williams & Wilkins.

[0085] References in the above text (1) Fergusson DA, Hebert PA, Mazer CD, Fremes S, MacAdams C, Murkin JM, et al. A comparison of aprotinin and lysine analogues in high-risk cardiac surgery. N Engl J Med 2008; 358: 2319-2331. (2) Karkouti K, Wijeysundera DN, Yau TM, McCluskey SA, Tait G, Beattie WS. The risk-benefit profile of aprotinin versus tranexamic acid in cardiac surgery. Anesth Analg 2010; 110:21-9. (3) Martin K, Knorr J, Breuer T, Gertler R, Macguill M, Lange R, Tassani P, Wiesner G. Seizures after open heart surgery: comparison of ε-aminocaproic acid and tranexamic acid. J Cardiothorac Vasc Anesth. 2011; 25:20-25. (4) Lecker I, Wang DS, Whissell PD, Avramescu S, Mazer CD, Orser BA. Tranexamic acid-associated seizures: Causes and treatment. Ann Neurol. 2016; 79:18-26. (5) Stevens LM, Noiseux N, Prieto I, Hardy JF. Major transfusions remain frequent despite the generalized use of tranexamic acid: an audit of 3322 patients undergoing cardiac surgery. Transfusion 2016; 56:1857-65. (6) Faraoni D, Levy JH. Development of a novel blood-sparing agent in cardiac surgery: do we need another agent? Anesth Analg. 2014; 119:11-12. (7) Bokesch PM, Szabo G, Wojdyga R, Grocott HP, Smith PK, Mazer CD, Vetticaden S, Wheeler A, Levy JH. A phase 2 prospective, randomized, double-blind trial comparing the effects of tranexamic acid with ecallantide on blood loss from high-risk cardiac surgery with cardiopulmonary bypass (CONSERV-2 Trial). J Thorac Cardiovasc Surg. 2012; 143:1022-1029. (8) Englberger L, Dietrich W, Eberle B, Erdoes G, Keller D, Carrel T. A novel blood-sparing agent in cardiac surgery? First in-patient experience with the synthetic serine protease inhibitor MDCO-2010: a phase II, randomized, double-blind, placebo-controlled study in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass. Anesth Analg. 2014; 119:16-25. (9) Earl ST, Masci PP, de Jersey J, Lavin MF, Dixon J. Drug development from Australian elapid snake venoms and the Venomics pipeline of candidates for haemostasis: Textilinin-1 (Q8008), Haempatch TM (Q8009) and CoVase TM (V0801). Toxicon. 2012; 59:456-463. (10) Beith JG. In vivo significance of kinetic constants of protein proteinase inhibitors. Biochem Med 1984; 32: 387-97. (11) Morrison JF, Walsh CT. The behavior and significance of slow‐binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 1988; 61: 201-301. (12) Kawamura K, Yamada T, Kurihara K, Tamada T, Kuroki R, Tanaka I, Takahashi H, Niimura N. X-ray and neutron protein crystallographic analysis of the trypsin-BPTI complex. Acta Crystallogr D Biol Crystallogr. 2011; 67:140-148. (13) Fujikawa K, Chung DW, Hendrickson LE, Davie EW. Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein. Biochemistry. 1986; 25:2417-2424. (14) Leytus SP, Foster DC, Kurachi K, Davie EW. Gene for human factor X: a blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C. Biochemistry. 1986; 25:5098-5102. EXAMPLES

[0086] Example 1: Enhanced antifibrinolytic efficacy of the plasmin-specific Kunitz-inhibitor of human tissue factor pathway inhibitor type 2 domain 1, a 60-residue Y11T / L17R double mutant with C-terminal IEK Current antifibrinolytic agents reduce blood loss by inhibiting the plasmin active site (e.g., aprotinin) or by preventing plasminogen / tissue plasminogen activator (tPA) from binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed a 60-residue Kunitz domain 1 (KD1) mutant of human tissue factor pathway inhibitor type 2 that can inhibit plasmin and plasminogen activation. A single (KD1-L17R-K T ) and the double mutant (KD1-Y11T / L17R-K T ) were expressed in Escherichia coli as His-tagged constructs (each with an enterokinase cleavage site). T The KD1-Y11T / L17R-K was also expressed in Pichia pastoris. T inhibits plasmin comparable to aprotinin, with K of ∼50 nM and ∼35 nM for the kringle domains of plasminogen / plasmin and tPA, respectively. d Importantly, compared to aprotinin, the KD1-L17R-K T and KD1-Y11T / L17R-K T does not inhibit kallikrein. T The antifibrinolytic potential of KD1-L17R-K was confirmed in plasma clot lysis assays T In thromboelastography experiments, KD1-Y11T / L17R-K T inhibits fibrinolysis in a dose-dependent manner, comparable to aprotinin at higher concentrations. T does not induce cytotoxicity in primary human endothelial cells or fibroblasts. T We conclude that this inhibitor rivals aprotinin, the most potent known inhibitor of plasmin, and can be produced in large quantities using Pichia.

[0087] 1. Introduction In severe trauma and during major surgical procedures (e.g., cardiac surgery), the fibrinolytic system is overactivated, resulting in massive bleeding (1-3). Excessive bleeding poses significant mortality risks and costs in battlefield, accident, and hospital settings. Uncontrolled bleeding is the number one cause of preventable deaths in trauma and often leads to the need for massive blood transfusions during surgery (4,5). Fibrinolytic drugs reduce transfusion requirements by inhibiting fibrinolysis and thereby fibrin degradation products (6,7). Aprotinin (bovine pancreatic trypsin inhibitor, BPTI), a potent inhibitor of the plasmin active site, has been a potent antifibrinolytic agent for reducing blood loss during cardiac surgery and limb trauma (8). However, its use has been associated with severe side effects (e.g., kidney damage, myocardial infarction, and stroke) (9-10). Moreover, because aprotinin is of bovine origin, its potential for anaphylaxis is a major concern (11). For these reasons, aprotinin was temporarily removed from the clinical market in 2008 (12). The currently approved therapeutic agents, tranexamic acid (TXA) and ε-aminocaproic acid (EACA), are lysine analogs that prevent the binding of plasminogen and tissue plasminogen activator (tPA) to fibrin clots (13,14). As a result, local activation of plasminogen to plasmin is prevented, preventing fibrinolysis. However, EACA and TXA are not as effective as aprotinin in reducing blood loss (15). Moreover, like aprotinin, they also cause renal failure (16), and recent evidence indicates that TXA, and to a lesser extent EACA, are associated with a significant incidence of seizures (16,17). Thus, improved antifibrinolytic agents that lack the adverse effects of aprotinin and lysine analogs are needed.

[0088] Several active site plasmin inhibitors have been reported in the literature (18-25), but the development stage of most of them is unknown. Textilinin-1 (Q8008), a Kunitz domain plasmin active site inhibitor derived from Pseudonaja textilis (19,26), is in preclinical development. Q8008 inhibits plasmin with 10- to 15-fold weaker affinity than aprotinin, but it also slightly inhibits kallikrein (19,26). In a mouse tail bleeding model, Q8008 was reported to be as effective as aprotinin in reducing blood loss (26). However, because Q8008 is derived from snake venom, it may cause anaphylactic responses in humans, similar to those observed for aprotinin. Furthermore, using the sunflower trypsin inhibitor-1 scaffold, a highly potent cyclic peptide active site inhibitor of plasmin with a Ki of 0.05 nM has been designed and proposed to be a candidate for drug development (25). In addition, there are allosteric synthetic fibrinolytic inhibitors proposed to reduce perioperative bleeding, but they are in very early stages of development (27, 28). Furthermore, a highly potent plasmin inhibitor (DX-1000) is being developed as an antitumor agent instead of as an antifibrinolytic agent (29).

[0089] Notably, when aprotinin was banned in 2008, a pharmacological agent, ecallantide (DX-88), which inhibits both kallikrein and plasmin, was clinically evaluated (30). This trial was terminated prematurely due to increased mortality observed in the ecallantide arm. Another agent, MDCO2010, which inhibits plasmin, factor Xa (F), FXIa, and activated protein C (APC), was also clinically evaluated (31). This trial was also prematurely terminated due to an increased number of serious adverse events in the treatment group. The cause of the safety issues and their potential relevance to drug use are under further investigation.

[0090] In this example, we describe an antifibrinolytic agent that inhibits plasmin with a potency comparable to aprotinin, but is a much weaker inhibitor of kallikrein. The agent was designed using the Kunitz domain 1 (KD1) of human tissue factor pathway inhibitor type 2 (TFPI-2) as a scaffold. A new 60-residue plasmin inhibitor, KD1-Y11T / L17R-K T The heterologous single mutant KD1-L17R (KD1-L17R-K COOH ) with one additional mutation at a different C-terminal lysine (IEK T ) (32). T The current single mutant KD1-L17R-K T In addition to plasmin, it also binds to the kringle domains of plasminogen and tPA with a dissociation constant of 35-50 nM. At therapeutic doses of 2 μM in plasma, the lowest Hammersmith regimen of aprotinin (33), KD1-Y11T / L17R-K T KD1-Y11T / L17R-K is therefore predicted to effectively inhibit fibrinolysis by inhibiting the plasmin active site and by blocking the binding of plasminogen and tPA to fibrin clots. T appears to be a promising candidate to replace aprotinin in the clinical setting. T The experimental details comparing and are presented herein. Furthermore, modeling was used to evaluate the effect of Tyr11 to Thr mutation and C-terminal IEK in the TFPI-2 KD1 inhibitor scaffold. The structural information obtained from such modeling is considered to elaborate the enhanced antifibrinolytic activity of the above mutants.

[0091] 2. Experimental Section 2.1. Materials Escherichia coli (E. coli) strain BL21(DE3), pLysS and pET28a expression vector were obtained from Novagen Inc. (Madison, WI). Amicon centrifugal filter devices (3000 Mr cutoff) were purchased from Millipore (Bedford, MA). QSepharose FF, Superdex 200, and His-Trap HP columns were obtained from Amersham Biosciences. Diisopropyl fluorophosphate (DFP) was obtained from Calbiochem (San Diego, CA). TXA, EACA, kanamycin and isopropyl thiogalactopyranoside (IPTG) were obtained from Sigma (St. Louis, MO). Caspase-Glo 3 / 7 Assay kit and CellTox TMGreen Cytotoxicity Assay kit was obtained from Promega (Madison, WI). Purified human FXIa, thrombin (IIa) and plasmin were purchased from Hematologic Technologies Inc (Essex Junction, VT). Plasma kallikrein (pKLK) was obtained from Enzyme Research Laboratories (South Bend, IN). Alteplase (tPA) was purchased from Genentech (South San Francisco, CA). Recombinant enterokinase was obtained from Novogen, EMD Chemicals (San Diego CA). Pooled normal plasma (NPP) was purchased from George King Bio-Medical Inc. (Overland Park, Kansas). Delta plasmin (recombinant plasmin containing the protease domain and the first kringle domain) was obtained from Dr. Victor Marder (University of California, Los Angeles, CA). Taxol was kindly provided by Dr. Zhenfeng Duan (University of California, Los Angeles, CA). Aprotinin (BPTI) was received from ZymoGenetics (Seattle, WA), and human factor VIIa (FVIIa) was prepared as previously described (34). Soluble tissue factor (sTF, residues 1–219) was obtained from Tom Girard (Washington University, St. Louis, Missouri). Plasmin substrate S-2251 (HD-Val-Leu-Lys-p-nitroanilide), pKLK, and FXIa substrate S-2366 (pyroGlu-Pro-Arg-p-nitroanilide), and FVIIa substrate S-2288 (H-DIle-Pro-Arg-p-nitroanilide) were obtained from Diapharma Inc (West Chester, OH). Fresh normal human citrated blood was purchased from Nebraska Medical Center (Omaha).Partial thromboplastin time (PT) and activated partial thromboplastin time (aPTT) were normal for each blood donor.

[0092] 2.2. KD1-L17R-K in E. coli T and KD1-Y11T / L17R-K T Expression and purification KD1-L17R-K T and KD1-Y11T / L17R-K with C-terminal IEK T The cDNA sequence of was cloned and overexpressed as an amino-terminal His6-tagged fusion protein using the T7 promoter system in E. coli strain BL21(DE3) pLysS. It is derived from pET28a and contains a His6 leader sequence followed by an enterokinase cleavage site, KD1-L17R-K T Or KD1-Y11T / L17R-K T A recombinant plasmid containing the cDNA encoding KD1-L17R-K was prepared according to standard procedures (35). The sequence of the expressed construct is shown in Figure 10. T and KD1-Y11T / L17R-K T was expressed in E. coli grown in Luria broth containing 15 mg / liter kanamycin and induced with 1 mM IPTG at mid-log phase (A600 approx. 0.9) for 5-6 hours at 37°C. T and KD1-Y11T / L17R-K T was purified from inclusion bodies using a nickel-charged His-Trap column. The His-Trap purified protein was refolded using a reduced and oxidized glutathione system and further purified using a Q-Sepharose FF column as previously described (32,36).

[0093] 2.3. KD1-Y11T / L17R-K T Clone construction and expression in Pichia pastoris Pichia pastoris strain X-33 and secretory expression vector pPICZαA were purchased from Invitrogen (San Diego, Calif.). KD1-Y11T / L17R-K, corresponding to the amino acid sequence (Figure 10), T cDNA was synthesized by IDT (Coralville, IA). The cDNA was amplified by PCR, and the product was linearized and subcloned into the XhoI and NotI restriction sites of pPICZαA. Further vector amplification was performed in DH5α competent cells. The extracted cDNA was introduced into P. pastoris X-33 via electroporation using a Bio-Rad Gene Pulser electroporator. Transformants were plated on YPD plates supplemented with 500 μg Zeocin / mL. Colonies were transformed with KD1-Y11T / L17R-K in BMM medium. T Expression was assessed by SDS-PAGE. Fermentation inoculum shake flasks were prepared using buffered minimal glycerol medium (BMGY) pH 6.0. First, KD1-Y11T / L17R-K T A single colony expressing KD1-Y11T / L17R-K was inoculated into 50 mL for 12 h, and 0.5 mL of the resulting culture was transferred into 300 mL BMGY pH 6.0 for 20 h. The latter was then inoculated into a 15 L NLF BioEngineering Bioreactor (Wald, Switzerland) containing 3 L Basal Salts Medium (BSM) pH 5.0. Protein expression was induced with methanol and carried out for 48 h at 30° C., pH 5.0 and 40% dissolved oxygen. The fermentation broth was centrifuged at 7200 rpm and 4° C. T The supernatant containing the was collected and stored at -30°C until further processing.

[0094] 2.4. KD1-Y11T / L17R-K from Pichia pastoris T purification 100 mL of the fermentation supernatant was adjusted to pH 8.5 and centrifuged at 1500 RFC for 5 min at room temperature. The supernatant was then collected, adjusted to pH 3.0, and mixed with 0.5 mL Triton® X-100 for 30 min. Urea was added to a final concentration of 4 M and incubated at room temperature for 2.5 h. After incubation, the solution was diluted to a final conductivity of 12 mS. Purification was performed using a Biocad Vision workstation at a constant flow rate of 120 cm / h. The sample was loaded onto a SP-Sepharose (GE Healthcare Bio-Sciences Pittsburgh, PA) column pre-equilibrated with 50 mM phosphate buffer pH 2.8 (wash buffer). The column was then washed with 2 column volumes of wash buffer and the protein was eluted with 50 mM phosphate buffer pH 2.8 containing 1.0 M NaCl. KD1-Y11T / L17R-K T Fractions containing were pooled and supplemented with L-arginine to 0.5 M and mannitol to 7%. After incubation at room temperature for 1 h, it was dialyzed against 10 mM phosphate buffer pH 8.0 using 3.5 kDa MW cutoff membrane dialysis tubing (Spectrum, NJ).

[0095] 2.5. SDS-PAGE SDS-PAGE was performed using the Laemmle buffer system (37). The acrylamide concentration used was 15%, and gels were stained with Coomassie Brilliant Blue dye.

[0096] 2.6. Protease inhibition assay All reactions were carried out in TBS, pH 7.5 (50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl) containing 0.1 mg bovine serum albumin / ml (TBS / BSA) and 2 mM Ca2+ (TBS / BSA / Ca2+, pH 7.5). Each enzyme (plasmin, pKLK, FXIa, or FVIIa / sTF) was added at various concentrations (10-1 to 2 × 103 nM) of KD1-WT, KD1-L17R-K, or KD1-L17R-K. T, KD1-Y11T / L17R-K T or aprotinin (BPTI) for 1 h at room temperature in a 96-well microtiter plate (total volume 100 μl / well). Synthetic substrates appropriate for each enzyme (5 μl) were then added at a final concentration of 1K M The residual amidolytic activity was measured using a Vmax kinetic microplate reader (Molecular Devices). i * was determined using the nonlinear regression data analysis program Grafit. Aprotinin, KD1-WT, KD1-L17R-K T and KD1-Y11T / L17R-K T The data were analyzed with the equation for tight-binding inhibitors (Equation 1), where vi and v0 are the percent inhibition and non-inhibition, respectively, and (I)0 and (E)0 are the total concentrations of inhibitor and enzyme, respectively (38, 39).

number

number

[0097] 2.7. Preparation of DIP-δ-plasmin Active site-blocked delta plasmin was generated by treating delta plasmin with an equal volume of 1 M Tris-HCl, pH 8.0 and 1 M DFP (final concentration 1 mM DFP) for 20 min at room temperature, followed by incubation on ice for several hours. An equal volume of 1 M Tris-HCl, pH 8.0 and 1 M DFP (final concentration 2 mM) was added and the reaction was incubated for 20 min at room temperature and then overnight at 4° C. DFP-inhibited delta plasmin (DIP-delta plasmin) was dialyzed against 20 mM HEPES pH 7.5 containing 150 mM NaCl and assayed for residual activity using S-2251 synthetic substrate hydrolysis. Based on the residual activity, >99% of delta plasmin was inactivated. DIP-delta plasmin revealed no proteolysis when analyzed using SDS-PAGE.

[0098] 2.8. KD1-Y11T / L17R-K to tPA and DIP-δ plasmin using surface plasmon resonance (SPR) T join Binding studies were performed on a Biacore T100 flow biosensor (Biacore, Uppsala, Sweden) at 25°C. DIP-δ-plasmin (approximately 98% pure using SDS-PAGE) or tPA (>98% pure using SDS-PAGE) was immobilized on a carboxymethyl-dextran flow cell (CM5 sensor chip, GE Healthcare) using amine coupling chemistry. The flow cell surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide for 5 min (flow rate 10 μl / min) before the protein (20 μg / ml in 10 mM sodium acetate, pH 5.5) was injected over the surface. Unreacted sites were blocked with 1 M ethanolamine for 5 min. Analyte, KD1-Y11T / L17R-K T(100-2000 nM) was perfused through the flow cell for 6 min at 10 μl / min in HBS-P buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.005% (v / v) P20). After exchange into protein-free HBS-P buffer, analyte dissociation was monitored for 10 min. The flow cell was regenerated with HBS-P containing 20 mM EACA. Data were corrected for non-specific binding by subtracting the signal obtained with analyte injected through the flow cell without coupled protein. Binding was analyzed with BIAevaluation software (Biacore) using a 1:1 binding model. K d The value is the derived dissociation (k d ) and meeting (k a ) was calculated from the quotient of the rate constants.

[0099] 2.9. Fibrinolysis (Clot Lysis) Assay The method of Sperzel and Huetter (40) was followed with minor modifications as previously outlined (32,41). Briefly, IIa was used to initiate fibrin formation in NPPs, and dissolution of the formed clot (fibrinolysis) was induced by the simultaneous addition of tPA. Clot formation and lysis were monitored with a Molecular Devices microplate reader (SPECTRAmax 190) measuring optical density at 405 nm. Briefly, 10 μL of each test compound (KD1-L17R-K T , KD1-Y11T / L17R-K T, aprotinin) or saline control was added to 240 μL of NPP. 225 μL of this mixture was then added to 25 μL IIa and tPA in TBS / BSA containing 25 mM CaCl2. In a final volume of 250 μL, the concentration of IIa was 0.15 μg / ml and that of tPA was 1 μg / ml. Under control conditions (zero tPA and zero test compound), OD405 increased immediately, indicating that following clotting, a very slow decrease represents fibrinolysis. Fibrinolysis induced by tPA was assessed as a relative decrease in OD405 for up to 60 minutes, since clotting was nearly complete after 5 minutes. KD1-L17R-K T was tested at final concentrations ranging from 0.5 μM to 5 μM, while KD1-Y11T / L17R-K T and aprotinin were tested at final concentrations ranging from 0.5 μM to 3 μM.

[0100] 2.10. Thromboelastography Different concentrations of KD1-WT, KD1-L17R-K on fibrinolysis T , KD1-Y11T / L17R-K TThe effects of , aprotinin or EACA were assessed by thromboelastography (TEG) using a TEG 5000 Thrombelastograph (Haemonetics Corp, Braintree, MA). Each clot formation / lysis assay contained 300 μl of citrated whole blood; plasmin (1.5 μM final concentration), CaCl2 (10 mM final concentration) and various concentrations of each antifibrinolytic agent in Ringer's solution to a final volume of 360 μL. Plasmin and CaCl2 were added last to initiate simultaneous clotting and fibrinolysis. The 1.5 μM plasmin concentration was selected based on plasmin effects on clot strength and lysis. Each experiment was performed for 180 min to establish LY60 values. The thromboelastograph was calibrated each day and each inhibitor concentration was tested in duplicate. TEG Analytical Software (version 4.2.2; Haemonetics Corporation, Braintree, MA) was used to calculate the time to onset of clotting (R), maximum clot strength (maximum amplitude (MA), which was directly related to the shear modulus strength, G), and percent lysis after 60 min of MA (LY60) (42).

[0101] 2.11. Cytotoxicity Assay 2.11.1. Cells and culture conditions Primary human pooled umbilical vein endothelial cells (HUVECs) were obtained from ATCC. The cells were maintained in Vascular Cell Basal Medium (ATCC) supplemented with Endothelial Cell Growth Kit-BBE (ATCC) and penicillin-streptomycin-amphotericin B (ATCC). Primary human dermal fibroblasts were obtained from LONZA and maintained in Fibroblast Basal Medium (FBMTM, LONZA) supplemented with growth factor cocktail, fetal bovine serum, and antibiotics (FGMTM-2 SingleQuotsTM, LONZA). All cells were maintained at 37°C in a humidified 5% CO2 atmosphere and passaged once they reached 80% confluence. All experiments were performed on cells in logarithmic growth phase.

[0102] 2.11.2. Antifibrinolytic agents (KD1-Y11T / L17R-K T , aprotinin, EACA and TXA) Stock solutions of antifibrinolytic agents were prepared in phosphate buffer. For toxicity testing, cells were seeded into 96-well or 24-well cell culture plates at 3,500 cells / cm2 and used for experiments once the cells reached 80% confluence. Cells were incubated with antifibrinolytic agents for 24 hours at the following concentrations - aprotinin and KD1-Y11T / L17R-K. T at 0.1 μM, 1 μM, 10 μM and 30 μM; EACA at 1 mM, 5 mM, 20 mM and 60 mM, and TXA at ​​0.2 mM, 2 mM, 10 mM and 30 mM.

[0103] 2.11.3. Resazurin reduction assay Resazurin reduction assay (Fisher Scientific) was used to evaluate the potential cytotoxicity of antifibrinolytic agents on primary human endothelial cells and dermal fibroblasts. Phosphate buffer used to lyse samples was included as a negative control. The assay is based on the reduction of the non-fluorescent dye resazurin to the highly fluorescent resorufin by viable cells. The fluorescent signal is proportional to the number of viable cells, since non-viable cells cannot reduce the dye and do not produce a fluorescent signal. Briefly, cells in 96-well cell culture plates were treated with different concentrations of antifibrinolytic compounds (as described above). After 24 hours, resazurin reagent was added to each well and the plates were incubated at 37°C for 4 hours. Fluorescence was measured by FLUOstar Omega Microplate Reader (BMG Labtech) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was performed in duplicate with three replicates each. Viability was evaluated based on comparison with untreated cells.

[0104] 2.11.4. Caspase 3 / 7 Assay The effect of antifibrinolytic agents on cell apoptosis was detected using the Caspase-Glo 3 / 7 Assay kit (Promega). Caspases 3 and 7 are activated in cells undergoing apoptosis. The assay provides a luminescent substrate for caspases 3 and 7. Enzyme activity results in luminescence, which is proportional to the amount of caspase activity present. Cells were seeded in 96-well plates and treated with antifibrinolytic agents or phosphate buffer (solvent control). Taxol was included as a positive control. 24 hours after treatment, caspase reagent was added to each well, mixed, and incubated for 1 hour at room temperature. Luminescence was measured using a FLUOstar Omega Microplate Reader (BMG Labtech).

[0105] Cytotoxicity Assays Cytotoxicity and cell death measured using CellTox TM Cell death was assessed with the Green Cytotoxicity Assay (Promega). This assay measures the changes in membrane integrity that occur as a result of cell death. The dye used in this system is excluded from live cells, but binds to DNA in injured cells, resulting in a fluorescent signal. We measured cell death in HUVEC and primary fibroblasts treated with antifibrinolytic agents after 24 hours of exposure in 24-well plates at four concentrations (as described above) in triplicate per concentration. Hoechst (Thermo Fisher Scientific) was used to stain all nuclei. Images of cells were captured using an inverted microscope (Nicon; Edipse T2000 TE). Green fluorescent cells (FITC filter) and Hoechst stained cells (DAPI filter) were counted using Image J software. Fluorescent cells were expressed as a percentage of total cells.

[0106] 2.12. Statistical methods One-way analysis of variance (ANOVA) was used to evaluate the efficacy of antifibrinolytic agents (KD1-WT, KD1-L17R-KD1) in inhibiting fibrinolysis in the plasma clot lysis assay. T , KD1-Y11T / L17R-K T The effects of IL-1, IL-2, IL-1, IL-2, IL-1, and IL-1 (aprotinin) were compared. p values ​​for comparing any two means were calculated using post-hoc tests and adjusted for multiple comparisons using Tukey's adjustment. For TEG data, Levene's F-test revealed that homogeneity of variance was not met. Thus, Welch's F-test was used with Games-Howell post-hoc procedure to determine which pairs of mean MA and mean LY60% levels were significantly different. For cytotoxicity assays, pooled data sets were analyzed by ANOVA and individual groups were compared using Student's t-test. All experiments were repeated two or three times with similar results. Quantitative values ​​are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM) as indicated in the figure legends. A p value of 0.05 or less was considered statistically significant. All statistical analyses were performed using SPSS V27 (IBM Corp., Armonk, NY, USA).

[0107] 2.13. Molecular Modeling Using the crystal structures of δ-plasmin (43), plasminogen kringle domain 1 (14), and wild-type KD1 (36) as templates, we synthesized KD1-Y11T / L17R-K T We modeled the complexes of KD1 with δ-plasmin and with plasmin kringle domain 1. Protocols for modeling these complexes have been described previously (41,44). Because the C-terminal residues are disordered in the wild-type KD1 crystal structure, we used the MODELLER program (45) to model KD1-Y11T / L17R-K T This part of the molecule was constructed using the AMBER program (46) to calculate the 10 kcal.mol -1 .Å -2 This was further refined by subjecting it to 1000 steps of minimization with the harmonic constraints.

[0108] 3. Results 3.1. KD1-L17R-K in E. coli T and KD1-Y11T / L17R-K T Expression and purification 60 residues His6 tagged KD1-L17R-K T and KD1-Y11T / L17R-K T were expressed in E. coli strain BL21(DE3) pLysS with an enterokinase cleavage site (Figure 10). These constructs were expressed as KD1-L17R with IEKVPK at the C-terminus (KD1-L17R-K COOH ), which is 9 residues shorter at the N-terminus and is IEK T The fusion protein was refolded and purified using a Q-Sepharose FF column. The purified KD1 mutant protein was incubated with enterokinase to remove the His6 tag; however, cleavage was unsuccessful at a 1:50 enzyme:substrate ratio. The reason for the unsuccessful His6 tag removal may be due to inhibition of enterokinase by the KD1 mutant, similar to that described for the inhibition of enterokinase by aprotinin (47). Purified KD1-L17R-K T and KD1-Y11T / L17R-K T (each containing an enterokinase cleavage site and a His6 tag at the NH2 terminus) is shown in FIG.

[0109] 3.2. KD1-Y11T / L17R-K in P. pastoris T Expression and purification Since the His6 tag could not be removed by enterokinase in the E. coli expressed mutant, we used P. pastoris to express the 60 residue double mutant KD1-Y11T / L17R-K T was expressed and purified to homogeneity as described in the experimental section. Approximately 50 mg of KD1-Y11T / L17R-KT was purified from 100 ml of culture medium. Purified P. pastoris KD1-Y11T / L17R-K T The SDS-PAGE analysis of P. pastoris expressing KD1-Y11T / L17R-K is shown in FIG. T but the corresponding E. coli expression KD1-Y11T / L17R-K containing a His6 tag and an enterokinase cleavage sequence T Note the slightly lower MW when compared to (Figure 11).

[0110] 3.3. KD1-L17R-K T and KD1-Y11T / L17R-K T Inhibition profile of Wild-type KD1 (KD1-WT), which contains the IIa cleavage site, binds plasmin to K i KD1-L17R-K, which has the IEK C-terminus containing the enterokinase cleavage site, inhibited the IEK-dependent cleavage at 6.0±0.5 nM (32, FIG. 12A). T The previously described KD1-L17R-K has a VPK C-terminus that contains the cleavage site. COOH Similar to plasmin, i Both E. coli and P. pastoris expressing KD1-Y11T / L17R-K T Aprotinin (K i 0.49±0.1) and inhibited plasmin with similar affinity (K i 0.59 ± 0.1) (Figure 12A). The K i The values ​​are provided in Table 1. Thus, the enterokinase cleavage sequence and the His6 tag do not affect the inhibitory activity. COOH Similar to (32,41), KD1-Y11T / L17R-K T (Example 1) weakly inhibited FVIIa / sTF, FXIa and pKLK with Ki>3 μM (FIG. 12B). [Table 1]

[0111] 3.4. KD1-Y11T / L17R-K to DIP-δ plasmin and tPA T join We used SPR to detect the binding of KD1-Y11T / L17R-K to immobilized DIP-δ plasmin (FIG. 13A) and tPA (FIG. 13B). T The binding of KD1-Y11T / L17R-K T k for DIP-δ plasmin binding to on is 1.49±0.3×10 3 M -1 s -1 It was;k off is 7.13±0.9×10 -5 s -1 And K d was 47.6±7nM. T k for tPA binding to on is 2.91±0.4×10 3 M -1 s -1 It was;k off is 1.05±0.7×10 -4 s -1 And K d was 35.4±5 nM.

[0112] 3.5. Fibrinolysis (clot lysis) assay These experiments were carried out to evaluate the efficacy and safety of KD1-L17R-K in inhibiting tPA-induced plasma clot fibrinolysis. T , KD1-Y11T / L17R-K TThe effectiveness of IIa, OD405, and aprotinin was compared. Addition of IIa to NPP caused fibrin formation, reflected by an increase in OD405 (curve IIa, zero tPA, Figure 14A-C). Concurrent addition of tPA caused initial clot formation followed by dissolution of fibrin induced by tPA-mediated conversion of plasminogen to plasmin (curve IIa, tPA; Figure 14A-C); the midpoint of fibrinolysis was between 6 and 7 min in each case in the absence of fibrinolysis inhibitors. All three agents inhibited fibrinolysis in a dose-dependent manner. The maximum OD405, OD405 at 60 min and time to reach the midpoint of fibrinolysis for each concentration of inhibitor used are provided in Table 2. The maximum OD405 did not differ between the inhibitors and between the concentrations of inhibitor used. The maximum OD405 reflects the IIa-inducible strength of the fibrin clot that formed, which was rapidly achieved before subsequent lysis was initiated by tPA-generated plasmin at the clot site. Therefore, it is expected that the maximum OD405 at the different concentrations of each inhibitor used is similar. Furthermore, the OD405 at 60 minutes, which indicates the degree of fibrinolysis, was relatively similar at low concentrations for each inhibitor; however, it was significantly higher for KD1-L17R-K when compared to aprotinin at higher concentrations. T Regarding the KD1-Y11T / L17R-K, T was moderately reduced (Figure 14, Table 2).

[0113] Importantly, KD1-L17R-K T increased the fibrinolysis midpoint from approximately 7 to 10 min at 0.5 μM, approximately 13 min at 1 μM, approximately 17 min at 1.5 μM, approximately 31 min at 3 μM, approximately 43 min at 4 μM, and approximately 55 min at 5 μM (Figure 14A, Table 2). TKD1-Y11T / L17R-K increased the midpoint of fibrinolysis from 7 to 12 min at 0.5 μM, 28 to 28 min at 1 μM, 43 to 43 min at 1.5 μM, and >60 min at 2 and 3 μM, respectively (FIG. 14B, Table 2). Aprotinin increased the midpoint of fibrinolysis from 7 to 13 min at 0.5 μM, 40 to 40 min at 1 μM, and >60 min at 1.5 and >1.5 μM concentrations, respectively (FIG. 14C, Table 2). Cumulatively, the statistical analysis presented in FIG. 15 indicates that KD1-Y11T / L17R-K increased the midpoint of fibrinolysis from 7 to 12 min at 0.5 μM, 40 to 43 min at 1.5 μM, and >60 min at 1.5 and >1.5 μM concentrations, respectively (FIG. 14C, Table 2). T But, KD1-L17R-K T Aprotinin was more effective in increasing the fibrinolysis midpoint compared with KD1-Y11T / L17R-K. T The results revealed that the 10-fold increase in the 10-fold increase in the 10-fold increase was slightly more effective than the 10-fold increase in ....

[0114] 3.6. Thromboelastography Thromboelastography experiments were performed to evaluate the effect of KD1-WT(31), KD1-L17R-K on plasmin-induced lysis of clots formed in whole blood by the addition of CaCl2. T , KD1-Y11T / L17R-K T The effects of aprotinin and EACA were evaluated. These data are shown in FIG. 16 and summarized in Table 3. FIG. 16A shows TEG traces at various concentrations of plasmin on CaCl2-initiated clot formation. In the absence of plasmin, the mean maximum amplitude (MA) achieved was about 47 mm at a shear modulus intensity G of about 4620 dyn / cm2, and no clot lysis was detected in 60 min (LY60<0.1%). At 1.5 μM plasmin, the MA reached was about 7 mm at a G value of about 401 dyn / cm2, and 100% clot lysis occurred within 30 min (Table 3). At >1.5 μM plasmin, no clot formation was observed. FIG. 16B-F shows the effect of KD1-WT, KD1-L17R-K on clot formation and lysis in the presence of 1.5 μM plasmin. T , KD1-Y11T / L17R-K TThe mean TEG tracings are illustrated for various concentrations of KD1-Y11T / L17R-K and aprotinin (1 μM to 7.5 μM). The data show that all antifibrinolytic drugs tested improved clot firmness (MA), shear strength (G), and inhibited fibrinolysis in a concentration-dependent manner (Table 3). Notably, at an inhibitor concentration of 5 μM (corresponding to the high dose of the Hammersmith regimen, an established clinical dosing regimen for aprotinin) (33), KD1-Y11T / L17R-K improved fibrinolysis. T KD1-Y11T / L17R-K improved clot strength MA to about 80% (37.5 mm) and G to about 65% (about 3004 dyn / cm2), whereas aprotinin improved MA to about 69% (32.9 mm) and G to about 53% (about 2453 dyn / cm2). However, about 12% of LY60 was improved with KD1-Y11T / L17R-K compared to 0.2% with aprotinin. T At 7.5 μM, KD1-Y11T / L17R-K T Both KD1-Y11T / L17R-K and aprotinin had similar MA (-83% and -80%) and G (-70% and -65%), and LY60 (0.2% each). EACA also improved MA, G, and LY60 in a dose-dependent manner (Figure 16G). However, at a 3 mM concentration of EACA (the dose used in the clinical setting), it only improved MA and G by -67% and -50%, respectively. Cumulatively, the TEG data suggest that EACA significantly reduced MA and G by -67% and -50%, respectively, in the absence of KD1-Y11T / L17R-K. T Furthermore, KD1-WT and KD1-L17R-K are not as effective as aprotinin. T Also available is KD1-Y11T / L17R-K T Importantly, at higher concentrations (≥7.5 μM), KD1-Y11T / L17R-K was not as effective as aprotinin. T restored MA and G and inhibited fibrinolysis similarly to aprotinin.

[0115] In TEG experiments, KD1-WT, KD1-L17R-K T , KD1-Y11T / L17R-KT Multiple comparison analyses performed on the concentration-dependent enhancement of maximum amplitude (MA), shear modulus strength (G), and LY60 by aprotinin are shown in Figures 17-19. At 1 μM inhibitor concentration, MA was not significantly different between the control and each Kunitz inhibitor, except for KD1-WT (Figure 17A). At inhibitor concentrations of 2 μM or 3 μM, MA enhancement by aprotinin was significantly higher for KD1-WT, KD1-L17R-K, and KD1-L17R-K. T and KD1-Y11T / L17R-K T The effect was statistically significant (p<0.05) when compared with KD1-L17R-K (FIGS. 17B-C). Above 3 μM, the enhancement of MA was T , KD1-Y11T / L17R-K T and aprotinin; however, it was significantly lower for KD1-WT (FIG. 17D-E). For EACA, a Student's t-test was performed to compare MA between the control and each EACA concentration tested (FIG. 17F). At 500 μM, 1000 μM, or 3000 μM EACA concentrations, MA enhancement was statistically significant when compared to the control.

[0116] Notably, up to 3 μM, aprotinin significantly enhanced G compared to the KD1-based inhibitor (FIGS. 18A-C). Surprisingly, at inhibitor concentrations of 5 μM and 7.5 μM, KD1-Y11T / L17R-K T The enhancement of G by KD1-Y11T / L17R-K was significantly higher when compared to other inhibitors (Figure 18D-E). T This observed improvement in clot shear strength G versus aprotinin is likely due to the KD1-Y11T / L17R-K TThis may be due to inhibition of FXIa and kallikrein by aprotinin, whereas essentially no inhibition by KD1. Furthermore, multiple comparison analysis of LY60 for each inhibitor at selected concentrations is shown in FIG. 19. At 1 μM or 5 μM, aprotinin was significantly better at preventing fibrinolysis when compared to each KD1 inhibitor, whereas KD1-WT was inferior to each inhibitor at all concentrations tested. At 2 μM or 3 μM, KD1-Y11T / L17R-K was significantly better at preventing fibrinolysis than each inhibitor. T and aprotinin, KD1-L17R-K T At 7.5 μM concentration, no LY60 was observed with any of the inhibitors. Overall, aprotinin and KD1-Y11T / L17R-K T appears to be superior to other inhibitors in inhibiting fibrinolysis in TEG experiments.

[0117] 3.7. Cytotoxicity Testing Here, we compare KD1-Y11T / L17R-K with aprotinin and the currently used antifibrinolytic agents EACA and TXA. T We wanted to gain insight into the potential toxicity of antifibrinolytic agents. Patients are typically treated with antifibrinolytic agents via intravenous injection while undergoing major surgery, or via topical application in trauma situations. We thus tested cytotoxicity in endothelial cells and dermal fibroblasts; the cells were KD1-Y11T / L17R-K TThe patients are most likely to be exposed to therapeutic doses of TXA. The plasma half-life of TXA in humans, rats and dogs is about 120 min (48). The half-life of each of its two KD1 variant homologs (aprotinin and ecallantide) is also about 120 min in humans (49,50), whereas the half-life of aprotinin in mice, rats or dogs is about 70 min (51). The half-life of each KD1 variant is not known, but is likely to be short and will be determined in a planned manner. Since the in vivo half-life of each of the antifibrinolytic agents is short, the infusion is usually continuous for the entire duration of the operation. Therefore, the treatment duration was set at 24 h, and the dose range selected includes the equivalent of about three times the clinical dose for each of the tested agents.

[0118] KD1-Y11T / L17R-K T Resazurin assays of HUVECs treated for 24 hours with or without aprotinin did not produce any significant changes in cell viability compared to cells treated with phosphate buffer control over the entire dose range of 0.1 μM to 30 μM (Figure 11A in Vadivel et al., J Clin Med. 2020 Nov 17;9(11):3684. doi:10.3390 / jcm9113684). The same results were obtained after treatment with EACA (dose range 1-60 mM) and TXA (dose range 0.2-30 mM). Cell viability was equally unchanged in primary human dermal fibroblasts (Figure 11B in Vadivel et al., J Clin Med. 2020 Nov 17;9(11):3684. doi:10.3390 / jcm9113684). This indicates that none of the antifibrinolytic agents tested caused measurable cytotoxicity within the 24 hour duration of treatment.

[0119] Viability is an endpoint of cell injury. Therefore, we tested the induction of apoptosis resulting from caspase activation. Caspase 3 / 7 assays were performed in HUVEC cells after treatment with antifibrinolytic agents (Vadivel et al., J Clin Med. 2020 Nov 17;9(11):3684. Figure 11C in doi:10.3390 / jcm9113684). Caspase 3 / 7 activity was significantly increased when the cells were treated with the two higher concentrations of TXA (10 mM and 30 mM) and to a lesser extent after exposure to EACA (20 mM and 60 mM). In contrast to TXA and EACA, KD1-Y11T / L17R-K T and aprotinin did not induce caspase activity above baseline at any concentration. Taxol was included as a positive control. None of the antifibrinolytic drugs increased caspase activity above baseline in primary fibroblasts across all doses.

[0120] To confirm the above results using a different assay, we performed a CellTox green cytotoxicity assay in HUVEC cells and primary fibroblasts. The CellTox green dye binds to DNA and produces fluorescent staining only when membrane integrity is impaired. KD1-Y11T / L17R-K was found to be cytotoxic in both endothelial cells (Vadivel et al., J Clin Med. 2020 Nov 17;9(11):3684. doi:10.3390 / jcm9113684, Fig. 11D) and fibroblasts (Vadivel et al., J Clin Med. 2020 Nov 17;9(11):3684. doi:10.3390 / jcm9113684, Fig. 11E). T No significant increase in the percentage of fluorescent cells was detected 24 hours after treatment with the highest dose (30 μM) of . Cell cytotoxicity also did not increase significantly above baseline when cells (HUVEC or fibroblasts) were treated with other antifibrinolytic agents tested. For brevity, data for aprotinin, TXA and EACA are not shown.

[0121] In summary, 0.1 to 30 μM KD1-Y11T / L17R-K T Treatment of HUVEC cells and primary human fibroblasts with TXA or aprotinin for 24 hours did not reduce viability, induce apoptosis, or show any signs of cytotoxicity. However, TXA and EACA induced apoptosis (cell death) at higher concentrations in HUVEC cells, as inferred from an increase in caspase 3 / 7 activity.

[0122] 4. Discussion Previously, based on structural information and S2'-subsite specificity, we designed a 73-residue Kunitz domain plasmin inhibitor from TFPI-2 KD1 (32). KD1-WT inhibits plasmin, as well as pKLK, FXIa, and FVIIa / TF with comparable affinity, whereas KD1-L17R inhibits only plasmin. A Leu to Arg change at residue 17 (BPTI numbering) made KD1-L17R specific for plasmin and dramatically reduced pKLK and FXIa inhibition. The current 60-residue KD1-L17R-K T Compared to the previously expressed KD1-L17R, the previously expressed KD1-L17R had 13 additional residues at the N-terminus (9 from the TFPI-2 sequence and 4 from the IIa cleavage site) and 4 residues (VPKV) at the C-terminus, distant from the core Kunitz domain. These additional residues do not interfere with KD1-L17R function, but they are flexible and may be disordered as inferred from the crystal structure of KD1-WT (36). Thus, the new 60-residue KD1-L17R-K T The mutants were expressed and their inhibition profiles were characterized. T None of the active site inhibition profiles of KD1-L17R-KD1 were altered from the previously expressed 73 residue KD1-L17R. T is highly effective in reducing blood loss and appears to be comparable to aprotinin in the two mouse bleeding models tested (liver laceration and tail cut) (32,41,52).

[0123] The 73-residue KD1-L17R has an IEKVPKV at the C-terminus, and the valine can be removed by prolonged incubation with IIa (41). Removal of the Val residue at the C-terminus results in a C-terminal lysine, which makes KD1-L17R a dual-reactive inhibitor of fibrinolysis by inhibiting the plasmin active site and plasminogen activation (41). Furthermore, prolonged incubation with IIa produced a heterogeneous population of KD1-L17R with different N-terminal residues (41). Structural analysis of a modeled complex of plasmin and KD1-L17R showed that the change of residue Tyr11 to Thr is beneficial for plasmin inhibition. KD1-Y11T / L17R-K T The threonine in makes an additional hydrogen bond with residue Q192 of plasmin (Figure 9A). Interestingly, the 73-residue KD1-L17R contains two lysine residues in the C-terminal segment (IEKVPKV), either of which could serve as the C-terminal residue. Furthermore, the 60-residue KD1-L17R-KD1 with the C-terminal IEK sequence T Modeling of IEK shows that it enhances interactions with the kringle domains of plasminogen and tPA (FIGS. 9A and B). Without being bound by any particular theory or mechanism of action, it is believed that, compared to the VPK sequence, the IEK sequence has two additional interactions resulting from Arg57 and Glu59 of the Kunitz domain and plasmin kringle residues Glu151 and Arg153, respectively (FIG. 9B). Similar interactions are presumed to occur with the kringle domain of tPA as well.

[0124] New E. coli expression KD1-L17R-K T and KD1-Y11T / L17R-K with the C-terminal IEK sequence T Both KD1-L17R-KD1-L17R constructs contain a His6 tag and an enterokinase cleavage sequence; however, these additional residues could not be removed by enterokinase. Similar to the 73 residue KD1-L17R construct, the presence of the additional residuesT and KD1-Y11T / L17R-K T The 60 residues KD1-Y11T / L17R-K did not affect the inhibitory properties of the mutant. T was expressed in P. pastoris. As expected, KD1-Y11T / L17R-K T is KD1-L17R-K T In addition, the IEK C-terminal 60 residue KD1-Y11T / L17R-KD1 inhibited plasmin with increased affinity (0.59 nM vs. 0.9 nM) compared to the control. T KD1-L17R-K with C-terminal VPK COOH KD1-Y11T / L17R-K binds to the kringle domains of tPA and plasmin with increased affinity (35 nM-50 nM) compared to the kringle domains of plasminogen and tPA (Figure 13) (41). The modest increase in plasmin active site inhibition and significantly improved affinity for the kringle domains of plasminogen and tPA were confirmed by KD1-Y11T / L17R-K in plasma clot lysis assays. T This was reflected in the strong inhibition of fibrinolysis by IL-1 (Fig. 14B) and the recovery of MA, G and LY60 in TEG experiments (Fig. 16B-F).

[0125] A KD1 double mutant (KD1-Y11T / L17R-K) was generated in P. pastoris. T ) is a small, homogeneous, and effective specific plasmin inhibitor of human origin. T The properties of KD1-Y11T / L17R-K are comparable to those of aprotinin in plasmin inhibition assays, plasma clot lysis assays and TEG experiments. T does not inhibit pKLK, FXIa, and FVIIa / sTF. Tdid not induce any measurable cytotoxicity in primary endothelial cells or dermal fibroblasts. However, TXA and EACA caused apoptosis in these cells at higher concentrations, which may be achieved during renal clearance of these antifibrinolytic drugs. These results indicate that KD1-L17R-K did not induce nephrotoxicity, seizures, or any detectable histopathological changes in monkey kidneys. COOH (C-terminal VPK) single variant (32). In the case of aprotinin, its acidic nature and pKLK inhibition lead to altered renal activity, which results in renal damage (32, 53). Current antifibrinolytic drugs, EACA and TXA, cause stroke by inhibiting glycine receptors (54). Because lysine analogs are not as effective as aprotinin, higher doses of EACA and TXA increase the risk of renal failure, as these drugs reach very high concentrations during clearance by glomerular filtration (55, 56). The KD1Y11T / L17R-K variant found in this disclosure T The data are encouraging; however, it needs to be evaluated in an appropriate animal bleeding model before it can be considered for clinical trials. [Table 2] [Table 3]

[0126] Example 1 References 1. Kashuk, JL; Moore, EE; Sawyer, M.; Wohlauer, M.; Pezold, M.; Barnett, C.; Biffl, WL; Burlew, CC; Johnson, JL; Sauaia, A. Primary fibrinolysis is integral in the pathogenesis of the acute coagulopathy of trauma. Ann. Surg. 2010, 252, 434-442. 2. Raza, I.; Davenport, R.; Rourke, C.; Platton, S.; Manson, J.; Spoors, C.; Khan, S.; De’Ath, H.D.; Allard, S.; Hart, D.P.; Pasi, K.J.; Hunt, B.J.; Stanworth, S.; MacCallum, P.K.; Brohi, K. The incidence and magnitude of fibrinolytic activation in trauma patients. J. Thromb. Haemost. 2013, 11, 307-314. 3. Cardenas, J.C.; Wade, C.E.; Holcomb, J.B. Mechanisms of trauma induced coagulopathy. Curr. Opin. Hematol. 2014, 21, 404-409. 4. Evans, J.A.; van Wessem, K.J.; McDougall, D.; Lee, K.A.; Lyons, T.; Balogh, Z.J. Epidemiology of traumatic deaths: comprehensive population-based assessment. World J. Surg. 2010, 34, 158-163. 5. Oyeniyi, B.T.; Fox, E.E.; Scerbo, M.; Tomasek, J.S.; Wade, C.E.; Holcomb, J.B. Trends in 1029 trauma deaths at a level 1 trauma center: Impact of a bleeding control bundle of care. Injury 2017, 48, 5-12. 6. Ker, K.; Roberts, I.; Shakur, H.; Coats, T.J. Antifibrinolytic drugs for acute traumatic injury. Cochrane Database Syst. Rev. 2015, CD004896. 7. Koster, A.; Faraoni, D.; Levy, J.H. Antifibrinolytic therapy for cardiac surgery: an update. Anesthesiology 2015, 123, 214-221. 8. Royston, D.; Van Haaften, N.; De Vooght, P. Aprotinin; friend or foe? A review of recent medical literature. Eur. J. Anaesthesiol. 2007, 24, 6-14. 9. Mangano, D.T.; Tudor, I.C.; Dietzel, C.; Multicenter Study of Perioperative Ischemia Research Group; Ischemia Research and Education Foundation. The risk associated with aprotinin in cardiac surgery. N. Engl. J. Med. 2006, 354, 353-365. 10. Immer, F.F.; Jent, P.; Englberger, L.; Stalder, M.; Gygax, E.; Carrel, T.P.; Tevaearai, H.T. Aprotinin in cardiac surgery: a different point of view. Heart Surg. Forum. 2008, 11, E9-12. 11. 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Crystal structures of the recombinant kringle 1 domain of human plasminogen in complexes with the ligands epsilon-aminocaproic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Biochemistry 1996, 35, 2567-2576. 15. Tempe, D.K.; Hasija, S. Are tranexamic acid and ε-aminocaproic acid adequate substitutes for aprotinin?. Ann. Card. Anaesth. 2012, 15, 4-5. 16. Martin, K.; Gertler, R.; Liermann, H.; Mayr, N.P.; MacGuill, M.; Schreiber, C.; Vogt, M.; Tassani, P.; Wiesner, G. Switch from aprotinin to ε-aminocaproic acid: impact on blood loss, transfusion, and clinical outcome in neonates undergoing cardiac surgery. Br. J. Anaest. 2011, 107, 934-939. 17. Martin, K.; Knorr, J.; Breuer, T.; Gertler, R.; MacGuill, M.; Lange, R.; Tassani, P.; Wiesner, G. Seizures after open heart surgery: Comparison of epsilon-aminocaproic acid and tranexamic acid. J. Cardiothorac. Vasc. Anesth. 2011, 25, 20-25. 18. Markland, W.; Ley, A.C.; Lee, S.W.; Ladner, R.C. Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin. Biochemistry 1996, 35, 8045-8057. 19. Flight, S.M.; Johnson, L.A.; Du, Q.S.; Warner, R.L.; Trabi, M.; Gaffney, P.J.; Lavin, M.F.; de Jersey, J.; Masci, P.P. Textilinin-1, an alternative anti-bleeding agent to aprotinin: importance of plasmin inhibition in controlling blood loss. Br. J. Haematol. 2009, 145, 207-211. 20. Dietrich, W.; Nicklisch, S.; Koster, A.; Spannagl, M.; Giersiefen, H.; van de Locht, A. CU-2010 - a novel small molecule protease inhibitor with antifibrinolytic and anticoagulant properties. Anesthesiology 2009, 110, 123-130. 21. Swedberg, J.E.; Harris, J.M. Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors. Biochemistry 2011, 50, 8454-8462. 22. Saupe, S.M.; Leubner, S.; Betz, M.; Klebe, G.; Steinmetzer, T. Development of new cyclic plasmin inhibitors with excellent potency and selectivity. J. Med. Chem. 2013, 56, 820-831. 23. Al-Horani, R.A.; Desai, U.R. Recent advances on plasmin inhibitors for the treatment of fibrinolysis-related disorders. Med. Res. Rev. 2014; 34: 1168-216. 24. de Veer, S.J.; Wang, C.K.; Harris, J.M.; Craik, D.J.; Swedberg, J.E. Improving the selectivity of engineered protease inhibitors: optimizing the P2 prime residue using a versatile cyclic peptide library. J. Med. Chem. 2015, 58, 8257-8268. 25. Swedberg, J. E.; Wu, G.; Mahatmanto, T.; Durek, T.; Caradoc-Davies, T.T.; Whisstock, J.C.; Law, R.; Craik, D.J. Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma. J. Med. Chem. 2019, 62, 552-560. 26. Earl, S.T.; Masci, P.P.; de Jersey, J.; Lavin, M.F.; Dixon, J. Drug development from Australian elapid snake venoms and the Venomics pipeline of candidates for haemostasis: Textilinin-1 (Q8008), Haempatch TM (Q8009) and CoVase TM (V0801). Toxicon. 2012, 59, 456-463. 27. Cheng, L.; Pettersen, D.; Ohlsson, B.; Schell, P.; Karle, M.; Evertsson, E.; Pahlen, S.; Jonforsen, M.; Plowright, A.T.; Bostroem, J.; Fex, T.; Thelin, A.; Hilgendorf, C.; Xue, Y.; Wahlund, G.; Lindberg, W.; Larsson, L. O.; Gustafsson, D. Discovery of the fibrinolysis inhibitor AZD6564, acting via interference of a protein-protein interaction. ACS Med. Chem. Lett. 2014, 5, 538-543. 28. Jankun, J.; Keck, R.; Selman, S.H; Skrzypczak-Jankun, E. Systemic or topical application of plasminogen activator inhibitor with extended half-life (VLHL PAI-1) reduces bleeding time and total blood loss. Int. J. Mol. Med. 2010, 26, 501-504. 29. Devy, L.; Rabbani, S.A.; Stochl, M.; Ruskowski, M.; Mackie, I.; Naa, L.; Toews, M.; van Gool, R.; Chen, J.; Ley, A.; Ladner, R.C.; Dransfield, D.T.; Henderikx, P. PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor. Neoplasia 2007, 9, 927-937. 30. Bokesch, P.M.; Szabo, G.; Wojdyga, R.; Grocott, H.P.; Smith, P.K.; Mazer, C.D.; Vetticaden, S.; Wheeler, A.; Levy, J.H. A phase 2 prospective, randomized, double-blind trial comparing the effects of tranexamic acid with ecallantide on blood loss from high-risk cardiac surgery with cardiopulmonary bypass (CONSERV-2 Trial). J. Thorac. Cardiovasc. Surg. 2012, 143, 1022-1029. 31. Englberger, L.; Dietrich, W.; Eberle, B.; Erdoes, G.; Keller, D.; Carrel, T. A novel blood-sparing agent in cardiac surgery? First inpatient experience with the synthetic serine protease inhibitor MDCO-2010: a phase II, randomized, double-blind, placebo-controlled study in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass. Anesth. Analg. 2014, 119, 16-25. 32. Bajaj, M.S.; Ogueli, G.I.; Kumar, Y.; Vadivel, K.; Lawson, G.; Shanker, S.; Schmidt, A.E.; Bajaj, S.P. Engineering Kunitz domain 1 (KD1) of human tissue factor pathway inhibitor-2 to selectively inhibit fibrinolysis: properties of KD1-L17R variant. J. Biol. Chem. 2011, 286, 4329-4340. 33. Royston, D.; Bidstrup, B.D.; Taylor, K.M.; Sapsford, R.N. Aprotinin decreases the need for post-operative blood transfusions in patients having open heart surgery. Bibl. Cardiol. 1988, 43, 73-82. 34. Bajaj, S.P.; Schmidt, A.E.; Agah, S.; Bajaj, M.S.; Padmanabhan, K. High resolution structures of p-aminobenzamidine- and benzamidine-VIIa / soluble tissue factor: unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+, and Zn2+ sites in factor VIIa. J. Biol. Chem. 2006, 281, 24873-24888. 35. Sambrook, J.; Russel, D.W. Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001. 36. Schmidt, A.E.; Chand, H.S.; Cascio, D.; Kisiel, W.; Bajaj, S.P. Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition. J. Biol. Chem. 2005, 280, 27832-27838. 37. Laemmli, U.K. Cleavage of structural proteins during assembly of head of bacteriophage-t4. Nature 1970, 227, 680-685. 38. Beith, J.G. In vivo significance of kinetic constants of protein proteinase inhibitors. Biochem. Med. 1984, 32, 387-397. 39. Morrison, J.F.; Walsh, C.T. The behavior and significance of slowbinding enzyme inhibitors. Adv. Enzymol. Relat. Areas Mol. Biol. 1988, 61, 201-301. 40. Sperzel, M.; Huetter, J. Evaluation of aprotinin and tranexamic acid in different in vitro and in vivo models of fibrinolysis, coagulation and thrombus formation. J. Thromb. Haemost. 2007, 5, 2113- 2118. 41. Kumar, Y.; Vadivel, K.; Schmidt, A.E.; Ogueli, G.I.; Ponnuraj, S.M.; Rannulu, N.; Loo, J.A.; Bajaj, M.S.; Bajaj, S.P. Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain. 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Current Protocols in Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30. 46. ​​Case, DA; Betz , RM ; Botello-Smith, W.; Cerutti , DS ; Cheatham III, TE; Darden, TA; Duke, RE; Giese , TJ ; Gohlke , H. ; Goetz , AW ; Homeyer, N.; Izadi, S.; Janowski , P. ; Kaus, J.; Kovalenko, A.; Lee, TS; LeGrand , S. ; Li, P.; Lin , C. ; Luchko , T. ; Luo, R.; Madej, B.; Mermelstein , D. ; Merz , KM ; Monard, G.; Nguyen , H. ; Nguyen , HT ; Omelyan, I.; Onufriev, A.; Roe, DR; Roitberg , A. ; Sagui, C.; Simmerling , CL ; Swails , J. ; Walker , RC ; Wang, J.; Wolf , RM ; Wu, X.; Xiao, L.; York , DM ; Kollman, PA AMBER, University of California, San Francisco, USA, 2018. 47. Light, A.; Fonseca, P. The Preparation and Properties of the Catalytic Subunit of Bovine Enterokinase. J. Biol. Chem. Rev. 1984, 259, 13195–13198. 48. Australian public assessment report for tranexamic acid. Cyclocapran. https: / / www.tga.gov.au / sites / default / files / auspar-cyclocapron.pdf Accessed 4 October 49. Trasylol. http: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2006 / 020304s022lbl.pdf Accessed 4 October 2020. 50. Farkas, H.; Varga, L. (2011) Ecallantide is a novel treatment for attacks of hereditary angioedema due to C1 inhibitor deficiency. Clin. Cosmet. Investig. Dermatol. 2011, 4, 61-68. 51. Markwardt, F. Naturally occurring inhibitors of fibrinolysis. Fibrinolytics and Anti-fibrinolytics, Ed. F. Markwardt, Springer-Verlag, 1978, pages 487-509. 52. Vadivel, K.; Kumar, Y.; Ogueli, G.I.; Ponnuraj, S.M.; Wongkongkathep, P.; Loo, J.A.; Bajaj, M.S.; Bajaj SP. S2'-subsite variations between human and mouse enzymes (plasmin, factor XIa, kallikrein) elucidate inhibition differences by tissue factor pathway inhibitor -2 domain1-wild-type, Leu17Arg-mutant and aprotinin. J. Thromb. Haemost. 2016, 14, 2509-2523. 53. 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[0127] Example 2: A novel plasmin-specific Kunitz inhibitor polypeptide containing the 60-residue KD1-Y11T / R15K / L17R triple mutant (rHUKD1-TM) with an unexpected functional profile The antifibrinolytic polypeptide variants previously tested were single and double mutants of the Kunitz domain 1 (KD1) of human tissue factor pathway inhibitor type 2 (KD1 L17R , KD1 Y11T / L17R ) (see, e.g., U.S. Patent No. 7,585,842 and U.S. Patent Publication Nos. 20080026998 and 20140288). These were successful in preventing blood loss in two mouse injury models (liver laceration and tail cut models). However, the efficacy of these polypeptide variants has limitations, for example, in that they show somewhat reduced activity in plasma clot lysis assays over extended incubation with tissue plasminogen activator when compared to similar tests with aprotinin.

[0128] In an attempt to increase the potency of such variants in plasma clot lysis assays, additional Kunitz domain 1 (KD1) variants of human tissue factor pathway inhibitor type 2 were generated and tested. As part of these studies, a truncated triple mutant (KD1) with a unique set of amino acid mutations was generated. Y11T / R15K / L17R ) was developed. This variant contains: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1). Below is provided a sequence comparison of single, double and triple KD1 mutants and their characteristics. [ka]

[0129] The KD1 triple mutant (sequence NH2-1NAEI …The IEK60-COOH (60 residues, BPTI numbering) was expressed in Pichia and purified, revealing a molecular weight of 7.1 KDa (Figure 1). As noted above, this variant was found to be a potent inhibitor of plasmin (Figure 2), similar to aprotinin, without the adverse effects of inhibiting other coagulation serine proteases, including kallikrein (Figure 2). Thus, compared to aprotinin, which is a broad specificity protease inhibitor, the KD1 triple mutant disclosed herein is of human origin and highly specific for inhibiting plasmin.

[0130] The data presented in Tables 4-6 below illustrate certain unexpected pharmacokinetic properties and combinations of unexpected properties of the 60-residue triple mutant polypeptide variants of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) disclosed herein. [Table 4] K for each inhibitor i Values ​​were calculated using the tight-binding equation. KD1TM inhibits kallikrein with a K of 18 nM. i Kallikrein inhibition by aprotinin is not observed up to 25 μM, compared to aprotinin, which has a . The inhibition of kallikrein by aprotinin is associated with renal damage. The lysine analogs tranexamic acid (TXA) and .epsilon.-aminocaproic acid (EACA) function through a different mechanism and do not inhibit the plasmin active site or other proteases.

[0131] Table 5 and Table 6: Human TFPI-2 KD1 mutant binding to the kringle domains of tPA and plasminogen Binding constants obtained from surface plasmon resonance experiments. [Table 5] [Table 6]

[0132] For example, as shown in the data provided in Tables 4-6 and the schematic diagram in FIG. 9, this 60-residue triple mutant polypeptide variant of the Kunitz domain 1 (KD1) of human tissue factor pathway inhibitor type 2 has been discovered to have a highly desirable pharmacokinetic profile. This profile includes a greater ability to inhibit the activity of plasmin when compared to aprotinin, a previously utilized but problematic antifibrinolytic agent. Moreover, the plasmin inhibitory activity of this new polypeptide variant is more favorable compared to that of aprotinin, while the polypeptide variant disclosed herein further avoids certain adverse side effects observed with aprotinin and related molecules. In one illustration of this, the polypeptide variant disclosed herein is observed to exhibit minimal inhibitory activity against other coagulation serine proteases (e.g., kallikrein, factor XIa, and factor VIIa / tissue factor).

[0133] As noted above, the 60-residue polypeptide variants disclosed herein contain a unique grouping of amino acid residues that includes a C-terminal lysine structure / moiety. Without being bound by any particular theory or mechanism of action, this C-terminal structure appears to function by facilitating binding of the 60-residue polypeptide variant to plasmin or plasminogen via its kringle domain in a manner that inhibits plasminogen binding to fibrin clots. The polypeptide variants disclosed herein contain a group of three amino acid mutations that include a lysine amino acid substitution at position 15 ("KD1"). Y11T / R15K / L17R"). Surprisingly, this Y11T / R15K / L17R triple mutant containing the C-terminal lysine is observed to be 4-5 times more potent in inhibiting plasmin when compared to a control 60 residue polypeptide variant having only the double mutation Y11T / L17R (see, e.g., the data presented in Figure 2 and Tables 4-6). Without being bound by a particular theory or mechanism of action, this triple mutant with a lysine amino acid substitution at position 15 appears to function by promoting the interaction of this variant polypeptide with residues Asp189 and Ser190 in plasmin.

[0134] Unexpectedly, this 60-residue Y11T / R15K / L17R triple mutant polypeptide further exhibits at least 10-fold weaker inhibition of kallikrein, factor XIa and factor VIIa / tissue factor when compared to the control 60-residue polypeptide variant with only the double mutation Y11T / L17R, and its functional profile limits undesirable side effects (e.g., those observed with aprotinin). The Arg residue at position 15 (BPTI numbering) of the wild-type human tissue factor pathway inhibitor type 2 molecule is important for inhibiting factor XIa and kallikrein. However, since factor XIa and kallikrein have an alanine residue at position 190, the lysine at position 15 of the Kunitz domain 1 polypeptide inhibitors disclosed herein apparently cannot interact with Ala190 in factor XIa and kallikrein. In conclusion, these 60 residue polypeptides with a lysine at this position are extremely poor inhibitors of factor XIa and kallikrein activity.

[0135] For the reasons noted above, the 60-residue variant polypeptides disclosed herein exhibit surprising and highly desirable pharmacokinetic / material profiles, including, for example, the ability to bind strongly to plasmin while simultaneously avoiding certain side effects associated with similar inhibitory molecules in the art. Such functional properties make these polypeptides optimized for use as therapeutic agents in vivo. The polypeptides disclosed herein also possess several other desirable properties. For example, the 60-residue variant polypeptides have been shown to bind to tissue plasminogen activator (tPA) and inhibit its binding to fibrin clots, thereby attenuating plasminogen activation at the site of coagulation.

[0136] Further aspects of the 60 residue variant polypeptide are discussed below.

[0137] KD1 Y11T / R15K / L17R -K T Modeling interactions with In FIG. 9A, KD1 with plasmin Y11T / R15K / L17R -K T The modeled complex of the interaction is shown. Subpart (A) shows KD1 Y11T / R15K / L17R -K T The modeled interactions of KD1 with the plasmin protease domain are shown. Y11T / R15K / L17R -K T A cartoon representation (light green) of the KD1 with plasmin is shown. Y11T / R15K / L17R -K T The interacting P1 (Lys15), P5 (Thr11) and P2' (Arg17) residues are shown in stick representation. On the electrostatic surface, blue represents positive charge, red represents negative charge and white represents neutral charge. Subpart (B) represents the KD1 Y11T / R15K / L17R -K T The modeled interactions of KD1 with the plasmin kringle domain are shown. Y11T / R15K / L17R -K T The cartoon representation (light green) of the Kringle domain and KD1Y11T / R15K / L17R -K T Residues that form hydrogen bonds and salt bridges (shown as dashed lines) between are shown in stick representation. Carbon atoms are shown in green for the Kringle domain and in red for KD1. Y11T / R15K / L17R -K T As shown in subpart (A), oxygen atoms are shown in red and nitrogen atoms in blue. Y11T / R15K / L17R -K T Residues are denoted with the suffix I. In the electrostatic surface, blue represents a positive charge, red represents a negative charge, and white represents a neutral charge.

[0138] Residue 190 Ser in plasmin is the same as that of rHuKD1-TM (KD1 Y11T / R15K / L17R -K T (Note that KD1 interacts with lysine 15 in plasmin, which is not possible with Ala 190 in kallikrein or factor XIa. Thus, plasmin active site affinity is increased for plasmin and decreased for kallikrein and factor XIa. Furthermore, without being bound by any particular theory or mechanism of action, KD1 Y11T / R15K / L17R -K T The IEK C-terminus in the polypeptide variant appears to have a higher affinity for the plasmin kringle domain, since it has a greater interaction with plasmin than a polypeptide with the VPK C-terminal sequence. Y11T / R15K / L17R -K T The polypeptide variant interaction occurs at the kringle domain of tissue plasminogen activator.

[0139] FIG. 9A shows the mechanism by which the IEK C-terminal motif binds to the plasmin ringle domain of KD1, including why the IEK C-terminal motif binds better to the plasmin ringle domain compared to polypeptides with the VPK C-terminal motif. Y11T / R15K / L17R -K TThis provides information regarding the level of plasmin inhibition observed with the polypeptide variants. This finding is supported by testing against a 73 residue long (plus 4 residues from thrombin cleavage) kuniz domain prior art construct with an Arg to Lys at position 15 (same as position 24) and the VPKV terminal sequence disclosed in US Patent Publication 20080026998. This prior art construct does not have a Lys amino acid at the C-terminus of the polypeptide and does not bind to the plasmin kringle domain. Furthermore, 9 residues at the N-terminus and 4 residues at the C-terminus of this specific 73 residue prior art molecule are solvent exposed and highly disordered, which may impair aspects of the binding ability of this prior art variant. In contrast, the triple mutant construct of the present invention has a 60 residue long ((KD1) with a C-terminal IEK. Y11t / R15K / L17R -K T ) and is observed to bind to the kringle domain with very high affinity (about 40 nM). Furthermore, as discussed in the following section, the polypeptide variant of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) disclosed herein (SEQ ID NO: 1) exhibits a highly desirable stability profile.

[0140] Stability testing The polypeptide variants of human tissue factor pathway inhibitor type 2 Kunitz domain 1 (KD1) disclosed herein exhibit unexpected and desirable stability profiles. For example, in certain embodiments of the present invention, the 60-residue polypeptide variants disclosed herein are placed into a composition, where the plasmin inhibition constant (Ki) of the polypeptide changes by less than 10% (or less than 5%) when the polypeptide composition is incubated in Tris-buffered saline (TBS) containing 0.1 mg / mL bovine serum albumin (BSA) and 2 mM calcium at 37° C. for at least 1 week.

[0141] In the stability test, KD1 Y11T / R15K / L17R -K TSamples (0.59 mg / ml) were kept at 4°C, room temperature, and 37°C for one week, and their plasmin inhibition was tested each day. rHuKD1-TM is stable and its plasmin inhibitory properties do not appear to change. The rHuKD1-TM mutant, similar to aprotinin, is a slow and tight-binding plasmin inhibitor. Reactions were carried out in TBS / BSA and 2 mM calcium. Human plasmin was incubated with various concentrations of rHuKD1-TM for 1 hour at room temperature in a 96-well microtiter plate. A synthetic substrate for plasmin was then added at a final concentration of 1K M and the residual amidolytic activity was measured in a kinetic microplate reader (Molecular Devices). In Table 7 below, the apparent inhibition constants, K i * was determined using a nonlinear regression data analysis program. Data were analyzed using the equation for tight binding inhibitors, K i Values ​​are obtained by correcting for the effect of substrate concentration. [Table 7]

[0142] KD1 Y11T / R15KL17R -K T The equilibrium dissociation constants (K i ) The equilibrium dissociation constants (K i ) was calculated using the slow tight binding inhibition equation (10,11). K iThe values ​​ranged from 50 to 150 picomolar in different experiments, compared to 400 to 500 picomolar for aprotinin (Figure 2). Furthermore, the C-terminal 60-lysine should enable binding of rHuKD1-TM to the kringle domains of plasminogen / plasmin and tissue plasminogen activator (tPA) and inhibit their binding to fibrin clots. This property of rHuKD1-TM further inhibits fibrinolysis by plasmin. Thus, rHuKD1-TM (KD1 Y11T / R15K / L17R -K T ) is a highly potent and specific agent for inhibiting fibrinolysis.

[0143] The Arg15 to Lys change in the KD1 double mutant (Y11T / L17R) effectively interacts with the S1 site Asp189 of plasmin, including the interaction of Lys15 of rHuKD1-TM with Ser190 of plasmin, as defined in the structures of trypsin and aprotinin (12). Such an interaction of Lys15 is not possible with kallikrein or with factors XIa and Xa, which have Ala190 instead of Ser (13,14). As a result, the KD1 Y11T / R15K / L17R -K T inhibits these three enzymes very slightly (Figure 2), a desirable outcome for use in patients. However, factor VIIa has Ser190 and is slightly inhibited by the triple mutant, similar to the single or double mutants (Figure 2).

[0144] Effect of rHuKD1-TM and aprotinin on fibrinolysis in normal pooled human plasma Experiments were performed to demonstrate that the triple mutant KD1 inhibits tPA-induced plasma clot fibrinolysis. Y11T / R15K / L17R -K Tand aprotinin were compared. These data are shown in Figure 3A for the triple mutant and in Figure 3B for aprotinin. Addition of thrombin (IIa) to human normal pooled plasma (NPP) caused fibrin formation, reflected by an increase in OD405 (black curve, Figures 3A and 3B). Concurrent addition of tPA caused first clot formation, followed by dissolution of fibrin induced by tPA-mediated conversion of plasminogen to plasmin (black curve with black circles, Figures 3A and 3B); the midpoint of fibrinolysis was around 8 min in each case. Both antifibrinolytic agents inhibited fibrinolysis in a dose-dependent manner. KD1 Y11T / R15K / L17R -K T Both KD1 (triple mutant) and aprotinin increased the fibrinolysis midpoint to approximately 15 min at 0.5 μM, and at 3 μM, both inhibitors completely prevented tPA-induced fibrinolysis (magenta curves in Figures 3A and 3B). Importantly, addition of 3 μM of each inhibitor in the absence of tPA had no effect on either clot formation or fibrinolysis (brown curves, Figures 3A and 3B). Thus, KD1 Y11T / R15K / L17R -K T and aprotinin are essentially equivalent in preventing tPA-induced plasma clot lysis over the long term.

[0145] Effect of antifibrinolytic agents on primary cell viability The effect of each antifibrinolytic agent, rHuKD1-TM, aprotinin, EACA, and TXA, was tested on the viability of human umbilical vein endothelial cells (HUVEC) and dermal fibroblasts. The data are shown in Figure 4 for HUVEC and Figure 5 for dermal fibroblasts. None of the antifibrinolytic agents tested had a detectable effect on viability within 24 hours for the two cell types tested. Note that viable cells maintain a reducing environment in the cytosol. Viable cells reduce the substrate resazurin to fluorescent resofurin, and fluorescence is directly proportional to cell number. The substrate resazurin is cell permeable (enters the cell) regardless of cell state. Therefore, this assay does not detect any cell apoptosis or necrosis.

[0146] Effect of antifibrinolytic agents on cell apoptosis Apoptosis, sometimes referred to as "cell suicide," is the normal programmed process of cell self-destruction. During apoptosis, cells shrink and become detached from their neighbors. Caspase 3 and 7 are proteases that are activated only when cells are undergoing apoptosis. In the assay used here, specific substrates are converted by caspase 3 and caspase 7 into substrates for luciferase. Luciferase then produces a luminescent signal. Luminescence is directly proportional to caspase activity. During apoptosis, the membrane integrity of the cell remains intact. In the assay used, EACA and TXA induce a significant increase in caspase activity in HUVECs but not in fibroblasts (Figure 6). Thus, EACA and TXA were able to induce programmed cell death in endothelial cells lining blood vessels in vivo. rHuKD1-TM and aprotinin (BPTI) did not induce apoptosis in either cell, HUVEC or dermal fibroblasts, under the conditions used (FIG. 6).

[0147] Effect on cytotoxicity caused by antifibrinolytic agents The cell toxicity green assay used here measures changes in membrane integrity that occur as a result of cell injury. Cyanine dyes cannot enter cells through intact membranes. If membrane integrity is compromised, the dyes enter the cells and stain the DNA, resulting in a fluorescent signal. The fluorescence observed is therefore proportional to cell injury. In the assay used, TXA induced toxicity in both HUVECs and dermal fibroblasts (Figures 7 and 8), whereas with EACA, significant toxicity was observed only in dermal fibroblasts. rHuKD1-TM and aprotinin (Trasylol, BPTI) did not induce toxicity in these cells under the conditions tested.

[0148] Thromboelastography Experiments KD1-Y11T / R15K / L17R-K for fibrinolysis COOHThe effect of different concentrations of (KD1™) was assessed by thromboelastography (TEG) using a TEG 5000 Thrombelastograph (Haemonetics Corp, Braintree, MA, USA). Each clot formation / lysis assay contained 300 μL of citrated whole blood, thrombin (0.15 μM, final concentration), tPA (2 nM, final concentration), CaCl2 (10 mM, final concentration) and various concentrations of KD1™ in TBS / BSA (containing 50 mM Tris-HCl, 100 mM NaCl, 0.1 mg bovine serum albumin / mL) to a final volume of 360 μL. Thrombin, tPA and CaCl2 were added last to initiate simultaneous clot formation and fibrinolysis. The concentration of tPA was chosen such that plasmin generation to nearly complete clot lysis occurred over 90 min, allowing the effect of plasmin inhibitors to be monitored. Each experiment was performed for at least 60 min after the maximum amplitude was reached to establish the LY60 value. TEG Analytical Software (version 4.2.3; Haemonetics Corporation, Braintree, MA, USA) was used to calculate the time to onset of clotting (R), maximum clot strength (maximum amplitude (MA), which was directly proportional to shear modulus strength, G), and percent lysis after MA60 min (LY60). Thromboelastography experiments were performed to evaluate the effect of KD1TM on tPA-induced lysis of clots formed in whole blood by the addition of thrombin and CaCl2. These data are shown in Figure 20 and summarized in Table 8. Figure 20 shows TEG traces at different concentrations of KD1TM for tPA-initiated clot lysis. In the absence of tPA, the maximum amplitude (MA) achieved was 4174 dyn / cm 2At 45.5 min, there was no detectable clot lysis (LY60<0.1%) at 180 min (Curve 1). Curve 2 illustrates the TEG traces for inhibitor-free clot formation and lysis in the presence of 2 nM tPA. Curves 3 and 4 illustrate the TEG traces at different concentrations of KD1TM (2 μM and 4 μM) for clot formation and lysis in the presence of 2 nM tPA. The data show that KD1TM improved clot firmness (MA) and shear strength (G) and inhibited fibrinolysis in a concentration-dependent manner (Table 8). Remarkably, at 4 μM, KD1TM improved clot strength MA to 98% (44.2 mm) and G to 96% (4011 dyn / cm 2 ) (curve 4). Furthermore, no LY30 was observed when compared to the control (LY30, 34.6%, curve 2). Similarly, only 3.7% or 1.7% of LY60 was observed at 2 μM and 4 μM concentrations of KD1TM when compared to the control (LY60 65%). Importantly, the TEG data show that KD1TM effectively restores MA and G in tPA-induced fibrinolysis. [Table 8]

[0149] The disclosure provided herein leads to a number of conclusions, including: KD1 Y11T / R15K / L17R -K T (rHuKD1-TM) is KD1-L17R-K COOH (KD1SM) and KD1 Y11T / L17R -K T (KD1DM) and equivalent to aprotinin (Trasylol) in inhibiting the active site of plasmin. rHuKD1-TM is a 60-residue Kunitz domain (BPTI numbering) that begins with the NAEIC and has a C-terminal IEK. Moreover, it is superior to the previous single mutant (KD1-L17R-K COOH) does not have the additional 9 residues at the N-terminus. The single mutant also has 3 additional residues at the C-terminus terminating in IEKVPK. The disadvantage of the single mutant is that the two hydrophobic residues, Val and Pro, reduce its solubility. rHuKD1-TM is highly soluble. rHuKD1-TM is also superior to KD1SM and KD1DM in inhibiting plasmin and in plasma clot lysis assays, and is comparable to aprotinin over long incubation periods. Furthermore, compared to the single mutant (KD1SM) and double mutant (KD1DM), the triple mutant (rHuKD1-TM) is a very weak inhibitor of kallikrein, factor XIa, factor Xa, and factor VIIa / tissue factor.

[0150] Extensive testing (e.g., Figures 4-8) shows that rHuKD1-TM does not cause damage to HUVECs or fibroblasts and possibly other cells. EACA and TXA (which are eliminated via the kidney) may possibly be present in high concentrations in the kidney and cause cell damage. This may be the reason for renal failure as an observed side effect of TXA and EACA.

[0151] As disclosed above, rHuKD1-TM (SEQ ID NO: 1) was discovered to be a strong inhibitor of plasmin, as well as a very weak inhibitor of kallikrein and factor XIa (and has no anticoagulant activity). This is in contrast to aprotinin, which is of bovine origin and causes kidney damage due to inhibition of kallikrein. Furthermore, the two inhibitors inhibit kallikrein (7,8) very strongly and failed in phase III cardiac bypass surgery trials. Thus, the very weak inhibition of kallikrein and factor XIa by rHuKD1-TM is a very advantageous property of the polypeptide of the present disclosure. Thus, the 60-residue variant polypeptide disclosed herein was discovered to have a surprising set of material properties that fulfill a long-standing need that has been recognized, persisted and not addressed by others.

[0152] All publications mentioned herein (e.g., those numerically listed above, U.S. Pat. No. 8,993,719, U.S. Patent Publication Nos. 20040126856, 20040110688, 20080026998, 20090018069, and 20140288000, and Vadivel et al., J Clin Med. 2020 Nov 17;9(11):3684. doi: 10.3390 / jcm9113684) are incorporated by reference herein to disclose and describe aspects, methods, and / or materials in connection with the cited publications. In certain embodiments, for example, the following are provided: (Item 1) a polypeptide having the sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO:1); and a pharma- ceutically acceptable excipient. 23. A pharmaceutical composition comprising: (Item 2) 2. The composition of claim 1, wherein the pharma- ceutically acceptable excipient is selected from at least one of a preservative, a tonicity adjusting agent, a detergent, a hydrogel, a viscosity adjusting agent, or a pH adjusting agent. (Item 3) The composition according to item 2, wherein the composition comprises a pharma- ceutically acceptable composition comprising a pharma- ceutically acceptable excipient selected for use in intravenous injection or infusion. (Item 4) 4. The composition according to any one of items 1 to 3, wherein the plasmin inhibition constant (Ki) of the polypeptide changes by less than 10% after incubation of the composition in Tris-buffered saline (TBS) containing 0.1 mg / mL bovine serum albumin (BSA) and 2 mM calcium at 37° C. for at least 2 days, 4 days, or 1 week. (Item 5) A composition comprising a polynucleotide encoding the polypeptide sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO:1). (Item 6) The polynucleotide has the sequence: AACGCGGAGATCTGTCTGCCCCTAGACACCGGACCCTGCAAAGCCAGACTTCTCCGTTACTACTACGACAGGTACACGCAGAGCTGCCGCCAGTTCCTGTACGGGGGCTGCGAGGGCAACGCCAACAATTTCTACACCTGGGAGGCTTGCGACGATGCTTGCTGGAGGATAGAAAAA (SEQ ID NO: 2). 6. The composition according to item 5, comprising: (Item 7) 7. The composition of claim 6, wherein the polynucleotide is placed in a vector comprising one or more regulatory sequences for expressing the polypeptide in a cell. (Item 8) A cell comprising the vector according to item 7. (Item 9) 8. The cell according to item 7, wherein the cell is a bacterial cell, a yeast cell, an insect cell or a mammalian cell. (Item 10) 5. A method for inhibiting at least one activity of plasmin, the method comprising contacting plasmin with a composition according to any one of claims 1 to 4 in an amount sufficient to inhibit at least one activity of plasmin. (Item 11) 11. The method of claim 10, comprising inhibiting fibrinolysis in a patient by administering to the patient an amount of the composition sufficient to inhibit fibrinolysis, such that fibrinolysis is inhibited. (Item 12) 5. A method for inhibiting bleeding in a subject, the method comprising administering to the subject an effective amount of the composition according to any one of items 1 to 4 such that bleeding is inhibited. (Item 13) 13. The method of claim 12, wherein the bleeding results from a traumatic injury. (Item 14) 14. The method of claim 13, wherein the traumatic injury is a traumatic brain injury. (Item 15) 13. The method of claim 12, wherein the bleeding results from surgery. (Item 16) 16. The method of claim 15, wherein the bleeding is in a patient undergoing cardiac surgery and in need of reduced blood loss, the method comprising administering a therapeutically effective amount of the composition before, during, or after the surgery. (Item 17) 17. The method of claim 16, wherein the cardiac surgery is cardiopulmonary bypass surgery. (Item 18) 13. The method of claim 12, wherein the bleeding is in a patient undergoing treatment for traumatic hemorrhagic shock. (Item 19) 19. The method according to any one of items 10 to 18, wherein the composition is disposed in a matrix. (Item 20) 20. The method of claim 19, wherein the matrix is ​​a patch or a compression bandage. (Item 21) A non-naturally occurring isolated polypeptide having the sequence NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO:1). (Item 22) 22. The non-naturally occurring isolated polypeptide of claim 21, wherein the polypeptide has at least 3-fold greater potency in inhibiting plasmin when compared to a 60-residue polypeptide variant having only the double mutation Y11T / L17R of KD1 of human tissue factor pathway inhibitor type 2.

Claims

1. A polypeptide consisting of the sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO: 1); and a pharma- ceutically acceptable excipient.

23. A pharmaceutical composition comprising:

2. 10. The composition of claim 1, wherein the pharma- ceutically acceptable excipient is selected from at least one of a preservative, a tonicity adjusting agent, a detergent, a hydrogel, a viscosity adjusting agent, or a pH adjusting agent.

3. 3. The composition of claim 2, wherein the composition comprises a pharma- ceutically acceptable composition comprising a pharma- ceutically acceptable excipient selected for use in intravenous injection or infusion.

4. 4. The composition of any one of claims 1 to 3, wherein the plasmin inhibition constant (Ki) of the polypeptide changes by less than 10% after incubation of the composition in Tris-buffered saline (TBS) containing 0.1 mg / mL bovine serum albumin (BSA) and 2 mM calcium at 37°C for at least 2 days, 4 days, or 1 week.

5. A composition comprising a polynucleotide encoding the polypeptide sequence: NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO:1).

6. The polynucleotide has the sequence: AACGCGGAGATCTGTCTCCTGCCCCTAGACACCGGACCCTGCAAAGCCAGACTTCTCCGTTACTACTACGACAGGTACACGCAGAGCTGCCGCCAGTTCCTGTACGGGGGCTGCGAGGGCAACGCCAACAAATTCTACACCTGGGAGGCTTGCGACGATGCTTGCTGGAGGATAGAAAA (SEQ ID NO: 2). The composition of claim 5 , comprising:

7. The composition of claim 6, wherein the polynucleotide is placed in a vector comprising one or more regulatory sequences for expressing the polypeptide in a cell.

8. A cell comprising the vector of claim 7.

9. The cell of claim 7 , wherein the cell is a bacterial cell, a yeast cell, an insect cell or a mammalian cell.

10. 5. A composition according to any one of claims 1 to 4 for use in a method of inhibiting at least one activity of plasmin, the method comprising contacting plasmin with an amount of the composition sufficient to inhibit at least one activity of plasmin.

11. The composition of claim 10, wherein the method comprises inhibiting fibrinolysis in a patient by administering to the patient an amount of the composition sufficient to inhibit fibrinolysis, such that fibrinolysis is inhibited.

12. The composition of any one of claims 1 to 4 for inhibiting bleeding in a subject.

13. The composition of claim 12 , wherein the bleeding results from a traumatic injury.

14. The composition of claim 13 , wherein the traumatic injury is a traumatic brain injury.

15. The composition of claim 12 , wherein the bleeding results from surgery.

16. 16. The composition of claim 15, wherein the bleeding is in a patient undergoing cardiac surgery and in need of reduced blood loss, and the composition is administered before, during, or after the surgery.

17. 17. The method of claim 16, wherein the cardiac surgery is cardiopulmonary bypass surgery.

18. The composition of claim 12, wherein the bleeding is in a patient undergoing treatment for traumatic hemorrhagic shock.

19. The composition of any one of claims 10 to 18, wherein the composition is disposed in a matrix.

20. 20. The composition of claim 19, wherein the matrix is ​​a patch or a compression bandage.

21. An isolated, non-naturally occurring polypeptide consisting of the sequence NAEICLLPLDTGPCKARLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRIEK (SEQ ID NO:1).

22. 22. The non-naturally occurring isolated polypeptide of claim 21, wherein the polypeptide has at least 3-fold greater potency in inhibiting plasmin when compared to a 60-residue polypeptide variant having only the double mutation Y11T / L17R of KD1 of human tissue factor pathway inhibitor type 2.

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