Combinations of plasminogen activator mutants and lysine analogs for inhibiting coagulation and associated conditions

Abstract

The present disclosure provides combinations of tissue plasminogen activator mutant / s and lysine analogs and compositions thereof for inhibiting fibrinolysis induced by free plasmin. The present disclosure further provides kits, uses and methods for treating coagulopathy associated with anti-fibrinolytic therapy.

Description

[0001] COMBINATIONS OF PLASMINOGEN ACTIVATOR MUTANTS AND LYSINE ANALOGS FOR INHIBITING COAGULATION AND ASSOCIATED CONDITIONS

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to the field of combination therapy. More specifically, the present disclosure relates to combinations of tissue plasminogen activator mutant / s and lysine analogs and compositions thereof for inhibiting fibrinolysis induced by free plasmin and treating coagulopathy associated with anti-fibrinolytic therapy.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. Hijazi, N., et al., Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice. Blood, 2015. 125(16): p. 2558-67.

[0007] 2. Armstead, W.M., et al., tPA-S481A prevents neurotoxicity of endogenous tPA in traumatic brain injury. J Neurotrauma, 2012. 29(9): p. 1794-802.

[0008] 3. Nassar, T., et al., Regulation of airway contractility by plasminogen activators through N- methyl-D-aspartate receptor-1. Am. J. Respir Cell Mol Biol, 2010. 43(6): p. 703-11.

[0009] 4. Armstead, W.M., et al., Neutralizing the neurotoxic effects of exogenous and endogenous tPA. Nat Neurosci, 2006. 9(9): p. 1150-5.

[0010] 5. W02014 / 006613.

[0011] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0012] BACKGROUND

[0013] The two main initiators of endogenous fibrinolysis are tissue type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). The activity of both plasminogen activators is largely stimulated by fibrin [Collen, D. et al. Thromb Haemost, 1995. 74(1): p. 167-71; Hoylaerts, M., et al. J Biol Chem, 1982. 257(6): p. 2912-9], a fact that targets their activity to the surface of blood clots.

[0014] The inventors have previously shown that tPA mutants are capable of directly binding to plasminogen in the presence of fibrin or lysine analogs, thereby inhibiting tPA- and uPA-mediated plasminogen activation and the subsequent generation of plasmin, and thus inhibiting fibrinolysis in vitro and in vivo [1, 5]. Specifically, they have found that catalytically inactive tPA variant 481 produced by substitution of the Ser residue to Ala at the catalytic site of tPA (tPASer481Ala) competes with the Wild type (WT) tPA and uPA in terms of affinity to plasminogen in the presence of fibrin and thereby interferes with the tPA- and uPA- mediated plasminogen activation to plasmin and the subsequent fibrinolysis [1, 5]. The mutant tissue type plasminogen activator tPAS481Avariant [1] inhibits the activation of plasminogen to plasmin by tPA and urokinase plasminogen activator (uPA) in a competitive pattern, and the activation of NMDA-R by tPA [2, 3]. The inhibition of plasmin by tPAS481Ais not competitive, it is allosteric and unlike as in the case of NMD AR activation by tPA, inhibition of plasmin by tPAS481A, requires the presence of fully active docking site in the tPAS481A[4],

[0015] Lysine analogs such as tranexamic acid (TXA) and a-aminocaproic acid (EACA) have been used extensively as anti-fibrinolytics to inhibit clot lysis and by that to prevent bleeding. TXA and EACA bind to the LBS of plasminogen, competing with the N-terminal lysine residues on fibrin, thereby inhibiting in a competitive manner, the binding of plasminogen to fibrin and its activation by tPA and uPA on the fibrin surface [Astedt, B. J Gastroenterol Suppl, 1987. 137: p. 22-5; Violand, B.N., et al. J Biol Chem, 1978. 253(15): p. 5395-401]. Furthermore, TXA and EACA bind to the plasmin’s LBS, preventing its binding to fibrin and by that exert a second anti fibrinolytic activity.

[0016] Besides inhibiting the binding of plasminogen to fibrin, in the case of uPA, binding of lysine analogs (TXA and EACA) to plasminogen mimics the effect of fibrin on plasminogen conformation and enhances its susceptibility to be activated by uPA [Markus, G. et al. J Biol Chem, 1978. 253(3): p. 733-9; Markus, G. et al. J Biol Chem, 1979. 254(4): p. 1211-6; Violand, B.N., et al. J Biol Chem, 1978. 253(15): p. 5395-401]. Based on these principles, it would be expected that lysine analogs would inhibit the uPA mediated generation of plasmin on the fibrin clot surface, leading to decreased fibrinolysis. However, on the other hand, this would enhance generation of free plasmin in a fibrin-independent mode. The enhanced activation of plasminogen bound to TXA or EACA by uPA leads to the generation of free plasmin, thereby limiting its beneficial anti- fibrinolytic effect.

[0017] Furthermore, plasmin generated in presence of lysine analogs (TXA and EACA) is enzymatically active and is partially protected from inactivation by a2- antiplasmin [Rakoczi, I., et al., Biochim Biophys Acta, 1978. 540(2): p. 295-300; Higazi, A.A. et al. iochem J, 1990. 269(2): p. 299-302]. This increases potential side effects on coagulation factors and platelets. Indeed, free active plasmin can induce coagulopathy by proteolyzing coagulation factors, that include fibrinogen [Rourke, C., et al. J Thromb Haemost, 2012. 10(7): p. 1342-51;], Factor V (FV) and Factor VIII (FVIII) [Nogami, K., et al. J Biol Chem, 2007. 282(8): p. 5287-95;] and can further damage the blood-brain barrier [Niego, B. et al., J Cereb Blood Flow Metab, 2014. 34(8): p. 1283-96]. In addition, free plasmin consumes the anti-fibrinolytic a2-antiplasmin, thereby leading to the formation of upcoming fragile blood clots [Eongstaff, C. et al., J Thromb Haemost, 2019. 17(1): p. 195-205],

[0018] GENERAL DESCRIPTION

[0019] The two main initiators of endogenous fibrinolysis are tissue type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). The activity of both plasminogen activators is largely stimulated by fibrin [Collen, D., et al. 1995. id ibid', Hoylaerts, M., et al. 1982. id ibid], a fact that targets their activity to the surface of blood clots.

[0020] The stimulation of the activity of each one of the two plasminogen activators by fibrin follows a unique mechanism. tPA exerts only modest plasminogen activation activity in the absence of fibrin [Hoylaerts, M., et al. 1982. id ibid]. Fibrinolysis by tPA is initiated when tPA and the pro-enzyme plasminogen bind to the surface of fibrin [Hoylaerts, M., et al. 1982. id ibid]. This increases the local concentrations of the reactants on the clot surface [Hoylaerts, M., et al. 1982. id ibid] and promoting plasmin generation by tPA by approximately 1000- fold [Mutch, N.J. J Thromb Haemost, 2023. 21(10): p. 2645-2647]. tPA cleaves the Arg560-Val561 bond in plasminogen on the surface of the fibrin clot, generating fibrin-bound active plasmin [Zamarron, C. et al. J Biol Chem, 1984. 259(4): p. 2080-3]. Plasmin is a potent serine protease, with broad specificity, that cleaves fibrin into soluble degradation products (FDPs). uPA also activates plasminogen to plasmin by cleaving the Arg560-Val561 bond on the fibrin surface, but without binding to fibrin [Stump, D.C., et al J Biol Chem, 1986. 261(3): p. 1274-8]. Plasminogen binds to N-terminal lysine residues on fibrin through its lysine binding sites (LBS) present in its kringles domains; Fibrin bound plasminogen undergoes a conformational change, which makes it a better substrate for uPA [astellino, F.J., et al. Haemostasis, 1988. 18 Suppl 1: p. 15-23]. The conformational changes in plasminogen induced by fibrin do not affect significantly its activation by tPA [Hoylaerts, M., et al. 1982 id ibid}.

[0021] Besides its cleaving effect on fibrin clots, and as result of its broad specificity, plasmin can inactivate several essential coagulation factors [Genet, G.F., et al. J Neurotrauma, 2013. 30(4): p. 301-6] and platelets [Quinton, T.M., et al. J Biol Chem, 2004. 279(18): p. 18434-9], leading to several undesired and life-threatening side effects.

[0022] Taken together, the resulting free active plasmin is considered a major side effect of the use of lysine analogs such as TXA and EACA, supporting the contention that a direct inhibition of plasmin by exogenous inhibitors will increase its anti-fibrinolytic activity and prevents the TXA and EACA induced coagulopathy and other life-threatening side effects. Indeed, the use of a direct plasmin inhibitor was in routine practice with great success in the past. More specifically, aprotinin, a small bovine protein that inhibits several serine proteases such as chymotrypsin, trypsin and plasmin was widely used as anti-fibrinolytic treatment alone or in combination with TXA or EACA.

[0023] However, due to accumulation of negative data related to side effects such as acute renal and vascular safety concerns and increased risk of long-term mortality [Mangano, D.T., et al. AMA, 2007. 297(5): p. 471-9] associated with its use, the aprotinin withdrew from the market in 2008.

[0024] Therefore, there is a need for antifibrinolytic compounds that effectively reduce fibrinolysis by free plasmin, in particular. More importantly, compounds for safe therapeutic use with reduced or no negative effects on coagulations factors, are most valuable.

[0025] In the present application the inventors show that the mutant tPAS481Ainhibits directly the activity of the fully active enzyme plasmin. Furthermore, the inventors show that beside inhibiting plasmin mediated fibrinolytic activity, tPAS481Aprevents the inactivation of the coagulation factors V and VIII and fibrinogen by plasmin. The inventors also found that tPAS481Aexerts a synergistic antifibrinolytic effect with TXA without the combined coagulopathy. Thus, according to one aspect, the present disclosure relates to a combination comprising the following components. Component (a) comprises at least one tissue plasminogen activator (tPA) mutant that carries a point mutation at position 481 of the wild type tPA molecule (WT tPA). The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2. The disclosed combination further comprises component (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin. The disclosed combination exerts at least one of: increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity.

[0026] A further aspect of the present disclosure relates to a composition comprising: (a) at least one tissue tPA mutant that carries a point mutation at position 481 of the WT tPA. It should be noted that the WT tPA comprises the amino acid sequence as denoted by SEQ ID NO: 2. The disclosed composition further comprises component (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin. In some embodiments the composition may comprise (c), a combination of (a) and (b), that exerts at least one of: increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity. In some optional embodiments, the disclosed compositions may optionally further comprise (d), at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

[0027] A further aspect of the present disclosure relates to a method for inhibiting the fibrinolytic activity of plasmin on at least one substrate thereof, the method comprising contacting a media or any substance comprising the substrate and / or free plasmin, with at least one tPA mutated molecule. In some optional embodiments, the substrate in the media may be further contacted with at least one lysin analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin, or with a composition comprising the same. In some embodiments, the tPA mutant carries a point mutation at position 481 of the WT tPA. It should be noted that the WT tPA comprises the amino acid sequence as denoted by SEQ ID NO: 2.

[0028] A further aspect of the present disclosure relates to a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder and / or any disease, disorder, or condition associated with fibrinolysis in a subject in need. More specifically, the disclosed therapeutic methods comprise the step of administering to the subject a therapeutically effective amount of: (a), at least one tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2; and (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasmin. Alternatively, the method may comprise administering (c), a combination of (a) and (b), or any composition comprising (a), (b) or (c). As shown by the present disclosure, this combination exerts at least one of: increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

[0029] A further aspect of the present disclosure relates to a method of treatment, amelioration, inhibition or prophylaxis of a hemostatic disease, disorder, or condition associated with fibrinolysis in a subject treated with at least one lysine analog. The method comprising the step of administering to the subject a therapeutically effective amount of at least one tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, or any composition thereof.

[0030] A further aspect of the present disclosure relates to an effective amount of a combination or composition comprising the same, for use in a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder or any disease, disorder, or condition associated with fibrinolysis. The combination comprising: (a), at least one tissue tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprises the amino acid sequence as denoted by SEQ ID NO: 2. The combination further comprises component (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin. The disclosed combination exerts at least one of: increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity.

[0031] A further aspect of the present disclosure relates to an effective amount of at least one tPA mutated molecule that carries a point mutation at position 481 of the WT tPA (the WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2), or a composition comprising the same, for use in a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder associated with fibrinolysis in a subject treated with at least one lysine analog.

[0032] A further aspect of the present disclosure relates to a kit comprising:

[0033] (a) at least one tPA mutant that carries a point mutation at position 481of the WT tPA. The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, optionally, in a first dosage form; and (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen, optionally in a second dosage form.

[0034] The combination of (a) and (b), exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

[0035] These and other aspects of the present disclosure will become apparent by the hand of the following disclosure.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0038] Figures 1A-1B: Effect of tPAS481Aon plasmin activity

[0039] The figures show measurement of the activity of plasmin in absence (C.) or the presence of the tPAS481Amutant. Plasmin 25 nM was incubated in phosphate buffered saline, pH 7.4, containing increasing concentrations of the chromogenic substrate (S-2251). The OD at 405 nm was measured continuously over a period of 20 minutes. Reaction rates (V) were calculated from the linear portion of the slope of the absorbance-time curves and expressed in nmol / min. The initial velocity data were plotted against substrate concentration to generate a Michaelis-Menten kinetic curve. To linearize the data, an inverse transformation was applied, and the reciprocal values (1 / V versus 1 / [S]) were plotted, yielding a Lineweaver-Burk plot.

[0040] Fig. 1A. shows the amidolytic activity of plasmin in the presence or absence of tPAS481A(25 nM) for increasing concentrations of the chromogenic substrate (0-0.9 mM).

[0041] Fig. IB shows the inverse transformation of the data presented in Fig. 1 A.

[0042] Figure 2: Effect of tPAS481Aand TXA on plasmin activity

[0043] The figure shows the amidolytic activity of plasmin, assayed as described in Fig. 1A, in the presence or absence (C.) of tPAS481A(25 nM) and in the presence or absence of TXA (0.1 mM). Figure 3: The role of the tPA docking site in plasmin inhibition

[0044] The figure shows the amidolytic activity of plasmin, assayed as described in Figure 1A, in the absence (C.) or the presence of each of two tPA mutants: the tPAS481Amutant and a variant carrying an additional mutation in its docking site (DS), referred to herein as tPAS481A-DS.

[0045] Figure 4: Effect of tPAS481Aon plasmin-mediated fibrinolysis of fibrin clots

[0046] Fibrin clots were formed in 24-well tissue culture plates.

[0047] 50 pl of PBS alone (well A) or PBS containing plasmin (25 nM), (wells B and C) was added to the clot surface for 2 hours at 37°C in the absence (well B) or presence of tPAS481A(100 nM) (well C). Clots were then washed with PBS, incubated overnight with 0.2% trypan blue, rinsed with PBS, and photographed.

[0048] Figure 5: Effect of tPAS481Aon plasmin-mediated fibrinolysis of fibrin clots

[0049] The figure shows evaluation of the amount of fibrinolysis (digestion) of the clots, extrapolated from color intensity in each well of Figure 4, calculated from the photographs scanned. The data shown represents the mean ± SEM of three experiments. C.=control.

[0050] Figure 6: Effect of TXA on plasmin-mediated fibrinolysis of fibrin clots

[0051] Fibrin clots were formed in 24-well tissue culture plates.

[0052] 50 pl of PBS containing plasmin (25 nM) in the absence (Well A) or presence of TXA (100 pM) (Well B) or PBS alone (Well C) was added to the clot surface for 2 hours at 37°C. Clots were then washed with PBS, incubated overnight with 0.2% trypan blue, rinsed with PBS, and photographed.

[0053] Figure 7: Effect of TXA on plasmin-mediated fibrinolysis of fibrin clots

[0054] The figure shows evaluation of the amount of fibrinolysis (digestion) of the clots, extrapolated from color intensity in each well of Figure 6, calculated from the photographs scanned (as in Figure 5). C.=control.

[0055] Figure 8: tPAS481Aand TXA synergistically inhibit plasmin-mediated fibrinolysis of fibrin clots

[0056] Fibrin clots were formed in 24-well tissue culture plates (as in Figure 5).

[0057] PBS containing plasmin (25 nM) was added to the clot surface in the presence of 25 nM tPAS481Aonly (Well A), 25 pM TXA only (Well B) or both tPAS481A(25 nM) and TXA (25 pM) (Well C). Well D, is a negative control, where PBS was added alone. Clots were then washed with PBS, incubated overnight with 0.2% trypan blue, rinsed with PBS, and photographed (as in Figure 5).

[0058] Figure 9: tPAS481Aand TXA synergistically inhibit plasmin-mediated fibrinolysis of fibrin clots

[0059] The figure shows evaluation of the amount of fibrinolysis (digestion) of the clots, extrapolated from color intensity in each well of Figure 8, calculated from the photographs scanned. C.=control

[0060] Figure 10: Human blood clots formation and lysis in the absence of tPA

[0061] Blood clots were formed from fresh human blood. TEG system was used to monitor and quantify the clot formation and lysis. The figure shows a control (C.) experiment of blood clots that were formed in the absence of tPA, with Ly 30 of 0.5%.

[0062] Figure 11: Human blood clots lysis in the presence of tPA

[0063] The activity of tPA (10 nM) was determined on blood clots formed from fresh human blood by TEG analysis. The used concentrations of tPA induced strong fibrinolysis with Ly 30 of 91%.

[0064] Figures 12: Inhibitory effect of TXA on tPA mediated fibrinolysis of human blood clots

[0065] The inhibitory effect of low dose of TXA on tPA mediated fibrinolysis was determined on blood clots formed from fresh human blood by TEG analysis. tPA (10 nM) was added in the presence of TXA (25 pM). A minimal inhibitory effect on clot lysis initiated by tPA was exerted by low dose of TXA, with Ly 30 of 88.8%.

[0066] Figure 13: Inhibitory effect of tPAS481Aon tPA mediated fibrinolysis of human blood clots

[0067] The inhibitory effect of low dose of tPAS481A(20 nM) on tPA mediated fibrinolysis was determined on blood clots formed from fresh human blood by TEG analysis. tPA (10 nM) was added in the presence of tPAS481A(20 nM). A minimal inhibitory effect on clot lysis initiated by tPA was exerted by low dose of tPAS481A, with Ly 30 of 76.8%.

[0068] Figure 14: Synergistic inhibitory effect of tPAS481Aand TXA on tPA mediated fibrinolysis of human blood clots

[0069] The inhibitory effect of both, tPAS481Aand TXA on tPA mediated fibrinolysis was determined on blood clots formed from fresh human blood by TEG analysis. The clots were formed in presence of tPA (10 nM), TXA (25 Mj and tPAS481A(20 nM). The combination of low concentrations of tPAS481Aand TXA indued strong inhibition of tPA, with Ly 30 of 1.7%.

[0070] Figure 15: Human blood clots formation and lysis in the absence of uPA

[0071] Blood clots were formed from fresh human blood as indicated for Figure 10. The figure shows a control (C.) experiment of blood clots that were formed in the absence of uPA, with Ly 30 of 0.0%.

[0072] Figure 16: Human blood clots lysis in the presence of uPA The activity of uPA (5 nM) was determined on blood clots formed from fresh human blood by TEG analysis. uPA induced storing fibrinolysis with Ly 30 of 95.7%.

[0073] Figure 17: Inhibitory effect of TXA on uPA mediated fibrinolysis of human clots

[0074] The inhibitory effect of low dose of TXA on uPA mediated fibrinolysis was determined on blood clots formed from fresh human blood by TEG analysis. uPA (5 nM) was added in the presence of TXA (25 pM). A minimal inhibitory effect on clot lysis initiated by uPA was exerted by low dose of TXA, with Ly 30 of 76.1%.

[0075] Figure 18: Inhibitory effect of tPAS481Aon uPA mediated fibrinolysis of human clots

[0076] The inhibitory effect of low dose of tPAS481A(20 nM) on uPA mediated fibrinolysis was determined on blood clots formed from fresh human blood by TEG analysis. uPA (5 nM) was added in the presence of tPAS481A(20 nM). A minimal inhibitory effect on clot lysis initiated by uPA was exerted by low dose of tPAS481A, with Ly 30 of 79.9%.

[0077] Figure 19: synergistic inhibitory effect of tPAS481Aand TXA on uPA mediated fibrinolysis of human blood clots

[0078] The synergistic inhibitory effect between of tPAS481Aand TXA on uPA mediated fibrinolysis was determined on blood clots formed from fresh human blood by TEG analysis. In this set of experiments the clots were formed in presence of uPA (5 nM), TXA (25 pM) and tPAS481A(20 nM). The combination of low concentrations of tPAS481Aand TXA indued strong inhibition of uPA, with Ly 30 of 2.3%. Figure 20: A histogram summarizing the effect of co-presence of tPAS481Aand TXA on tPA mediated fibrinolysis

[0079] The histogram illustrates the effect of the co-presence of 20 nM tPAS481Aand 25 pM TXA on tPA mediated fibrinolysis as determined on blood clots formed from fresh human blood by TEG analysis. The data is the summary of the data presented in Figures 10-14.

[0080] Figure 21: A histogram summarizing the effect of co-presence of tPAS481Aand TXA on uPA mediated fibrinolysis

[0081] The histogram illustrates the effect of the co-presence of 20 nM tPAS481Aand 25 pM TXA on uPA mediated fibrinolysis as determined on blood clots formed from fresh human blood by TEG analysis. The data is the summary of the data presented in Figures 15-19.

[0082] Figure 22: The preventive effect of tPAS481Aon TXA and uPA mediated fibrinogen inactivation uPA (50 ng / ml) was added to fresh human plasma in the presence or absence (C.) of TXA (100 pM) and with or without tPAS481A(100 nM). After 120 min of incubation at 37° C, the concentration of fibrinogen was determined.

[0083] Figure 23: The preventive effect of tPAS481Aon TXA and uPA mediated Factor V inactivation uPA (50 ng / ml) was added to fresh human plasma in the presence or absence (C.) of TXA (100 pM) and with or without tPAS481A(100 nM). After 120 min of incubation at 37° C, the concentration of Factor V was determined.

[0084] Figure 24: The preventive effect of tPAS481Aon TXA and uPA mediated Factor VIII inactivation uPA (50 ng / ml) was added to fresh human plasma in the presence or absence (C.) of TXA (100 pM) and with or without tPAS481A(100 nM). After 120 min of incubation at 37° C, the concentration of Factor VIII was determined. DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0085] Previous studies showed that the withdrawal of aprotinin from the combined anti-fibrinolytic therapy was associated with increased blood loss, transfusion requirements and reoperations in cardiac surgery. Since the withdraw of aprotinin, anti-fibrinolytic therapy is now days achieved solely by administration of lysine analogs only, mainly TXA.

[0086] There have been several attempts to renew the use of aprotinin, in various clinical settings that evidenced the clinical need of aprotinin by proving its advantages over TXA alone or as combination of both. It was reported that aprotinin reduced the need for blood transfusion in cardiac surgery and that aprotinin exert a more potent anti-bleeding effect than TXA in patients undergoing a coronary artery bypass graft (CABG). Furthermore, the combined use of TXA and aprotinin shown to be more effective than TXA alone. Although the clinical benefit of the aprotinin is clear, there are still concerns regarding safety that prevent its use.

[0087] The findings that aprotinin, either alone or in combination with TXA, exhibits a more potent antibleeding effect than TXA alone, highlights the untapped potential for improving management of bleeding risk and the opportunity to expand the use of inhibitors of fibrinolysis to additional indications in clinical practice. To achieve this objective, novel approaches, including those that can be integrated with TXA or used alone, should be explored.

[0088] In the present application the inventors show that the mutant tPA tPAS481Ainhibits the activity of the enzyme plasmin, exerts an antifibrinolytic effect on plasmin mediated fibrinolysis and has a synergistic effect on TXA mediated inhibition of fibrinolysis. Furthermore, it is shown that tPAS481Aprevents the inactivation of the coagulation factors V and VIII and the digestion of fibrinogen by plasmin. The combination and combined therapy provided by the present disclosure is safe and effective.

[0089] Thus, a first aspect of the present disclosure relates to a combination comprising the following components. Component (a), comprises at least one tissue plasminogen activator (tPA) mutant that carries a point mutation at position 481 of the wild type tPA molecule (WT tPA). The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2. The disclosed combination further comprises component (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin. The disclosed combination exerts at least one of: increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity.

[0090] A major component of the disclosed combination is a mutant of tissue plasminogen activator. The "tissue plasminogen activator (tPA)" is a trypsin-like serine protease that consists of five domains: a fibronectin finger-like domain (F, residues 23-50), an epidermal growth factor domain (EGF, residues 51-87), two kringle domains (KI, residues 88-176, and K2, residues 177-261), and a protease domain (P, residues 262-527). In addition to the binding to its substrate plasminogen, tPA binds also to fibrin. Binding of tPA to fibrin is predominantly mediated by its finger domain and with some participation of KI that also mediates its rapid clearance from the circulation. More specifically, the term tPA used herein for the tissue plasminogen activator (also known as PLAT ; enzyme entry EC 3.4.21.68,) relates to a secreted serine protease that converts and activates the proenzyme plasminogen to a potent fibrinolytic enzyme plasmin. tPA is synthesized in vascular endothelial cells as a single polypeptide chain that undergoes proteolytic cleavage by plasmin or trypsin at a centrally located arginine-isoleucine bond, resulting in a 2-chain disulfide-linked form composed of the N-terminally derived heavy chain and the C-terminal light chain. The tPA gene (DNA acc. NT_167187.1 mapped to chr. 8pl 1.21) contains 14 exons encoding the heavy chain domain including two kringle regions (KI and K2) and regions homologous to growth factors and the light chain domain comprising the serine protease catalytic site. Alternative splicing of the tPA gene results in multiple transcript variants encoding different isoforms taking part in multiple biological processes, apart from fibrinolysis, such as cell migration and tissue remodeling. Increased tPA activity causes hyperfibrinolysis manifested as excessive bleeding; decreased tPA activity leads to hypofibrinolysis which can result in thrombosis or embolism. tPA linked phenotypes include familial hyperfibrinolysis (due to increased tPA release) and familial thrombophilia (due to decreased tPA release (OMIM num. 612348). As indicated above, in some embodiments, the tPA is the human tPA. In yet some further embodiments, the wild type tPA comprise the amino acid sequence as denoted by SEQ ID NO: 2. The disclosed combination comprises a tPA mutant that carries at least one mutation in the wild type tPA sequence.

[0091] Still further, plasmin is produced in an inactive form (proenzyme), plasminogen, in the liver. Both, tPA and urokinase plasminogen activator ( uPA ), are multi-functional serine proteases (Nassar T et al, JBC 2002, 277:40499-40504; Nassar T et al, Blood 2004, 106:897-902) that, among others, 560 561 cleave a single peptide bond between the amino acid residues Arg -Vai in the pro-enzyme plasminogen thereby producing a potent proteolytic enzyme - "plasmin" . Efficient activation of plasminogen depends on its binding to fibrin, which in turn dramatically stimulates tPA and uPA activation of plasminogen.

[0092] Although in the process of plasminogen activation, tPA and uPA cleave the same single peptide bond between the amino acids Arg560 and Val561 in plasminogen, the mechanism of their fibrinolytic activities is totally different. This major difference results in an undesired effect of uPA, as will be discussed herein after. The tPA mutant of the disclosed composition provides an effective remedy for uPA deleterious effects.

[0093] The tPA of the present disclosure is a mutated tPA molecule that carries at least one mutation. It should be noted that the term "mutation" referred to herein relates to induced genetic variations which have shown to alter function of the encoded protein product. Mutations may be point mutations that are substitutions, deletions or insertions of a single nucleotide in the DNA sequence, or deletion / insertion mutations - changes in a number of nucleotides in the DNA sequence; or duplication mutations - abnormal repetitions of a DNA sequence compared to the wild-type sequence. It is well known in the art that point mutations may be missense mutations or nonsense mutations or frameshift mutations. In specific embodiments of the present disclosure, the disclosed mutants carry at least one point mutation substituting or replacing a specific amino acid residue with another, due to a point mutation in the codon encoding the specific amino acid residue. It should be noted that the mutated tPA molecule of the present disclosure may carry at least one mutation at any domain of the tPA molecule.

[0094] Still further, in addition to the tPA mutant, the disclosed combination comprises at least one lysine analog. A "lysine analog" as used herein, refers to a molecule that shares structural similarities with the amino acid lysine but possesses certain key differences. These analogues typically retain the fundamental structure of lysine, including the a-amino and a-carboxyl groups and a basic side chain bearing a terminal amino group. However, changes may be introduced in the length or configuration of the side chain, the nature of attached functional groups, or the rigidity and stereochemistry of the molecule. As indicated above, differences between lysine and lysine analogs can be in the side chain attached to the central carbon backbone. Lysine analogs can be shorter or longer as compared to lysine's side chain. For example, a lysine analog might have a shorter chain with two or three carbons, instead of a chain with four carbons or a longer chain with additional functional groups. In addition, the functional group on the side chain might be chemically altered compared to lysine's primary amine group. This can involve changes like adding a methyl group or replacing it with an aromatic ring. Structurally, lysine analogues are designed to mimic or interfere with the biological roles that lysine normally fulfills in proteins and metabolic processes. In many cases, these analogues can bind to the same receptors or enzymes that recognize lysine, acting as competitive inhibitors or biochemical probes. In some embodiments, of particular interest are lysine analogues capable of competing with lysine binding to plasminogen.

[0095] Functionally, Lysine analogues act as competitive inhibitors, binding to the kringle domains of plasminogen and preventing its association with fibrin or tissue-type plasminogen activator (tPA). This reduces plasmin generation and inhibits fibrinolysis, helping to stabilize clots. Non-limiting examples of lysine analogs that may be used in the combinations, methods and kit of this disclosure include 4-(aminomethyl)-cyclo-hexane-carboxylic acid (tranexamic acid or TXA), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl-cyclohexanecarboxylic acid. "Lysine" (symbol Lys or K) is an a-amino acid that is a precursor to many proteins. It contains an a-amino group (which is in the protonated-NH+ form when dissolved in water), an a-carboxylic acid group (which is in the deprotonated -COO- form when dissolved in water), and a side chain lysyl ((CH2)4NH2), classifying it as a basic, charged (at physiological pH), aliphatic amino acid. Its formula is C6H14N2O2. It should be appreciated that although lysine analogs are used by the disclosed combinations, the present disclosure further encompasses the option of using the amino acid lysine in the combination.

[0096] As indicated above, in some embodiments, any lysine analog (or in some embodiments, lysine residues) can be used in the disclosed combinations, provided that such analog is capable of binding the lysine binding site (LBS) of plasminogen and / or plasmin. The "lysine binding site (LBS) of plasminogen ” , as used herein, is a specific region on the plasminogen molecule that interacts with lysine residues. It is located within certain kringle domains, which are finger-like loops with a specific folded structure. These LBSs are found in four out of the five kringle domains (KI, K4, and K5) of plasminogen. The interaction between LBS and lysine plays a crucial role in regulating the activation of plasminogen. When ligands, like lysine analogs, bind to the LBS, they may induce conformational changes in plasminogen. This alters its shape and makes it easier for plasminogen activators to convert it into its active form, plasmin. The "kringle domain" (K) as referred to herein, relates to autonomous protein domains that fold into large loops stabilized by three disulfide linkages and are important in protein-protein interactions, most notably with blood coagulation factors. Similarly, the LBSs present within the kringle domains of plasmin (notably those corresponding to the kringle domains inherited from the precursor plasminogen) mediate binding to lysine residues exposed on the surface of fibrin clots, cellular receptors, or extracellular matrix proteins.

[0097] Binding of lysine to the lysine binding site (LBS) of plasminogen, as referred to herein, involves a specific molecular interaction between the a-amino group of lysine and structural features within the kringle domains of plasminogen, particularly kringle 1 and kringle 5. These LBS regions contain negatively charged and aromatic residues that form a complementary surface to recognize and bind the positively charged side chain of lysine. This interaction is mediated through hydrogen bonding and cation-71 interactions, which together stabilize the binding. Functionally, the binding of lysine or lysine-containing sequences on proteins such as fibrin induces a conformational change in plasminogen from a closed, inactive state to an open, activatable form. This conformational shift enhances the accessibility of plasminogen to tissue-type or urokinase-type plasminogen activators (tPA or uPA), facilitating its conversion to plasmin. Furthermore, lysine binding localizes plasminogen to the surface of fibrin clots, where C-terminal lysine residues are exposed during clot formation and remodeling. This localization enhances targeted plasmin generation and promotes efficient fibrinolysis. The importance of this interaction is clinically evident in the use of lysine analogs such as tranexamic acid and s- aminocaproic acid, which act as competitive inhibitors by mimicking lysine and blocking the LBS, thereby preventing excessive plasminogen activation and fibrinolysis.

[0098] The combination of the present disclosure inhibits fibrinolytic activity of free plasmin, as well as of tPA and uPA. In some embodiments, the fibrinolytic activity and / or free plasmin activity is caused by at least one of urokinase plasminogen activator (uPA) and / or tissue plasminogen activator (tPA) in the presence of the lysine analog. The term "fibrinolysis" or "fibrinolytic activity" used herein in relation to the disclosure denotes a physiological process wherein blood clots are dissolved. In this process, fibrin, the structural protein forming blood clots, is enzymatically degraded, primarily through the action of plasmin. This process plays a central role in maintaining vascular patency and preventing pathological thrombosis. Fibrinolysis is fundamentally a hydrolytic process, as it involves the cleavage of peptide bonds within the fibrin polymer through the enzymatic action of plasmin, a serine protease. Plasmin catalyzes the hydrolysis of specific peptide bonds in fibrin using water molecules, leading to the production of soluble fibrin degradation products such as fragments D and E. A major reaction in fibrinolysis is the activation of the proenzyme plasminogen to plasmin. . Fibrinolysis is important for maintenance of homeostasis to enable accessibility of biological factors responsible for repair and regeneration (angiogenesis) of to the damaged blood vessels. Inhibition or reduction of fibrinolysis by fibrinolytic inhibitors is important in surgery and trauma to control blood loss and even more strongly indicated for patients with bleeding disorders. To assess fibrinolytic activity (and the inhibition of such activity by the disclosed combination), various in vitro methods are employed. One classical method is the fibrin plate assay, in which samples are applied to a fibrin-containing gel and zones of lysis are measured to indicate fibrin degradation. Chromogenic or Anorogenic substrate assays are also used, where plasmin activity is detected based on the cleavage of synthetic substrates that generate a measurable signal. Clot lysis assays monitor the rate or extent of clot dissolution over time in the presence of fibrinolytic agents. Additionally, enzyme-linked immunosorbent assays (ELISAs) can quantify fibrin degradation products or fibrinolytic enzymes, and global assays such as thromboelastography (TEG) or rotational thromboelastometry (ROTEM) provide real-time assessments of clot formation and breakdown in whole blood, offering functional insight into fibrinolytic capacity.

[0099] "Anti- fibrinolytic activity " refers to a process, which prevents, inhibit, or decrease the fibrinolytic activity. The disclosed combination exerts in some embodiments, a decrease, reduction, decline, drop, diminishment, shrinkage, downturn, cutback, attenuation, lessening, and abatement of the fibrinolytic activity of free plasmin, in about 1% to about 100%, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, as compared with the fibrinolytic activity of free plasmin in the absence of the tPA mutant or the disclosed combination. In yet some additional or alternative embodiments, the disclosed combination exerts an increase in the anti-fibrinolytic activity of the lysine analogs. More specifically, enhancement, improvement, augmentation, amplification, refinement, advancement, elevation, enrichment, boost, intensification, and upgrade of the anti-fibrinolytic activity of the lysine analogs in about 1% to about 100%, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, as compared with the anti-fibrinolytic activity of the lysine analogs in the absence of the tPA mutant, or any combination thereof.

[0100] As noted above, the disclosed combination exerts reducing free plasmin activity. "Free plasmin" refers herein to an active plasmin that is not bound to fibrinogen and is not bound or only partially bound to its natural inhibitors such as alpha-2-antiplasmin (a2-AP) and alpha-2-macroglobulin (a2-M), which render the free plasmin inactive. "Free plasmin activity” refers to the activity of the active pool of free plasmin which is available to break down clots, induce fibrinolysis and / or is free to degrade coagulation factors, such as fibrinogen, Factor V (FV) and factor VIII (FVIII), that may lead to serious side effects by inhibiting coagulation and the formation of new blood clots.

[0101] In some embodiments, the fibrinolytic activity is caused by urokinase plasminogen activator (uPA). In some other embodiments, the free plasmin activity is caused by uPA. In some further embodiments, the fibrinolytic activity and the free plasmin activity is caused by uPA. uPA is expressed as a single-chain molecule (scuPA) composed of an N-terminal fragment (ATF; amino acids 1-135) and a protease domain (amino acids 136-411), also known as low molecular weight uPA (LMW-uPA). The amino-terminal fragment (ATF) is itself composed of 2 independent domains, the amino-terminal growth factor-like domain (GFD; amino acids 1-43), which is known to bind to the uPA receptor, and a single kringle (K; amino acids 47-135).

[0102] Further, the term "uPA " used herein for the "urinary plasminogen activator" (also known as PLAU; EC 3.4.21.73) relates to another enzyme that converts plasminogen to plasmin. uPA may occur as a single-chain form (scuPA) or as a 2-chain derivative (also called HMW uPA) generated by cleavage of the single-chain form by plasmin. HMW uPA can be further processed into LMW uPA which is proteolytically active but does not bind to the uPA receptor. The uPA gene (Gene acc. NT_030059.13 mapped to chr. 10q22.2) produces several alternatively spliced transcript variants encoding different uPA isoforms. uPA is involved in degradation of the extracellular matrix and angiogenesis, and possibly tumor cell migration and proliferation.

[0103] As noted above, the disclosed combinations reduce and even inhibits free plasmin activity. In some embodiments, “reducing” may be up to, and including, inhibiting free plasmin activity. More specifically, as used herein, the terms "inhibiting" and "reducing" relate to the elimination, reduction, decreasing and attenuating free plasmin activity by any one of about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, or even 100% (complete inhibition). Still further, in some embodiments, the disclosed combinations exert increasing the anti- fibrinolytic activity of the lysine analog. As used herein, the terms "increasing", "enhancing" and "promoting" relate to the augmentation or amplification of the anti-fibrinolytic activity of the lysine analog by any one of about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.

[0104] In some embodiments, the mutant is a tPA mutant that carries a point mutation in position 481 of the wild type molecule as denoted by SEQ ID NO: 2. In yet some further embodiments, such mutation results in a substitution of serine 481 to alanine.

[0105] In more specific embodiments, the tPA mutant of the disclosed combination may comprise the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives or homologs thereof. In more specific embodiments, the mutant being tPASer481Ala.

[0106] Both the recombinant wild type tPA and the S481A mutant protein sequences (SEQ ID NO: 2 and 1, respectively) contain 2 extra amino acids, RS (arginine and serine), at the N- terminal end, as a result of introduction of Bgl II cloning site into the original cDNA sequence encoding the human tPA. Thus, in some embodiments, "variant" of the mutant of SEQ ID NO: 1, may be any variant that does not contain the two N-terminal residues RS.

[0107] With respect to amino acid sequences, for example, the amino acid sequence of the any one of the tPA mutants of the invention, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the present disclosure.

[0108] More specifically, homologs of the disclosed tPA mutant, display certain percentage of homology. "Homology" with respect to a reference polypeptide, specifically, the disclosed tPA mutant, and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. This term encompasses similarity as well as identity. Neither N- nor C-terminal extensions nor insertions or deletions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. In some embodiments, the present disclosure also encompasses polypeptides which are variants of, or analogues to, the polypeptides specifically defined in the present disclosure by their amino acid sequence. With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence thereby altering, adding or deleting a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant”, where the alteration results in the substitution of an amino acid with a chemically similar amino acid.

[0109] Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles and analogous peptides of the present disclosure.

[0110] For example, substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

[0111] 1) Alanine (A), Glycine (G);

[0112] 2) Aspartic acid (D), Glutamic acid (E);

[0113] 3) Asparagine (N), Glutamine (Q);

[0114] 4) Arginine (R), Lysine (K);

[0115] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0116] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0117] 7) Serine (S), Threonine (T); and

[0118] 8) Cysteine (C), Methionine (M).

[0119] The term "derivative" as used herein refers to any biologically active molecule that is structurally based on, or originates from, the tPASer481Ala mutant (SEQ ID NO: 1), and which may include modifications, substitutions, deletions, insertions, additions, chemical alterations, or combinations thereof. In some embodiments, "biologically active molecule” refers to a derivative of tPASer481Ala mutant that in combination with at least one lysin analog, exerts at least one of increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity.

[0120] In some embodiments, the tPA mutant of the present disclosure, specifically the mutant as disclosed in SEQ ID NO: 1, lacks any fibrinolytic activity, or display reduced fibrinolytic activity, and moreover, does not activate plasminogen.

[0121] It should be noted that the term "amino acid(s)" as used herein refers to all naturally occurring L- amino acids, e.g. and including D-amino acids. The amino acids are identified by either the well- known single-letter or three-letter designations.

[0122] As mentioned above, the other component (b), of the disclosed combination is at least one lysine analog. In some embodiments, the lysine analog of the disclosed combination is at least one of 4- (aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl-cyclohexanecarboxylic acid. In some specific embodiments, the lysine analog used in the disclosed combination may be 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid). As used herein, "Tranexamic acid" or "4- (aminomethyl)-cyclo- hexane-carboxylic acid" is a synthetic analog of the amino acid lysine. Structurally, tranexamic acid consists of a cyclohexane ring substituted with an aminomethyl group at the 4-position and a carboxyl group at the 1 -position. TXA has the molecular formula CsHisNCX and a molecular weight of approximately 157.21 g / mol. TXA serves as an antifibrinolytic by reversibly binding four to five lysine binding sites on plasminogen. This prevents the binding of plasminogen to fibrin and by that, decreases the conversion of plasminogen to plasmin, preventing fibrin degradation and preserving the breakdown of fibrin's matrix structure. TXA is identified by DrugBank Accession Number DB00302.

[0123] In yet some further specific embodiments, the lysine analog used in the disclosed combination may be e-amino caproic acid (EACA). The " s-aminocaproic acid (EACA)” also known as Aminocaproic acid, s-Ahx, or 6-aminohexanoic acid is a derivative and analogue of the amino acid lysine, which makes it an effective inhibitor for enzymes that bind that particular residue such as plasmin. EACA has the molecular formula CeHnNCX and a molecular weight of approximately 131.17 g / mol. EACA is structurally analogous to lysine but lacks the amino group at the a-position, thus behaving as a straightforward antifibrinolytic agent. For this reason, EACA is effective in treatment of certain bleeding disorders. EACA is sold for example under the brand name Amicar®. In some further specific embodiments, the lysine analog used in the disclosed combination may be cyclohexanecarboxylic acid. More specifically, "cyclohexanecarboxylic acid”, is a saturated cyclic carboxylic acid comprising a cyclohexane ring substituted with a single carboxyl group. Cyclohexanecarboxylic acid has the molecular formula CeHnCChH and a molecular weight of approximately 128.17 g / mol. Cyclohexanecarboxylic acid is the carboxylic acid of cyclohexane. It is a colorless oil that crystallizes near room temperature.

[0124] In some further specific embodiments, the lysine analog used in the disclosed combination may be 4-methyl-cyclohexanecarboxylic acid. The "4-methyl-cyclohexanecarboxylic acid" is a modified cyclohexanecarboxylic acid in which a methyl group is substituted at the 4-position of the cyclohexane ring relative to the carboxyl group. 4-methyl-cyclohexanecarboxylic acid has the molecular formula CH3C6H10CO2H and a molecular weight of approximately 142.20 g / mol.

[0125] It should be appreciated that in some further embodiments the amino acid lysine may be used in the combination of the present disclosure.

[0126] In some embodiments, the lysine analog is at least one of 4-(aminomethyl)-cyclo- hexanecarboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

[0127] In some particular embodiments, the lysine analog of the disclosed combination may be 4- (aminomethyl)-cyclo- hexane-carboxylic acid (TXA), or any salt, base, ester or amide thereof, any enantiomer, stereoisomer or diasterioisomerdisterioisomer thereof, or any combination or mixture thereof. The phrase "tranexamic acid and any salt, base, ester, or amide thereof, any enantiomer, stereoisomer or diasterioisomerdisterioisomer thereof, or any combination or mixture thereof" refers broadly to tranexamic acid in any pharmaceutically acceptable chemical form that retains its antifibrinolytic activity. This includes the free base form of tranexamic acid as well as its pharmaceutically acceptable salts, which may improve solubility or stability. It also encompasses ester and amide derivatives in which the carboxylic acid group of tranexamic acid is chemically modified, for example to enhance pharmacokinetic properties, bioavailability, or tissue targeting, while preserving therapeutic efficacy. These forms are considered equivalent in the context of pharmaceutical compositions, ensuring that all therapeutically active variants of tranexamic acid are included within the scope of the disclosure Thus, in some embodiments, the disclosed combination may comprise TXA and the tPASer481Alamutant. In yet some specific embodiments, the disclosed combination consists of in some embodiments, TXA and the tPASer481Alamutant.

[0128] According to some embodiments, the disclosed combination leads to reduction or inhibition of free plasmin activity. Free plasmin activity, as used herein, refers in some embodiments, to the enzymatic activity of unbound plasmin, the active serine protease derived from plasminogen, when it is not inhibited by circulating inhibitors such as a2-antiplasmin. Free plasmin cleaves fibrin and other plasma proteins, leading to the dissolution of blood clots in a process known as fibrinolysis. The term specifically distinguishes active plasmin in its unbound, functional state from plasmin that is complexed with inhibitors and thereby rendered inactive. It should be noted that in some embodiments, free plasmin activity comprises and / or results in at least one of: (i) cleavage and / or inactivation of at least one coagulation factor; (ii) damaging the blood brain barrier (BBB); and (iii) modulation of platelets activity. More specifically, in some embodiments, free plasmin activity comprises and / or results in cleavage and / or inactivation of at least one coagulation factor. As used herein, "cleavage" refers to the breaking of peptide bonds (the chemical bonds linking amino acids) within the protein chain. This process splits the protein into smaller fragments (degradation), which may lead to fragments with specific function or breakdown of the proteins, leading to the loss of their functions and forwarding proteins for recycling. The term "inactivation " refers herein to the loss of a protein's biological function. In some embodiments, the protein structure may still be intact, but it cannot perform its intended activity. Thus, in some embodiments, free plasmin activity results in cleaved and inactivated coagulation factors that cannot perform their coagulation activity. Coagulation activity, in the context of coagulation factors, refers to the functional ability of a coagulation factor to participate in the biochemical cascade that leads to blood clot formation. This activity typically involves the activation of downstream factors through proteolytic cleavage, the formation of multiprotein complexes on phospholipid surfaces in the presence of calcium ions, and ultimately the generation of thrombin and conversion of fibrinogen to fibrin. The term encompasses both the enzymatic activity of serine protease factors (e.g., Factor Xa or thrombin) and the cofactor function of non-enzymatic proteins (e.g., Factors V and VIII), all of which are essential for the orderly progression and amplification of the coagulation cascade. It should be understood that this term further encompasses the maintenance and stability of a blood clot. More specifically, " coagulation factors" , also known as clotting factors, are a collection of plasma glycoproteins (proteins with attached sugar molecules) that function in a highly orchestrated cascade to achieve hemostasis, the physiological process of maintaining blood vessel integrity and preventing excessive blood loss after injury. There are several principal coagulation factors, designated by Roman numerals. Non limiting examples of coagulation factors encompassed by the present disclosure include Factor I (Fibrinogen), Factor II (prothrombin), Factor III (tissue factor), Factor V, Factor VII, Factor VIII (Antihemophilic factor (AHF)), Factor IX, Factor X, Factor XI, Factor XII and Factor XIII. Coagulation factors function in a tightly regulated cascade system. Activation of one factor by another in a specific sequence is crucial for efficient clot formation. Each factor possesses specific enzymatic activity or serves as a cofactor, facilitating the activation of downstream factors. These interactions often involve calcium ions (Ca2+) as essential cofactors. Tissue factor (TF) plays a pivotal role, initiating the extrinsic pathway upon vascular injury by forming a complex with Factor Vila, leading to activation of Factor X. Activation cascades (intrinsic and extrinsic) converge on the activation of Factor X, which subsequently activates prothrombin (Factor II) to thrombin (Factor Ila). Thrombin, the central enzyme in coagulation, cleaves fibrinogen (Factor I) into fibrin monomers, which then polymerize to form an insoluble fibrin mesh, the structural backbone of a blood clot. Factor XIII (a transglutaminase) covalently cross-links fibrin polymers, strengthening and stabilizing the clot. The intrinsic pathway of coagulation is initiated within the vascular system when blood comes into contact with negatively charged surfaces, such as exposed subendothelial collagen or artificial biomaterials. This contact activates Factor XII (Hageman factor), which undergoes conformational change and, in the presence of high-molecular-weight kininogen (HMWK) and prekallikrein, catalyzes the activation of Factor XI to Factor Xia. Activated Factor Xia subsequently cleaves and activates Factor IX to Factor IXa. Factor IXa, in complex with its cofactor, activated Factor VIII (Factor Villa), calcium ions, and phospholipid surfaces, forms the intrinsic tenase complex. This complex efficiently catalyzes the activation of Factor X to Factor Xa, thus linking the intrinsic pathway to the common coagulation cascade. Like the extrinsic pathway, activation of Factor X leads to the conversion of prothrombin (Factor II) to thrombin (Factor Ila), with thrombin serving as the central enzyme in coagulation. Thrombin cleaves fibrinogen (Factor I) to generate fibrin monomers, which polymerize and are subsequently crosslinked by Factor XIII to produce a stable, insoluble fibrin clot. Notably, the intrinsic pathway amplifies the coagulation response and can independently sustain thrombin generation following initial tissue factor exposure, ensuring robust hemostatic plug formation. Still further, it should be understood that, in addition to the classical coagulation factors defined herein, the phrase 'inactivation of at least one coagulation factor' also encompasses any factor or protein that participates, whether directly or indirectly, in the coagulation process. For example, Von Willebrand factor (vWF) that is not considered as a coagulation factor in the classical numbered sense, but it is critically involved in blood coagulation. Von Willebrand factor (vWF) is a large multimeric glycoprotein that plays a central role in blood clotting by mediating the adhesion of platelets to sites of vascular injury. It is synthesized primarily by endothelial cells and megakaryocytes and is stored in Weibel-Palade bodies and platelet a-granules. In addition to its adhesive function, vWF also serves as a carrier protein for coagulation Factor VIII, protecting it from rapid degradation and extending its half-life in circulation. In addition to von Willebrand factor (vWF), several other components enhance coagulation but are not part of the classically numbered coagulation factors and are thus encompassed by the scope of the present disclosure. Tissue factor (TF), sometimes referred to historically as Factor III, is a membrane-bound protein expressed by subendothelial tissues and activated endothelial cells. Although not an enzyme or a numbered coagulation factor in the modern classification, tissue factor is essential for initiating the extrinsic pathway of coagulation through its complex formation with activated Factor VII (Factor Vila).

[0129] High-molecular-weight kininogen (HMWK) is another non-numbered factor that functions as a cofactor in the contact activation (intrinsic) pathway. It facilitates the activation of Factor XII and prekallikrein on negatively charged surfaces, thereby initiating the intrinsic coagulation cascade. Prekallikrein, also known as Fletcher factor, is the zymogen form of kallikrein. It contributes to coagulation by activating Factor XII and participating in the generation of bradykinin, which links coagulation with inflammation. Although not a classical coagulation factor, its role in amplifying the intrinsic pathway is well recognized.

[0130] Platelet-activating factor (PAF) is a phospholipid mediator that promotes platelet aggregation and degranulation.

[0131] Thrombomodulin is a transmembrane protein expressed on endothelial cells. While it primarily functions to regulate coagulation by altering thrombin activity, it plays an indirect but essential role in modulating clot formation and resolution. Specifically, it converts thrombin from a procoagulant enzyme to one that activates protein C, a natural anticoagulant, thus integrating pro- and anticoagulant mechanisms.

[0132] In addition to these proteins, small molecules like adenosine diphosphate (ADP) and thromboxane A2 (TXAz), which are released from activated platelets, significantly enhance coagulation. ADP promotes the recruitment and activation of additional platelets, while TXA2 induces vasoconstriction and amplifies platelet aggregation. As indicated above, in some embodiments, free plasmin activity comprises and / or results in damaging the blood brain barrier (BBB). In the context of the present disclosure, "damaging the blood brain barrier (BBB)'' , refers to any action or process that results in the compromise, alteration, or disruption of the integrity and selective permeability of the blood-brain barrier, a physiological barrier that separates the circulating blood from the brain extracellular fluid in vertebrates. The BBB is formed by tightly joined endothelial cells lining the blood vessels in the brain, which restrict the movement of large molecules, charged particles, and pathogens from the bloodstream into the brain tissue. The BBB is supported by astrocytes, whose foot processes encase the blood vessels, and by pericytes, which contribute to the regulation of blood flow. Such damage may include but is not limited to physical disruption, biochemical alterations, or pathological conditions leading to increased permeability, compromised functionality, or structural changes within the blood-brain barrier, thereby allowing substances that are normally restricted from freely entering the brain to penetrate or cross the barrier. Damaging the blood-brain barrier (BBB) also includes increasing its permeability, which can be measured by assessing the leakage of substances such as contrast agents, proteins, or other biomarkers that typically do not cross the intact barrier. Techniques such as magnetic resonance imaging (MRI) with contrast agents, cerebrospinal fluid (CSF) analysis, or the measurement of specific protein levels in the bloodstream (e.g., serum SIOOB or albumin) are commonly used to evaluate BBB disruption and permeability.

[0133] In yet some further embodiments, free plasmin activity comprises and / or results in modulation of platelets activity. In the context of the present disclosure, "modulation of platelet activity " , refers to altering, regulating, or influencing the function, behavior, or physiological responses of platelets, including but not limited to aggregation, adhesion, activation, degranulation, and thrombus formation. Such modulation may encompass both enhancement and suppression of platelet activity. More specifically, by modulation, it is meant that plasmin, once formed, also binds to the platelets surface and, at low concentrations, renders the platelet dysfunctional by cleaving glycoprotein Illa selectively in the presence of bound fibrinogen. At higher concentrations (e.g., approximately 1 caseinolytic unit / ml), plasmin activates the platelet directly. This, in turn, directly or indirectly leads to activation / inactivation and clearance of platelets. As indicated above, the disclosed combination leads to a reduction of free plasmin activity, more specifically, reduction as used herein refers to any decrease, reduction, decline, drop, diminishment, shrinkage, downturn, cutback, attenuation, lessening, and abatement of the of the free plasmin activity, in about 1% to about 100%, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, as compared with free plasmin activity in the absence of the combination of the present disclosure, or at least one component thereof.

[0134] Free plasmin activity may result in cleavage and / or inactivation of at least one coagulation factor. In some specific embodiments, such coagulation factor / s may comprise at least one of fibrinogen, Factor V (FV) and factor VIII (FVIII). Thus, the disclosed combination inhibits the cleavage and / or inactivation of at least one of fibrinogen, FV and FVIII, as shown by the present disclosure.

[0135] "Fibrinogen” or "Factor I” , as used herein, is a glycoprotein complex, produced in the liver, that circulates in the blood of all vertebrates. During tissue and vascular injury, it is converted enzymatically by thrombin to fibrin and then to a fibrin-based blood clot. Fibrin clots function primarily to occlude blood vessels to stop bleeding. Fibrin also binds and reduces the activity of thrombin.

[0136] Still further, "Factor V (FV)", is a protein of the coagulation system, rarely referred to as proaccelerin or labile factor. In contrast to most other coagulation factors, it is not enzymatically active but functions as a cofactor. The gene for factor V is located on the first chromosome ( 1 q24).

[0137] "Factor VIII (FVIII)" is an essential blood-clotting protein, also known as anti-hemophilic factor (AHF). Defects in this gene result in hemophilia A, an X-linked coagulation disorder. This protein circulates in the bloodstream in an inactive form, bound to another molecule called von Willebrand factor, until an injury that damages blood vessels occurs. In response to injury, coagulation factor VIII is activated and separates from von Willebrand factor. The active protein (sometimes written as coagulation factor Villa) interacts with another coagulation factor called factor IX. This interaction sets off a chain of additional chemical reactions that form a blood clot.

[0138] It should be understood that the disclosed combination, which in some embodiments comprises at least two active components, specifically, the tPA mutant Ser481Ala and tranexamic acid (TXA), may include any effective ratio (e.g., molar ratio) of these components. For example, the ratio may range from approximately 1:1 to about 1:1000 or greater, for either of the components, depending on the intended application and therapeutic effect. More specifically, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1: 15, 1:16, 1:17, 1: 18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,

[0139] 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58,

[0140] 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75,

[0141] 1:76. 1:77. 1:78. 1:79. 1:80. 1:81. 1:82. 1:83. 1:84. 1:85. 1:86. 1:87. 1:88. 1:89. 1:90. 1:91. 1:92.

[0142] 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109, 1:110, 1:111, 1:112, 1:113, 1:114, 1:115, 1:116, 1:117, 1:118, 1:119, 1:120, 1:121, 1:122, 1:123, 1:124, 1:125, 1:126, 1:127, 1:128, 1:129, 1:130, 1:131, 1:132, 1:133, 1:134, 1:135, 1:136, 1:137, 1:138, 1:139, 1:140, 1:141, 1:142, 1:143, 1:144, 1;145, 1:146, 1:147, 1:148, 1:149, 1:150, 1:151, 1:152, 1:153, 1:154, 1:155, 1:156, 1:157, 1:158, 1:159, 1:160, 1:161, 1:162, 1:163, 1:164, 1:165, 1:166, 1:167, 1:168, 1:169, 1:170, 1:171, 1:172, 1:173, 1:174, 1:175, 1:176, 1:177, 1:178, 1:179, 1:180, 1:181, 1:182, 1:183, 1:184, 1:185, 1:186, 1:187, 1:188, 1:189, 1:190, 1:191, 1:192, 1:193, 1:194, 1:195, 1:196, 1:197, 1:198, 1:199, 1:200, 1:201, 1:202, 1:203, 1:204, 1:205, 1:206, 1:207, 1:208, 1:209, 1:210, 1:211, 1:212, 1:213, 1:214, 1:215, 1:216, 1:217, 1:218, 1:219, 1:220, 1:221, 1:222, 1:223, 1:224, 1:225, 1:226, 1:227, 1:228, 1:229, 1:230, 1:231, 1:232, 1:233, 1:234, 1:235, 1:236, 1:237, 1:238, 1:239, 1:240, 1:241, 1:242, 1:243, 1:244, 1:245, 1:246, 1:247, 1:248, 1:249, 1:250, 1:251, 1:252, 1:253, 1:254, 1:255, 1:256, 1:257, 1:258, 1:259, 1:260, 1:261, 1:262, 1:263, 1:264, 1:265, 1:266, 1:267, 1:268, 1:269, 1:270, 1:271, 1:272, 1:273, 1:274, 1:275, 1:276, 1:277, 1:278, 1:279, 1:280, 1:281, 1:282, 1:283, 1:284, 1:285, 1:286, 1:287, 1:288, 1:289, 1:290, 1:291, 1:292, 1:293, 1:294, 1:295, 1:296, 1:297, 1:298, 1:299, 1:300, 1:301, 1:302, 1:303, 1:304, 1:305, 1:306, 1:307, 1:308, 1:309, 1:310, 1:311, 1:312, 1:313, 1:314, 1:315, 1:316, 1:317, 1:318, 1:319, 1:320, 1:321, 1:322, 1:323, 1:324, 1:325, 1:326, 1:327, 1:328, 1:329, 1:330, 1:331, 1:332, 1:333, 1:334, 1:335, 1:336, 1:337, 1:338, 1:339, 1:340, 1:341, 1:342, 1:343, 1:344, 1:345, 1:346, 1:347, 1:348, 1:349, 1:350, 1:351, 1:352, 1:353, 1:354, 1:355, 1:356, 1:357, 1:358, 1:359, 1:360, 1:361, 1:362, 1:363, 1:364, 1:365, 1:366, 1:367, 1:368, 1:369, 1:370, 1:371, 1:372, 1:373, 1:374, 1:375, 1:376, 1:377, 1:378, 1:379, 1:380, 1:381, 1:382, 1:383, 1:384, 1:385, 1:386, 1:387, 1:388, 1:389, 1:390, 1:391, 1:392, 1:393, 1:394, 1:395, 1:396, 1:397, 1:398, 1:399, 1:400, 1:401, 1:402, 1:403, 1:404, 1:405, 1:406, 1:407, 1:408, 1:409, 1:410, 1:411, 1:412, 1:413, 1:414, 1:415, 1:416, 1:417, 1:418, 1:419, 1:420, 1:421, 1:422, 1:423, 1:424, 1:425, 1:426, 1:427, 1:428, 1:429, 1:430, 1:431, 1:432, 1:433, 1:434, 1:435, 1:436, 1:437, 1:438, 1:439, 1:440, 1:441, 1:442, 1:443, 1:444, i:445, 1:446, 1:447, 1:448, 1:449, 1:450, 1:451, 1:452, 1:453, 1:454, 1:455, 1:456, 1:457, :458, 1:459, 1:460, 1:461, 1:462, 1:463, 1:464, 1:465, 1:466, 1:467, 1:468, 1:469, 1:470, 1:471,:472, 1:473, 1:474, 1:475, 1:476, 1:477, 1:478, 1:479, 1:480, 1:481, 1:482, 1:483, 1:484, 1:485,:486, 1:487, 1:488, 1:489, 1:490, 1:491, 1:492, 1:493, 1:494, 1:495, 1:496, 1:497, 1:498, 1:499,:500, 1:501, 1:502, 1:503, 1:504, 1:505, 1:506, 1:507, 1:508, 1:509, 1:510, 1:511, 1:512, 1:513,:514, 1:515, 1:516, 1:517, 1:518, 1:519, 1:520, 1:521, 1:522, 1:523, 1:524, 1:525, 1:526, 1:527,:528, 1:529, 1:530, 1:531, 1:532, 1:533, 1:534, 1:535, 1:536, 1:537, 1:538, 1:539, 1:540, 1:541,:542, 1:543, 1:544, 1:545, 1:546, 1:547, 1:548, 1:549, 1:550, 1:551, 1:552, 1:553, 1:554, 1:555,:556, 1:557, 1:558, 1:559, 1:560, 1:561, 1:562, 1:563, 1:564, 1:565, 1:566, 1:567, 1:568, 1:569,:570, 1:571, 1:572, 1:573, 1:574, 1:575, 1:576, 1:577, 1:578, 1:579, 1:580, 1:581, 1:582, 1:583,:584, 1:585, 1:586, 1:587, 1:588, 1:589, 1:590, 1:591, 1:592, 1:593, 1:594, 1:595, 1:596, 1:597,:598, 1:599, 1:600, 1:601, 1:602, 1:603, 1:604, 1:605, 1:606, 1:607, 1:608, 1:609, 1:610, 1:611,:612, 1:613, 1:614, 1:615, 1:616, 1:617, 1:618, 1:619, 1:620, 1:621, 1:622, 1:623, 1:624, 1:625,:626, 1:627, 1:628, 1:629, 1:630, 1:631, 1:632, 1:633, 1:634, 1:635, 1:636, 1:637, 1:638, 1:639,:640, 1:641, 1:642, 1:643, 1:644, 1:645, 1:646, 1:647, 1:648, 1:649, 1:650, 1:651, 1:652, 1:653,:654, 1:655, 1:656, 1:657, 1:658, 1:659, 1:660, 1:661, 1:662, 1:663, 1:664, 1:665, 1:666, 1:667,:668, 1:669, 1:670, 1:671, 1:672, 1:673, 1:674, 1:675, 1:676, 1:677, 1:678, 1:679, 1:680, 1:681,:682, 1:683, 1:684, 1:685, 1:686, 1:687, 1:688, 1:689, 1:690, 1:691, 1:692, 1:693, 1:694, 1:695,:696, 1:697, 1:698, 1:699, 1:700, 1:701, 1:702, 1:703, 1:704, 1:705, 1:706, 1:707, 1:708, 1:709,:710, 1:711, 1:712, 1:713, 1:714, 1:715, 1:716, 1:717, 1:718, 1:719, 1:720, 1:721, 1:722, 1:723,:724, 1:725, 1:726, 1:727, 1:728, 1:729, 1:730, 1:731, 1:732, 1:733, 1:734, 1:735, 1:736, 1:737,:738, 1:739, 1:740, 1:741, 1:742, 1:743, 1:744, 1:745, 1:746, 1:747, 1:748, 1:749, 1:750, 1:751,:752, 1:753, 1:754, 1:755, 1:756, 1:757, 1:758, 1:759, 1:760, 1:761, 1:762, 1:763, 1:764, 1:765,:766, 1:767, 1:768, 1:769, 1:770, 1:771, 1:772, 1:773, 1:774, 1:775, 1:776, 1:777, 1:778, 1:779,:780, 1:781, 1:782, 1:783, 1:784, 1:785, 1:786, 1:787, 1:788, 1:789, 1:790, 1:791, 1:792, 1:793,:794, 1:795, 1:796, 1:797, 1:798, 1:799, 1:800, 1:801, 1:802, 1:803, 1:804, 1:805, 1:806, 1:807,:808, 1:809, 1:810, 1:811, 1:812, 1:813, 1:814, 1:815, 1:816, 1:817, 1:818, 1:819, 1:820, 1:821,:822, 1:823, 1:824, 1:825, 1:826, 1:827, 1:828, 1:829, 1:830, 1:831, 1:832, 1:833, 1:834, 1:835,:836, 1:837, 1:838, 1:839, 1:840, 1:841, 1:842, 1:843, 1:844, 1:845, 1:846, 1:847, 1:848, 1:849,:850, 1:851, 1:852, 1:853, 1:854, 1:855, 1:856, 1:857, 1:858, 1:859, 1:860, 1:861, 1:862, 1:863,:864, 1:865, 1:866, 1:867, 1:868, 1:869, 1:870, 1:871, 1:872, 1:873, 1:874, 1:875, 1:876, 1:877,:878, 1:879, 1:880, 1:881, 1:882, 1:883, 1:884, 1:885, 1:886, 1:887, 1:888, 1:889, 1:890, 1:891,:892, 1:893, 1:894, 1:895, 1:896, 1:897, 1:898, 1:899, 1:900, 1:901, 1:902, 1:903, 1:904, 1:905,:906, 1:907, 1:908, 1:909, 1:910, 1:911, 1:912, 1:913, 1:914, 1:915, 1:916, 1:917, 1:918, 1:919, 1:920, 1:921, 1:922, 1:923, 1:924, 1:925, 1:926, 1:927, 1:928, 1:929, 1:930, 1:931, 1:932, 1:933,

[0143] 1:934, 1:935, 1:936, 1:937, 1:938, 1:939, 1:940, 1:941, 1:942, 1:943, 1:944, 1:945, 1:946, 1:947,

[0144] 1:948, 1:949, 1:950, 1:951, 1:952, 1:953, 1:954, 1:955, 1:956, 1:957, 1:958, 1:959, 1:960, 1:961,

[0145] 1:962, 1:963, 1:964, 1:965, 1:966, 1:967, 1:968, 1:969, 1:970, 1:971, 1:972, 1:973, 1:974, 1:975,

[0146] 1:976, 1:977, 1:978, 1:979, 1:980, 1:981, 1:982, 1:983, 1:984, 1:985, 1:986, 1:987, 1:988, 1:989,

[0147] 1:990, 1:991, 1:992, 1:993, 1:994, 1:995, 1:996, 1:997, 1:998, 1:999, 1:1000.

[0148] It should be appreciated that the disclosed combination display in some embodiments, a synergistic action, as exerting functions that are not provided by each of the components individually. For example, increasing the anti-fibrinolytic activity of lysine analog and / or reducing free plasmin activity. Accordingly, in some embodiments, the disclosed combination may be referred to herein as a synergistic combination. The term "synergism" refers to interaction of discrete agents (as drugs), such that the total effect is greater than the sum of the individual effects.

[0149] It should be understood that all definitions provided in connection with this aspect are equally applicable to each and every aspect of the present disclosure.

[0150] A further aspect of the present disclosure relates to a composition comprising:

[0151] (a) at least one tissue tPA mutant that carries a point mutation at position 481 of the WT tPA. It should be noted that the WT tPA comprises the amino acid sequence as denoted by SEQ ID NO: 2. The disclosed composition further comprises component (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin. In some embodiments the composition may comprise (c), a combination of (a) and (b), that exerts at least one of: increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity. In some optional embodiments, the disclosed compositions may optionally further comprise (d), at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

[0152] In some embodiments, the fibrinolytic activity and / or free plasmin activity is caused by at least one of urokinase plasminogen activator (uPA) and / or tissue plasminogen activator (tPA) in the presence of the lysine analog.

[0153] In some embodiments, the fibrinolytic activity and / or free plasmin activity is caused by urokinase plasminogen activator (uPA). As used herein, the term "composition” refers broadly to any combination or mixture of two or more components, which may exist in various physical forms, such as solid, liquid or gel, and is not necessarily intended for administration to a living subject. Compositions, as defined herein, may be formulated for use in industrial, diagnostic, research, agricultural, veterinary, or therapeutic settings, without limitation to medical or clinical applications. Specifically, a "pharmaceutical composition" refers to a composition that is formulated and intended for administration to a living organism, typically a human or animal, for the purpose of diagnosing, preventing, treating, ameliorating, or curing a disease, disorder, or pathological condition. Pharmaceutical compositions comprise active pharmaceutical ingredients (APIs) together with one or more pharmaceutically acceptable carriers, excipients, diluents, or adjuvants, which ensure that the final product is suitable for safe and effective administration.

[0154] In some embodiments, the disclosed composition may comprise any of the combinations as defined by the present disclosure.

[0155] In some embodiments, the tPA mutant of the combination of the disclosed composition may comprise at least one point mutation that results in a substitution of serine 481 to alanine.

[0156] In more specific embodiments, the tPA mutant of the combination of the disclosed composition may comprise the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof. In more specific embodiments, the mutant being tPASer481Ala.

[0157] Still further in some embodiments, the lysine analog of the combination of the disclosed composition is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl- cyclohexanecarboxylic acid.

[0158] It should be appreciated that in some further embodiments the amino acid lysine may be used in the combination of the disclosed compositions.

[0159] In some embodiments, the lysine analog is at least one of 4-(aminomethyl)-cyclo- hexanecarboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA). In some particular embodiments, the lysine analog of the combination of the disclosed composition may be 4-(aminomethyl)-cyclo- hexane-carboxylic acid (TXA).

[0160] According to some embodiments, the combination of the disclosed composition leads to reduction or inhibition of free plasmin activity. It should be noted that free plasmin activity comprises and / or results in at least one of: (i) cleavage and / or inactivation of at least one coagulation factor; (ii) damaging the blood brain barrier (BBB); and (iii) modulation of platelets activity.

[0161] As indicated above, free plasmin activity may result in cleavage and / or inactivation of at least one coagulation factor. In some embodiments, such coagulation factor / s may comprise at least one of fibrinogen, Factor V (FV) and factor VIII (FVIII). Thus, the combination of the disclosed composition inhibits the cleavage and / or inactivation of at least one of fibrinogen, FV and FVIII, as shown by the present disclosure.

[0162] In some embodiments of the disclosed compositions, at least one of: (a) the tPA mutant carry a substitution of serine 481 to alanine. Such mutant may comprise the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof. In some embodiments, the mutant being tPASer481Ala; and / or (b), the lysine analog is 4-(aminomethyl)-cyclo- hexane-carboxylic acid (TXA). Thus, in some embodiments, the disclosed combination may comprise as active ingredients TXA and the tPASer481Alamutant.

[0163] It should be appreciated that the compositions of the present disclosure may comprise the active compound / s in free form and be administered directly to the subject to be treated. Alternatively, depending on the size of the active molecule / s (specifically, TXA and the tPASer481Alamutant), it may be desirable to conjugate it to a pharmaceutically acceptable carrier prior to administration. Therapeutic formulations may be administered in any conventional dosage formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more pharmaceutically and physiologically acceptable carriers in the sense of being compatible with the other ingredients and not injurious to the patient.

[0164] It should be understood that in some embodiments, the disclosed combinations and compositions thereof, may further include additional plasmin inhibitors, for example, a2-antiplasmin. Alpha-2- antiplasmin (a2-antiplasmin) is a serine protease inhibitor (serpin) that serves as the primary physiological inhibitor of plasmin, the main enzyme responsible for fibrinolysis, or the breakdown of fibrin clots. It is produced mainly by the liver and circulates in the plasma, where it binds rapidly and irreversibly to free plasmin, forming an inactive complex that prevents excessive degradation of fibrin.

[0165] Still further, in some alternative embodiments, the disclosed combinations and / or compositions may be provided together with, at least one blood product, for example, whole blood unit, fresh plasma unit, plasma, fresh frozen plasma (FFP), and cryoprecipitate.

[0166] In some specific embodiments, the composition or the pharmaceutical composition of the present disclosure may be suitable for injection. The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0167] The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0168] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0169] In the case of sterile powders for the preparation of the sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The compositions of the present disclosure generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and / or additives as known in the art. Supplementary active ingredients can also be incorporated into the compositions. The pharmaceutically acceptable carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Thus, as used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0170] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0171] Still further, it should be understood that each of the active components of the disclosed composition (or of the combination of the present disclosure) may be provided in at least one dosage unit form. As used herein, the term 'dosage unit form' refers to a physically discrete unit suitable for administration to a subject, which contains a predetermined quantity of an active component calculated to produce a desired therapeutic effect. More specifically, each active component may be formulated individually or together in a dosage unit form selected from, for example, a tablet, capsule, injectable solution, suspension, or any other pharmaceutically acceptable format appropriate for the route of administration.

[0172] The compositions or pharmaceutical compositions of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0173] The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. The compositions of the present disclosure also include, but are not limited to, emulsions and liposome containing formulations.

[0174] Formulations include those suitable for topical, oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intraperitoneal (IP), intravenous (IV) and intradermal) administration. The nature, availability and sources, and the administration of all such compounds including the effective amounts necessary to produce desirable effects in a subject are well known in the art. The preparation of pharmaceutical compositions is well known to the skilled man of the art and has been described in many articles and textbooks, see e.g., Remington’s Pharmaceutical Sciences, Gennaro A. R. ed., Mack Publishing Co., Easton, PA, 1990, and especially pp. 1521- 1712 therein.

[0175] In some specific embodiments, the pharmaceutical composition of the present disclosure may be applicable for topical administration or transdermal delivery. Such composition and formulations may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0176] Transdermal delivery may be accomplished in various ways. By "transdermal" herein is meant the passing through the skin and into a subject's blood stream, whereby to provide a systemic effect. Whilst the term embraces transmucosal, i.e. passing through mucosal tissue so as to embrace sublingual, buccal, vaginal and rectal delivery, typically transdermal delivery is affected through a subject's skin. For this reason, references are generally made herein to skin for simplicity's sake only although it will be appreciated that the transdermal delivery described herein may also be transmucosal.

[0177] According to some embodiments, a transdermal delivery system is provided comprising a composition of the present disclosure. Such compositions may be presented in a number of different ways, a typical presentation being one that permits transdermal delivery. For example, the compositions may be contained within an adhesive patch designed to be affixed to the skin of a patient or formulated into a capsule or sachet susceptible to easy rupture (e.g. by rubbing or squeezing between fingers) for release of a calculated dose of the tPA mutants and lysin analogs formulations onto the skin into which it may be rubbed. Other formulations, such as topically applied gels, are known to the skilled person. Typically, the compositions of the present invention are presented as adhesive transdermal patches. Such patches comprising the compositions of the present disclosure constitute a delivery system for transdermal delivery of the composition of the disclosure contained within them.

[0178] Transdermal patches comprising the compositions of this disclosure contain a quantity of tPA mutants and lysine analogs to be delivered and an adhesive to allow contact between the patch and the skin to be maintained in absence of external pressure. Typically, the patches comprise a first face that contacts the skin, and a protective backing layer on a second face of the patch opposing the first face, one face of the backing layer being exposed to the environment during use and comprising a material that is impervious to the components present in the patch. Attached to the first face of a transdermal patch is typically a releasable protective layer that protects the patch prior to its use, and which may be released from the adhesive disposed at the first face of the patch prior to the patch being affixed to the skin.

[0179] By a patch or adhesive patch herein is meant material adapted for adhesion to a subject's skin or mucosal tissue. Typically patches herein have a substantial degree of rigidity and, in use, comprise a backing layer exposed to the environment and a composition of the invention beneath the backing layer. However, the patches of the present disclosure may also be of a non-rigid nature.

[0180] An exemplary transdermal delivery system comprises: (a) at least one drug reservoir containing the at least one tPA mutant and at least one lysine analog , or any salt, base, ester or amide, or any combination or mixture thereof and, optionally, a pharmaceutically acceptable inorganic or organic base in an amount effective to enhance the flux of the active ingredient through the body surface without causing damage thereto; (b) a means for maintaining the system in active ingredient transmitting relationship to the body surface and forming a body surface-system interface; and (c) a backing layer that serves as the outer surface of the device during use. In one embodiment, the drug reservoir comprises a polymeric matrix of a pharmaceutically acceptable adhesive material that serves to affix the system to the skin during drug delivery; typically, the adhesive material is a pressure-sensitive adhesive (PSA) that is suitable for long-term skin contact, and which should be physically and chemically compatible with the active ingredient, inorganic or organic base, and any carriers, vehicles or other additives that are present. Examples of suitable adhesive materials include, but are not limited to, the following: polyethylenes; poly siloxanes; polyisobutylenes; polyacrylates; polyacrylamides; polyurethanes; plasticized ethylene-vinyl acetate copolymers; and tacky rubbers such as poly isobutene, polybutadiene, polystyrene-isoprene copolymers, polystyrene-butadiene copolymers, and neoprene (polychloroprene). Preferred adhesives are polyisobutylenes.

[0181] The transdermal patches that may be used in transdermal delivery of the at least one tPA mutant and at least one lysine analog or any salt, base, ester or amide, or any combination or mixture thereof comprise the backing layer, the active ingredient-containing layer and the release liner. The release-control membranes to control the transdermal absorption of the active ingredient or the adhesive layers to adhere to the skin can be added, if desired. Furthermore, reservoir-type patches can be adopted. In some embodiments, in the active ingredient-containing layer is a matrix-type adhesive layer containing the active ingredient and an adhesive agent as a base agent. By providing the patches as the matrix-type patches, the patches can be easily designed and the additional layer such as the adhesive layers is not needed so that the cost for manufacturing the patches can be reduced.

[0182] The adhesive agents contained in the drug-containing layers of the transdermal patches of the present disclosure are according to specific embodiments, non-aqueous adhesive agents, and are inclusive of rubber adhesive agents, acryl polymers and silicone polymers.

[0183] The rubber adhesive agents are inclusive of one or not less than two agents selected from styrene- isoprene-styrene block copolymer, styrene-butadiene-styrene block copolymer, styrene-butadiene rubber, polyisobutylene, polybutene, butyl rubber, natural rubber and isoprene rubber and can be used in the present disclosure. The acryl polymers include, but are not limited to, polymers or copolymers containing at least one kind of the (meth)acrylate represented by 2-ethylhexyl acrylate, methyl acrylate, butyl acrylate, 2- hydroxyethyl acrylate, 2-ethylhexyl methacrylate and the like as a monomer unit, for example, adhesive polymers such as acrylic acid-octyl acetate copolymer, 2-ethylhexyl acrylate-N-vinyl-2- pyrrolidone-l,6-hexaneglycolyl dimethacrylate copolymer, 2-ethylhexyl acrylate-vinyl acetate copolymer, 2-ethylhexyl acrylate-vinyl acetate- acrylic acid copolymer, 2-ethylhexyl acrylate-2- ethylhexyl methacrylate-dodecyl methacrylate copolymer, methyl methacrylate-2-ethylhexyl acrylate copolymerization resin emulsion, acryl polymer contained in acryl resin, alkanolamine solution can be used and the commercially available DURO-TAK® (registered trademark) acrylate adhesive agents series (provided by Henkle Japan Ltd.), GELVA® (registered trademark) acrylate adhesive agents series (provided by Monsanto Co.), SK-DYNE MATRIDERM® (provided by Soken Chemical and Engineering Co., Ltd) or EUDRAGIT® (registered trademark) series (provided by Higuchi Inc.) and can be used in the present disclosure.

[0184] The silicone polymers include derivatives of polysiloxane (for example, silicone polymer such as poly dimethylsiloxane and amine-resistant poly dimethylsiloxane).

[0185] Still further, in some embodiments, the disclosed compositions may be formulated in a nanoparticle form. More specifically, in some embodiments, the formulations of the disclosed compositions may be adapted for use as nano- or micro-particles. Nanoscale drug delivery systems using liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity. A particularly desired solution allows for externally triggered release of encapsulated compounds. Externally controlled release can be accomplished if drug delivery vehicles, such as liposomes or polyelectrolyte multilayer capsules, incorporate nanoparticle (NP) actuators.

[0186] More specifically, controlled drug delivery systems (DDS) have several advantages compared to the traditional forms of drugs. A drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects. Cell-specific targeting can be accomplished by attaching drugs to specially designed carriers. Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles, have been tested as carriers in drug delivery systems. Polymeric nanoparticles are one technology being developed to enable clinically feasible oral delivery.

[0187] The pharmaceutical composition of the present disclosure can be administered and dosed by the methods of the invention, in accordance with good medical practice. More specifically, the compositions used in the methods and kits of the invention, described herein after, may be adapted for administration by systemic, parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0188] A further aspect of the present disclosure relates to methods for inhibiting the fibrinolytic activity of plasmin on at least one substrate thereof, the method comprising contacting a media or any substance comprising the substrate and / or free plasmin, with at least one tPA mutated molecule. In some optional embodiments, the substrate in the media, may be further contacted with at least one lysin analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin, or with a composition comprising the same. In some embodiments, the tPA mutant carries a point mutation at position 481 of the WT tPA. It should be noted that the WT tPA comprises the amino acid sequence as denoted by SEQ ID NO: 2.

[0189] Plasmin is a serine protease that plays a central role in the process of fibrinolysis, the physiological mechanism responsible for the dissolution of fibrin clots. The "fibrinolytic activity of plasmin" refers to its enzymatic capability to recognize, bind, and cleave substrates, such as fibrin, the major structural protein forming the matrix of a thrombus or a fibrin clot.

[0190] In the context of the present disclosure, "contacting the substrate" or "contacting a solution, media or any substance comprising the substrate" entails bringing the substrate contained within the medium or any material containing the substrate, into physical or chemical interaction with the at least one tPA mutated molecule. As indicated herein, the "solution" , "media" or "substance" may refer to any fluid, or solid material capable of serving as a carrier or environment for the substrate. Examples of solutions or substances include, but are not limited to, aqueous solutions, organic solvents, colloidal suspensions, emulsions, and biological fluids such as blood, serum, or tissue culture media. Still further, the term "substrate" as used herein refers to a molecule that an enzyme acts upon. The enzyme (i.e. plasmin) binds and catalyzes a chemical reaction (e.g. cleavage) on the substrate molecule. The substrate is then transformed into one or more products during the reaction. Non limiting examples of a substrate related to the present disclosure includes fibrin clot (also referred herein as a coagulation clot), coagulation factors (as disclosed by the present disclosure, for example, fibrinogen, FVIII and FV), platelets, etc.

[0191] A "fibrin clot" refers to a dense, cross-linked network formed by the polymerization of fibrin monomers, which are generated through the enzymatic cleavage of fibrinogen by thrombin during the coagulation cascade.

[0192] "Platelets” , also known as thrombocytes, refers to small, anucleate cell fragments derived from megakaryocytes in the bone marrow that play a critical role in hemostasis. Upon vascular injury, platelets adhere to the exposed extracellular matrix, become activated, aggregate with one another, and provide a phospholipid surface essential for the assembly of coagulation factor complexes. Platelets can be isolated from whole blood by centrifugation using a two-step process, wherein an initial low-speed centrifugation separates platelet-rich plasma (PRP) from red and white blood cells, followed by a subsequent higher- speed centrifugation to pellet the platelets for further collection and use.

[0193] In yet some further embodiments, any synthetic substrate that is cleaved by plasmin may be applicable in the present disclosure. In some embodiments, the substrate may be a chromogenic substrate that when cleaved by plasmin, generates a detectable product. In some non-limiting embodiments, a chromogenic substrate applicable in the present disclosure may be the (S-2251) that upon cleaved by plasmin results in a product having a yellow color that is detected and quantified using spectrophotometer at a wavelength of 405 nm.

[0194] In some embodiments, the tPA mutant used by the disclosed methods may carry a point mutation that results in a substitution of serine 481 to alanine. In yet some further embodiments, the tPA mutant may comprise the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof. Such mutant being tPASer481Ala.

[0195] In some embodiments, the plasmin substrate is at least one of: a coagulation clot, at least one coagulation factor, and platelets. Thus, the disclosed methods may be used for inhibiting the fibrinolytic activity of plasmin on a coagulation clot, at least one coagulation factor, and / or on platelets.

[0196] In more specific embodiments, the disclosed methods may be used for inhibiting the fibrinolytic activity of plasmin on such at least one coagulation factor, specifically, at least one of fibrinogen, Factor V (FV) and factor VIII (FVIII).

[0197] In some embodiments, the contacting step of the substrate or any media or substance comprising such substrate, with the tPA mutant, is performed in a subject in need, thereby inhibiting the fibrinolytic activity of plasmin on a coagulation clot, at least one coagulation factor, and / or on platelets in the subject.

[0198] In some embodiments, such subject is suffering from at least one hemostatic disorder and / or any disease, disorder, or condition associated with fibrinolysis.

[0199] In some embodiments, the methods of the present disclosure relate to, and are applicable for, disease, disorder, or condition associated with fibrinolysis. The term "associated with" refers to a relationship between two features, where one feature (i.e. disease, disorder or condition) is related or caused by the other (i.e. fibrinolysis). It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, “disease”, “disorder”, “condition”, “pathology” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

[0200] "Fibrinolysis" is a tightly regulated physiological mechanism responsible for the degradation of fibrin clots, thereby maintaining vascular patency and preventing pathological thrombus propagation. Fibrinolysis can be classified into two distinct forms: primary fibrinolysis, which occurs as a natural homeostatic process, and secondary fibrinolysis, which results from pharmacological intervention, pathological conditions, or external stimuli leading to clot breakdown. During fibrinolysis, the fibrin meshwork, the principal structural component of a thrombus formed during the coagulation cascade, is enzymatically cleaved. The central protease mediating this process is plasmin, a serine protease that proteolytically degrades fibrin by cleaving specific peptide bonds within the fibrin polymer. This proteolysis generates soluble fibrin degradation products (SFDPs), which are subsequently removed from circulation by secondary proteolytic systems or are cleared via renal and hepatic pathways.

[0201] In more specific embodiments of the disclosed methods, the hemostatic disorder, disease, disorder, or condition comprises at least one of: coagulopathy, thrombocytopenia, hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

[0202] As indicated above, in some optional embodiments, the disclosed methods may further use a lysine analog, for example, at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl- cyclohexanecarboxylic acid.

[0203] In yet some further embodiments, the lysine analog that may be used in the disclosed methods is 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid).

[0204] As indicated herein, in some embodiments, the inhibition of the fibrinolytic activity of plasmin on at least one of its substrates may occur within a subject, specifically, the diseased subject. According to such embodiments, the disclosed methods may further comprise an additional step of administering to the subject each of the components of the disclosed combination, specifically, at least one lysine analog, such as tranexamic acid (TXA), and the disclosed tPA mutant, or any combination or composition comprising the same.

[0205] A further aspect of the present disclosure relates to a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder and / or any disease, disorder, or condition associated with fibrinolysis in a subject in need. More specifically, the disclosed therapeutic methods comprise the step of administering to the subject a therapeutically effective amount of: (a), at least one tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2; and (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasmin. Alternatively, the method may comprise administering (c), a combination of (a) and (b), or any composition comprising (a), (b) or (c). As shown by the present disclosure, this combination exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

[0206] Still further, in some embodiments the tPA mutant used in the disclosed therapeutic methods comprises at least one point mutation that results in a substitution of serine 481 to alanine in said tPA mutant.

[0207] In yet some further embodiments, the tPA mutant used in the disclosed therapeutic methods may comprise the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof. In more specific embodiments, such mutant being designated tPASer481Ala.

[0208] In some embodiments, the lysine analog used in the disclosed methods is at least one of 4- (aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl-cyclohexanecarboxylic acid. It should be appreciated that in some embodiments, also the amino acid lysine may be used.

[0209] In yet some further specific embodiments, the lysine analog used in the therapeutic methods of the present disclosure may be 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid).

[0210] In some embodiments, the fibrinolytic activity and / or free plasmin activity is caused by, associated with, enhanced by, resulting from, due to, arising from, as a consequence of, associated with, linked to, related to, correlated with, in connection with, enhanced by, amplified by, augmented by, increased through, potentiated by, at least one of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) in the presence of the lysine analog.

[0211] In some embodiments, the fibrinolytic activity and / or free plasmin activity is caused by urokinase plasminogen activator (uPA).

[0212] In certain embodiments, the methods of the invention may be particularly applicable for subjects suffering from a hemostatic disorder that may be hereditary or acquired bleeding disorders.

[0213] Hemostatic disorders are bleeding disorders classified as either hereditary or acquired. Acquired bleeding disorders are disorders where bleeding is induced by an external (acquired) cause such as trauma, surgery or fibrinolytic treatment, as will be discussed herein after. Bleeding disorders caused by inherited deficiencies of one or more coagulation factors are rare disorders distributed worldwide. Homozygotes or compound heterozygotes for the mutant genes responsible for these defects exhibit bleeding manifestations that are of variable severity and usually related to the extent of the decreased activity of the particular coagulation factor.

[0214] In yet further embodiments the methods of the invention are applicable for the treatment, prophylaxis, amelioration, inhibition or delaying the bleeding associated with hereditary hemostatic disorder and undefined bleeding tendency.

[0215] "Hereditary hemostatic disorder" as used herein relates to a hereditary deficiency in at least one coagulation factor. More specifically, numerous mutations have been identified in genes encoding coagulation factors I, II, V, VII, X and XI, that lead to deficiency of at least one of said factors or to impaired activity thereof. Homozygotes for these mutations exhibit bleeding tendency either spontaneously or following trauma / surgery. Heterozygotes for the various deficiencies rarely display a bleeding tendency.

[0216] Thus, the disclosed methods may be applicable for any form of bleeding that accompanies hereditary hemostatic disorders caused by a deficiency in at least one of: factor XI, factor X, factor V, factor VII, factor II (prothrombin) and factor I (fibrinogen) as disclosed herein.

[0217] In yet some further embodiments, the methods of the present disclosure may be applicable for treating disorders characterized by hereditary deficiencies of the coagulation factors I, II, V, VII, X and XI that include at least one of or any bleeding tendency associated therewith.

[0218] Still further, in some embodiments, the disclosed methods may be applicable for Acquired bleeding disorders. "Acquired bleeding disorders", are conditions characterized by abnormal bleeding that develop after birth due to non-genetic causes. Unlike inherited bleeding disorders, which result from congenital deficiencies or defects in clotting factors or platelets, acquired bleeding disorders arise from external or physiological factors that impair the normal hemostatic process. These may include liver disease, vitamin K deficiency, autoimmune conditions such as acquired hemophilia, the use of anticoagulant or antiplatelet medications, disseminated intravascular coagulation (DIC), or certain cancers. The underlying mechanisms often involve reduced production or increased destruction of clotting factors, platelet dysfunction, or the presence of inhibitors that interfere with normal coagulation.

[0219] In some embodiments, the therapeutic methods of the present disclosure may be applicable for at least one hemostatic disorder, disease, disorder, or condition. More specifically, at least one of: coagulopathy, thrombocytopenia, hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

[0220] It should be appreciated that "coagulopathy" (also known as clotting or bleeding disorder) as referred herein relates to a condition in which the blood’s ability to clot is impaired. More specifically in this context, hypocoagulability is an unusual susceptibility to bleeding, that is, an increased bleeding diathesis, due to an abnormality in coagulation. Coagulopathy may be caused by reduced levels or absence of blood clotting proteins, known as coagulation factors. A specific group within coagulopathies are congenital coagulopathies wherein the deficiency of coagulation factors is genetically determined and inherited. Coagulopathy may also occur as a result of dysfunction or reduced levels of platelets. Coagulopathies may severely complicate blood loss from injury, childbirth, surgery, and other conditions in which bleeding can occur. In some specific embodiments, coagulopathy is induced by free plasmin. In some other specific embodiments, coagulopathy is induced by a coagulation factor. The term "induced by " as used herein means caused or triggered by something (e.g. plasmin or an inactivated coagulation factor) and describes a situation where one factor (the inducer, e.g. plasmin) affects another factor (e.g. coagulopathy). This might be a result for example of the plasmin cleavage activity on fibrin clots and / or inactivation of clotting factors.

[0221] As the present invention is also relevant to "congenital coagulopathies", it should be further appreciated that the most common congenital coagulopathies include: hemophilia; Von Willebrand's disease (vWD), a hereditary disorder with prolonged bleeding time due to a clotting factor deficiency and impaired platelet function; hypoprothrombinemia, another congenital deficiency of clotting factors that can lead to hemorrhages; other disorders which include factor XI deficiency (hemophilia C) or factor VII deficiency (also called Serum Prothrombin Conversion Accelerator (SPCA) deficiency). More specifically, "hemophilia" is a congenital coagulation disorder characterized by a deficiency or dysfunction of specific clotting factors, leading to a pronounced tendency toward prolonged or spontaneous bleeding. It is inherited in an X-linked recessive pattern. The hallmark clinical manifestation of hemophilia is spontaneous musculoskeletal bleeding, particularly into joints (hemarthroses) and muscles, which can result in chronic joint disease and significant morbidity if untreated. There are two primary forms of hemophilia: Hemophilia A results from a deficiency or dysfunction of coagulation factor VIII. Hemophilia B (also known as Christmas disease) results from a deficiency of coagulation factor IX. Although the clinical presentation of hemophilia B is similar to that of hemophilia A, hemophilia B is less common and its severity may be influenced by vitamin K status, given the vitamin K dependence of factor IX synthesis.

[0222] "Disseminated Intravascular Coagulation" ( DIC , also known as consumption coagulopathy) is a systemic process caused by pathologic thrombin generation that may ironically result in hemorrhage due to depletion of clotting. DIC etiologies include sepsis, snakebites, burns, amniotic fluid embolism, trauma, acute leukemias and other malignancies.

[0223] In certain embodiments, the present disclosure is particularly relevant for the treatment and amelioration and possibly prevention of "thrombocytopenia" which is the most common coagulation disorder. Thrombocytopenia is defined as a platelet count below the normal range (between 150,000 to 450,000 / pl ± 2 standard deviation). Most notably, when the platelet count is below 50,000 / pl, risks for spontaneous mucocutaneous bleedings (gingival bleed, epistaxis, menorrhagia, petechiae, ecchymoses) and life threatening spontaneous intracranial hemorrhages or gastrointestinal bleeding increase rapidly. Etiologies of thrombocytopenia include: immune thrombocytopenic purpura (ITP also idiopathic thrombocytopenic purpura) - an autoimmune condition caused by the presence of antiplatelet antibodies; thrombotic thrombocytopenic purpura or hemolytic uremic syndrome (TTP-HUS), a relatively uncommon life-threatening form of thrombocytopenia caused by inherited or acquired conditions; drug-induced thrombocytopenia which is relatively common, specifically the chemotherapy-induced thrombocytopenia and heparin-induced thrombocytopenia (HIT); sepsis / infection- induced thrombocytopenia, specifically resulting from bacterial or viral infections (e.g. HIV, EBV or CMV); DIC and hypersplenism, which may cause thrombocytopenia due to sequestration of blood elements; other causes include pregnancy-induced thrombocytopenia and bone marrow transplant, blood transfusion or metastatic carcinoma-induced thrombocytopenia.

[0224] Further, the invention is exceptionally applicable to a large and versatile group of conditions referred herein under general terms as "hemorrhages " or "bleedings" . According to the American College of Surgeons' advanced trauma life support (ATLS) classification, hemorrhages include: Class I - loss of up to 15% of blood volume with no change in vital signs; Class II -15-30% loss with symptoms of tachycardia, peripheral vasoconstriction and paleness of the skin; Class III - 30- 40% loss with significant drop of blood pressure, increase of heart rate and peripheral hypoperfusion (shock); Class IV - >40% loss reaching the limit of the body's compensation wherein aggressive resuscitation is required to prevent death. According to the World Health Organization (WHO), the standardized grading scale to measure the severity of bleeding consists of Grade 0 - no bleeding; Grade 1 - petechial bleeding; Grade 2 - mild blood loss (clinically significant); Grade 3 - gross blood loss requiring transfusion (severe); Grade 4 - debilitating blood loss including cerebral associated with fatality.

[0225] Hemorrhage -prone sites include mouth (hematemesis, hemoptysis), nose (epistaxis); anus (hematochezia), urinary tract (hematuria), upper head (intracranial, cerebral and intracerebral hemorrhages) and subarachnoid hemorrhage (SAH) within the subarachnoid space usually results from some pathologic non-traumatic process, such as rupture of a berry aneurysm or arteriovenous malformation (AVM); lungs (pulmonary hemorrhage); gynecologic (vaginal bleeding, postpartum hemorrhage, breakthrough bleeding and ovarian bleeding); internal bleedings (spleen and liver); and gastrointestinal (upper gastrointestinal bleed).

[0226] Hemorrhages may be also caused by either traumatic injury or other medical conditions (e.g. internal bleedings induced by Warfarin / Coumadin) or a combination of both. "Trauma-induced hemorrhages" occur as a direct consequence of mechanical injury to blood vessels, tissues, or organs. These hemorrhages can arise from blunt force, penetrating trauma, or other forms of physical insult that disrupt the integrity of the vascular system, leading to the extravasation of blood into surrounding tissues or body cavities.

[0227] Among trauma-induced hemorrhages, specifically relevant " noncompressible hemorrhages" to the torso (chest, abdomen, pelvis and back), in which compression cannot be applied, unlike extremity wounds that are more amenable to compression to stop bleeding. Especially for noncompressible hemorrhages, control of bleeding and limitation of blood loss is the only way to avoid a massive hemorrhage. The combinations and composition of the present disclosure, when administered intravenously, provide potential treatment for truncal hemorrhages that are particularly resistant to the currently available treatments. Specifically pertinent to this context is hemorrhagic stroke involving bleeding within the brain including: as deep intracerebral hemorrhage in the thalamus, basal ganglia, or cerebellum; lobar intracerebral hemorrhage predominantly in the cerebrum; SAH caused by cerebral aneurysm; or hypertensive intracerebral hemorrhage due to high blood pressure.

[0228] Pertinent to the present context are "gynecological hemorrhages" that include menorrhagia, pregnancy or parturition and obstetric bleeding, postpartum hemorrhage.

[0229] Still further, in some embodiments the disclosed methods may be applicable for GI bleeding. "Gastrointestinal (GI) bleeding", also known as gastrointestinal hemorrhage, as used herein, relates to all forms of bleeding in the gastrointestinal tract, from the mouth to the rectum. "Acute gastrointestinal bleeding" means that there is a significant blood loss over a short time causing acute blood loss and hemorrhagic shock. Symptoms may include vomiting (hemathemesis) either red blood or black blood, bloody stool, or black stool (digested blood called melena). In contrast, chronic gastrointestinal bleeding is bleeding of small amounts of blood over a long time. In this case the symptoms are of iron-deficiency anemia.

[0230] GI bleeding is typically divided into two main types: upper gastrointestinal bleeding and lower gastrointestinal bleeding. Causes of upper GI bleeds include: peptic ulcer disease, esophageal varices, that may occur in some embodiments, due to liver cirrhosis and cancer, among others. Causes of lower GI bleeds include: hemorrhoids, cancer, and inflammatory bowel disease among others. Endoscopy of the lower and upper gastrointestinal track may locate the area of bleeding. Medical imaging may be useful in cases that are not clear.

[0231] Acute upper GI bleed is more common than lower GI bleed. An upper GI bleed occurs in 50 to 150 per 100,000 adults per year. A lower GI bleed is estimated to occur in 20 to 30 per 100,000 per year. It results in about 300,000 hospital admissions a year in the United States. Risk of death from a GI bleed is between 5% and 30%. Risk of bleeding is more common in males and increases with age.

[0232] In other particular embodiments, the disclosed combinations and methods may be suitable for treatment of burns, and specifically, bleeding associated with burns.

[0233] A burn is a type of injury to skin, or other tissues, caused by heat, cold, electricity, chemicals, friction, or radiation. In some specific embodiments, the methods of the invention may be suitable for treating lung injury associated with emphysema and COPD.

[0234] The present disclosure may be also relevant for treating and preventing bleeding resulting from "minor surgery ” , i.e. surgical procedures that do not involve anesthesia or respiratory assistance such as circumcision, dental surgery; as well as for "major surgery” i.e. any surgery in which a patient must undergo general anesthesia, for example orthopedic surgery, urological surgery, heart surgery, liver surgery, and any surgery in any organ or tissue.

[0235] Still, further, the invention may further be utilized for the treatment and prevention of "bleeding associated with jibrinolytic or thrombolytic therapy ". Bleeding associated with fibrinolytic or thrombolytic therapy occurs when pharmacologic agents designed to dissolve pathological fibrin clots or blood clots (thrombi) inadvertently impair physiological hemostasis. Fibrinolytic / thrombolytic therapy, involves the use of anti-coagulants or anti-coagulating agents. As used herein, the term "anticoagulant agent" is intended to mean any agent which interferes with the clotting of blood. Some anticoagulants, such as the coumarin derivatives bishydroxycoumarin (Dicumarol) and warfarin (Coumadin) inhibit synthesis of prothrombin, a clot-forming substance, and other clotting factors. Anticoagulants can include but are not limited to compounds acting as beta2 Adrenoreceptor Antagonists, Neuropeptide V2 Antagonists, prostacyclin analogs, thromboxane synthase inhibitors, calcium agonists, coumarin derivatives, elastase inhibitors, nonsteroidal anti-inflammatories thrombin inhibitors, lipoxygenase inhibitors, Factor Vila inhibitors, Factor Xa inhibitors, phosphodiesterase III inhibitors, Heparins, and fibrinogen glucoprotein Ilb / IIIa Antagonists.

[0236] Coumarins are vitamin K antagonists. A prominent member of this class is Warfarin (Coumadin). These anticoagulants are used to treat patients with deep- vein thrombosis (DVT), pulmonary embolism (PE) and to prevent emboli in patients with atrial fibrillation (AF), and mechanical prosthetic heart valves. Other examples are acenocoumarol, phenprocoumon, atromentin, and phenindione.

[0237] Heparin works by activating antithrombin III, which blocks thrombin from clotting blood. Low molecular weight heparin, a more highly processed product, is useful as it does not require monitoring of the APTT coagulation parameter and has fewer side effects as for example Enoxaparin (Clexane). Fondaparinux is a synthetic sugar composed of the five sugars (pentasaccharide) in heparin that bind to antithrombin and is an inhibitor of factor Xa. It is a smaller molecule than low molecular weight heparin. Another example is Idraparinux sodium which has a similar chemical structure and method of action as fondaparinux.

[0238] Drugs such as rivaroxaban, apixaban and edoxaban work by inhibiting factor Xa directly (unlike the heparins and fondaparinux, which work via antithrombin activation).

[0239] Further examples include but are not limited to betrixaban from Portola Pharmaceuticals, darexaban (YM150) from Astellas, and more recently letaxaban (TAK-442) from Takeda and eribaxaban (PD0348292) from Pfizer.

[0240] Another type of anticoagulant is the direct thrombin inhibitor. Current members of this class include but are not limited to the bivalent drugs hirudin, lepirudin, and bivalirudin; and the monovalent drugs argatroban and dabigatran.

[0241] The antithrombin protein itself is used as a protein therapeutic anticoagulant agent that can be purified from human plasma or produced recombinantly (for example, Atryn, which is produced in the milk of genetically modified goats).

[0242] As indicated above, anti-coagulants administration for example, heparin, is the standard antithrombotic therapy indicated for acute venous thrombosis, for prophylaxis of thrombosis in the post-surgical (especially orthopedic) and immobile patient, and for flushing of intravenous lines to maintain patency. However, due to their potency, heparin and LMWH suffer drawbacks. Uncontrolled bleeding as a result of the simple stresses of motion and accompanying contacts with physical objects or at surgical sites is the major complication.

[0243] Additional agents, such as recombinant tissue plasminogen activator (tPA), streptokinase, and urokinase, enhance the conversion of plasminogen to plasmin, leading to the degradation of fibrin clots and, consequently, an increased risk of hemorrhage. Non-limiting examples of bleeding events associated with fibrinolytic or thrombolytic therapy include intracerebral hemorrhage, gastrointestinal bleeding, and postoperative bleeding, all of which represent significant complications during or following thrombolytic treatment. The present disclosure may additionally be applicable in managing bleeding arising from "coagulopathy associated with anti-fibrinolytic" . Coagulopathy associated with anti-fibrinolytic therapy arises from the excessive inhibition of the physiological fibrinolytic system, resulting in an imbalance between clot formation and clot degradation. Anti-fibrinolytic agents, such as tranexamic acid, a-aminocaproic acid, and aprotinin, act by preventing plasminogen activation or inhibiting plasmin activity, thereby stabilizing fibrin clots. However, when fibrinolysis is excessively suppressed, there is an increased risk of pathological clot persistence and propagation, leading to thrombotic complications. Non-limiting examples of disorders associated with such coagulopathy include deep vein thrombosis (DVT), pulmonary embolism, myocardial infarction, and ischemic stroke, each representing serious clinical manifestations of impaired fibrin turnover and excessive clot stabilization.

[0244] In the context of hemorrhages, the combinations of the present disclosure may be administered in conjunction with an additional therapeutic agent. A specific example for such additional agent may be at least one coagulation promoting agent. Such coagulation promoting agent may be selected from thrombin, fibrinogen, platelet activating agent, plasma preparations and platelets reach plasma preparations and vitamin K. In addition, the disclosed combinations, compositions and kits can be administered with styptics that work by contracting tissue to seal injured blood vessels or other locally-acting agents that work by causing vasoconstriction or promoting platelet aggregation.

[0245] As shown by the present disclosure, the disclosed combination, effectively inhibit also the fibrinolytic activity of uPA. The involvement of uPA has been recently demonstrated in modulation of angiogenesis. More specifically, uPA has been implicated in tissue proliferation and cell adhesion, as it regulates proteolytic degradation of the extracellular matrix, liberating growth factors and matrix metalloproteases. Moreover, uPA has been shown as required for both endogenous and vascular endothelia growth factor (VEGF)-augmented angiogenesis. On the other hand, uPAhas been shown as involved in the generation of angiostatin from plasminogen. It should be noted that angiostatin is an inhibitor of angiogenesis. The inhibition of the activity of uPA by the mutants of the invention may be therefore useful in modulating angiogenesis. It should be noted that modulation refer to either enhancing or inhibiting angiogenesis. In more specific embodiments, the mutants of the present disclosure may be used in enhancing angiogenesis in a subject in need thereof. Therefore, the present disclosure may further provide according to some embodiments, the use of the combinations, compositions and kits of the present disclosure in a method for modulating angiogenesis.

[0246] "Angiogenesis" as referred herein relates to a physiological process of formation of new blood vessels from pre-existing vessels. This is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. Angiogenesis is a normal and vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. It is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Specifically, relevant to this context are conditions related to insufficient angiogenesis, such as coronary artery disease, stroke, chronic wounds and during surgery.

[0247] It has been demonstrated that angiogenesis is controlled by a net balance between molecules that have positive and negative regulatory activity. This observation has led to the concept of "angiogenic switch", in which the endothelial activation status is determined by the induction of positive regulators and / or loss of negative regulators. As the present disclosure affects the balance between bioavailability of plasminogen and uPA, it may further provide means to modulate angiogenesis together with other known factors as vascular endothelial growth factor (VEGF) cytokines and angiopoietins.

[0248] In the same way, the present disclosure provides the combinations, compositions and kits for use in a method for promoting and enhancing wound healing.

[0249] It should be appreciated that "wound healing" (or cicatrization) as referred herein relates to an intricate process in which the skin or another organ-tissue repairs itself after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) exist in steady-state equilibrium, forming a protective barrier against the external environment. Once the protective barrier is broken, the normal (physiologic) process of wound healing is immediately set in motion. The classic model of wound healing is divided into three or four sequential, yet overlapping, phases: (1) hemostasis (not considered a phase by some authors), (2) inflammatory, (3) proliferative and (4) remodeling. Upon injury to the skin, a set of complex biochemical events takes place in a closely orchestrated cascade to repair the damage. Within minutes post-injury, platelets (thrombocytes) aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding (hemostasis).

[0250] In the inflammatory phase, bacteria and debris are phagocytosed and removed, and factors are released that cause the migration and division of cells involved in the proliferative phase.

[0251] The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction. In angiogenesis, new blood vessels are formed by vascular endothelial cells.

[0252] In the maturation and remodeling phase, collagen is remodeled and realigned along tension lines and cells that are no longer needed are removed by apoptosis.

[0253] In yet other embodiments, the invention provides various composition as described herein for use in a biological glue.

[0254] As used herein, the expression "biological glue" refers to compositions as described above that comprise as an active ingredient at least one tPA mutated molecule and at least one lysine analog, which can be used for a variety of purposes, for example, as sealants, delivery agents and adhesive compounds. More particularly, the composition of the invention may be used as biological glue for controlling bleeding and for the tight sealing of vessels, lungs or skin incisions, but also in case of intracavitary injuries. For instance, the disclosed combinations and compositions may be used for sealing or reinforcing wounds that have been sutured or stapled, with pressure over an injured area, or to reduce blood loss and post-operative bleeding. When used as biological glue, the disclosed combination is preferably eliminated after the cicatrization of the wound, by biodegradation, absorption or by simple detachment in the form of scabs.

[0255] More specifically, the disclosed therapeutic methods may be applicable for treating hemostatic disorder associated with fibrinolysis, for example, at least one of coagulopathy induced by free plasmin and coagulopathy induced by inactivation of at least one coagulation factor (e.g., Fibrinogen, Factor V and factor VIII).

[0256] It should be noted that in some embodiments of the disclosed methods, the level of free plasmin in the subject is increased in the presence of the at least one lysine analog.

[0257] In yet some further embodiments, the increase in the level of plasmin results in a decrease in the level of at least one coagulation factor in the subject. However, the combination with the pASer481Alamutant increases the anti-fibrinolytic activity of the lysine analog and reduces the free plasmin activity in the treated subject, thereby preventing or reducing the decrease in the level of at least one coagulation factor in the subject. This provides a safe combined therapy that reduces the major side effects, specifically, coagulopathy caused by lysine analog treatment. Moreover, in addition to reduction of the side effect, the combined therapy disclosed herein further increases the anti-fibrinolytic activity of the lysine analog treatment. Accordingly, a combination therapy is hereby provided.

[0258] Thus, a further aspect of the present disclosure relates to a method of treatment, amelioration, inhibition or prophylaxis of a hemostatic disease, disorder, or condition associated with fibrinolysis in a subject treated with at least one lysine analog. The method comprising the step of administering to the subject a therapeutically effective amount of at least one tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, or any composition thereof.

[0259] In some embodiments, the tPA mutant used in the disclosed methods carry a point mutation that results in a substitution of serine 481 to alanine. In more specific embodiments, the tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof. In yet some further embodiments, the mutant used may be the tPASer481Ala.

[0260] Still further, in some embodiments, the subject administered with the tPASer481Alamutant, is a subject treated with at least one lysine analog, for example, at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

[0261] A further aspect of the present disclosure relates to an effective amount of a combination or composition comprising the same, for use in a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder or any disease, disorder, or condition associated with fibrinolysis. The combination comprising: (a), at least one tissue tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprises the amino acid sequence as denoted by SEQ ID NO: 2. The combination further comprises component (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin. The disclosed combination exerts at least one of: increasing the anti-fibrinolytic activity of the lysine analog and reducing free plasmin activity. In some embodiments, the combination used herein, is any one the combinations defined by the present disclosure.

[0262] In some embodiments of the disclosed effective amount of a combination for use in accordance with the present disclosure, the tPA mutant carries at least one point mutation that results in a substitution of serine 481 to alanine. In some embodiments, the tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, being tp^Ser481Ala

[0263] In some embodiments, the lysine analog of the combination for use herein, is at least one of 4- (aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

[0264] Still further, in some embodiments, the combination in accordance with the present disclosure is applicable for use in methods for treating at least one hemostatic disorder, disease, disorder, or condition. More specifically, at least one of: coagulopathy, thrombocytopenia, hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

[0265] Still further aspect of the present disclosure relates to an effective amount of at least one tPA mutated molecule that carries a point mutation at position 481 of the WT tPA (the WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2), or a composition comprising the same, for use in a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder associated with fibrinolysis in a subject treated with at least one lysine analog.

[0266] In some embodiments, the tPA mutant used herein, carries a point mutation that results in the substitution of serine 481 to alanine. In more specific embodiments, the tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, the mutant beingtPASer481Ala. In some embodiments of the tPA mutant for use as disclosed herein, the subject is treated with at least one lysine analog, for example, at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

[0267] In some embodiments of the tPA mutant for use disclosed herein, the method is applicable for treating hemostatic disorder, disease, disorder, or condition, that may be at least one of: coagulopathy, thrombocytopenia, hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

[0268] In some embodiments, the disease, disorder, or condition associated with fibrinolysis is at least one of: coagulopathy induced by free plasmin and coagulopathy induced by inactivation of at least one coagulation factor.

[0269] It should be noted that the combinations, compositions as well as any of the methods, uses and kits of the present disclosure may be applicable for any of the disorders described herein.

[0270] In some embodiments of the disclosed tPA mutated molecule for use, the level of the free plasmin in subject is increased in the presence of at least one lysine analog.

[0271] In yet some further embodiments, the increase in the level of plasmin results in a decrease in the level of at least one coagulation factor in the subject. Use of the tPA mutated molecule in accordance with the present disclosure increases the anti-fibrinolytic activity of the lysine analog and more importantly, reduces the free plasmin activity.

[0272] As used herein, the term “therapeutically effective amount” means an amount of a compound or composition which is administered to a subject in need thereof, necessary to effect a beneficial change in the severity of a disease or disorder, or prevent such disease, in said subject. This amount should also be within specific pharmacological ranges, to avoid toxic effects by over-dosing. For example, in the present invention, a therapeutically effective amount of at least one of the tPA mutant and at least one lysine analog of the present disclosure, for the treatment of coagulopathy would be the amount of these molecules administered to a subject which would induce a beneficial change in the subject, alleviating, ameliorating, or preventing the recurrence of said hyperfibrinolysis, without causing detrimental side effects, or causing only mild side-effects. It is understood that the therapeutically effective amount is not an absolute term and depends on subjective circumstances, such as the subject's age, health, weight, and various other statistics, as described in the and specifically determined by the attendant physician or other person skilled in the art after an evaluation of the subject’s conditions and requirements.

[0273] It should be noted that the mutants of the present disclosure as well as any compositions thereof as described herein may be presented in unit dose forms containing a predetermined amount of each active ingredient per dose. Such a unit may be adapted to provide O.Ol-lOOmg / Kg of body weight of each of the tPA mutant and the lysine analog of the invention. Specifically, either 0.1- lOmg / Kg, 5-15mg / Kg, 10-30mg / Kg, 25-50mg / Kg 40-80mg / Kg or 60-100mg / Kg. More specifically, said effective dosage is about 0.01 to about 100 mg / Kg of each of the tPA mutant and the lysin analog, about 0.1 to about 90 mg / Kg, about 0.3 to about 8 mg / Kg, about 0.4 to about 70 mg / Kg, about 0.5 to about 60 mg / Kg, about 0.7 to about 50 mg / Kg, about 0.8 to about 40 mg / Kg, about 0.9 to about 30 mg / Kg, about 1 to about 20 mg / Kg, specifically, about 1 to about 10 mg / Kg. Such doses can be provided in a single dose or as a number of discrete doses. In case a single dose may be administered, a dosage unit form may comprise an amount of about 0.01 mg to about lOOOmg, that may be administered one a day, a week or a month. The ultimate dose will of course depend on the condition being treated, the route of administration and the age, weight and condition of the patient and will be at the doctor's discretion.

[0274] It should be further noted that for the method of treatment and prevention provided in the present disclosure, the therapeutic effective amount, or dosage, is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. In general, dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the tPA mutant and lysine analog used by the present disclosure or any composition of the invention in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the combined composition of the disclosure is administered in maintenance doses.

[0275] It should be appreciated that the combination of the invention may be effective when administered to an injured subject after 10’, 20’, 30’, 45’, 50’, 60’, 90’, 150’, 180’, 4hr, 5hr, 6hr, 7hr, 8hr, 9hr, lOhr, l lhr, 12hr, 13hr, 14hr, 15hr, 16hr, 17hr, 18hr, 19hr,20hr, 21hr, 22hr, 23hr, 24hr, 2 days, 3 days 4 days, 5 days, 6 days and even 7 days or more after the occurrence of the injury, or any applicable therapeutic window.

[0276] As used herein, the term "treatment" refers to an improvement of at least one undesired manifestation of the disorder, condition or disease wherein inhibition of fibrinolysis is beneficial. The present disclosure may also be conceived as a prophylactic treatment before the disorder, condition or disease occurs.

[0277] The term "amelioration " as referred to herein, relates to the inhibition or reduction of fibrinolysis.

[0278] The term "prophylaxis" refers to prevention or reduction the risk of occurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician.

[0279] The term "inhibition / reduction " as referred to herein, relates to retardation, attenuation, retraining or reduction of a process. More specifically, it is understood that the combination and / or composition of the invention inhibits fibrinolysis and / or hemostatic disorders by at least about 1 % to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.

[0280] In the same way, it is understood that the combination and compositions of the present disclosure inhibit at least one of tPA- and uPA-dependent plasminogen activation, and / or free plasmin activity, by any one of about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.

[0281] The term "decrease" or "reduce", as referred to herein, relates to a decrease in value, amount, or rate. A decrease or a reduction as referred to herein, relate to the a reduction or lessening of a quantity or process by any one of about 1% to 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9% compared to a reference value.

[0282] The term "increase" or "enhance" , as referred to herein, relates to a measurable elevation in value, amount, or rate. An increase or enhancement as referred to herein, relate to the elevation of a quantity or level by any one of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or greater compared to a reference value.

[0283] As used herein, the term "increase" refers to a measurable enhancement or elevation of a specified parameter, characteristic, or biological activity relative to a baseline or control level. An increase may be characterized quantitatively by a range of percentages, depending on the context of the application. For example, an increase may comprise a relative enhancement of at least 5%, 10%, 25%, 50%, 75%, or greater compared to the reference value. In certain embodiments, the increase may range from about 5% to about 20%, from about 20% to about 50%, or from about 50% to about 100% or more.

[0284] As used herein, the term "subject in need" relates to a mammalian subject, such as human, bovine, equine, murine, feline, canine or other, suffering from at least one of pathologic fibrinolysis, or hyper-fibrinolysis, and neural trauma as described, the treatment of which with any of the therapeutic tPA mutants of the invention, combinations and compositions thereof according to the invention, would ameliorate or decrease the severity of the disorder, both in intensity of the symptoms, rate of progression of the disorder, and time frame of the disorder. In some embodiments, the methods of the present disclosure may be applicable for any subject or organism of the biological kingdom Animalia. Still further, in some embodiments, the methods of the present disclosure may be applicable for any vertebrate organism or subject, specifically, any organism derived from any of the vertebrates groups that include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans). In some particular embodiments, the methods of the present disclosure may be applicable for a mammal (specifically, at least one of a human, cattle, rodent, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and camels).

[0285] More specifically, in some embodiments, as indicated herein, the methods of the present disclosure may be applicable for a vertebrate organism.

[0286] Still further, in some embodiments, the subject of the present disclosure may be any one of a human or non-human mammal, an avian and a fish.

[0287] In yet some further embodiments, the present disclosure may be applicable for any organism of the order primates. More specifically, primates are divided into two distinct suborders, the first is the strepsirrhines that includes lemurs, galagos, and lorisids. The second is haplorhines that includes tarsier, monkey, and ape clades, the last of these including humans. In yet some further embodiments, the present disclosure may be applicable for any organism of the subfamily Homininae, that includes gibbons, orangutans and homininae [gorillini (gorilla) and hominini ((panina(chimpanzees) and hominina (humans))]. Thus, in some embodiments, a subject as disclosed herein relates to a human subject. In some embodiments, the human subject may be of any gender, ethnic group, age or physical or mental condition.

[0288] In some specific embodiments, the tPA mutant and each of the lysine analog may be administered in the form of a single pharmaceutical composition comprising a combination of the tPA mutant and at least one lysine analog, together with a pharmaceutically acceptable carrier or diluent. Alternatively, the tPA mutant and each of the lysine analogs may be stored in a separate compartment. In such a case, the disclosed combination may be provided in a kit. Thus, a further aspect of the present disclosure relates to a kit comprising:

[0289] (a) at least one tPA mutant that carries a point mutation at position 481 of the WT tPA. The WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, optionally, in a first dosage form; and (b), at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen, optionally in a second dosage form.

[0290] In some embodiments, the kit comprising a combination of (a) and (b). The combination of (a) and

[0291] (b), exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

[0292] In some embodiments, the kit includes means for containing both separate compositions, such as a divided bottle or a divided foil packet. However, the separate compositions may also be contained within a single, undivided container comprising several compartments.

[0293] Typically, the kit includes instructions for the administration of the separate components. The kit form is particularly advantageous when the separate components are administered in different dosage forms, at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0294] It should be appreciated that when the kit is comprised of at least two dosage forms, both dosage forms may be administered simultaneously.

[0295] Alternatively, the first dosage form and said second dosage form are administered sequentially in either order.

[0296] In yet some further embodiments, the tPA mutated molecule of (a) and at least one lysine analog of (b) may be provided as a kit in separated components, which may include the tPA mutant with at least one, two, three, four, five or more different lysine analogs.

[0297] Still further, in some embodiments According to specific embodiments, the kit of the invention may be suitable for use as a sealant, delivery agent or adhesive, and may be used for controlling bleeding, for the tight sealing of vessels, the sealing of lungs or skin incisions, and for treating intracavitary injuries.

[0298] In some embodiments, the tPA mutant of the disclosed kit carries a point mutation that results in the substitution of serine 481 to alanine and wherein said tPA mutant comprising the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tp^Ser481Ala

[0299] In some embodiments, of the disclosed kits, the lysine analog is at least one of 4-(aminomethyl)- cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

[0300] In yet some further embodiments, the kit is adapted for use in the treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder and / or any disease, disorder, or condition associated with fibrinolysis in a subject in need thereof.

[0301] Still further, in some embodiments, the disclosed kit is adapted for use in any of the methods as defined the present disclosure.

[0302] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0303] The term "about" as used herein indicates values that may deviate up to between 1% to 10%, thus used herein the term "about" refers to ± 10 %.

[0304] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". Thus, in some embodiments, in case limitation is desired, replacement of the term "comprising" with "consisting of" cannot be considered as not supported by the present disclosure, or as adding a new matter. Accordingly, the present disclosure further provides combinations consisting of any of the disclosed tPA mutants, specifically, the Se481Ala mutant, and the at least one lysine analog, specifically TXA, and any compositions, formulations and kits thereof. The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0305] It should be noted that various embodiments of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[0306] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0307] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0308] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0309] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0310] The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

[0311] EXAMPLES

[0312] Experimental Procedure

[0313] Activity of plasmin

[0314] The amidolytic activity of plasmin was assayed, as was previously reported [1] [ Higazi, A.A. et al. Biochem J, 1990. 269(2): p. 299-302, Higazi, A. A., et al. J Biol Chem, 1995. 270(16): p. 9472- 7], by measuring the cleavage of the chromogenic substrate, Tf-D-norleucyl-hexahydrotyrosyl- lysine-p-nitroanilide (S-2251), which releases chromogen p-nitroaniline detected at the WL of 405 nm. Plasmin 25 nM was incubated in phosphate buffered saline, pH 7.4, containing increasing concentrations of the chromogenic substrate (0-0.9 mM). The optical density (OD) at 405 nm was measured continuously over a period of 20 minutes. Reaction rates (V) were calculated from the linear portion of the slope of the absorb ance-time curves and expressed in nmol / min. The initial velocity data were plotted against substrate concentration to generate a Michaelis-Menten kinetic curve. To linearize the data, an inverse transformation was applied, and the reciprocal values (1 / V versus 1 / [S]) were plotted, yielding a Lineweaver-Burk plot. In vitro fibrin clot formation

[0315] Fibrin clots were formed in 24-well tissue culture plates, as was previously described [1] [Higazi, A.A., et al. Blood, 1998. 92(6): p. 2075-83] , by adding human thrombin (0.2 NIH u / mL final concentration) for 1 hour at 37°C to fibrinogen that had been reconstituted to a concentration of 3 mg / mL in phosphate-buffered saline (PBS), pH 7.4.

[0316] Ex vivo system of blood clots

[0317] Fresh human blood taken from healthy voluntaries was used to produce blood clots by adding kaolin as previously reported [Chitlur, M., et al. Haemophilia, 2011. 17(3): p. 532-7; bu-Fanne, R„ et al. Blood, 2019. 133(5): p. 481-493] .

[0318] Plasminogen activators, tPA or uPA were used to generate plasmin from endogenous plasminogen, mimicking physiological fibrinolysis.

[0319] TEG system was used to monitor and quantify the clot formation and lysis, as previously described [bu-Fanne, R., et al. 2019. id ibid . Thromboelastography (TEG) is a viscoelastic hemostatic assay that measures the global viscoelastic properties of whole blood clot formation under low shear stress. TEG shows the interaction of platelets with the coagulation cascade (aggregation, clot strengthening, fibrin cross-linking and fibrinolysis). The parameters shown in Figs. 10-19 represent as follows: R = R time, the time to start forming clot; K=K time, time until clot reaches a fixed strength; Alpha= Alpha angle, the speed of fibrin accumulation; MA=maximum amplitude, the highest vertical amplitude of the TEG; PMA=Platclct-Mcdiatcd Aggregation, the platelet contribution to the overall clot strength; G= G-value is a log-derivation of the MA; EPL= estimated percent lysis (EPL); A = Amplitude, the maximum strength of the clot, CL= Clot Lysis, the degree of clot breakdown (fibrinolysis); LY30=Lysis at 30 minutes, percentage of amplitude reduction 30 minutes after maximum amplitude.

[0320] Measurement of fibrinogen concentration and of Factor V and Factor VIII activity

[0321] The commercially available Sysmex Analyzer CS-5100 was used for determining the concentrations of fibrinogen and the activity of Factors V and VIII, in accordance with the manufacturer instructions. EXAMPLE 1

[0322] Assessing the effect of tPAS481Aon plasmin activity

[0323] The inventors first examined the effect of the tissue plasminogen activator mutant, tPAS481A, on plasmin activity. For that, the amidolytic activity of plasmin was assayed in the absence or presence of tPAS481A(25 nM) by measuring the cleavage of the chromogenic substrate, H-D- norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide (S-2251) as described in the experimental procedure. Figures 1A and IB show that tPAS481Ainhibits the plasmin activity. More specifically, tPAS481Adecreased Vmax from 0.183 to 0.032 nmol / min and increased Km from 72 to 333 pM (Figures 1A and IB).

[0324] The fact that the tPAS481Asignificantly decreased the Vmax of plasmin indicates that the inhibition is not competitive. Indeed, kinetic analysis shows that the inhibition of plasmin by tPAS481Ais of a mixed pattern where Vmax was decreased and Km was increased (Figure IB), showing a novel mode of plasmin regulation and inhibition.

[0325] EXAMPLE 2

[0326] Identifying the mechanism of plasmin activity inhibition by tPAS481A

[0327] Plasmin is composed of two subunits: the catalytic subunit, that contains the active site and a second unit that contains the kringle domains [Castellino, F.J. and S.G. McCance Ciba Found Symp, 1997. 212: p. 46-60; discussion 60-5]. The kringle domains play a regulatory role in the conformation and activation of plasmin parent’s molecule, plasminogen. Binding of lysine or its synthetic analog TXA to plasminogen induce conformation changes in plasminogen and facilitate its activation by uPA. Furthermore, the lysine binding sites (LBSs) mediates the binding of plasmin to fibrin and its binding to its inhibitor a2-anti-plasmin. The inventor's earlier publication indicates that the plasmin’s LBSs regulate the activity of the catalytic subunit, where binding of fibrinogen to LBSs inhibit the cleavage of S-2551 by plasmin. The inhibitory effect of fibrinogen on plasmin activity was prevented by the lysine analog EACA [Higazi, A.A. et al. Biochem J, 1990. 269(2): p. 299-302],

[0328] To examine the role of the lysine binding sites in plasmin inhibition by tPAS481A, the amidolytic activity of plasmin was assayed as in EXAMPLE 1 , in the presence of tPAS481A(25 nM) and in the presence or absence of TXA (0.1 mM). Figure 2 shows that the lysine analog TXA had no effect on the inhibition of plasmin by tPAS481A. Furthermore, Figure 2 shows that TXA by itself has no significant effect on plasmin activity on the synthetic substrate S-2251 , in line with the inventor's earlier findings mentioned above.

[0329] The incapacity of the lysine analog TXA to prevent the inhibitory effect of tPAS481Aon plasmin activity together with the mixed pattern of the inhibition seen in Figure IB, supports the contention that the inventors are describing a novel mechanism of plasmin inhibition.

[0330] EXAMPLE 3

[0331] The structure of tPAS481Ais involved in plasmin inhibition

[0332] In addition to the catalytic site, tPA and uPA have another binding site that mediates their binding to their inhibitor Plasminogen Activator Inhibitor-1 (PAI-1), called docking site (DS). Furthermore, the inventors reported that the DS mediates the binding of tPA to the N-Methyl-D- Aspartate Receptor (NMDA-R). tPA variant that lacks the DS failed to bind the NMDA-R and PAI-1.

[0333] To further study the mechanism of plasmin activity inhibition by tPAS481A, the inventors used another tPA variant that has the same mutation used in Figure 1 (tPAS481A) in addition to other mutations in its DS (tPAS481A-DS) that disable its capacity to bind PAI-1 and NMDA-R, described earlier. Figure 3 shows that tPAS481A-DS failed to inhibit the plasmin activity, supporting the contention of a novel interaction between tPA and plasmin.

[0334] EXAMPLE 4

[0335] Effect of tPAS481Aon plasmin activity using fibrinogen derived clots

[0336] To evaluate the effect of tPAS481Aon plasmin fibrinolytic activity on fibrin clots, fibrin clots were formed in 24-well tissue culture plates as described in the experimental procedure. Lysis of fibrin clots by plasmin was then assessed as described earlier [Higazi, A.A., et al. Blood, 1998. 92(6): p. 2075-83] by adding 50 pl phosphate buffered saline (PBS) alone (Figure 4, well A) or PBS containing plasmin (25 nM), (Figure 4, well B and well C) to the clot surface for 2 hours at 37°C in the absence (Figure 4, well B) or presence (Figure 4, well C) of tPAS481A(100 nM). Clots were then washed with PBS, incubated overnight with 0.2% trypan blue, rinsed with PBS, and photographed. Photographs were scanned in order to evaluate the amount of fibrinolysis (digestion) extrapolated from color intensity in each well. The calculated data (Figure 5) shows about 96% inhibition of plasmin activity by tPAS481A. The experiment results shown in Figure 5 are representative of three repeats. The lytic areas generated by plasmin in this set of experiments (Figure 4, well B) were 1.74 +- 13 cm2, that was considered as 100% plasmin activity. The data shown in Figure 5 is the mean ± SEM of the performed experiments.

[0337] As can be seen, plasmin digested the fibrin clot, an effect that was almost totally inhibited by tPAS481A(Figures 4 and 5).

[0338] EXAMPLE 5

[0339] The synergistic inhibitory effect between tPAS481Aand TXA on plasmin activity using fibrinogen derived clots

[0340] TXA is the main inhibitor of fibrinolysis in clinical use. In contrast to tPAS481A, TXA inhibits the activity of plasmin only on fibrin by preventing its binding to the fibrin; TXA do not inhibit plasmin catalytic site activity as can be seen by its inability to inhibit the cleavage of the small synthetic substrate S-2251 by plasmin (See Figure 2).

[0341] Taken together, TXA and tPAS481Ainhibit the plasmin mediated cleavage of fibrin clots using two different mechanisms. Therefore, the inventors asked if both inhibitors have any additive or synergistic effects on fibrin clot cleavage by plasmin.

[0342] To answer the above question, the inventors first examined the effect of TXA on plasmin mediated fibrinolysis. The fibrinolytic activity of plasmin was evaluated as in the previous experiment (EXAMPLE 4). Plasmin was added alone (Figure 6, well A) or together with TXA (100 pM) (Figure 6, well B). In well C of Figure 6 only PBS was added and served as a negative control.

[0343] As shown herein, the used concentration of TXA inhibits almost totally the fibrinolytic activity of plasmin (Figures 6 and 7).

[0344] To examine the possible synergistic effect between tPAS481Aand TXA on fibrin cleavage by plasmin, low concentrations of TXA and tPAS481Awere used to induce minimal inhibition of plasmin mediated fibrin clot lysis. PBS containing plasmin (25 nM) was added to the clot surface in the presence of 25 nM tPAS481A(Figure 8, well A), 25 pM TXA (Figure 8, well B) or both (25 nM) tPAS481Aand TXA (25 M) (Figure 8, well C). In well D of Figure 8 PBS was added alone and served as a negative control.

[0345] Figures 8 and 9 show a synergistic effect between the two inhibitors, TXA and tPAS481A, where the combination of minimally inhibitory concentrations of both inhibitors leads to almost totally inhibition of plasmin mediated clot lysis.

[0346] EXAMPLE 6

[0347] The synergistic inhibitory effect between tPAS481Aand TXA on fibrinolysis using human blood clots

[0348] To examine further the synergistic effect between tPAS481Aand TXA and in more physiological setting, the inventors used ex vivo system of fresh human blood to produce blood clots as described in the experimental procedure.

[0349] Clots were formed in the absence of tPA and uPA (Figures 10 and 15) with almost no fibrinolysis, Ly 30 of 0.5% and 0% (Figures 10 and 15, respectively).

[0350] In a second set of experiments, clots were formed in the presence of tPA (10 nM) (Figure 11) or uPA (5 nM) (Figure 16). As can be seen, the used concentrations of tPA and uPA induced storing fibrinolysis with Ly 30 of 91% and 95.7% respectively.

[0351] In a third set of experiments, the clots were formed in the presence of tPA or uPA and low concentrations of TXA (25 pM) (Figures 12 and 17) or low concentrations of tPAS481A(20 nM) (Figures 13 and 18). That exerts minimal inhibitory effects on clot lysis initiated by tPA with Ly 30 of 88.8% and 76.8% for TXA and tPAS481A, respectively (Figures 12 and 13, respectively) or uPA Ly 30 of 76.1% and 79.9%, respectively (Figures 17 and 18, respectively).

[0352] In the fourth set of experiments, tPA (Figure 14) or uPA (Figure 19) were added in the presence of low concentrations of both tPAS481A(15 nM) and TXA (25 pM). The combination of low concentrations of tPAS481Aand TXA indued strong inhibition of tPA and uPA, Ly 30 of 1.7% and 2.3%, respectively.

[0353] The data summarized in Figures 20 and 21 show that the co-presence of 15 nM tPAS481Aand 25 pM TXA exerted more potent inhibition on tPA (Figure 20) and uPA (Figure 21) mediated fibrinolysis, supporting the contention described in Figures 8 and 9 of synergistic effect between tPAS481Aand TXA in inhibiting plasmin mediated fibrinolysis. Effect of tPAS481Aon plasmin generation induced uPA

[0354] One of the side effects of TXA is its paradoxical ability to stimulate the generation of free plasmin. Free plasmin is responsible for the coagulopathy associated with TXA use. It has been shown that treating patients with TXA increases the concentrations of free plasmin in their circulation. Free plasmin mediates the cleavage of several coagulation factors including fibrinogen, FV and FVIII. Moreover, in ex-vivo experiments, adding uPA and TXA to human plasma induced the cleavage of fibrinogen and depletion of a2-antiplasmin.

[0355] To test the ability of tPAS481Ato prevent the TXA mediated increase of coagulation factors cleavage, uPA (50 ng / ml) was added to a fresh human plasma in the presence or absence of TXA (100 pM) and with or without tPAS481A(100 nM). After 120 min of incubation at 37°C, the concentrations of several coagulation factors were determined as described in the experimental procedure.

[0356] The used concentration of uPA had no significant effect on the measured coagulations factors, fibrinogen, FV and FVIII (Figures 22-24). In contrast, adding uPA (50 ng / ml) together with TXA (100 pM), significantly decreased the concentrations of fibrinogen (Figure 22) and the activity of factor V (Figure 23) and factor VIII (Figure 24). Adding tPAS481Atogether with the uPA and TXA neutralized their anti-coagulation effects by preventing the cleavage of fibrinogen (Figure 22) and inactivation of factors V (Figure 23) and VIII (Figure 24).

[0357] The inventors further show that adding tPAS481Aneutralizes the effect of TXA and uPA on fibrinogen.

[0358] EXAMPLE 7

[0359] Synergistic effect of tPAS481Aand tranexamic acid in bleeding liver in rats

[0360] The objective of this study is to evaluate the synergistic effect of tPAS481Aand tranexamic acid in bleeding liver, by removing a piece of the liver, for controlling bleeding in SD rats.

[0361] A total of 24 rats (approximately 10-12 weeks old) are utilized and divided into 4 groups of 6 animals in each group.

[0362] Each Test Item and vehicle formulation is administered to 6 animals per group, according to Table 1. The TXA is provided ready for use and suitable for Intravenous (IV) injection. If necessary, it is diluted with sterile 0.9% sodium chloride (normal saline) or sterile water for injection to adjust the working concentration.

[0363] Table 1: Study design

[0364] * Animals with a liver piece weighing less than 150 mg or gauze pads with a post-bleeding weight increase of less than 0.5 mg are excluded from the study.

[0365] The study variables and endpoints include daily assessments of mortality and morbidity, as well as body weight measurements conducted during the acclimation period and immediately prior to surgery.

[0366] Clinical observations are performed before surgery to establish baseline health status. Observations document any irregularities, such as changes in local injection site, skin, fur, eyes, mucous membranes, respiratory, occurrence of secretions and excretions (e.g. diarrhea) and autonomic activity (e.g. decreased motor activity, salivation, piloerection, ptosis, unusual respiratory pattern). Changes in gait, posture and response to handling, as the presence of bizarre behavior, tremors, convulsions, sleep and coma are also included. All observed abnormalities, toxic signs, moribund conditions and pre-terminal deaths are recorded.

[0367] Rats are anesthetized using a combination of ketamine and xylazine. Once anesthetized, animals receive tail vein injections of saline, tranexamic acid, the tested item at a dose of 30 mg / kg, or a combination of the tested item and tranexamic acid. Five minutes post-injection, a midline incision is made to open the abdominal cavity and expose the liver, and a portion of the liver (about 150 - 12 - mg of the left medial liver lobe) is excised and weighed. Blood loss is assessed by weighing gauze applied to the site of liver excision, and bleeding time is also recorded. Blood samples (approximately 1 mL per time point) are collected both one day before the procedure (to establish baseline values) and after surgery. Samples are drawn into K3EDTA tubes, and plasma is separated and transferred into new tubes for subsequent analysis. At the conclusion of the experiment, animals are terminally bled, and the liver is collected for further evaluation.

Claims

CLAIMS:

1. A combination comprising:(a) at least one tissue plasminogen activator (tPA) mutant that carries a point mutation at position 481 of the wild type tPA molecule (WT tPA), said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2; and(b) at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin; wherein said combination exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

2. The combination according to claim 1 , wherein said point mutation results in a substitution of serine 481 to alanine in said tPA mutant.

3. The combination according to any one of claims 1 and 2, wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tPASer481Ala.

4. The combination according to any one of claims 1 to 3, wherein said at least one lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl-cyclohexanecarboxylic acid.

5. The combination according to any one of claims 1 to 4, wherein said lysine analog is 4- (aminomethyl)-cyclo- hexane-carboxylic acid (TXA).

6. The combination according to any one of claims 1 to 5, wherein said free plasmin activity comprises at least one of: (i) cleavage and / or inactivation of at least one coagulation factor; (ii) damaging the blood brain barrier (BBB); and (iii) modulation of platelets activity.

7. The combination according to claim 6, wherein said at least one coagulation factor comprises at least one of fibrinogen, Factor V (FV) and factor VIII (FVIII).

8. A composition comprising:(a) at least one tPA mutant that carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2; and(b) at least one lysine analog that binds to the lysine binding site (LBS) of plasmin; or(c) a combination of (a) and (b), wherein said combination exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity; and optionally,(d) at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

9. The composition according to claim 8, wherein said combination is as defined in any one of claims 1 to 7.

10. The composition according to any one of claims 8 and 9, wherein at least one of:(a) said tPA mutant carries a substitution of serine 481 to alanine, and comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tPASer481Ala; and(b) said lysine analog is 4-(aminomethyl)-cyclo- hexane-carboxylic acid (TXA).

11. A method for inhibiting the fibrinolytic activity of plasmin on at least one substrate thereof, the method comprising contacting a substrate and / or free plasmin with at least one tPA mutated molecule, and optionally, with at least one lysin analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin, or a composition comprising the same; wherein said tPA mutant carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2.

12. The method according to claim 11, wherein said point mutation results in a substitution of serine 481 to alanine, wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1, or any variants or derivatives thereof, and wherein said mutant being tPASer481Ala.

13. The method according to any one of claims 11 to 12, wherein said substrate is at least one of: a coagulation clot, at least one coagulation factor, and platelets.

14. The method according to claim 13, wherein said at least one coagulation factor comprises at least one of: fibrinogen, factor V (FV) and factor VIII (FVIII).

15. The method according to any one of claims 11 to 14, wherein said contacting step is performed in a subject in need.

16. The method according to claim 15, wherein said subject is suffering from at least one hemostatic disorder associated with fibrinolysis.

17. The method according to claim 16, wherein said hemostatic disorder comprises at least one of: hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, coagulopathy, thrombocytopenia, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

18. The method according to any one of claims 11 to 17, wherein said lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl-cyclohexanecarboxylic acid.

19. The method according to any one of claims 11 to 18, wherein said lysine analog is 4- (aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid).

20. A method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder associated with fibrinolysis in a subject, comprising the step of administering to said subject a therapeutically effective amount of:(a) at least one tPA mutant that carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2; and(b) at least one lysine analog that binds to the lysine binding site (LBS) of plasmin; or(c) a combination of (a) and (b), wherein said combination exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity; or any composition comprising (a), (b) or (c).

21. The method according to claim 20, wherein said point mutation results in a substitution of serine 481 to alanine in said tPA mutant.

22. The method according to any one of claims 20 and 21, wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being designated tPASer481Ala.

23. The method according to any one of claims 20 to 22, wherein said lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid), e-amino caproic acid (EACA), cyclohexanecarboxylic acid and 4-methyl-cyclohexanecarboxylic acid.

24. The method according to any one of claims 20 to 23, wherein said lysine analog is 4- (aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid).

25. The method according to any one of claims 20 to 24, wherein said hemostatic disorder comprises at least one of: hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, coagulopathy, thrombocytopenia, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

26. The method according to any one of claims 20 to 25, wherein said hemostatic disorder associated with fibrinolysis is at least one of coagulopathy induced by free plasmin and coagulopathy induced by inactivation of at least one coagulation factor.

27. The method according to any one of claims 20 to 26, wherein the level of said free plasmin in said subject is increased in the presence of said at least one lysine analog.

28. The method according to any one of claims 26 to 27, wherein increase in the level of plasmin results in a decrease in the level of at least one coagulation factor in said subject.

29. A method of treatment, amelioration, inhibition or prophylaxis of a hemostatic disorder associated with fibrinolysis in a subject treated with at least one lysine analog, comprising the step of administering to said subject a therapeutically effective amount of at least one tPA mutant that carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, or any composition thereof.

30. The method according to claim 29, wherein said point mutation results in a substitution of serine 481 to alanine, and wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tPASer481Ala.

31. The method according to any one of claims 29 and 30, wherein said lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

32. An effective amount of a combination or composition comprising the same, for use in a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder or any disorder associated with fibrinolysis, wherein said combination comprises:(a) at least one tPA mutant that carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2; and;(b) at least one lysine analog that binds the lysine binding site (LBS) of plasmin; wherein said combination exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

33. The effective amount of a combination for use according to claim 32, wherein said combination is as defined by any one of claims 1 to 7.

34. The effective amount of a combination for use according to any one of claims 32 or 33, wherein said point mutation results in a substitution of serine 481 to alanine, and wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tPASer481Ala.

35. The effective amount of a combination for use according to any one of claims 32 and 34, wherein said lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

36. The effective amount of a combination for use according to any one of claims 32 to 35, wherein said hemostatic disorder comprises at least one of: hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, coagulopathy, thrombocytopenia, congenital coagulopathy, hemophilia,disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

37. An effective amount of at least one tPA mutated molecule that carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, or a composition comprising the same, for use in a method of treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder associated with fibrinolysis in a subject treated with at least one lysine analog.

38. The effective amount of the tPA mutated molecule for use according to claim 37, wherein said point mutation is a substitution of serine 481 to alanine, and wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tPASer481Ala.

39. The effective amount of the tPA mutated molecule for use according to any one of claims 37 and 38, wherein said lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

40. The effective amount of the tPA mutated molecule for use according to any one of claims 37 to 39, wherein said hemostatic disorder comprises at least one of: hemorrhage, noncompressible hemorrhage, trauma induced hemorrhage, gynecological hemorrhage, minor surgery or major surgery bleeding, coagulopathy, thrombocytopenia, congenital coagulopathy, hemophilia, disseminated intravascular coagulation (DIC) and conditions related to angiogenesis, bleeding associated with fibrinolytic or thrombolytic therapy, coagulopathy associated with anti-fibrinolytic therapy.

41. The effective amount of the tPA mutated molecule for use according to any one of claims 37 to 40, wherein said disease, disorder, or condition associated with fibrinolysis is at least one of coagulopathy induced by free plasmin and coagulopathy induced by inactivation of at least one coagulation factor.

42. The effective amount of the tPA mutated molecule for use according to any one of claims 37 to 41, wherein the level of said free plasmin in said subject is increased in the presence of said at least one lysine analog.

43. The effective amount of the tPA mutated molecule for use according to any one of claims 37 to 42, wherein increase in the level of plasmin results in a decrease in the level of at least one coagulation factor in said subject.

44. A kit comprising:(a) at least one tissue plasminogen activator (tPA) mutant that carries a point mutation at position 481 of the WT tPA, said WT tPA comprising the amino acid sequence as denoted by SEQ ID NO: 2, optionally, in a first dosage form; and(b) at least one lysine analog that binds to the lysine binding site (LBS) of plasminogen and / or plasmin, optionally in a second dosage form; or(c) a combination of (a) and (b), wherein said combination exerts at least one of increasing the anti-fibrinolytic activity of said lysine analog and reducing free plasmin activity.

45. The kit according to claims 43, wherein said point mutation results in a substitution of serine 481 to alanine, and wherein said tPA mutant comprises the amino acid sequence as denoted by SEQ ID NO: 1 or any variants or derivatives thereof, said mutant being tPASer481Ala.

46. The kit according to any one of claims 43 and 44, wherein said lysine analog is at least one of 4-(aminomethyl)-cyclo- hexane-carboxylic acid (tranexamic acid or TXA) and e-amino caproic acid (EACA).

47. The kit according to any one of claims 43 to 46, wherein said kit is adapted for use in the treatment, amelioration, inhibition or prophylaxis of at least one hemostatic disorder associated with fibrinolysis in a subject in need thereof.

48. The kit according to any one of claims 43 to 46, wherein said kit is adapted for use in a method as defined by any one of claims 20 to 27.

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