Agents for promoting hemostasis

Abstract

The present invention relates to the use of pretrombin-1 as a medicament.

Description

Technical Field

[0001] The present invention relates to agents for promoting hemostasis and treating bleeding.

Background Art

[0002] Hemostasis is the result of a cascade-like proteolytic activation of inactive enzyme precursors. The coordinated interaction of activated coagulation factors and their cofactors results, as the second-to-last step of hemostasis, in the activation of prothrombin to thrombin, which, when present in sufficient concentration, coagulates fibrinogen present in plasma at the site of injury and forms an insoluble fibrin matrix together with activated platelets there.

[0003] Therefore, the formation of thrombin is an important element of hemostasis, together with the production of thrombin from prothrombin, which is a complex and tightly regulated enzymatic process. Other coagulation factors and cofactors, as well as platelets and certain endothelial factors, are required for the entire hemostasis process.

[0004] The enzymatic activation of prothrombin occurs via factor Xa, an enzyme that is expressed via the activation of factor X. The activity of factor Xa is enhanced by orders of magnitude when it forms the enzyme complex prothrombinase together with factor Va as a cofactor. Prothrombinase occurs in the presence of calcium ions on the phospholipid-containing membrane surface formed by activated platelets or endothelium.

[0005] Factor Xa is formed by two different tenases: the intrinsic and extrinsic tenases. The intrinsic tenase is characterized by the enzyme factor IXa and its cofactor VIIIa. Next, the extrinsic tenase constitutes the initiation of the coagulation cascade and consists of the enzyme factor VIIa and tissue factor that comes into contact with blood upon endothelial injury. Factor Xa formed by the extrinsic tenase is rapidly inhibited by TFPI. A very small amount of thrombin formed by exogenously produced factor Xa activates cofactors V and VIII, as well as platelets, at the site of the coagulation process (1).

[0006] Factor V or the shortened form of factor V (short factor V) is a cofactor for TFPI in addition to protein S. When factor V or short factor V is activated by thrombin, respectively, a decrease in the activity of its cofactor function and a concomitant decrease in the inhibitory effect of TFPI occur (2).

[0007] Hemophilia A and B are pathological changes characterized by the dysfunction of the components of the intrinsic tenase. This dysfunction of the intrinsic tenase leads to an interruption of hemostasis. The treatment of their coagulation disorders is carried out by replacing the lost coagulation factors. A common problem with this replacement therapy is the formation of alloantibodies against the replaced coagulation factors. These inhibitors can prevent further replacement or even render it completely ineffective. Avoiding these inhibitors has become an important issue in the treatment of hemophilia. In the early 1970s, prothrombin complex was developed as a drug for the treatment of bleeding from various causes. The prothrombin complex was also found to be very effective in cases of hemophilia (Hemmkoerperhaemophilie). The activated prothrombin complex resulting from further development gradually replaced the non-activated prothrombin complex in the treatment of hemophilia (Hemmkoerperhaemophilie verdraengten) (3, 4). Factor VIIa as a component of the extrinsic tenase has also been found to be an option for the potential treatment of hemophilia (Hemmkoerperhaemophilie).

[0008] The activated factors, particularly factor Xa, were hypothesized to be an effective component of activated prothrombin complex concentrate when combined with prothrombin (5). In blood, prothrombin has a relatively long half-life, such that the prothrombin concentration can be increased to 2 to 3 times the normal value by repeated administration of activated prothrombin complex.

[0009] In addition to the antithrombin concentration, the prothrombin concentration in blood has long been recognized as the most important parameter for normal blood coagulation (6).

[0010] In mouse models of hemophilia A and B, highly purified prothrombin was found to be effective in hemostasis (7). The prothrombin concentration in plasma is approximately 80 - 90 mg / L. The half-life is 48 - 70 hours. To significantly increase the prothrombin concentration in plasma, application of high-content prothrombin is essential, and due to the long half-life, a long-lasting effect can also be obtained. Nevertheless, the use of prothrombin as a drug has not been investigated. Clinical studies evaluating the safety, toxicity, and pharmacology of recombinant human prothrombin were prematurely terminated (8).

[0011] Prothrombin is a vitamin K-dependent enzyme precursor and has a complex structure characterized by an N-terminal Gla domain, kringle 1 domain (fragment 1), kringle 2 domain (fragment 2), and the actual enzyme domain. For the formation of thrombin, prothrombin must be cleaved at two sites: R271 and R320. Cleavage at the R320 site results in the formation of enzymatically active meizothrombin. Subsequent cleavage at the R271 site results in the release of thrombin. If the R271 cleavage site of prothrombin is cleaved first, enzymatically inactive prethrombin-2 is formed, which can subsequently be converted to thrombin by a second cleavage at the R320 site.

[0012] In the presence of cofactor Va, the R320 cleavage site is preferentially cleaved, and meizothrombin is produced as an intermediate. However, in the absence of factor Va, the R271 cleavage site is preferentially cleaved, mainly producing prethrombin-2 (9).

[0013] Most of the thrombin essential for blood coagulation is formed on the phospholipid-containing surface by the prothrombinase complex consisting of factor Xa and its cofactor Va in a concerted mechanism (10). However, thrombin also plays an important role in platelet activation and the formation of important cofactors Va and VIIIa for the two types of tenases.

[0014] Prethrombin-1 is an enzymatically inactive cleavage product that results from the cleavage of fragment 1 from prothrombin by thrombin in a feedback reaction (11, 12). Both prethrombin-1 and prethrombin-2 lack the Gla domain required for membrane binding ability, and thus are not efficiently activated by prothrombinase bound to phospholipid vesicles in vitro (13). In the absence of factor Va or phospholipids, free factor Xa activates prothrombin and prethrombin-1 at similar rates to form thrombin.

[0015] When prethrombin-1 is produced on the surface of activated platelets, activation to form thrombin occurs rapidly (14).

[0016] Fragment 2 present in prethrombin-1 has a sequence that binds to factor Va. In prethrombin-2, this sequence located in kringle 2 is lost.

[0017] McDuffie et al. (16) used a specific immunoassay to measure the concentrations of prothrombin, thrombin, and prothrombin fragments in the plasma of healthy subjects and patients suspected of having disseminated intravascular coagulation (DIC). From the results obtained, it was concluded that the measurement of prethrombin-1 levels is not useful for the diagnosis of DIC nor for its therapeutic management.

[0018] Owen et al. (17) examined the catabolism of prothrombin and its fragments in healthy dogs. To determine whether prethrombin-1 could serve as a degradation product of prothrombin, radiolabeled prethrombin-1 was injected into dogs. Based on the determined half-life and plasma concentration of prethrombin-1 measured in healthy individuals, it was found that prothrombin is not catabolized via the release of prethrombin-1.

[0019] Lanchantin et al. (18) early recognized that in a coagulation test performed using prothrombin-deficient plasma, prothrombin activity is lost due to the action of thrombin on prothrombin. Two cleavage products resulting from the action of thrombin on prothrombin were ultimately called prethrombin-1 and fragment 1. Prethrombin-1 is enzymatically inactive.

[0020] Heldebrandt et al. (19) reported that prothrombin and prethrombin-1 (intermediate product 1) can be activated by factor Xa to form thrombin at approximately the same rate in a diluted aqueous buffer. However, due to the absence of fragment 1, prethrombin-1 cannot bind to the phospholipid membrane, and thus is converted by prothrombinase at a much lower rate than prothrombin. This is also the reason why prethrombin-1 has little activity in the coagulation test using prothrombin-deficient plasma. This test method is based on a coagulation test in which all coagulation factors other than prothrombin are present in the blood plasma. The activity of prothrombin is measured by adding calcium and thromboplastin. Prothrombin is a limiting factor, and the clotting time is inversely proportional to the concentration of prothrombin.

[0021] Seegers et al. (20) and Baker et al. (21) also showed that prethrombin-1 can be barely converted to thrombin only when phospholipid, factor Xa, factor V, and calcium ions are used as coagulation accelerators.

[0022] Seegers et al. (22, 23) described a previously unknown coagulation accelerator called protein M. This protein promotes the formation of thrombin in a five-component system consisting of prothrombin enzyme precursor, factor Xa, factor V, phospholipid, and calcium ions.

[0023] The topical application of thrombin for hemostasis is widely used clinically (24, 25).

[0024] Disorders of the coagulation system are not an option for treatment because direct administration of thrombin in hemorrhagic diathesis has a high risk of fatal systemic coagulation, and can cause severe dysfunction of hemostasis.

[0025] Therefore, it is very interesting that it is possible to provide treatment options using appropriate agents that normalize the prolonged bleeding time, especially in hemophilia A or B in the presence of inhibitors.

[0026] Currently available treatment options basically include activated prothrombin complex (FEIBA®, Takeda) or activated factor VII (NovoSeven®, Novo Nordisk). Activated factor VII has a very short half-life and thus prophylactic use is not considered. The activated prothrombin complex has a complex composition using zymogen precursors present in a balanced ratio and anticoagulants such as trace amounts of active coagulation factors II, VII, IX, X, protein C and TFPI that promote coagulation, and small amounts of cofactors FV, FVIII, and protein S (26).

Summary of the Invention

Problems to be Solved by the Invention

[0027] An object of the present invention is to provide an agent for promoting hemostasis and treating bleeding, which contains a molecularly clearly defined active ingredient and has a composition that is easy to formulate. Furthermore, the agent is expected to be an alternative to currently commonly used agents for promoting hemostasis such as activated prothrombin complex and activated factor VIIa.

Means for Solving the Problems

[0028] This object is achieved by the use of an agent containing prothrombin-1 as an active ingredient. Therefore, the present invention is, respectively, the use of prothrombin-1 as an agent or the use of prothrombin-1 for the manufacture of an agent.

Modes for Carrying Out the Invention

[0029] Preferred embodiments of the present invention each consist of the specific use of prothrombin-1 for promoting hemostasis or the use of prothrombin-1 for the manufacture of a medicament for promoting hemostasis.

[0030] Even more preferred embodiments of the present invention are: - the use of prothrombin-1 for use in the treatment of bleeding or the use of prothrombin-1 for the manufacture of a medicament for treating bleeding, - the use of prothrombin-1 for use in the treatment of bleeding in patients with a coagulation disorder or the use of prothrombin-1 for the manufacture of a medicament for treating bleeding in patients with a coagulation disorder, - the use of prothrombin-1 for use in the treatment of bleeding due to trauma or internal injury or the use of prothrombin-1 for the manufacture of a medicament for treating bleeding due to trauma or internal injury, - the use of prothrombin-1 for use in the treatment of breakthrough bleeding under alternative therapy using a coagulation factor, a therapy mimicking factor VIII, or gene therapy or the use of prothrombin-1 for the manufacture of a medicament for treating breakthrough bleeding under alternative therapy using a coagulation factor, a therapy mimicking factor VIII, or gene therapy, and - the use of prothrombin-1 for use in the reversal of anticoagulation by an orally administered direct-acting anticoagulant or the use of prothrombin-1 for the manufacture of a medicament for reversing the anticoagulation by an orally administered direct-acting anticoagulant.

[0031] Furthermore, the present invention provides - a method of treating a patient by promoting hemostasis, - a method of treating bleeding, - a method of treating a coagulation disorder, - a method of treating bleeding due to trauma or internal injury, - A method for treating breakthrough bleeding under replacement therapy using a coagulation factor, a therapy mimicking factor VIII, or gene therapy, and - A method for reversing the anticoagulant effect of an orally administered direct-acting anticoagulant which also includes a method comprising administering an effective amount of prothrombin-1 to a patient.

[0032] As described in the following examples, prothrombin-1 can be prepared in a simple manner from prothrombin by cleavage with thrombin followed by purification by chromatography. Prothrombin-1 can be formulated using a suitable pharmaceutical solvent, sterilized by filtration, and filled aseptically. Lyophilization is a preferred pharmaceutical preparation method.

[0033] For the use of prothrombin-1 for the manufacture of a medicament from prothrombin, various methods of virus inactivation or virus removal can be applied. Such examples are known to those skilled in the art.

[0034] As an alternative to extraction from blood plasma, prothrombin-1 can also be prepared by using suitable molecular biological methods, specifically recombinant DNA technology. For this reason, an additional specific embodiment of the present invention consists of using recombinant prothrombin-1 instead of natural prothrombin-1 for the above-mentioned indications.

[0035] Prothrombin complex concentrates cannot be prepared using recombinant DNA technology. Their composition is extremely complex. Prothrombin, which mainly acts as a blood coagulation promoting factor, must be balanced by a sufficient amount of anticoagulant components. Since these have a shorter half-life than prothrombin, an excess of prothrombin and the accompanying undesirable thrombogenic effects can occur.

[0036] Prothrombin-1 is defined by the protein sequence obtained when fragment 1 is cleaved from prothrombin at position R155.

[0037] The dosage of prothrombin-1 is based on the cause and severity of bleeding, but preferably reaches 100 units / kg body weight, where 1 unit of prothrombin-1 is defined as the amount molecularly equivalent to 1 unit of prothrombin. Even lower dosages can promote hemostasis. If bleeding cannot be stopped with the standard dosage, higher dosages up to 300 units / kg may be effective. Plasma levels can be further increased by repeated administration of prothrombin-1.

[0038] Prothrombin-1 has been shown to have only a slight tendency to cause unwanted thromboembolic events.

[0039] The efficacy of prothrombin-1 as a drug having hemostatic activity in the FVIII inhibitor mouse model is shown below. The inventors used this model to examine the promotion of hemostasis by prothrombin-1. As demonstrated in the following examples, it was surprisingly shown that prothrombin-1 has excellent hemostatic effects in this model.

[0040] Prothrombin-2 has no hemostatic effect in vivo.

[0041] Therefore, the present invention is based, inter alia, on the recognition that prothrombin-1 promotes hemostasis in vivo.

[0042] Prothrombin-1 can be used, in particular, for the following types of bleeding that are difficult to control: - Severe acute bleeding in patients with coagulation disorders - Acute life-threatening bleeding due to trauma or internal injury - Reversal of anticoagulation under therapy with direct-acting oral anticoagulants (NOACs) for life-threatening or uncontrolled bleeding. NOACs (novel oral anticoagulants) are anticoagulants and antithrombotic agents. This anticoagulant effect is based on the direct inhibition of blood coagulation factors. - Other critical bleeding situations where activated or inactivated prothrombin complex concentrate or recombinant factor VIIa is applied.

[0043] "Severe" type bleeding is understood to be bleeding that cannot be stopped by conventional methods.

[0044] Prothrombin-1 will have an effect when applied intravenously as well as subcutaneously.

[0045] The present invention will be described in more detail using the following examples. Example

Example

[0046] Preparation of Prothrombin-1 1 mL of prothrombin concentrate having an activity of 425 U prothrombin / mL was mixed with 50 U thrombin. This mixture was incubated at room temperature for about 24 hours. After incubation, this mixture was diluted to 10 mL using a buffer of 10 mM citrate, 137 mM NaCl, pH 7.0, and applied to 15 mL Heparin Sepharose FF. Prothrombin-1 was isolated from the fraction of the second UV280 nm (18 mL) signal peak of isocratic elution using a buffer of 15 mM citrate, 150 mM NaCl, pH 7.0, concentrated to 5 mL through a 5 kDa UF membrane, diluted to 10 mL using distilled water, and applied to 10 mL AIEX CaptoQ ImpRes. Prothrombin-1 was isolated from the fraction of the second UV280 nm (22 mL) signal peak of isocratic elution using a buffer of 15 mM citrate, 150 mM NaCl, pH 7.0. This prothrombin-1 fraction was further concentrated to 1 mL through a 5 kDa UF membrane. The prothrombin-1 thus obtained was adjusted to a protein concentration of 5.0 mg / mL.

[0047] Prothrombin-1 purified from the band produced by this method was formulated using a suitable pharmaceutically acceptable solvent and formulated into a medicament using a method well known per se. Prothrombin-1 produced by this method had only about 1% of the activity of prothrombin in a prothrombin assay using prothrombin-deficient plasma. Using a complex prepared from factor Xa and its cofactor Va, 1 unit of the resulting prothrombin-1 was able to form about 200 NIH units of thrombin.

Example

[0048] Determination of Hemostatic Effect in FVIII Inhibitor Mouse Model FVB mice were anesthetized and, after cutting the tail, were treated with an FVIII antibody via the tail vein to enhance the bleeding tendency. When the test substance was applied via the femoral vein, the tip of the tail was cut with a scalpel 3 mm apart. The blood loss was determined by weighing. The time elapsed until bleeding stopped was defined as the bleeding time. After bleeding stopped, slight secondary bleeding may occur. In all specimens, the test period was 30 minutes.

[0049] The results obtained are clearly shown in the following table using the abbreviation "AK" representing the antibody against factor VIII.

[0050]

Table 1

[0051] This table shows the following:

[0052] The placebo group administered isotonic saline to 5 animals had a bleeding time > 30 minutes, which was accompanied by a blood loss of 424.8 - 823.6 mg.

[0053] When pretrombin-1 was administered at a dose of 100 U / kg body weight, the bleeding time was shortened to 6 minutes, 9 minutes, 2 minutes, or 1 minute 30 seconds in 4 animals. In only 1 animal, the bleeding time was longer than 30 minutes with very slight bleeding. In all animals, the blood loss was significantly reduced, and in 3 of them, no blood loss was measured at all. In 1 animal, the final blood loss was 3.7 mg, and in another 1 animal, the final blood loss was 10.1 mg.

[0054] When the dose of pretrombin-1 was increased to 300 mg / kg body weight, the bleeding times were 5 minutes 10 seconds, >30 minutes, 4 minutes 30 seconds, 1 minute 30 seconds, and 1 minute 40 seconds. Also in this case, bleeding was extremely less compared to the placebo group. No blood loss was measured in 5 animals, and in 1 animal, the blood loss was 51.4 mg.

[0055] This experiment showed that regardless of whether pretrombin-1 at a dose of 100 mg / kg body weight or 300 mg / kg body weight was used, the bleeding characteristics were significantly lower compared to the placebo group, and in particular, the blood loss was significantly reduced compared to the placebo group.

Example

[0056] Thrombogenic ability of pretrombin-1 in the modified Wessler test The Wessler test has been used for many years to determine the thrombogenicity of various substances in in vivo models (27, 28).

[0057] White New Zealand rabbits (body weight 2.5 - 3.5 kg, Charles River, Germany) were used. These animals were anesthetized and a venous access was established in the ear vein. Then, the contralateral common carotid vein was prepared, and the animals were made in a temporary hemophilic state using factor VIII antibody.

[0058] After intravenous application of the test substance, a part of the vein was ligated with a length of 1.5 cm. After a waiting time of 20 minutes, the ligated part of the vein was excised and incised in isotonic sodium citrate buffer. The surface of the vein lumen was observed macroscopically, and if thrombus was present, it was excised and weighed. The following criteria were used to evaluate the thrombus: 0 ······ No thrombus 1 ······ Small thrombus with a weight of less than 2 mg 2 ······ One or more thrombi 3 ······ Thrombus completely occluding the vein

[0059]

Table 2

[0060] This result indicates that in the Wessler test, prothrombin-1 is not thrombogenic even at high doses.

Example

[0061] Effectiveness of prothrombin-1 in reversing the anticoagulant effect of an oral Xa inhibitor The effectiveness of prothrombin-1 in reversing the anticoagulant effect of an oral Xa inhibitor was demonstrated in a rivaroxaban rabbit model. In this case, anesthetized New Zealand White rabbits were treated with rivaroxaban, a factor Xa inhibitor, to obtain an anticoagulant effect. The increased bleeding tendency associated with the anticoagulant effect was determined by cutting the claws. When prothrombin-1 was administered intravenously, the blood loss was measured after cutting the claws again.

[0062] The results obtained are summarized in the following table.

[0063]

Table 3

[0064] The dose of rivaroxaban was increased up to 7 - 70 mg / kg body weight. Since a sufficient bleeding tendency was obtained at a dose of 70 mg / kg body weight, no further increase in dose was necessary. When prothrombin-1 was administered intravenously, no blood loss was measured in 10 animals. Blood loss was measured in 2 animals, but it was less than the blood loss after anticoagulation.

[0065] From these measurements, it can be concluded that prothrombin-1 can reverse the bleeding-promoting effect of oral Xa inhibitors when reversal of the anticoagulant effect is essential due to lethal or uncontrolled bleeding.

[0066] Literature: (1) Orfeo T, Butenas S, Brummel-Ziedins KE, Mann KG. The tissue factor requirement in blood coagulation. J Biol Chem. 2005; 280(52):42887-96. (2) Petrillo T, Ayombil F, Van't Veer C, Camire RM. Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding. J Biol Chem. 2021 Jan-Jun; 296:100234. (3) Eibl H, Schwarz O and Elsinger F. Patent AT 350726 (1976) Verfahren zur Herstellung einer blutgerinnungsfordernden Praparation aus menschlichem Blutplasma (4) Eibl H, Schwarz O and Elsinger F. Patent AT 350726 USP 4,160,025 (1979) Method of producing a blood - coagulation - promoting preparation from human blood plasma. (5) Turecek P. and Schwarz HP. (2013) “Factor eight inhibitor bypassing activity” in Production of Plasma Proteins for Therapeutic use, publisher by John Wiley & Sons inc. (6) Butenas S, van't Veer C, Mann KG "Normal" thrombin generation. Blood. 1999 Oct 1;94(7):2169 - 78. (7) Hansson KM, Lindblom A, Elg M, Lovgren A. Recombinant human prothrombin (MEDI8111) prevents bleeding in haemophilia A and B mice. Haemophilia. 2016 May;22(3):453 - 61. (8) To Assess Safety, Tolerability and Pharmacodynamics of Intravenous MEDI8111 After Single Ascending Doses. - Study Results - ClinicalTrials.gov (9) Stojanovski BM, Di Cera E. Role of sequence and position of the cleavage sites in prothrombin activation. J Biol Chem. 2021 Aug; 297(2):100955. (10) Mann KG, Elion J, Butkowski RJ, Downing M, Nesheim ME. Prothrombin. Methods Enzymol. 1981;80 Pt C:286-302. (11) Haynes LM, Bouchard BA, Tracy PB, Mann KG. Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism. J Biol Chem. 2012 Nov 9;287(46):38647-55. (12) Chen Z, Pelc LA, Di Cera E. Crystal structure of prethrombin-1. Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19278-83. (13) Bukys MA, Orban T, Kim PY, Nesheim ME, Kalafatis M. The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase. Thromb Haemost. 2008 Mar;99(3):511-22 (14) Ayombil F, Wood et al. Prethrombin-1, the Gla-domainless prothrombin intermediate, is activated efficiently to thrombin by prothrombinase assembled on the activated platelet surface. July 2011. Conference: International Society of Thrombosis and Haemostasis. Japan Volume: Journal of Thrombosis and Haemostasis 9, 352-35 (15) Friedmann AP, Koutychenko A, Wu C, Fredenburgh JC, Weitz JI, Gross PL, Xu P, Ni F, Kim PY. Identification and characterization of a factor Va-binding site on human prothrombin fragment 2. Sci Rep. 2019 Feb 21;9(1):2436. (16) McDuffie FC, Giffin C, Niedringhaus R, Mann KG, Owen CA Jr, Bowie EJ, Peterson J, Clark G, Hunder GG. Prothrombin, thrombin, and prothrombin fragments in plasma of normal individuals and of patients with laboratory evidence of disseminated intravascular coagulation. Thromb Res. 1979;16(5-6):759-73. (17) Owen CA Jr, Mann KG, McDuffie FC. The turnover in normal dogs of prothrombin and its fragments; effect of induced intravascular coagulation. Thromb Haemost. 1979 Aug 31;42(2):548-55. (18) Lanchantin GF, Friedmann JA, Hart DW. On the occurrence of polymorphic human prothrombin. Electrophoretic and chromatographic alterations of the molecule due to the action of thrombin. Biol Chem. 1968 Feb 10;243(3):476-86. (19) Heldebrant CM, Butkowski RJ, Bajaj SP, Mann KG The activation of prothrombin. II. Partial reactions, physical and chemical characterization of the intermediates of activation. J Biol Chem. 1973 Oct 25;248(20):7149-63. (20) Seegers WH, Hassouna HI, Novoa E. Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas. 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Claims

1. A pharmaceutical composition containing prethrombin-1 for use as a drug.

2. A pharmaceutical composition comprising prethrombin-1 for specific use to promote hemostasis.

3. The pharmaceutical composition according to claim 2 for use in the treatment of bleeding.

4. The pharmaceutical composition according to claim 3, for use in the treatment of bleeding in patients with coagulation disorders.

5. The pharmaceutical composition according to claim 3, for use in the treatment of bleeding resulting from trauma or internal injury.

6. The pharmaceutical composition according to claim 3, for use in the treatment of break-even bleeding (Durchbruchsblutungen) under alternative therapy using coagulation factors, factor VIII mimicry therapy, or gene therapy.

7. A pharmaceutical composition comprising prethrombin-1 for use in the reversal (Umkehrung) of anticoagulant effects by orally administered direct-acting anticoagulants.

8. A pharmaceutical composition according to any one of claims 1 to 7, wherein the prethrombin-1 is recombinant prethrombin-1.