JP2013531651A - Treatment of coagulopathy with increased fibrinolysis - Google Patents

Abstract

The present invention relates to the use of thrombomodulin analogues for the manufacture of a medicament for the treatment of coagulation disorders with increased fibrinolysis, such as hemophilia. These thrombomodulin analogs exhibit an antifibrinolytic effect at therapeutically effective dosages. Novel protein modifications are disclosed along with their identification methods. The present invention provides particularly suitable pharmaceutically active proteins and peptides that can be used, for example, according to the present invention. In accordance with one embodiment of the present invention, a thrombomodulin analog of the present invention having reduced binding affinity for thrombin compared to rabbit lung thrombomodulin can be used for the treatment of coagulopathy with hyperfibrinolysis.

Description

  The present invention relates to the field of coagulation disorders with increased fibrinolysis. More particularly, the invention relates to the treatment of hemophilia diseases such as hemophilia A or hemophilia B. The present invention claims the priority of PCT application PCT / EP2009 / 004218, which is fully incorporated herein by reference for its disclosure.

  Hemophilia is a group of inherited genetic disorders that impair the body's ability to control blood clotting or coagulation used to stop bleeding when blood vessels are destroyed. The most common form, haemophilia A, occurs as a result of a gene mutation for factor VIII; haemophilia B, also known as Christmas disease, results from a gene mutation for factor IX. As follows. Hemophilia B, like hemophilia A, is X-linked and accounts for approximately 12% of hemophilia cases. Symptoms are identical to those of hemophilia A: excessive bleeding upon injury; and idiopathic bleeding, especially to load joints, soft tissues and mucous membranes. Repeated bleeding to the joints results in arthremia causing painful and severe arthropathy that often requires joint replacement. Hematomas in soft tissue can result in pseudotumors consisting of necrotic coagulated blood; they can occlude, compress, or rupture into adjacent organs Yes, it can cause infection. Once formed, hematomas are even difficult to treat with surgery. Nerve recovery after compression is poor, resulting in paralysis. Those bleeding episodes involving the gastrointestinal tract, central nervous system, or airway / retroperitoneal cavity can be fatal if not detected. Intracranial hemorrhage is the leading cause of death from hemophilia.

  There are an estimated 100,000 cases of congenital hemophilia in the United States. Of these, approximately 20,000 are cases of hemophilia B, and the blood of such patients is either completely free of factor IX or severely lacking plasma factor IX components. Thus, the disease exists with varying severity, generally requiring treatment from weekly to once or twice a year. Completely deficient cases require weekly replacement therapy; partially deficient cases require treatment only when bleeding episodes occur, which is less frequent than once a year There is also. Bleeding episodes in cases of congenital partial deficiency are generally caused by temporary acquired susceptibility as well as injury. Deficient subject deficiency is temporarily corrected by intravenous injection of a sufficiently large amount of fresh plasma or equivalent amount of fresh blood. This beneficial effect often lasts 2 or 3 weeks, but the coagulation deficiency appears to improve only for 2 or 3 days as measured by in vitro tests using the patient's blood.

  Although such treatment with fresh plasma or fresh blood is effective, it has several significant drawbacks: (1) it requires large amounts of fresh plasma to be readily available; (2) Requires hospitalization for the administration of plasma; (3) so many patients become sensitive to repeated blood or plasma infusions and eventually encounter a lethal transfusion reaction; (4) at most plasma Can only partially alleviate the deficiency; and (5) long-term treatment or surgery is not possible because the large volumes of blood or plasma required will cause acute and lethal edema. is there.

  Improved treatment includes intravenous replacement therapy with Factor VIII or Factor IX concentrate. However, this therapy also has several disadvantages: (1) When treating major bleeding episodes, tissue damage remains even after rapid detection and treatment; (2) very Many patients become resistant to coagulation factors and develop inhibitory antibodies against the coagulation factors (so-called haemophilia with inhibitors); (3) despite improved virus inactivation methods, HIV There is still an increased risk of contamination with lethal viruses such as hepatitis C (in the United States, more than 50% of the hemophilia population estimated to suffer from HIV from contaminated blood supplies) ); (4) Isolated clotting factors, especially recombinant clotting factors, are very expensive and generally available in the developing world. Absent.

  Treatment or prevention of bleeding beyond replacement therapy is a challenge. Because bleeding in hemophilia is (1) reduced thrombin generation by extrinsic pathways at low tissue factor concentrations, (2) reduced secondary burst of thrombin generation by intrinsic pathways, and (3) lines by intrinsic pathways. This is because it is a complex pathophysiological process that can be attributed to the triple defect of incomplete down-regulation of the solution system.

  It is generally accepted that reducing thrombin generation results in a reduced clotting tendency and thus an increased bleeding risk. However, research in the past decade has shown that incomplete down-regulation of fibrinolysis can also play a role in hemophilia. As a result, hemophilia can also be classified as a coagulation disorder with increased fibrinolysis.

  A recent announcement has shown that this assumption is that fibrinolysis is not sufficiently down-regulated when clots are formed in factor VIII-depleted plasma (FVIII-DP) and supplemented with tissue plasminogen activator tPA. This is supported by demonstrating in vitro that clots dissolve prematurely (Non-Patent Document 1; Non-Patent Document 2). Furthermore, this “premature lysis” is due to reduced or absent activation of thrombin-activated fibrinolysis inhibitor (TAFI), and in FVIII-DP. The activated TAFI-containing mixture could prove to increase the clot dissolution time. It was concluded that stabilized TAFI can be used for the treatment of hemophilia (Patent Document 1).

TAFI plays a critical role in fibrinolytic downregulation required for the formation of stable clots. TAFI, also known as plasma procarboxypeptidase B2 or procarboxypeptidase U, is a basic carboxypeptidase (TAFIa or activated that inhibits fibrinolysis by proteolysis at Arg ⁹² when exposed to a thrombin-thrombomodulin complex. It is a plasma zymogen that is converted to TAFI). It strongly attenuates fibrinolysis by removing from the fibrin the C-terminal lysine and arginine residues that are important for plasminogen binding and activation.

  As discussed above, thrombomodulin (TM) in complex with thrombin is responsible for TAFI activation. Thrombomodulin is a membrane protein that acts as a thrombin receptor on endothelial cells lining the blood vessels. Thrombin is a central enzyme in the coagulation cascade that converts fibrinogen to fibrin making up the matrix clot. Initially, a small amount of thrombin is generated from its inactive precursor prothrombin by local injury. Thrombin in turn activates platelets and then certain coagulation factors, including Factors V and VIII. The latter action produces a so-called thrombin burst, which is a massive activation of additional prothrombin molecules, and eventually results in the formation of a stable clot.

  However, binding to thrombomodulin alters the direction of thrombin activity: the main feature of the thrombin-thrombomodulin complex is its ability to activate protein C, which protein C is then the essential cofactor factor. By inactivating factor Va and factor VIIIa by proteolysis (Non-Patent Document 3) and thus providing anticoagulant activity, the coagulation cascade is downregulated. The thrombin-thrombomodulin complex can also activate thrombin activated fibrinolysis inhibitor (TAFI), where the TAFI antagonizes fibrinolysis (see above).

  Mature human TM consists of a single polypeptide chain of 559 residues and includes an amino-terminal “lectin-like” domain and a “six EGF-like repeat domain” that includes six epidermal growth factor (EGF) -like repeats. , O-glycosylation domain, transmembrane domain, and cytoplasmic domain, these domains are localized as follows (the given amino acid in SEQ ID NO: 1 or SEQ ID NO: 3) position):

  Various structure-function studies using proteolytic fragments of rabbit TM or deletion mutants of recombinant human TM localized its activity to the last three EGF-like repeats. The minimal mutant that could efficiently promote TAFI activation contained residues including the c-loop to EGF6 of epidermal growth factor-3 (EGF3). This mutant is 13 residues longer than the minimal mutant that activates C and consists of residues from the interdomain loop connecting EGF3 and EGF4 to EGF6.

  As discussed above, replacement therapies for treating clotting abnormalities such as hemophilia do not meet medical needs. Importantly, in addition to the coagulation factors used in the replacement therapy, no drugs are available that can prevent or treat hemophilia patients.

  Thus, despite the long-standing need for the development of treatments to prevent or treat coagulation disorders with increased fibrinolysis, particularly hemophilia, progress has been slow, safe and effective treatment The law has not yet been found.

International Publication No. 02/099098

Broze and Higuchi, Blood 1996, 88: 3815-3823. Mosnier et al., Thromb. Haemost. 2001, 86: 1035-1039 Esmon et al., Ann. N. Y. Acad. Sci. (1991), 614: 30-43.

  Accordingly, it is an object of the present invention to provide a novel means for the treatment of coagulopathy with increased fibrinolysis.

  This object is solved by providing a medicament for the treatment of coagulation disorders with hyperfibrinolysis in mammals, particularly in humans, comprising thrombomodulin analogs that exhibit antifibrinolytic effects at therapeutically effective dosages. . Also provided are particularly suitable pharmaceutically active proteins and peptides that can be used in accordance with the present invention.

  This novel approach is superior to its fibrinolytic activity even at high plasma concentrations, in particular at concentrations higher than 15 nM, in particular at concentrations higher than 20, 30, 40 or 50 nM (up to at least 100 nM). Based on the surprising discovery that thrombomodulin can be modified to exhibit lytic activity. These TM analogues therefore exhibit an antifibrinolytic effect and are therefore suitable for use according to the invention. In the most preferred embodiments, the TM analog may have superior antifibrinolytic activity at concentrations of up to 200 nM or more, even more preferred up to 300 nM or 500 nM.

  This antifibrinolytic effect was demonstrated in plasma from hemophilia patients (factor VIII depleted; FVIII-DP) and in whole blood or plasma of dogs with hemophilia A. In addition, it has been demonstrated that such thrombomodulin analogs can be used as therapeutic agents.

  To date, the therapeutic use of thrombomodulin for the treatment of hemophilia has not been considered a viable option. Because it was known from rabbit lung thrombomodulin (rlTM) that thrombomodulin always had both anti-fibrinolytic and fibrinolytic activity, even at fairly low concentrations (Mosnier and Bouma, Arterioscler. Thromb. Vasc). Biol., 2006; 26: 2445-2453; see especially FIG. At plasma concentrations below 15 nM, rlTM increased clot lysis time, but at plasma concentrations higher than 15 nM, a clear decrease in lysis time was demonstrated (Mosnier et al., 2001, Mosnier and Bouma, 2006) and the final results It had the effect of promoting fibrinolysis. This therapeutic effect of fibrinolysis at higher concentrations prohibits any therapeutic treatment in hemophilia. This is because possible overdose or individual differences in susceptibility can cause fatally worsening, prolonged or even bleeding events.

  According to the present invention, there are various options that result in TM analogs that exhibit an antifibrinolytic effect and are therefore suitable for treatment according to the present invention.

  In one embodiment, thrombomodulin analogs with reduced binding affinity to thrombin can be used. Consequently, they can prolong clot lysis in normal plasma and FVIII-DP, for example up to 100 nM (FIG. 4) or 500 nM (FIG. 10).

  The importance of this discovery is that these thrombomodulin analogs exhibit an antifibrinolytic effect without a harmful fibrinolytic effect even at high concentrations. This concentration is far beyond the therapeutically effective dosage. Thus, these TM analogs allow the treatment of coagulopathy with hyperfibrinolysis.

Without being bound by this theory, we clearly show that this therapeutic potential of the TM analog can be explained by the fact that the TM analog exhibits a significant reduction in affinity for thrombin. I made it. This, Bajzar et (J.Biol.Chem 1996; 271: 16603-16608) have been revealed by, they thrombin observed _{K D} values of 0.2nM for binding to rabbit lung thrombomodulin (Esmon al, Ann.NY.Acad.Sci, 1986,485:. 215-220) and found _{K D} values of 23nM different.

  Thus, in accordance with one embodiment of the present invention, a thrombomodulin analog of the present invention having reduced binding affinity to thrombin compared to rabbit lung thrombomodulin can be used to treat coagulopathy with hyperfibrinolysis. .

Specifically, for thrombin binding, a K higher than 0.2 nM, preferably higher than 1 nM, 2 nM, 4 nM, 5 nM, 7.5 nM, 10 nM, 12.5 nM, 15 nM, 17.5 nM, 20 nM, 22.5 nM or 25 nM. _D, and more preferably, it may be used in the range between 10nM and 30 nM, or 10nM and ranging between 100 nM, or thrombomodulin analog which presents more than _{K D} values 100 nM. In certain embodiments, using the TM analogs with _{K D} of about 50, 60 or 70nM for thrombin binding.

  In a further embodiment of the invention, the reduced fibrinolytic activity of the thrombomodulin analog may be due to a reduced ability to activate protein C (so-called “cofactor activity”). Since protein C activation results in fibrinolytic upregulation (Mosnier et al., 2001), reducing cofactor activity will prolong clot lysis time. Several strategies for reducing the cofactor activity of thrombomodulin, such as altering the primary structure, preferably by altering the glycosylation, secondary or tertiary structure of the protein, or preferably mutating one or more amino acids, for example. Such changes are known to those skilled in the art.

In yet another embodiment, a TM analog having reduced cofactor activity compared to the thrombomodulin analog TM _E M388L (where TM _E refers to an analog consisting of only 6 EGF domains). Can be used.

  According to the present invention, TAFI activation results in fibrinolytic down-regulation (Mosnier and Bouma, 2006), so that the ability to activate TAFI (so-called “TAFI activation activity”) is increased. The body can also be used. Those skilled in the art will recognize TAFI activation activity by thrombomodulin, such as altering the glycosylation, secondary or tertiary structure of a protein, or preferably changing the primary structure, for example by mutation of one or more amino acids. There are several strategies to increase.

In particular, the present invention also provides for thrombomodulin analogs in which the ratio of TAFI activating activity to cofactor activity is significantly increased compared to the thrombomodulin analog TM _E M388L.

Notably, in accordance with the present invention, the TM analog used in the treatment of coagulopathy has the features described above:
(I) is reduced as compared to the rabbit lung thrombomodulin, binding affinity to thrombin, and / or binding affinity to thrombin at 0.2nM larger k _D values,
(Ii) reduced cofactor activity compared to the cofactor activity of TM analog TMEM388L, or (iii) increased ratio of TAFI activation activity to cofactor activity compared to TM analog TM _E M388L ,
One or more of.

  In one embodiment of the invention, thrombomodulin can be used to treat a human patient who has developed any coagulopathy and significantly reduces fibrinolysis or slightly compared to healthy subjects. Even so, it can be reduced. Specifically, the following diseases can be treated with the thrombomodulin analogs: hemophilia A, hemophilia B, hemophilia C, von Willebrand disease (vWD), later Congenital von Willebrand disease, factor X deficiency, parahaemophilia, hereditary disorders of factor I, II, V or VII clotting factors, bleeding disorders due to circulating anticoagulants (coagulation factors such as factor VIII) Including autoantibodies to) or acquired coagulopathy.

  It will be appreciated that the therapeutic success that can be maintained or achieved by the treatment of the present invention will depend on the nature and extent of the disease in any individual patient.

  Certain embodiments of the invention relate to prophylactic treatment of coagulation disorders to prevent bleeding, or acute treatment when bleeding occurs ("on demand"). Bleeding events treated with said thrombomodulin analogs are most important in any organ or tissue in the organism, most importantly in the central nervous system, for example as intracranial hemorrhage, joint, muscle, gastrointestinal tract, respiratory tract, retroperitoneal cavity Or it can occur in soft tissue.

  For preventive measures, the TM analog may be given to the patient at regular intervals over a long period of time. However, multiple administrations ("sub-chronic treatment") for a fairly limited period are also possible.

  In one embodiment of the invention, the thrombomodulin analog is given prior to a higher bleeding risk (eg, surgery or extraction).

  In a further embodiment of the invention, the thrombomodulin analog is administered to a patient who is refractory to standard therapies (eg, blood transfusion or plasma transfusion, or replacement therapy using coagulation factors).

  In accordance with the present invention, the TM analog is administered in multiple doses, preferably once a day for a total period of less than 1 to 4 weeks, but also once every 2 days, or 3 days, 4 days, 5 days. , Every 6 or 7 days, more preferably as a chronic administration. Therefore, according to the present invention, there is provided a pharmaceutical composition suitable for allowing multiple administrations of said thrombomodulin analog.

  The TM analog is preferably given parenterally as a parenteral application, for example by intravenous or subcutaneous application. Intravenous or subcutaneous boluses are possible. Therefore, according to the present invention, a pharmaceutical composition suitable for parenteral administration of thrombomodulin is provided.

  In one embodiment of the invention, said thrombomodulin analog is a soluble TM analog, in particular a TM analog in which the cytoplasmic domain has been deleted and the transmembrane domain has been completely or partially deleted. It is.

  In a preferred embodiment of the invention, said thrombomodulin analogue is EGF3, EGF4, EGF5 or EGF6, preferably EGF domains EGF1 to EGF6, more preferably EGF domains EGF3 to EGF6, and most preferably EGF domains EGF4 to EGF6, and In particular, it comprises at least one structural domain selected from the group comprising a fragment comprising EGF6 from the c-loop of epidermal growth factor-3 (EGF3).

  Various forms of soluble thrombomodulin, such as the so-called ART-123 developed by Asahi Corporation (Tokyo, Japan), or recombinant soluble human thrombomodulin currently under development by PAION Deutschland GmbH, Aachen (Germany) Solulin is known to those skilled in the art. Recombinant soluble thrombomodulin, ie soluble thrombomodulin without amino acid sequence modification, is the subject of Asahi Kasei Corporation EP 0 312 598 B1.

  Solulin is soluble and is a protease and oxidation resistant analog of human thrombomodulin and therefore exhibits a long life span in vivo. Since Solulin does not exclusively inhibit thrombin, the main feature of Solulin resides in its wide mechanism of action. Solulin also activates TAFI and the native protein C / protein S pathway. As a result of its reduced thrombin binding, Solulin inhibits fibrinolysis even at high concentrations. Solulin and its modifications are special embodiments of the present invention.

  Solulin is the subject of, inter alia, European patents EP 0 641 215 B1, EP 0 544 826 B1 and EP 0 527 821 B1. Solulin contains modifications in the following positions compared to the sequence of native human thrombomodulin (SEQ ID NO: 1): G-3V, removal of amino acids 1-3, M388L, R456G, H457Q, S474A, and P490 terminated. . This numbering scheme is by the natural thrombomodulin of SEQ ID NO: 1 and SEQ ID NO: 3. The sequence of Solulin as one preferred embodiment of the present invention is shown in SEQ ID NO: 2.

  However, it is noteworthy that, according to the present invention, one of the characteristics mentioned above or as outlined in the aforementioned European patents EP 0 544 826 B1, EP 0 641 215 B1 and EP 0 527 821 B1. Thrombomodulin analogs containing only or more can also be used.

  In addition, according to the present invention, the properties described above, or Wnag et al. Biol CHem. 275: 22942-22947 can be used thrombomodulin analogs containing only one or more of the properties outlined in the presentation.

Particularly preferred thrombomodulin analogs that can be utilized in accordance with the present invention are those that have one or more of the following characteristics:
(I) they exhibit oxidation resistance;
(Ii) they exhibit protease resistance;
(Iii) they have a uniform N- or C-terminus,
(Iv) they are post-translationally modified, for example by glycosylation of at least part of the glycosylation site of natural thrombomodulin (SEQ ID NO: 1),
(V) they have linear double reciprocal thrombin binding properties;
(Vi) they are soluble in aqueous solutions in relatively small amounts of surfactants and they typically lack transmembrane sequences;
(Vii) they have no glycosaminoglycan chains.

  The production of these analogs for use in the present invention is disclosed in the aforementioned European patent relating to Solulin.

  In one embodiment of the invention, it is possible to use only the 6 EGF domains of Solulin, in particular Solulin fragments consisting of EGF4 to EGF6 domains.

In one embodiment, thrombomodulin analogs with reduced cofactor activity as known from WO 93/25675 A1 can be used. A series of thrombomodulin analogs having about 50% or less of the cofactor activity of a control human soluble thrombomodulin (TM _E M388L) are disclosed herein.

More particularly, said thrombomodulin analog exhibits less than 50% modified cofactor activity when bound to thrombin compared to binding with TM _E M388L, said analog comprising SEQ ID NO: 1 or SEQ ID NO: A given amino acid position in number 3:
ab) ³⁵⁵ Asn;
ae) ³⁵⁹ Gln;
af) ³⁶³ Leu;
ai) ³⁶⁸ Tyr;
aj) ³⁷¹ Val;
ak) ³⁷⁴ Glu;
al) ³⁷⁶ Phe;
am) ³⁸⁴ His;
an) ³⁸⁵ Arg;
ba) ³⁸⁷ Gln;
bb) ³⁸⁹ Phe;
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bf) ⁴⁰³ Thr;
bg) ⁴⁰⁸ Glu;
bh) ⁴¹¹ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
bn) ⁴²⁰ Ile;
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
ce) ⁴²⁸ Glu;
cf) ⁴²⁹ Asp;
cg) ⁴³² Phe;
ch) ⁴³⁴ Ser;
ci) ⁴³⁶ Val;
cj) ⁴³⁸ His;
ck) ⁴³⁹ Asp;
cl) ⁴⁴⁰ Leu;
cm) ⁴⁴³ Thr;
cn) ⁴⁴⁴ Phe;
co) ⁴⁴⁵ Glu;
cp) ⁴⁵⁶ Arg;
cq) ⁴⁵⁸ Ile; or cr) ⁴⁶¹ Asp
Have amino acid substitutions at one or more positions corresponding to

  Most preferred are TM analogs having only one of the substitutions listed above. In one embodiment of the invention, the TM analog is Solulin (SEQ ID NO: 2) having one or more, preferably one of the above mutations. Accordingly, the present invention also relates to the protein set forth in SEQ ID NO: 2 having at least one of the above mutations, and in certain embodiments exactly one. In one embodiment, one amino acid at a given position is deleted rather than substituted. In a further embodiment, the invention encompasses a Solulin fragment having one of the above mutations, in particular an Solulin fragment consisting of an EGF3 to EGF6 domain or an EGF3 c loop from EGF6. The Solulin or the Solulin fragment may contain at least one of the above mutations at amino acid positions 371 to 389, or exactly one (eg, a mutation at position 376). If the Solulin or the Solulin fragment contains a mutation at position 376 (eg, F376A), there is a possibility of a second mutation selected from the mutation.

  For convenience, the left symbols, eg, aa) are the same for each modification site. The first letter represents the EGF domain, a is EGF4, b is EGF5, and c is EGF6. The second letter represents the relative modification position relative to other residues in this list. Nucleic acids encoding the TM analogs described above are also provided herein.

The following analogs constitute a preferred subset of the analogs given above, and these analogs have 25% or less of the cofactor activity of the control, TM _E M388L. These analogs have one or more, preferably only one, amino acid substitution (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
ae) ³⁵⁹ Gln;
aj) ³⁷¹ Val;
ak) ³⁷⁴ Glu;
al) ³⁷⁶ Phe;
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bg) ⁴⁰⁸ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
bo) ⁴²³ Asp;
bp) ⁴²⁴ Ile;
bq) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
ce) ⁴²⁸ Glu;
ck) ⁴³⁹ Asp;
cn) ⁴⁴⁴ Phe; or cr) ⁴⁶¹ Asp.

  In one embodiment of the invention, the TM analog is Solulin (SEQ ID NO: 2) having one or more, preferably one of the above mutations. Accordingly, the present invention also relates to the protein set forth in SEQ ID NO: 2 having at least one of the above mutations, and in certain embodiments exactly one. In one embodiment, one amino acid at a given position is deleted rather than substituted. In a further embodiment, the invention encompasses a Solulin fragment, in particular an EGF3 to EGF6 domain, or a Solulin fragment consisting of EGF3 from the EGF3 c-loop, having one of the above mutations. The Solulin or the Solulin fragment may contain at least one of the above mutations at amino acid positions 371 to 389, or exactly one (eg, a mutation at position 376). If the Solulin or the Solulin fragment contains a mutation at position 376 (eg, F376A), there is a possibility of a second mutation selected from the mutation.

  The modifications described above with respect to protease activity, aliphatic substitution, oxidation resistance and uniform terminology also apply to the above analogs having less than 50% of the control cofactor activity.

Those listed above with less than 30% of the control activity are preferred. These analogs are represented by mutations in domain 4. These analogs have one or more (preferably only one) amino acid substitution (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
ae) ³⁵⁹ Gln;
aj) ³⁷¹ Val; or al) ³⁷⁶ Phe.

  In one embodiment of the invention, the TM analog is Solulin (SEQ ID NO: 2) having one or more, preferably one of the above mutations. Accordingly, the present invention also relates to the protein set forth in SEQ ID NO: 2 having at least one of the above mutations, and in certain embodiments exactly one. In one embodiment, one amino acid at a given position is deleted rather than substituted. In a further embodiment, the invention encompasses a Solulin fragment, in particular an EGF3 to EGF6 domain, or a Solulin fragment consisting of EGF3 from the EGF3 c-loop, having one of the above mutations. The Solulin or the Solulin fragment may contain at least one of the above mutations at amino acid positions 371 to 389, or exactly one (eg, a mutation at position 376). If the Solulin or the Solulin fragment contains a mutation at position 376 (eg, F376A), there is a possibility of a second mutation selected from the mutation.

Analogs with K _D values that are not essentially altered as compared to TM _E M388L also described herein. EGF5 and EGF6 are known to play an important role in high-affinity binding to thrombin, whereas EGF4, which plays a less heavy role in binding, is a cofactor activity on the TM / thrombin complex. It is indispensable to grant. For this reason, modification in EGF repeats 5 and 6, for example (S406a), analogues having _has substantially the same cofactor activity compared to TM E M388L, has a reduced _{K D.} Analogs with alterations in EGF repeats 5 and 6 that result in reduced cofactor activity are listed below. These analogs have one or more, preferably only one amino acid substitution (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bf) ⁴⁰³ Thr;
bg) ⁴⁰⁸ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
cf) ⁴²⁹ Asp;
ck) ⁴³⁹ Asp;
cn) ⁴⁴⁴ Phe; or cr) ⁴⁶¹ Asp.

The analogs may be grouped by their respective domains (ie, EGF4, EGF5 or EGF6) or by their respective relative activities. For example, a TM analog having an EGF4 sequence modification with approximately 50% of the control cofactor activity is (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
bb) ³⁵⁵ Asn;
ae) ³⁵⁹ Gln;
af) ³⁶³ Leu;
ai) ³⁶⁸ Tyr;
aj) ³⁷¹ Val;
ak) ³⁷⁴ Glu;
al) ³⁷⁶ Phe;
am) ³⁸⁴ His; or an) ³⁸⁵ Arg
It is.

  Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments.

For EGF4 having less than 25% of the control cofactor activity (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
ae) ³⁵⁹ Gln;
aj) ³⁷¹ Val; or al) ³⁷⁶ Phe;
It is.

  Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments.

For EGF5, the following modifications resulted in analogs with at least 50% reduction in cofactor activity (given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bf) ⁴⁰³ Thr;
bg) ⁴⁰⁸ Glu;
bh) ⁴¹¹ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp; or bn) ⁴²⁰ Ile.

Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments. These analogs include those with kCat / Km that are essentially unmodified as compared to TM _E M388L.

With respect to EGF5, the analogs can be further divided into subgroups (given amino acids in SEQ ID NO: 1 or SEQ ID NO: 3) by modifications that result in analogs having at least 75% reduction in cofactor activity. position):
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bg) ⁴⁰⁸ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp; or bm) ⁴¹⁷ Asp.

Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments. These analogs include those with kCat / Km that are essentially unmodified as compared to TM _E M388L. Nucleic acids encoding the analogs are also provided.

For EGF6, groups are provided below. Those that have a cofactor activity of less than 50% of the control (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
ce) ⁴²⁸ Glu;
cf) ⁴²⁹ Asp;
cg) ⁴³² Phe;
ch) ⁴³⁴ Ser;
ci) ⁴³⁶ Val;
cj) ⁴³⁸ His;
ck) ⁴³⁹ Asp;
cl) ⁴⁴⁰ Leu;
cm) ⁴⁴³ Thr;
cn) ⁴⁴⁴ Phe;
co) ⁴⁴⁵ Glu;
cp) ⁴⁵⁶ Arg;
cq) ⁴⁵⁸ Ile; or cr) ⁴⁶¹ Asp
It is.

  Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments.

Those that have a cofactor activity of less than 25% of the control (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
cf) ⁴²⁹ Asp;
ck) ⁴³⁹ Asp;
cn) ⁴⁴⁴ Phe; or cr) ⁴⁶¹ Asp
It is.

Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments. Preferred analogs are those mentioned above with additional modifications for solubility, protease resistance, oxidation resistance and uniform ends. Nucleic acids encoding these analogs are also part of the claimed invention. As with other groups, these analogs include those with kCat / Km that are essentially unmodified as compared to TM _E M388L.

The analogs can be further divided into subgroups by those having amino acid modifications at certain positions, in which case the analogs are directed to thrombin compared to analogs having a natural residue at the position. It has an essentially equivalent K _D, the position (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
bb) ³⁵⁵ Asn; or ae) ³⁵⁹ Gln
Corresponding to

  Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments. These analogs can have a modified kCat / Km of less than 30% of the control.

The following site has a K _D or KCAT / Km, which is modified as compared to the analog with natural residue in the position, which extends to analogs described, in this case, the position is (SEQ No. 1 or given amino acid position in SEQ ID NO: 3):
af) ³⁶³ Leu;
aj) ³⁷¹ Val;
ak) ³⁷⁴ Glu;
al) ³⁷⁶ Phe;
am) ³⁸⁴ His;
an) ³⁸⁵ Arg;
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp; or be) ⁴⁰² Asn
Corresponding to

Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments. These analogs having both modified K _D and modified KCAT / Km, particularly those which are modified at least 20%, further comprising.

The following sites when compared to analogs with natural residue in said position, is intended to be illustrative of the analogs with lower cofactor activity, and is essentially equivalent K _D or KCAT / Km, In this case, the position is (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
bg) ⁴⁰⁸ Glu;
bh) ⁴¹¹ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
bn) ⁴²⁰ Ile;
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
ce) ⁴²⁸ Glu;
cf) ⁴²⁹ Asp;
cg) ⁴³² Phe;
ch) ⁴³⁴ Ser;
ci) ⁴³⁶ Val;
cj) ⁴³⁸ His;
ck) ⁴³⁹ Asp;
cl) ⁴⁴⁰ Leu;
cm) ⁴⁴³ Thr;
cn) ⁴⁴⁴ Phe;
co) ⁴⁴⁵ Glu;
cp) ⁴⁵⁶ Arg;
cq) ⁴⁵⁸ Ile; or cr) ⁴⁶¹ Asp
Corresponding to

  Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments.

The following positions describe the subgroupings of modifications that result in at least 75% reduction in cofactor activity but essentially no changes in kcat / Km (SEQ ID NO: 1 or SEQ ID NO: A given amino acid position in 3):
bg) ⁴⁰⁸ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
cf) ⁴²⁹ Asp;
ck) ⁴³⁹ Asp;
cn) ⁴⁴⁴ Phe; or cr) ⁴⁶¹ Asp.

Most preferred are TM analogs having only one of the substitutions listed above, such as Solulin or Solulin fragments. For the modified K _D for thrombin to be modified at least 30%, it can be divided into further sub-group.
The invention further comprises a method. More specifically, described herein is a method useful for screening analogs of thrombomodulin that exhibits a modified Kd for thrombin binding, comprising:
a) Certain positions (given amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 3):
bg) ⁴⁰⁸ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
bn) ⁴²⁰ Ile;
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
ce) ⁴²⁸ Glu;
cf) ⁴²⁹ Asp;
cg) ⁴³² Phe;
ch) ⁴³⁴ Ser;
ci) ⁴³⁶ Val;
cj) ⁴³⁸ His;
ck) ⁴³⁹ Asp;
cl) ⁴⁴⁰ Leu;
cm) ⁴⁴³ Thr;
cn) ⁴⁴⁴ Phe;
co) ⁴⁴⁵ Glu;
cp) ⁴⁵⁶ Arg;
cq) ⁴⁵⁸ Ile;
cr) ⁴⁶¹ Asp
Comprising the step of comparing with a control molecule K _D to and b) thrombin; THAT step performing amino acid substitutions.

When used in these methods, TM analogs having only one amino acid substitution, such as Solulin or Solulin fragments, are preferred. Various embodiments of the present invention, the K _D of including those being modified at least 33%, or the modification is an amino acid substitution, or the control molecule is a TM _E M388L. A preferred grouping of modifications for use in the method is (a given amino acid position in SEQ ID NO: 1 or SEQ ID NO: 3):
bg) ⁴⁰⁸ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
ca) ⁴²³ Asp;
cb) ⁴²⁴ Ile;
cc) ⁴²⁵ Asp;
cd) ⁴²⁶ Glu;
cf) ⁴²⁹ Asp;
ck) ⁴³⁹ Asp;
cn) ⁴⁴⁴ Phe; or cr) ⁴⁶¹ Asp
It is.

  When used in these methods, TM analogs having only one amino acid substitution, such as Solulin or Solulin fragments, are preferred.

Another method useful for screening for analogs of thrombomodulin that has altered cofactor activity upon binding to thrombin,
a) Certain positions (given amino acid positions in SEQ ID NO: 1 or SEQ ID NO: 3):
bb) ³⁵⁵ Asn; or ae) ³⁵⁹ Gln
Described herein is a method comprising the steps of: making an amino acid substitution at; and b) comparing the rate of cofactor activity when bound to thrombin to the rate of a control molecule.

  When used in these methods, TM analogs having only one amino acid substitution, such as Solulin or Solulin fragments, are preferred.

In a preferred embodiment of the invention, the thrombomodulin analog has a modification of the phenylalanine residue at position 376 (Phe376X; SEQ ID NO: 1 or SEQ ID NO: 3). This residue can be chemically or biochemically modified or detected by methods well known to those skilled in the art. The phenylalanine residue is preferably substituted with an aliphatic amino acid, more preferably with glycine, alanine, valine, leucine or isoleucine, and most preferably with alanine. Substitution of Phe ³⁷⁶ with alanine (“F376A”) has been demonstrated to substantially reduce the cofactor activity of thrombomodulin analogs while retaining TAFI activation activity (see FIG. 7). As a result, the F376A-TM analog has an increased ratio of TAFI activation activity to cofactor activity. In one embodiment of the present invention, Solulin contains a Phe376X, in particular F376A substitution.

  In a further embodiment of the invention the thrombomodulin analog has a glutamine residue modification at position 387 (SEQ ID NO: 1 or SEQ ID NO: 3). The glutamine residues are preferably replaced with the following amino acids in order of increasing cofactor activity of the resulting mutant Gln387X-TM analog (see FIG. 8A): Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys. In one embodiment of the invention, Solulin contains this Gln387X substitution; in a further embodiment, this substitution within Solulin contains the F376X substitution together with a Gln387X substitution.

  In another embodiment of the invention, the thrombomodulin analog has a modification of the methionine residue at position 388 (SEQ ID NO: 1 or SEQ ID NO: 3). The methionine residues are preferably replaced with the following amino acids in order of increasing cofactor activity of the resulting mutant Met388X-TM analog (see FIG. 8B): Gln, Tyr, Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys. In one embodiment, Solulin contains one or both of the Phe376X and Gln387 substitutions along with this substitution.

  In a further embodiment of the invention the thrombomodulin analog has a phenylalanine residue modification at position 389 (SEQ ID NO: 1 or SEQ ID NO: 3). The phenylalanine residues are preferably substituted with the following amino acids in order of increasing cofactor activity of the resulting mutant Phe389X-TM analog (see FIG. 8C): Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu, Ile, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro. In one embodiment, Solulin contains this substitution and can further combine this substitution with one or more, preferably all, of the Phe376X, Gln387X or Met388X substitutions described above.

In another embodiment of the invention, the interdomain loop of TM, eg Solulin, consisting of the three amino acids Gln ³⁸⁷ , Met ³⁸⁸ and Phe ³⁸⁹ , is partially linked by one or more amino acids, preferably by alanine residues. Or one or more amino acids, preferably alanine residues, are inserted into the interdomain loop (see FIG. 8D).

For TM analogs with modifications at positions Phe ³⁷⁶ , Gln ³⁸⁷ , Met ³⁸⁸ or Phe ³⁸⁹ , the TM analog may be a full-length TM analog or comprises EGF domains EGF1 to EGF6, preferably May be a soluble TM analog comprising the EGF domains EGF3 to EGF6. In preferred embodiments, these analogs contain the substitutions described for the TM analog Solulin. In a further preferred embodiment, these Solulin-derived TM analogues consist exclusively of EGF1 to EGF6, in particular from EGF domains EGF3 to EGF6, or from the c-loop of EGF3 (these three fragments are referred to as Sollin fragments). ).

  In one embodiment of the invention, the thrombomodulin analog is used in oxidized form. Several techniques for the controlled oxidation of proteins are known to those skilled in the art. The TM analog is preferably oxidized using chloramine T, hydrogen peroxide or sodium periodate.

The invention further relates to methods that are useful for screening TM analogs for use in the treatment of coagulation disorders associated with hyperfibrinolysis. This method preferably comprises insertion, deletion or deletion of one or more amino acids in EGF domains EGF1 to EGF6, more preferably in EGF domains EGF3 to EGF6, and most preferably between amino acid positions Asp ³⁴⁹ and Asp ^461. A first step of altering the amino acid sequence of thrombomodulin by substitution. Several techniques for modifying protein sequences are known to those skilled in the art, such as by site-directed mutagenesis or random mutagenesis followed by selection.

In the second step, the modified TM analogs, binding affinity to thrombin _{(K D} value), cofactor activity, TAFI activation activity or TAFIa potential, the ratio of TAFI activation activity as cofactor activity, Protein One or more of the characteristics selected from the group consisting of the effect of oxidation, the effect on clot lysis over time in an in vitro assay, or the effect in a coagulation-related animal model is compared to a control protein.

  As a control protein, a thrombomodulin protein or analogue, preferably a rabbit lung thrombomodulin or human TM analogue comprising the six EGF domains is used. The TM analog may have a natural amino acid sequence, or alternatively may have one or more modifications such as an M388L substitution.

  The present invention further relates to a method of treating coagulopathy with hyperfibrinolysis, which comprises the administration of a therapeutically effective amount of a thrombomodulin analog that exhibits an antifibrinolytic effect.

In particular, this method of treatment, compared to control proteins containing TM analogs that exhibit one or more of the following characteristics: binding affinity decreased to thrombin, with 0.2nM larger k _D value Binding affinity to thrombin, significantly reduced cofactor activity, or increased ratio of TAFI activation activity to cofactor activity. As a control protein, a thrombomodulin protein or analogue, preferably a rabbit lung thrombomodulin or human TM analogue comprising the six EGF domains is used. The TM analog may have a natural amino acid sequence, or alternatively may have one or more modifications such as an M388L substitution.

In a further embodiment, the invention is a thrombomodulin analog having reduced cofactor activity when bound to thrombin compared to TM _E M338L, comprising:
a. B) the amino acid sequence set forth in SEQ ID NO: 4, or c) at least 90%, more preferably at least Amino acid sequence having 95%, most preferably at least 98% identity.
d) Six EGF-like repeat domains of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 EGF-like repeat domain 3 to EGF-like repeat domain 6 (amino acid positions 307 to 462 as numbered in SEQ ID NO: 1) or c-loop of EGF-like repeat domain 3 of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 Having a thrombomodulin fragment consisting essentially of repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1),
Phenylalanine at position 376 (when numbered according to SEQ ID NO: 1) is deleted or substituted by glycine, alanine, leucine, isoleucine or valine;
It relates to thrombomodulin analogues.

  In yet another embodiment of the invention, the thrombomodulin further comprises a deletion or substitution of a glutamine residue at position 387 (when numbered in SEQ ID NO: 1), wherein the substitution is preferably Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys, Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys.

  In a further embodiment, the present invention further comprises a deletion or substitution of a methionine residue at position 388 (when numbered in SEQ ID NO: 1), wherein the methionine residue is preferably Gln, Tyr, Ile, It relates to thrombomodulin substituted with Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys.

  In a further embodiment, the thrombomodulin further comprises a deletion or substitution of a phenylalanine residue at position 389 (when numbered in SEQ ID NO: 1), wherein the phenylalanine is preferably Val, Glu, Thr, Ala, Substituted with His, Trp, Asp, Gln, Leu, Ile, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro.

  In another embodiment, the thrombomodulin comprises a combination of first and second amino acid modifications as shown in Table 4.

  In yet another embodiment, the thrombomodulin comprises a combination of first, second and third amino acid modifications as shown in Table 5.

The present invention further relates to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1). As well as the following sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and one or more of the terminations at P490, whereby the thrombomodulin analog is linked to the amino acid sequence of the next amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1) Further comprising a sequence modification for one or more of:
a) ³⁵⁹ Gln;
b) ³⁹⁸ Asp;
c) ⁴⁰⁰ Asp;
d) ⁴⁰² Asn;
e) ⁴⁰⁸ Glu;
f) ⁴¹³ Tyr;
g) ⁴¹⁴ Ile;
h) ⁴¹⁵ Leu;
i) ⁴¹⁷ Asp;
j) ⁴³⁹ Asn.

  In a further preferred embodiment of the invention, the amino acids as listed in a) to j) are substituted with alanine.

The present invention further relates to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1). As well as the following sequence modifications:
a) removal of amino acids 1-3;
b) R456G;
c) H457Q;
d) S474A, and one or more of the terminations at P490, whereby the thrombomodulin analog is linked to the amino acid sequence of the mature thrombomodulin shown in SEQ ID NO: 1 Further comprising a sequence modification for one or more of:
a) with modifications other than changes to Leu, ³⁸⁸ Met,
b) ⁴¹⁶ Asp;
c) ⁴²³ Asp;
d) ⁴²⁵ Asp;
e) ⁴²⁶ Glu;
f) ⁴²⁹ Asp;
g) ⁴⁴⁰ Leu;
h) ⁴⁶¹ Asp.

  In one embodiment of the invention, the amino acids as listed in a) to h) are substituted with alanine.

The present invention further relates to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1). As well as the following sequence modifications:
a) removal of amino acids 1-3;
b) R456G;
c) H457Q;
d) S474A, and one or more of the terminations at P490, whereby this thrombomodulin analog comprises oxidation of the ³⁸⁸ Met residue (numbering is the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1) To be related).

The present invention relates preferably to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1) As well as the following modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) includes one or more of the terminations at S474A, and P490, whereby the analog comprises the group consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro It contains a mutation of amino acid ³⁸⁷ Gln, substituted or deleted by a more selected amino acid (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention further relates to a thrombomodulin analog and its medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (SEQ ID NO: 1). As well as the following modifications:
a) removal of amino acids 1-3;
b) R456G;
c) H457Q;
d) including one or more of the terminations at S474A and P490, whereby this analog is Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, A mutation of amino acid ³⁸⁸ Met, substituted or deleted by an amino acid selected from the group consisting of Gly, Glu, Asp or Cys (numbering is in the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1) Related).

The present invention relates preferably to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1) As well as the following sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) one or more of the terminations at S474A and P490, whereby the analog is by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro It contains a mutation of amino acid ³⁸⁹ Phe that is substituted or deleted (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention relates preferably to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1) As well as the following sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and one or more of the terminations at P490, whereby this analog comprises the following amino acid pairs: ³⁸⁶ Cys and ³⁸⁷ Gln, ³⁸⁷ Gln and ³⁸⁸ Leu, ³⁸⁸ Leu and ³⁸⁹ Phe, ³⁸⁹ It includes an insertion of a hydrophobic amino acid, preferably Ala, between one pair of Phe and ³⁹⁰ Cys (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention relates preferably to thrombomodulin analogs and their medical use for the treatment of coagulation disorders with hyperfibrinolysis, wherein the thrombomodulin analog is an amino acid corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1) As well as the following sequence modifications:
a) removal of amino acids 1-3;
b) M388L;
c) R456G;
d) H457Q;
e) S474A, and one or more of the terminations at P490, whereby this analog comprises a mutation of amino acid ³⁷⁶ Phe, preferably the Phe376Ala mutation (numbering is SEQ ID NO: 1 In relation to the amino acid sequence of mature thrombomodulin shown in FIG.

In a further preferred embodiment, the present invention provides the following amino acid positions (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) ³⁵⁹ Gln;
b) ³⁹⁸ Asp;
c) ⁴⁰⁰ Asp;
d) ⁴⁰² Asn;
e) ⁴⁰⁸ Glu;
f) ⁴¹³ Tyr;
g) ⁴¹⁴ Ile;
h) ⁴¹⁵ Leu,
i) ⁴¹⁷ Asp;
j) ⁴³⁹ Asn
Relates to Solulin as shown in SEQ ID NO: 2 comprising one or more, preferably exactly one of the sequence modifications.

  The above amino acids as listed under a) to j) are preferably substituted by alanine.

In one embodiment, Solulin as shown in SEQ ID NO: 2 is the following amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) with modifications other than changes to Leu, ³⁸⁸ Met,
b) ⁴¹⁶ Asp;
c) ⁴²³ Asp;
d) ⁴²⁵ Asp;
e) ⁴²⁶ Glu;
f) ⁴²⁹ Asp;
g) ⁴⁴⁰ Leu;
h) ⁴⁶¹ Asp
Including modifications in one or more (preferably one).

  In one aspect of the invention, amino acids as listed in a) to h) are substituted with alanine.

Solulin as used in the present invention is substituted or deleted by an amino acid selected from the group consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro. It may contain a mutation at amino acid ³⁸⁷ Gln (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In a further aspect of the invention, Solulin is by an amino acid selected from the group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys. It contains a mutation of amino acid ³⁸⁸ Met that is substituted or deleted (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The invention further relates to Solulin comprising a mutation of amino acid ³⁸⁹ Phe, which can be substituted or deleted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro ( The numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In addition, in one embodiment of the present invention, Solulin has the following amino acid pairs: ³⁸⁶ Cys and ³⁸⁷ Gln, ³⁸⁷ Gln and ³⁸⁸ Leu, ³⁸⁸ Leu and ³⁸⁹ Phe, ³⁸⁹ Phe and ³⁹⁰ Cys (numbering is SEQ ID NO: The insertion of a hydrophobic amino acid, preferably Ala, between one pair of mature thrombomodulin amino acids shown in FIG.

Solulin can be modified to include an oxidized ³⁸⁸ Met residue (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1). Furthermore, it may comprise a mutation of amino acid ³⁷⁶ Phe, preferably the Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In a preferred embodiment, the present invention is a thrombomodulin analog consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, Amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) ³⁵⁹ Gln;
b) ³⁹⁸ Asp;
c) ⁴⁰⁰ Asp;
d) ⁴⁰² Asn;
e) ⁴⁰⁸ Glu;
f) ⁴¹³ Tyr;
g) ⁴¹⁴ Ile;
h) ⁴¹⁵ Leu;
i) ⁴¹⁷ Asp;
j) ⁴³⁹ Asn
Relates to thrombomodulin analogues which may contain one or more, preferably one of the sequence modifications.

  In one aspect of the invention, amino acids as listed in a) to j) are substituted with alanine.

The TM analogs of the invention may also consist essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, although these fragments Each of the following amino acid positions (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) with modifications other than changes to Leu, ³⁸⁸ Met,
b) ⁴¹⁶ Asp;
c) ⁴²³ Asp;
d) ⁴²⁵ Asp;
e) ⁴²⁶ Glu;
f) ⁴²⁹ Asp;
g) ⁴⁴⁰ Leu;
h) ⁴⁶¹ Asp
May include sequence modifications in one or more (preferably one).

  Amino acids as listed in a) to h) are preferably substituted by alanine.

The present invention further relates to a TM analog consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, although each of these fragments Contains a mutation of amino acid ³⁸⁷ Gln that can be substituted or deleted by an amino acid selected from the group consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro (Numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention also relates to a TM analog consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, each of these fragments being , Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys can be substituted or deleted , Amino acid ³⁸⁸ Met may be included (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

Furthermore, the present invention relates to a TM analog consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, but each of these fragments May include a mutation of amino acid ³⁸⁹ Phe that may be substituted or deleted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro (numbering) Is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In addition, the present invention relates to TM analogs consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, although Each is associated with the following amino acid pairs: ³⁸⁶ Cys and ³⁸⁷ Gln, ³⁸⁷ Gln and ³⁸⁸ Met, ³⁸⁸ Met and ³⁸⁹ Phe, ³⁸⁹ Phe and ³⁹⁰ Cys (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1 ) Between one pair of)), preferably an insertion of a hydrophobic amino acid, preferably Ala.

Furthermore, the present invention relates to a TM analog consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, but each of these fragments May contain an oxidized ³⁸⁸ Met residue (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

Furthermore, the present invention relates to a TM analog consisting essentially of six EGF domains as given by residues 227 to 462 of SEQ ID NO: 1 or residues 224 to 459 of SEQ ID NO: 2, but each of these fragments May include a mutation of amino acid ³⁷⁶ Phe, preferably the Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In one embodiment of the invention, a TM analog consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2 is claimed. Each of these fragments has the following amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) ³⁵⁹ Gln;
b) ³⁹⁸ Asp;
c) ⁴⁰⁰ Asp;
d) ⁴⁰² Asn;
e) ⁴⁰⁸ Glu;
f) ⁴¹³ Tyr;
g) ⁴¹⁴ Ile;
h) ⁴¹⁵ Leu;
i) ⁴¹⁷ Asp;
j) ⁴³⁹ Asn
One or more, preferably one of the modifications.

  Amino acids as listed in a) to j) can be replaced by alanine.

In one embodiment of the invention, a TM analog consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2 is claimed. Each of these fragments has the following amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) with modifications other than changes to Leu, ³⁸⁸ Met,
b) ⁴¹⁶ Asp;
c) ⁴²³ Asp;
d) ⁴²⁵ Asp;
e) ⁴²⁶ Glu;
f) ⁴²⁹ Asp;
g) ⁴⁴⁰ Leu;
h) ⁴⁶¹ Asp
One or more, preferably one of the modifications.

  Amino acids as listed in a) to h) can be replaced by alanine.

The present invention further relates to thrombomodulin analogs consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2, although Each has a mutation of amino acid ³⁸⁷ Gln that can be replaced or deleted by an amino acid selected from the group consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro. (Numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention also relates to a thrombomodulin analog consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2, each of these fragments Can be substituted or deleted by an amino acid selected from the group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys The amino acid ³⁸⁸ Met mutation (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention further relates to thrombomodulin analogs consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2, although Each may include a mutation at amino acid ³⁸⁹ Phe, which may be substituted or deleted by an amino acid selected from the group consisting of Ser, Arg, Lys, Met, Tyr, Gly, Cys or Pro (No. The attachment is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In addition, the present invention relates to thrombomodulin analogs consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2, although these fragments Each of the following amino acid pairs: ³⁸⁶ Cys and ³⁸⁷ Gln, ³⁸⁷ Gln and ³⁸⁸ Met, ³⁸⁸ Met and ³⁸⁹ Phe, ³⁸⁹ Phe and ³⁹⁰ Cys (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1 The insertion of a hydrophobic amino acid, preferably Ala, between one pair of

In a further aspect of the invention, a thrombomodulin analog consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2, Each of the fragments may contain an oxidized ³⁸⁸ Met residue (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In a further aspect, the present invention relates to thrombomodulin analogs consisting essentially of EGF domains 3 to 6 as given by residues 311 to 462 of SEQ ID NO: 1 or residues 308 to 459 of SEQ ID NO: 2, Each of the fragments may contain a mutation of amino acid ³⁷⁶ Phe, preferably the Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In a preferred embodiment of the invention, a thrombomodulin consisting essentially of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Although asserting analogs, each of these fragments has the following amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) ³⁵⁹ Gln;
b) ³⁹⁸ Asp;
c) ⁴⁰⁰ Asp;
d) ⁴⁰² Asn;
e) ⁴⁰⁸ Glu;
f) ⁴¹³ Tyr;
g) ⁴¹⁴ Ile;
h) ⁴¹⁵ Leu;
i) ⁴¹⁷ Asp;
j) ⁴³⁹ Asn
One or more, preferably one of the modifications.

  Amino acids as listed in a) to j) can be replaced by alanine.

In one embodiment of the invention, consists essentially of the c-loop of EGF domain 3 and EGF domains 4 to 6, as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Although thrombomodulin analogs are claimed, each of these fragments has the following amino acid position (this numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1):
a) with modifications other than changes to Leu, ³⁸⁸ Met,
b) ⁴¹⁶ Asp;
c) ⁴²³ Asp;
d) ⁴²⁵ Asp;
e) ⁴²⁶ Glu;
f) ⁴²⁹ Asp;
g) ⁴⁴⁰ Leu;
h) ⁴⁶¹ Asp
One or more, preferably one of the modifications.

  Amino acids as listed in a) to h) can be preferably replaced by alanine.

The present invention further relates to a thrombomodulin analog consisting essentially of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Although related to the body, each of these fragments can be substituted or deleted by an amino acid selected from the group consisting of Lys, Gly, Ile, Trp, Tyr, Leu, Asn, Phe, Asp, Cys or Pro. , Amino acid ³⁸⁷ Gln mutation (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention also provides a thrombomodulin analog consisting essentially of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Although each of these fragments is by an amino acid selected from the group consisting of Ile, Phe, His, Arg, Pro, Val, Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp or Cys. It may contain a mutation of amino acid ³⁸⁸ Met, which may be substituted or deleted (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

Furthermore, the present invention relates to a thrombomodulin analog consisting essentially of the c-loop of EGF domain 3 and EGF domains 4 to 6, as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. It relates to the body, each of these fragments, Ser, Arg, Lys, Met , Tyr, Gly, may be replaced by an amino acid selected from the group consisting of Cys or Pro, or may be deleted, sudden amino ³⁸⁹ Phe It may contain mutations (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

In addition, the present invention relates to a thrombomodulin consisting essentially of a c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Analogue pairs of the following amino acid pairs: ³⁸⁶ Cys and ³⁸⁷ Gln, ³⁸⁷ Gln and ³⁸⁸ Met, ³⁸⁸ Met and ³⁸⁹ Phe, ³⁸⁹ Phe and ³⁹⁰ Cys (numbering is in the amino acid sequence of the mature thrombomodulin shown in SEQ ID NO: 1) Relates to a thrombomodulin analogue which comprises an insertion of a hydrophobic amino acid, preferably Ala, between one pair of

In a preferred embodiment of the invention, a thrombomodulin consisting essentially of the c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Although asserting analogs, each of these fragments may contain an oxidized ³⁸⁸ Met residue (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1).

The present invention preferably comprises a thrombomodulin consisting essentially of a c-loop of EGF domain 3 and EGF domains 4 to 6 as given by residues 333 to 462 of SEQ ID NO: 1 or residues 330 to 459 of SEQ ID NO: 2. Regarding analogs, each of these fragments may contain a mutation of amino acid ³⁷⁶ Phe, preferably the Phe376Ala mutation (numbering is related to the amino acid sequence of mature thrombomodulin shown in SEQ ID NO: 1) ).

  According to the present invention, the TM analog described in SEQ ID NO: 2 may contain two sequence modifications shown in Table 4 (“first amino acid modification” and “second amino acid modification”).

  In yet another embodiment of the invention, the TM analog set forth in SEQ ID NO: 4 contains the two sequence modifications shown in Table 4 (“first amino acid modification” and “second amino acid modification”). Sometimes.

  In yet another embodiment of the invention, the TM analog set forth in SEQ ID NO: 3 contains the two sequence modifications shown in Table 4 (“first amino acid modification” and “second amino acid modification”). Sometimes.

  6 EGF-like repeat domains of SEQ ID NO: 2 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domains 3 to EGF-like repeat domain 6 of SEQ ID NO: 2 (numbered in SEQ ID NO: 1) Consisting essentially of amino acid positions 307 to 462) or EGF-like repeat domain 3 of SEQ ID NO: 2 to EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1), In the case of yet another thrombomodulin fragment, each of these EGF domain-containing fragments may contain the two sequence modifications shown in Table 4 ("first amino acid modification" and "second amino acid modification"). is there.

  6 EGF-like repeat domains of SEQ ID NO: 3 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domains 3 to EGF-like repeat domain 6 of SEQ ID NO: 3 (numbered in SEQ ID NO: 1) Consisting essentially of amino acid positions 307 to 462) or EGF-like repeat domain 6 of SEQ ID NO: 3 to EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1), In the case of yet another thrombomodulin fragment, each of these EGF domain-containing fragments may contain the two sequence modifications shown in Table 4 ("first amino acid modification" and "second amino acid modification"). is there.

  6 EGF-like repeat domains of SEQ ID NO: 4 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domains 3 to EGF-like repeat domain 6 of SEQ ID NO: 4 (numbered in SEQ ID NO: 1) Consisting essentially of c-loop of EGF-like repeat domain 3 of SEQ ID NO: 4 to EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1). In the case of yet another thrombomodulin fragment, each of these EGF domain-containing fragments may contain the two sequence modifications shown in Table 4 ("first amino acid modification" and "second amino acid modification"). is there.

  According to the present invention, the TM analogs set forth in SEQ ID NO: 2 may each contain modifications as shown in Table 5 (“first, second and third amino acid modifications”). Table 5 shows the alanine substitution ("first amino acid modification") for Phe376. In certain embodiments of the invention, the “first amino acid modification” is constituted by glycine, valine, leucine or isoleucine. These modifications are each combined with a “second amino acid modification” and a “third amino acid modification” as given in Table 5. These further embodiments of the invention are collectively referred to as “modifications as shown in Table 5”.

  In yet another embodiment of the invention, the TM analog set forth in SEQ ID NO: 4 contains the two sequence modifications shown in Table 5 (“first amino acid modification” and “second amino acid modification”). Sometimes.

  In yet another embodiment of the invention, the TM analog set forth in SEQ ID NO: 3 contains the two sequence modifications shown in Table 5 (“first amino acid modification” and “second amino acid modification”). Sometimes.

  6 EGF-like repeat domains of SEQ ID NO: 2 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domains 3 to EGF-like repeat domain 6 of SEQ ID NO: 2 (numbered in SEQ ID NO: 1) Consisting essentially of amino acid positions 307 to 462) or EGF-like repeat domain 3 of SEQ ID NO: 2 to EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1), In the case of yet another thrombomodulin fragment, each of these EGF domain-containing fragments may contain the two sequence modifications shown in Table 5 ("first amino acid modification" and "second amino acid modification"). is there.

  6 EGF-like repeat domains of SEQ ID NO: 3 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domains 3 to EGF-like repeat domain 6 of SEQ ID NO: 3 (numbered in SEQ ID NO: 1) Consisting essentially of amino acid positions 307 to 462) or EGF-like repeat domain 6 of SEQ ID NO: 3 to EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1), In the case of yet another thrombomodulin fragment, each of these EGF domain-containing fragments may contain the two sequence modifications shown in Table 5 ("first amino acid modification" and "second amino acid modification"). is there.

  6 EGF-like repeat domains of SEQ ID NO: 4 (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domains 3 to EGF-like repeat domain 6 of SEQ ID NO: 4 (numbered in SEQ ID NO: 1) Consisting essentially of c-loop of EGF-like repeat domain 3 of SEQ ID NO: 4 to EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1). In the case of yet another thrombomodulin fragment, each of these EGF domain-containing fragments may contain the two sequence modifications shown in Table 5 ("first amino acid modification" and "second amino acid modification"). is there.

  The following table provides an overview of the reduction in cofactor activity that occurs as a result of certain amino acid modifications of thrombomodulin analogs.

Therapeutic treatment according to the invention In one aspect of the invention, a thrombomodulin analog as disclosed herein is used in the treatment of coagulation disorders with hyperfibrinolysis in patients with anti-factor VIII antibodies. These antibodies can inhibit factor VIII activity. In typical cases, they occur as allogeneic antibodies during replacement therapy in hemophilia A patients. They can cause failure of FVIII replacement therapy in hemophilia A patients. Thus, according to one aspect of the present invention, the thrombomodulin analogs disclosed herein can be used as an emergency drug in hemophilia patients who are non-responders to Factor VIII.

  In accordance with a further aspect of the invention, a patient who is deficient in factor VIII is treated with a combination of factor VIII and the TM analog of the invention. Factor VIII and the TM analog may be administered simultaneously or sequentially. Preferably, said patient is treated with recombinant factor VIII or a recombinant B domain deleted factor VIII molecule, more preferably with Octocog alpha or Moloccog alpha. In one embodiment, isolated human factor VIII, such as Aafact®, can be used.

  In accordance with the present invention, hemophilia patients who have previously been treated with Factor VIII or are currently receiving Factor VIII treatment can also be treated. Thus, these patients have therapeutically effective factor VIII levels.

  Since the presence of factor VIII causes a change in properties in the thromboelastogram, in a further aspect of the invention, the claimed thrombomodulin analog is used to identify hemophilia patients for the presence of factor VIII antibody. Can be screened (see FIG. 11A vs. FIG. 12A).

FIG. 1 shows the clot lysis profile and lysis time for factor VIII deficient plasma (FVIII-DP), normal plasma (NP) and FVIII-DP mixed with NP. The clot dissolution profiles were 0 (-), 1 (...), 6 (---), 10 (-------), 50 (------) and 100% ----) It shows about NP. From the clot dissolution profile, the dissolution time was determined using the time that the clot was reduced to one-half of its maximum optical density. The inset summarizes the dissolution time, but there is a general tendency for dissolution time to increase as the percentage of NP (and hence the amount of FVIII) increases. The effect of adding NP on dissolution time reaches a plateau at 10% NP. FIG. 2 shows thrombin-activated fibrinolysis inhibitor (TAFI) activation in plasma containing various percentages of FVIII. (A) When FVIII deficient plasma (FVIII-DP) is mixed with normal plasma (NP), TAFI activation is enhanced. In FVIII-DP, only 30 pM TAFIa was measured at its peak (•) compared to about 600 pM TAFIa in 50% NP (□) and 100% NP (Δ). These experiments were performed in triplicate and data represent the mean ± SE. The TAFIa potential (B), defined here as the area under the time course of the activation plot from the clot start time to the final time point (A), increases to a plateau at 50% NP as the percentage of NP increases. . The TAFIa potentials of 50% NP and 100% NP are similar (14,100 pM · min and 16,800 pM · min, respectively), despite the shape of their respective TAFI activation plots being completely different. Since the relationship between dissolution time (FIG. 1, inset) and TAFIa potential is related to FVIII level, it is presented using a plot of log dissolution time versus log TAFIa potential (FIG. 2B, inset). As expected, the data show a strong positive correlation between lysis time in plasma containing 0-100% FVIII and TAFIa potential. Warning 1: “■”, “〓”, “?”, “?” Exist. Note that “■”, “〓”, “?”, “?” May have been converted from external characters. FIG. 3 shows the activation of prothrombin in plasma containing various percentages of FVIII. The time course of prothrombin activation is shown for FVIIIDP mixed with 0 (●), 1 (■), 6 (▲), 10 (◯), 50 (□) and 100% NP (Δ). In general, the rate of prothrombin activation increases as the percentage of NP increases. At 50% NP, prothrombin activation occurs fast (determined by examining the slope of each plot) and appears to end within 15 minutes, whereas 100% NP has slower prothrombin activation over a longer period of time. Have speed. 4A-D shows sTM (0-100 nM) and tPA (0.25 nM in FIG. 4A, 0.75 nM in FIG. 4B, 0.75 nM in FIG. 4C) in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP). FIG. 5 shows the effect of sTM on thrombin activated fibrinolysis inhibitor (TAFI) activation at various concentrations, both 1.5 nM and 3 nM in FIG. 4D. The TAFIa-dependent defect with extended lysis in FVIII-DP is corrected by the addition of 100 nM sTM to plasma containing 0.25 nM tPA. In FVIII-DP, only partial correction of the lysis defect is observed in the presence of 100 nM sTM, even with increasing tPA concentration. In these experiments, potato tuber carboxypeptidase inhibitor (PTCI) was used to create the absence of functional TAFIa. Thus, any increase in dissolution as indicated by the dissolution time / dissolution time + PTCI ratio is TAFIa dependent. 4A-D shows sTM (0-100 nM) and tPA (0.25 nM in FIG. 4A, 0.75 nM in FIG. 4B, 0.75 nM in FIG. 4C) in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP). FIG. 5 shows the effect of sTM on thrombin activated fibrinolysis inhibitor (TAFI) activation at various concentrations, both 1.5 nM and 3 nM in FIG. 4D. The TAFIa-dependent defect with extended lysis in FVIII-DP is corrected by the addition of 100 nM sTM to plasma containing 0.25 nM tPA. In FVIII-DP, only partial correction of the lysis defect is observed in the presence of 100 nM sTM, even with increasing tPA concentration. In these experiments, potato tuber carboxypeptidase inhibitor (PTCI) was used to create the absence of functional TAFIa. Thus, any increase in dissolution as indicated by the dissolution time / dissolution time + PTCI ratio is TAFIa dependent. 4A-D shows sTM (0-100 nM) and tPA (0.25 nM in FIG. 4A, 0.75 nM in FIG. 4B, 0.75 nM in FIG. 4C) in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP). FIG. 5 shows the effect of sTM on thrombin activated fibrinolysis inhibitor (TAFI) activation at various concentrations, both 1.5 nM and 3 nM in FIG. 4D. The TAFIa-dependent defect with extended lysis in FVIII-DP is corrected by the addition of 100 nM sTM to plasma containing 0.25 nM tPA. In FVIII-DP, only partial correction of the lysis defect is observed in the presence of 100 nM sTM, even with increasing tPA concentration. In these experiments, potato tuber carboxypeptidase inhibitor (PTCI) was used to create the absence of functional TAFIa. Thus, any increase in dissolution as indicated by the dissolution time / dissolution time + PTCI ratio is TAFIa dependent. 4A-D shows sTM (0-100 nM) and tPA (0.25 nM in FIG. 4A, 0.75 nM in FIG. 4B, 0.75 nM in FIG. 4C) in normal plasma (NP) and factor VIII deficient plasma (FVIII-DP). FIG. 5 shows the effect of sTM on thrombin activated fibrinolysis inhibitor (TAFI) activation at various concentrations, both 1.5 nM and 3 nM in FIG. 4D. The TAFIa-dependent defect with extended lysis in FVIII-DP is corrected by the addition of 100 nM sTM to plasma containing 0.25 nM tPA. In FVIII-DP, only partial correction of the lysis defect is observed in the presence of 100 nM sTM, even with increasing tPA concentration. In these experiments, potato tuber carboxypeptidase inhibitor (PTCI) was used to create the absence of functional TAFIa. Thus, any increase in dissolution as indicated by the dissolution time / dissolution time + PTCI ratio is TAFIa dependent. FIG. 5 shows TAFI activity in normal plasma (NP) (A) and in FVIII-deficient plasma (FVIII-DP) (B) in the presence of 10 nM thrombomodulin (●) or without thrombomodulin (◯). FIG. 3 shows the crystallization and clot dissolution profiles. The accompanying clot dissolution profile is shown (-) and the clot dissolution profile for sTM free is shown (---). These experiments were performed in triplicate and data represent the mean ± SE. FIG. 6 shows thrombin binding to thrombomodulin. Titrate 1.5 mL of a solution consisting of thrombin (20 nM) and DAPA (20 nM) in 20 mM Tris.HCl, 150 mM NaCl, 5.0 mM Ca ²⁺ , 0.01% Tween 80 with 1.54 μM thrombomodulin in the same solution. The binding of thrombin to thrombomodulin was determined. The fluorescence intensity was measured (λ _ex = 280 nm, λ _em = 545 nm). FIG. 7 shows the relative cofactor activity of point mutants in TAFI and protein C activation. Alanine scanning mutagenesis was used to prepare point mutants of soluble thrombomodulin. For TAFI (black bars) and protein C (shaded bars) the rate of protein C and TAFI activation (relative to the activation rate with mutant TM _E M388L) is shown. FIG. 8 shows the mutation of the interdomain loop between EGF4 and EGF5. Three independent plasmids were constructed for each mutant in E. coli. Shots were prepared and assayed for cofactor activity by APC assay, and samples were analyzed by Western blot (not shown). Activity values are average values from 3 separate clones. Panel A, substitution mutant at Gln ³⁸⁷ ; Panel B, substitution at Met386; Panel C, substitution mutant at Phe389; Panel D, deletion and alanine insertion in the interdomain loop. Shown is the activity measured for a squirt from E. coli transfected with a control plasmid lacking the TM insert, pSelect. See Clarke et al. (J. Biol. Chem. 1993; 268: 6309-6315) for further details. FIG. 8 shows the mutation of the interdomain loop between EGF4 and EGF5. Three independent plasmids were constructed for each mutant in E. coli. Shots were prepared and assayed for cofactor activity by APC assay, and samples were analyzed by Western blot (not shown). Activity values are average values from 3 separate clones. Panel A, substitution mutant at Gln ³⁸⁷ ; Panel B, substitution at Met386; Panel C, substitution mutant at Phe389; Panel D, deletion and alanine insertion in the interdomain loop. Shown is the activity measured for a squirt from E. coli transfected with a control plasmid lacking the TM insert, pSelect. See Clarke et al. (J. Biol. Chem. 1993; 268: 6309-6315) for further details. FIG. 8 shows the mutation of the interdomain loop between EGF4 and EGF5. Three independent plasmids were constructed for each mutant in E. coli. Shots were prepared and assayed for cofactor activity by APC assay, and samples were analyzed by Western blot (not shown). Activity values are average values from 3 separate clones. Panel A, substitution mutant at Gln ³⁸⁷ ; Panel B, substitution at Met386; Panel C, substitution mutant at Phe389; Panel D, deletion and alanine insertion in the interdomain loop. Shown is the activity measured for a squirt from E. coli transfected with a control plasmid lacking the TM insert, pSelect. See Clarke et al. (J. Biol. Chem. 1993; 268: 6309-6315) for further details. FIG. 8 shows the mutation of the interdomain loop between EGF4 and EGF5. Three independent plasmids were constructed for each mutant in E. coli. Shots were prepared and assayed for cofactor activity by APC assay, and samples were analyzed by Western blot (not shown). Activity values are average values from 3 separate clones. Panel A, substitution mutant at Gln ³⁸⁷ ; Panel B, substitution at Met386; Panel C, substitution mutant at Phe389; Panel D, deletion and alanine insertion in the interdomain loop. Shown is the activity measured for a squirt from E. coli transfected with a control plasmid lacking the TM insert, pSelect. See Clarke et al. (J. Biol. Chem. 1993; 268: 6309-6315) for further details. FIG. 9 is a schematic representation of the fibrinolysis-promoting and anti-fibrinolytic effects of thrombomodulin (with some modifications of Mosnier and Bouma, Arterioscler. Thomb. Vasc. Biol. 2006; 26: 2445-2453). The increase in clot lysis time at low TM concentrations is attributed to stimulation of TAFI activation and illustrates the antifibrinolytic activity of TM. At higher rabbit lung TM concentrations, clot lysis time decreases due to protein C activation and inhibition of TFAI activation; this illustrates the fibrinolytic activity of rabbit lung TM (bold line). Note that above 15 nM, the fibrinolysis promoting activity of rabbit lung TM exceeds the antifibrinolytic activity, resulting in a comprehensive fibrinolysis promoting effect. In contrast, soluble TM analogs show only an antifibrinolytic effect (intermittent line). FIG. 10 is a diagram showing the relationship between TAFIa-dependent propagation fold of clot dissolution and addition of Solulin. The dose-dependent increase in clot dissolution time is due to stimulation of TAFI activation and illustrates the antifibrinolytic activity of TM. FIG. 11A shows a Solulin-dependent change in clot appearance (measured by thromboelastography) in whole blood from canines with hemophilia A. FIG. FIG. 11B shows a Solulin-dependent increase in clot hardness in whole blood from canines with hemophilia A. The area under the clot dissolution curve was measured by thromboelastography as a function of Solulin (sTM) concentration. FIG. 11C shows a Solulin-dependent dependent extension of lysis in whole blood from canines with hemophilia A. Clot dissolution time measured as a function of Solulin (sTM) concentration by thromboelastography. FIG. 12A shows Solulin-dependent prolongation of lysis (measured by thromboelastography) in whole blood from canines with hemophilia A and inhibitory anti-factor VIII antibodies. FIG. 12B shows a Solulin-dependent increase in clot hardness in whole blood from canines with hemophilia A in the presence of inhibitory anti-factor VIII antibodies. The area under the clot dissolution curve was measured by thromboelastography as a function of Solulin (sTM) concentration. FIG. 13 is a table showing a list of data used to generate FIG. 4, including the absolute dissolution time in the presence of PTCI to allow determination of the dissolution time under each condition. It is. In all cases, the dissolution times for those obtained in the presence of the TAFIa inhibitor, PTCI, are displayed. TAFI, thrombin activated fibrinolysis inhibitor; PTCI, potato tuber carboxypeptidase inhibitor. Figure 14 shows Table 2: Chloramine T oxidation of site-specific mutant analogs of TM _E (Sf9). The results after chloramine T treatment were expressed as percentage of activity after control treatment. ^* Average value of duplicate determination and deviation from the average. FIG. 15 shows the activation rates of TAFI (upper panel) and protein C (lower panel) with elevated concentrations of Solulin and Solulin mutants, Phe376Ala (both present at 25 nM). FIG. FIG. 16 shows Solulin compared to Solulin analogs with Phe376Ala mutant (light gray bars) as determined at TM concentrations of 0.25, 0.5, 0.75, 1, 1.5 and 2 μM. FIG. 5 shows the ratio of TAFI activation to protein C activation for (black bars). The rightmost bar shows the combined ratio from the measurements at all concentrations. FIG. 17 shows a thromboelastography of a blood sample from a dog with severe hemophilia A. Samples were taken at different time points after intravenous injection of Solulin at a dose of 500 μg / kg. FIG. 18 shows the bell-shaped concentration dependence of improved clot formation and stability in blood from severe hemophilia dogs. Solulin was injected at a dose of 10 μg / kg and plasma samples were collected at the indicated times for determination of thromboelastogram. Each thromboelastogram shows sample collection time and Solulin plasma concentration. FIG. 19 shows an overview of TAFI specific thrombomodulin analogs.

Embodiments of the Invention In one aspect of the invention, the TM analog claims the use of a thrombomodulin analog for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis, wherein the TM analog is a therapeutically effective dosage. It is characterized by presenting an antifibrinolytic effect in an amount.

In a further aspect of the invention, the use of a thrombomodulin analog for the manufacture of a medicament for the treatment of a coagulation disorder with hyperfibrinolysis is claimed, said thrombomodulin analog being one or more of the following features: Present:
(I) is reduced as compared to the rabbit lung thrombomodulin, binding affinity to thrombin, and / or binding affinity to thrombin at 0.2nM larger k _D values, and / or (ii) TM analog Reduced cofactor activity compared to TMEM388L cofactor activity,
(Iii) Increased ratio of TAFI activation activity to cofactor activity compared to TM analog TM _E M388L.

  In a further aspect of the present invention, the use of the thrombomodulin analog described above is claimed, but the coagulation disorder with hyperfibrinolysis is selected from the group of diseases as follows: hemophilia A, hemophilia Disease B, hemophilia C, von Willebrand disease (vWD), acquired von Willebrand disease, factor X deficiency, parahaemophilia, hereditary disorder of factor I, II, V or VII clotting factors Bleeding disorders due to circulating anticoagulants or acquired coagulopathy.

  In a further aspect of the invention, said thrombomodulin analog is selected from the group consisting of intracranial or other CNS bleeding, bleeding in joints, microcapillaries, muscles, gastrointestinal tract, respiratory tract, retroperitoneal cavity or soft tissue It can be used to treat one or more of the bleeding events. The intracranial hemorrhage event treated with a thrombomodulin analog of the invention can be intracerebral, extracerebral or subarachnoid hemorrhage (SAH) or epidural or subdural hematoma. Preferably, patients with SAH are treated, more preferably aneurysm bleeding after SAH is treated.

  In a preferred embodiment of the invention, the thrombomodulin analogues according to the invention are used to treat physical trauma, preferably CNS trauma, subsequent hyperfibrinolysis. Physical trauma as defined herein refers to a physical wound or shock caused by sudden physical injury, such as from violence or an accident. Physical trauma includes multiple trauma, head injury, chest trauma, abdominal trauma, extremity trauma, facial trauma, urogenital trauma, pelvic trauma and soft tissue injury.

  In a further aspect of the invention, a thrombomodulin analog for use in the treatment of a coagulation disorder associated with hyperfibrinolysis is provided in combination with an additional fibrinolysis inhibitor. Specifically, substances that modify standard platelet adhesion, such as Etamylate, can be used. Preferably, an inhibitor of proteolytic enzymes can be used, which is more preferably an inhibitor of plasmin such as aprotinin. In an even more preferred embodiment of the invention said further antifibrinolytic agent, eg epsilon-aminocaproic acid or tranexamic acid, blocks the lysine binding site of plasmin.

  In a preferred embodiment of the invention, the patient treated with said thrombomodulin analog has an anti-factor VIII antibody.

  In another aspect of the invention, a patient treated with said thrombomodulin analog is treated with a factor VIII, preferably a recombinant factor VIII or a recombinant B domain deleted factor VIII molecule, more preferably octocog alpha or Further treatment with Moloctocog Alpha.

  In a preferred embodiment of the invention, the thrombomodulin analogs set forth in SEQ ID NOs: 5 to 11 are given at the doses or dose ranges set forth above.

  In a further aspect of the invention, the thrombomodulin analog used for the treatment of coagulopathy with hyperfibrinolysis is a factor VIII, preferably a recombinant factor VIII or a recombinant B domain deleted factor VIII molecule, It is preferably given in combination with Octocog alpha or Moloccog alpha.

  In one embodiment of the invention, the thrombomodulin analog is administered during a bleeding event of a coagulopathy with hyperfibrinolysis or prior to an increased risk of bleeding (eg, surgery or extraction).

  In a further aspect of the invention, the thrombomodulin analog is administered to a patient who is refractory to blood transfusion / plasma transfusion or coagulation factor replacement therapy.

  In one aspect of the invention, the thrombomodulin analog is administered in multiple doses, preferably once a day, once every two days, or three days, four days over a total period of less than one week to four weeks. It is administered as a long-term administration every 5 days, 6 days or 7 days.

  In a preferred embodiment of the invention, the thrombomodulin analogs set forth in SEQ ID NOs: 5 to 11 are administered according to the dosing regime described above.

  In a preferred embodiment of the invention, the thrombomodulin is preferably given as a parenteral application and given as an intravenous or subcutaneous application.

  In one aspect of the invention, the thrombomodulin analog is an amount that produces a plasma concentration of less than 5 nM / L, preferably less than 3 nM / L, and more preferably less than 1.5 nM / L in the subject to be treated. Give in.

  In a further aspect of the invention, the thrombomodulin analog is dosed such that the plasma concentration is between 0.1 nM / L and 5 nM / L, preferably between 0.1 nM / L and 3 nM / L.

  In a preferred embodiment of the invention, the thrombomodulin analogs set forth in SEQ ID NOs: 5 to 11 are used to produce the plasma concentrations disclosed above.

  In another aspect of the invention, said thrombomodulin analog is administered to a subject to be treated at a dose between 0.1 μg / kg and 140 μg / kg, preferably between 0.5 μg / kg and 40 μg / kg. Given at a dose, more preferably at a dose between 0.5 μg / kg and 4 μg / kg, and in particular at a dose between 0.75 μg / kg and μg / kg (kg is kg of the body weight of the subject to be treated) ).

  In a further aspect of the invention, said thrombomodulin analog is 0.75, 1.4, 2.5 or 4.0 μg / kg equal to 0.6, 1, 3 or 4.0 mg / patient body weight adjusted dose. Given at a dose of

  In a further preferred embodiment of the invention, the thrombomodulin analogues according to SEQ ID NOs: 5 to 11 are given in the doses or dose ranges described above.

  In a further preferred embodiment of the invention, said thrombomodulin analogue is a soluble TM analogue.

  In an even more preferred embodiment of the invention, said thrombomodulin analog is a human soluble TM analog.

  In one embodiment of the invention, said thrombomodulin analogue is selected from the group comprising EGF3, EGF4, EGF5, EGF6, preferably comprising fragments EGF3 to EGF6, and more preferably comprising EGF domains 1 to 6. At least one structural domain.

  In a further aspect of the invention, the thrombomodulin analog consists of the EGF domains EGF1 to EGF6, and more preferably consists of the EGF domains EGF3 to EGF6.

In one embodiment of the invention, the thrombomodulin analog has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1 or SEQ ID NO: 3) and includes one or more of the following modifications: :
a) Removal of amino acids 1 to 3 b) M388L
c) R456G
d) H457Q
e) Termination at S474A and P490.

  In a further aspect of the invention, the thrombomodulin analog has an amino acid sequence comprising at least 85% or at least 90% or 95% sequence identity with SEQ ID NO: 2.

In a preferred embodiment of the invention, said thrombomodulin analogue is of the native sequence (described in SEQ ID NO: 1 or SEQ ID NO: 3):
ab) ³⁵⁵ Asn;
ae) ³⁵⁹ Gln;
af) ³⁶¹ Gln;
ag) ³⁶³ Leu;
ah) ³⁶⁴ Asn;
ai) ³⁶⁸ Tyr;
aj) ³⁷¹ Val;
ak) ³⁷⁴ Glu;
al) ³⁷⁶ Phe;
am) ³⁸⁴ His;
an) ³⁸⁵ Arg;
ba) ³⁸⁷ Gln;
bb) ³⁸⁹ Phe;
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bf) ⁴⁰³ Thr;
bg) ⁴⁰⁸ Glu;
bh) ⁴¹¹ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
bn) ⁴²⁰ Ile;
bo) ⁴²³ Asp;
bp) ⁴²⁴ Ile;
bq) ⁴²⁵ Asp;
br) ⁴²⁶ Glu;
ca) ⁴²⁸ Glu;
cb) ⁴²⁹ Asp;
cc) ⁴³² Phe;
cd) ⁴³⁴ Ser;
ce) ⁴³⁶ Val;
cf) ⁴³⁸ His;
cg) ⁴³⁹ Asp;
ch) ⁴⁴⁰ Leu;
ci) ⁴⁴³ Thr;
cj) ⁴⁴⁴ Phe;
ck) ⁴⁴⁵ Glu;
cl) ⁴⁵⁶ Arg;
cm) ⁴⁵⁸ Ile; or cn) ⁴⁶¹ Asp
With amino acid modifications at one or more positions corresponding to

  In a further aspect of the invention, the thrombomodulin analog is preferably substituted with an aliphatic amino acid at position 376 set forth in SEQ ID NO: 1 or SEQ ID NO: 3, more preferably with glycine, alanine, valine, leucine or isoleucine. It has a phenylalanine modification that is substituted and most preferably substituted with alanine.

In a further aspect of the invention, the thrombomodulin analog is the following amino acid set forth in SEQ ID NO: 1 or SEQ ID NO: 3:
a) ³⁸⁷ Gln;
b) ³⁸⁸ Met;
b) ³⁸⁹ Phe
Wherein one or more additional amino acids are inserted, or preferably the amino acids are replaced.

  In a further aspect of the invention, said thrombomodulin analogue is used in its oxidized form, preferably oxidized with chloramine T, hydrogen peroxide or sodium periodate.

  In a further aspect of the invention, a thrombomodulin analog is used, but one or more of the methionine residues in the TM analog is oxidized, preferably at a position (described in SEQ ID NO: 1 or SEQ ID NO: 3). 388 methionine residues are oxidized.

Another aspect of the invention claims a method of screening for analogs of thrombomodulin suitable for the treatment of coagulopathy with hyperfibrinolysis, wherein the thrombomodulin has the following characteristics:
(I) reduced binding affinity to thrombin,
(Ii) reduced cofactor activity,
(Iii) exhibit one or more of the increased TAFI activation activities, the method comprising:
a) making one or more amino acid substitutions of the thrombomodulin sequence (SEQ ID NO: 1 or SEQ ID NO: 3), preferably at the amino acid position according to claim 15;
b) The modified analog and a control molecule, preferably rabbit lung TM or soluble human TM analog, with the following properties:
ba) Binding affinity to thrombin (KD value);
bb) cofactor activity;
bc) TAFI activation activity or TAFIa potential;
bd) ratio of TAFI activating activity to cofactor activity;
be) the effect of protein oxidation;
bf) Effect on clot lysis over time in an in vitro assay;
bg) comparing for one or more of the effects in the coagulation-related animal model.

  In another aspect of the invention, a method for treating coagulopathy with hyperfibrinolysis is claimed, which method comprises administering a therapeutically effective amount of a thrombomodulin analog according to any of claims 1-20. including.

  In another aspect of the invention, at position 376 (numbering as set forth in SEQ ID NO: 1), preferably substituted with an aliphatic amino acid, more preferably substituted with glycine, alanine, valine, leucine or isoleucine. And most preferably claims a thrombomodulin analog according to SEQ ID NO: 2 having a modification of phenylalanine, which is substituted with alanine.

  In a preferred embodiment of the present invention, thrombomodulin analogs as shown in FIG. 19 and shown by the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 11 are used.

These preferred TM analogs are:
1. TM fragment spanning aa4 to aa490 with the following amino acid exchanges: Phe376Ala, Met388Leu, Arg456Gly, His457Gln and Ser474Ala (equal to SEQ ID NO: 5).
2. TM fragment spanning aa227 to aa462 (= EGF 1-6) with the following amino acid exchanges: Phe376Ala, Met388Leu, Arg456Gly and His457Gln (equal to SEQ ID NO: 6).
3. TM fragment spanning aa333 to aa462 with the following amino acid exchange: Phe376Ala, Met388Leu, Arg456Gly and His457Gln (equal to SEQ ID NO: 7).
4). TM fragment spanning aa227 to aa462 (equal to SEQ ID NO: 8) with the following amino acid exchange: Phe376Ala and Met388Leu.
5. TM fragment spanning aa333 to aa462 with the following amino acid exchange: Met388Ala, Arg456Gly and His457Gln (equal to SEQ ID NO: 9).
6). TM fragment spanning aa333 to aa462 (equal to SEQ ID NO: 10) with the following amino acid exchange: Met388Leu, Arg456Gly, His457Gln and Glu461Ala.
7). TM fragment spanning aa333 to aa462 (equal to SEQ ID NO: 11) with the following amino acid exchanges: Phe376Ala, Met388Ala, Arg456Gly and His457Gln.

Definitions As used in the context of the present invention, the term “antifibrinolytic effect” refers to thrombomodulin that prolongs clot lysis time (as described in Example I) compared to the same assay conditions without the addition of thrombomodulin analogs. Refers to the ability of an analog. The antifibrinolytic effect is due to the superior antifibrinolytic activity of the TM analog compared to its fibrinolytic promoting activity.

  As used herein, the term “fibrinolysis-promoting effect” refers to a significant increase in clot lysis time in an in vitro assay (as described in Example I) compared to the same assay conditions without the addition of a thrombomodulin analog. It shall refer to the ability of the thrombomodulin analog to reduce.

  As used herein, the terms “significantly reduce” and “significantly extend” refer to an increase or decrease in clot lysis time that is significantly different from the baseline value at the p = 0.1 level, and / Or refers to an extension or reduction of more than 10%, preferably 20%, more preferably 30% and most preferably more than 40%, 50%, 60%, 70%, 80%, 100%, 150% or 200%.

  As used in the context of the present invention, the terms “treat”, “treating” or “treatment” use the TM analogs of the present invention or any composition comprising them to prevent bleeding events. Refers to preventing or reducing, improving or stopping bleeding events. These terms encompass not only relief, remission or prevention, but also healing or repair, unless explicitly stated otherwise. Also, as used herein, the term “patient” refers to mammals including humans.

  As used herein, the term “coagulopathy with hyperfibrinolysis” refers to coagulopathy as a disease that affects blood coagulability, where a significant increase in fibrinolysis causes, exacerbates or prolongs the bleeding event. Shall be pointed to.

  As used in the context of the present invention, the term “thrombomodulin analog” refers to both proteins and peptides that have the same characteristic biological activity as membrane-bound or soluble thrombomodulin. Biological activity is the ability to act as a receptor for thrombin and increase activation of TAFI, or other biological activity associated with natural thrombomodulin.

The term "binding affinity" as used herein, refers to an affinity strength between the thrombomodulin analog with thrombin, as described by the association constant K _{D. K D} values for the binding affinity between thrombin and thrombomodulin, for example, by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE (TM) analysis) Can be determined. Preferably, binding affinity is analyzed using a kinetic assay as described in Example II of the present invention.

“K _D ” refers to the relative binding affinity between the TM analog and thrombin. Higher _KD values represent lower binding affinity. The exact assay and means for determining the K _D to provide in practice Example II.

  As used herein, the term “cofactor activity” refers to the ability of a thrombomodulin analog to form a complex with thrombin to enhance the ability of thrombin to activate protein C. The assay procedure used to measure cofactor activity is given in Example III of the present invention.

  As used herein, the term “TAFI activation activity” refers to the ability of a thrombomodulin analog to form a complex with thrombin to enhance the ability of thrombin to activate TAFI. The assay procedure used to measure TAFI activity is given in Example IV of the present invention.

“Km” refers to the Michaelis constant and is derived by standard techniques by measuring the rate of catalysts measured at different substrate concentrations. Km is equal to the substrate concentration when the reaction rate is half of its maximum value. "Km" for TM analogs of the present invention, the thrombin concentration constant level (e.g., 1 nM) to hold the and saturation level, depending on its _{K D} (e.g., 100 nM or higher) using a TM of Decide by. Reactions are performed using increasing concentrations of protein C (eg, 1-60 μM). Km and kcat are then determined using line weaver bark plotting or non-linear regression analysis.

“TM _E ” refers to an analog of TM consisting of 6 EFG repeats (amino acids 227 to 462 set forth in SEQ ID NO: 1 or SEQ ID NO: 3).

“TM _E M388L” refers to an analog of TM consisting of 6 EGF repeats (aa 277 to 462) with a substitution of the natural methionine at position 388 by a leucine residue (based on SEQ ID NO: 3).

  The term “therapeutically effective amount” is defined as the amount of active ingredient that will reduce symptoms associated with coagulopathy with increased fibrinolysis, such as bleeding events. “Therapeutically effective” also refers to any improvement in disability severity, frequency of occurrence or duration compared to no treatment.

  As used in the context of the present invention, the term “sequence modification” relates to modification of the primary amino acid sequence, in particular by amino acid substitution, deletion or insertion. If not otherwise clearly defined, the term means the replacement of an amino acid with another amino acid, said other amino acid having its polar, hydrophilic or hydrophobic properties, acidic or basic properties, Each differs substantially from the first amino acid in size or aromaticity.

  Considering the general class of amino acids, namely acidic, basic, polar, nonpolar, negatively charged, positively charged, aromatic and aliphatic amino acids, the concept in the present case of sequence modification is: Preferably, the substitution of one amino acid with another amino acid of a different amino acid class is sought. In a preferred embodiment, the amino acid is selected as a substituent having the “opposite characteristics” of the amino acid to be substituted. The following amino acid (aa) substitutions are specifically proposed: acidic aa vs basic aa, polar aa vs nonpolar aa, negative aa vs positive aa, aromatic aa vs aliphatic aa.

  A special embodiment of the sequence modification of the present invention is the substitution of amino acids with aliphatic amino acids (eg glycine, alanine, valine, leucine and isoleucine), with substitution with alanine being most preferred.

  Sequence modifications in the case of the present invention may also include substitution with unnatural amino acids. “Unnatural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. The term “unnatural amino acid” is naturally occurring by modification of naturally encoded amino acids, including but not limited to the 20 common amino acids or pyrrolysine and selenocysteine, but as such is a translation complex. Includes but is not limited to amino acids that are not incorporated into the growing polypeptide chain. Examples of naturally occurring amino acids that are not naturally encoded include N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine, ortinin, taurine. It is not limited to. Such sequence modifications also include linking other groups to amino acids. These groups include acetates, phosphates, various lipids and carbohydrates that preferably alter the amino acid chemistry. Said sequence modification further comprises oxidation or reduction of the respective amino acid, preferably the Met or Cys residues.

Example I
Clot lysis assay in human plasma An in vitro clot lysis model was used to test the ability of soluble thrombomodulin (Solulin) to reduce or increase clot lysis time in a mixture of normal and factor VIII deficient plasma.

1. Test System Clotting was initiated in vitro by mixing thrombin (factor IIa), calcium chloride and phosphatidylcholine / phosphatidylserine (PCPS) vesicles in the plasma composition. The time course of clotting and fibrinolysis was determined with a turbidity assay, and a “TAFIa potential” was determined using a functional assay.

2. Experimental procedure Materials. Thrombin and fibrinogen except that for the preparation of fibrinogen, the solution was made water containing 1.2% PEG-8000 instead of 2% PEG-8000 by addition of 40% (w / v) PEG-8000 after β-alanine precipitation. And prepared as described in Walker et al. (J. Biol. Chem. 1999; 274: 5201-5212). This change in protocol allowed a greater yield of fibrinogen. QSY-FDP (Fibrin degradation product covalently linked to quencher, QSY9 C5-maleimide) and TAFIa standards used in the TAFIa assay have been described (Kim et al., 2008; Anal. Biochem 372: 32). -40; Neill et al., 2004; Anal. Biochem. 330: 332-341), recombinant human Pg (S741C) and fluorescein derivative (5IAF-Pg) were prepared as described in Horrevoets et al. (J. Biol. Chem 1997; 272: 2176-2182). S525C-prothrombin was purified and fluorescently labeled with 5-iodoamidofluorescein (5IAF) as previously described by Brufato et al. (J. Biol. Chem. 2001; 276: 17663-17671). QSY9 C5-maleimide and 5-iodoamide fluorescein are available from Invtrogen Canada Inc. (Burlington, Ontario, Canada). Plasmin is available from Haematologic Technologies Inc. (Essex Junction, Vermont, USA) and recombinant human soluble thrombomodulin (Solulin; sTM) was provided by Paion Deutschland GmbH (Aachen, Germany). Normal human pooled plasma (NP) was obtained from healthy donors at a blood bank at Kingston General Hospital (KGH), Kingston, Ontario, Canada, and FVIII deficient plasma (FVIII-DP) was obtained from Affinity Biologicals, Inc. (Hamilton, Ontario, Canada). TAFI deficient plasma (TDP) is obtained by affinity chromatography of normal plasma on a column of immobilized anti-human TAFI monoclonal antibody as described by Schneider et al. (J. Biol. Chem. 2002; 277: 1021-1030). Prepared. The plasmin inhibitor D-Val-Phe-Lys chloromethyl ketone (VFKck), thrombin inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPAck) and potato tuber carboxypeptidase inhibitor (PTCI) were obtained from Calbiochem (California, USA). From San Diego). Tissue-type plasminogen activator (Activase; tPA) was purchased from a pharmacy in KGH (Kingston, Ontario, Canada). All other reagents were of analytical quality.

  3. Method.

  Clot lysis assay and sample preparation to determine the extent of TAFI activation.

FVIII-DP and NP were mixed so that the final percentage of NP was 0, 1, 6, 10, 50 or 100% (0-100% NP). Prior to mixing, each plasma was diluted to an optical density of 32 and added to an equal volume of solution containing 1.5 nM tPA, 40 μM PCPS and 20 mM CaCl _{2 in} the presence or absence of 20 nM thrombin (final concentration: 0. 75 nM tPA, 20 μM PCPS, 10 mM CaCl ₂ , ± 10 nM thrombin), the samples were divided into a number of Eppendorf tubes and placed in a 37 ° C. water bath. In these tubes, clotting and lysis were stopped at various time points by the addition of 10 μM PPAck and 10 μM VFKck to selectively inhibit thrombin and plasmin, respectively. The sample was mixed vigorously and then centrifuged at 16000 g (room temperature) for 30 seconds and immediately placed on ice to prevent thermal inactivation of TAFIa. The supernatant of each sample was serially diluted 5-fold with TAFI-deficient plasma and TAFIa was measured using the functional assay described by Kim et al. (Anal. Biochem 2008; 372: 32-40). The same experiment was performed in a 96-well plate with a cover, and the turbidity was monitored over time using a SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, Calif., USA) to determine the timing of clotting and fibrinolysis. Judged. Similar experiments were performed at 4 tPA concentrations (0.25, 0.75, 1.5 and 3 nM) in the presence or absence of soluble thrombomodulin (0-100 nM) to determine the effect of sTM on TAFI activation and lysis time. Was judged. These experiments were also performed in the presence of 5 μM PTCI to demonstrate a TAFIa-dependent prolongation of lysis in normal and FVIII deficient plasma.

  Determination of the time course of prothrombin activation in normal and FVIII deficient plasma.

Clotting was initiated by adding prothrombin derivative (5IAF-II; final 300 nM) and 20 μM PCPS and 10 mM CaCl ₂ in the presence of 10 nM thrombin to normal and FVIII deficient plasma (0-100% NP). These experiments were performed in opaque, plastic covered 96 well plates. SpectraMax GeminiXS (Molecular Devices, Sunnyvale, Calif., USA) was used to monitor fluorescence intensity over time at 37 ° C. using an excitation and emission wavelength of 495 nm and 535 nm, respectively, using a 530 nm emission cutoff filter. . Fluorescence was normalized (0-1) to represent baseline fluorescence and maximum fluorescence correlated with full prothrombin activation.

  Determination of TAFIa potential.

  The area under the TAFIa plot was selected as a parameter to quantify the effect of TAFIa over the course of the experiment. This parameter was called the “TAFIa potential”, similar to the “thrombin potential” defined by Hemker et al. (Thromb. Haemost. 1993; 85: 5-11). Like the thrombin potential, the TAFIa potential is proportional to the amount of substrate that degrades and can be described mathematically as follows:

(Where dS / dt is the substrate consumption rate and S is the substrate).
If S is constant (ie limited consumption of S),

For a certain interval 0 to t,

Specifically expressing that the integral on the right side in equation (4) is the area under the TFAIa plot,

4). Results Clot lysis time is increased by the addition of normal plasma to FVIII deficient plasma.

Clotting was initiated with 10 nM Factor IIa, 10 mM CaCl ₂ and 20 μM PCPS vesicles to create a model where the clot structure was insensitive to FVIII concentration. Since the clot structure is similar regardless of FVIII concentration, the effect of FVIII on tPA-dependent (0.75 nM) clot lysis can be determined. Using this lysis model, the lysis time increased as the percentage of normal plasma increased. FIG. 1 shows the clot dissolution profile for FVIII-DP with 0-100% normal plasma addition and their dissolution times are summarized in FIG. 1 (inset). The dissolution time in FVIII-DP is 37 minutes, which can be increased by approximately 50% by the addition of normal plasma.

  10% normal plasma is sufficient to restore clot lysis in FVIII-DP.

  In 10% normal plasma, the lysis time reduction associated with FVIII-DP was corrected to the lysis time observed in normal plasma (see FIG. 1, inset).

  A 50% TAFIa potential is sufficient to restore clot dissolution in FVIII-DP.

  TAFI activation was measured in normal plasma, FVIII deficient plasma and mixed plasma to quantify the effect of FVIII on the time course of activation. TAFIa was measured over the time course of clotting and lysis using a functional assay. Results are presented in FIG. Approximately 30 pM TAFIa was measured after 5 minutes when clotting was initiated in FVIII-DP using thrombin, calcium ions and PCPS. As the percentage of normal plasma increased, the peak concentration of TAFIa also increased. Although the lysis time was corrected by adding 10% normal plasma to FVIII-DP, this was not sufficient for a complete correction of TAFI activation. By calculating the area under the TAFIa time course plot (FIG. 2A), approximately the same TAFIa potential (FIG. 2B) was achieved over the first 50 minutes in normal and 50% normal plasma (16800 pM · min and 14100 pM, respectively). Fmin-DP plasma mixed with 10% normal plasma was determined to have only 50% TAFIa potential of TAFIa potential in normal plasma.

  There is a strong correlation between the dissolution time and the TAFIa potential.

  To quantitate the relationship between lysis time and TAFI activation over the range 0-100% FVIII, log lysis time versus log TAFIa potential was plotted (FIG. 2B, inset). As expected, the data show a strong positive correlation between lysis time and TAFIa potential in plasma containing 0-100% FVIII. Since thrombin is an activator of TAFI, analysis of prothrombin activation in plasma can rationalize the TAFI activation profile in FIG. 2A (FIG. 3). There is a general trend that as the percentage of normal plasma increases, the rate of prothrombin activation also increases (which can be determined by examining the slope of the curve in FIG. 3). An exception occurs in normal plasma. The rate of prothrombin activation in normal plasma is slower than in FVIII-DP mixed with 50% normal plasma. The rate is slower in normal plasma, but prothrombin activation persists for about twice the length in FVIII-DP mixed with 50% normal plasma. In all experiments, the timing of prothrombin activation is in good agreement with TAFI activation. Normal plasma was also clotted without using thrombin and using calcium ions and PCPS. Calcium-induced clotting does not occur immediately; it takes approximately 15 minutes for the clot to form in normal plasma. At this time, prothrombin activation enters the propagation phase, and as a result, TAFI is activated. The degree and timing of TAFI activation in the context of clot formation is the same whether clotting is initiated in the presence or absence of added thrombin, which is the same as TAFI. This suggests that activation is a result of thrombin generated in situ and not a thrombin added to induce clotting. In the presence of thrombin, there was a TAFIa potential of 16,800 pM · min compared to 14,150 pM · min in the absence of thrombin.

  Soluble thrombomodulin prolongs clot lysis in normal and FVIII deficient plasma.

  In normal plasma, peak TAFIa levels and TAFIa potentials increased from 600 pM and 16800 pM · min in the absence of sTM to approximately 6000 pM and 150,000 pM · min, respectively, in the presence of 10 nM sTM. This increase in TAFI activation resulted in a 70% increase in lysis time. The effect of 10 nM sTM on the relative extension of lysis in FVIII-DP was similar to normal plasma where lysis was extended by 65% when FVIII-DP was clotted and lysed in the presence of sTM. In the presence of 10 nM sTM, 750 pM TAFIa was present at the peak TAFIa concentration compared to 30 pM in the absence of sTM. The time from clot start to clot dissolution time, the TAFIa potential was measured to be 12800 pM · min in the presence of 10 nM sTM compared to 600 pM in the absence of sTM.

  The increase in clot lysis time in normal and FVIII deficient plasma is dependent on tPA and sTM concentrations.

  The effect of TAFI activation on lysis time was analyzed over a range of tPA and sTM concentrations to determine if the lysis defect in FVIII-DP can be corrected by stimulating TAFI activation. The dissolution times summarized in FIG. 4 are related to dissolution times from similar experiments containing PTCI, an inhibitor of TAFIa. Since there is no functional TAFIa in the presence of PTCI, the relative lysis time presented in FIG. 4 is representative of TAFIa-dependent lysis extension. When 1 nM sTM was added to normal plasma, the maximum TAFIa-dependent dissolution extension (2-fold) was observed at the lowest concentration of tPA (0.25 nM). Addition of sTM to FVIII-DP resulted in a dose-dependent extension of lysis time (Figure 4). When 100 nM sTM was added to FVIII-DP, the lysis time correlated perfectly with that seen in normal plasma. As tPA concentration increased, higher concentrations of sTM were required to achieve maximum TAFIa-dependent dissolution extension. For example, when 1.5 nM tPA (FIG. 4) is present, 25 nM sTM is required to maximize TAFIa-dependent lysis extension in normal plasma and 100 nM sTM is required in FVIII-DP. Also, as tPA is increased in these clot dissolution experiments, TAFIa has an even greater effect on dissolution time (5 for 1.5 nM tPA compared to 2.3 times for 0.25 nM tPA). .. up to 2 times). It appears that as the tPA concentration is increased, the concentration of sTM required to achieve any TFAIa-dependent dissolution extension is increased. 0.25 nM tPA did not require sTM to achieve lysis extension in normal plasma, but when 3 nM tPA was added to normal plasma (Figure 4), 25 nM sTM was used to achieve lysis extension. I needed it. To clarify how the actual lysis time is affected by tPA and sTM, the lysis times in TAFIa-inhibited normal and FVIII-deficient plasma are presented in Table 1.

  Thrombomodulin greatly accelerates TAFI activation and prolongs lysis, both in normal plasma and in FVIII-deficient plasma.

  TAFI activation in normal plasma is significantly increased in the presence of 10 nM sTM (●; 6000 pM TAFIa at its peak level) compared to absence of sTM (◯; 600 pM TAFIa; see FIG. 5A). Prove it. The accompanying clot dissolution profile shows that the addition of 10 nM sTM resulted in a 70% increase in dissolution time. In FVIII-DP supplemented with 10 nM sTM, TAFIa was measured to be 750 pM at its peak compared to 30 pM in the absence of sTM (see FIG. 5B). Increased TAFI activation resulted in 60% prolonged dissolution compared to FVIII-DP deficient sTM.

Example II
Using analytical fluorescence kinetic assay of binding affinity between thrombin and thrombomodulin, has an affinity, expressed as K _D values determined for binding of thrombin and thrombomodulin analogs.

1. The affinity of the binding between the test system thrombin thrombomodulin analogs were determined using fluorescence kinetic assay, expressed as K _D values.

2. Experimental procedure Materials.

  Human thrombin was isolated from plasma as described by Bajzar et al. (J. Biol. Chem. 1995; 270: 14477-14484). Recombinant soluble thrombomodulin (Solulin) was obtained from PAION Deutschland GmbH (Aachen, Germany). All other reagents were obtained from Sigma in analytical quality.

  Method.

Measurement of thrombin binding to thrombomodulin and TAFI The binding of thrombin to thrombomodulin was measured as an equilibrium binding assay. Thrombin (20 nM), thrombomodulin (1.54 μM) and DAPA (20 nM, dansyl arginine N) in 0.02 M Tris-HCl, 0.15 M NaCl, 5.0 mM CaCl ₂ , 0.01% Tween 80, pH 7.4. A solution containing -3- (ethyl-1,5-pentanediyl) amide, a fluorescent, reversible thrombin inhibitor) was continuously added in portions to the same solution except lacking thrombomodulin. Added to a cuvette fitted with a magnetic stirrer in the sample compartment of a Perkin-Elmer model LS50B spectrofluorometer. Intensity values were recorded continuously at excitation and emission wavelengths of 280 and 545 nm, respectively. A 430 nm cut-off filter was used for the emission beam. The data was analyzed as follows. The intensity of fluorescence, I, was assumed to be the sum of intensities from thrombin-DAPA (T · D) and thrombin-thrombomodulin-DAPA (T · TM · D). That is, I = i ₁ · [T · D] + i ₂ · [T · TM · D], where i ₁ and i ₂ are fluorescence coefficients for T · D and T · TM · D ( Since the excitation was at 280 nm, the emission from free DAPA was negligible). TM does not appreciably modify the K _m for either protein C activation or TAFI activation (see Bajzar et al., 1996; J. Biol. Chem. 271: 16603-16608), so the thrombin-DAPA reciprocal It can be assumed that the affinity of action is not altered.
Therefore, [T · D] = ([T] + [T · D]) / (1 + K _DAPA / [DAPA])
And [T.TM.D] = ([T.TM] + [T.TM.D]) / (1 + K _DAPA / [DAPA])
(In these formulas, K _DAPA is the dissociation constant for the thrombin- _DAPA interaction).
Therefore,
I = i ₁ · ([T] + [T · D]) / (1 + K _DAPA / [DAPA]) + i ₂ ([T · TM] + [T · TM · D]) / (1 + K _DAPA / [DAPA] ).

Define f and b as the fraction of thrombin that is free and bound to thrombomodulin, respectively, and if [T] ₀ is the total concentration of thrombin, f = ([T] + [T D]) / [T] ₀ , b = ([T · TM] + [T · TM · D]) / [T] ₀ and f + b = 1. At this time, the fluorescence intensity is given by I = i ₁ · f [T] ₀ / (1 + K _DAPA / [DAPA]) + i ₂ · b [T] ₀ / (1 + K _DAPA / [DAPA]). If I ₀ is defined as the initial intensity when no thrombomodulin is added, f = 1 and I ₀ = i ₁ [T] ₀ / (1 + K _DAPA / [DAPA]). Similarly, if I _max is defined as the intensity of thrombin at saturation with thrombomodulin, b = 1 and I _max = i ₂ [T] ₀ / (1 + K _DAPA / [DAPA]). Therefore, I = I ₀ · f + I _max · b. Next, by substituting 1-b for f, I = I ₀ + (I _max −I ₀ ) · b or ΔI = ΔI _max · b is obtained. By normalizing to the initial intensity, (ΔI / I ₀ ) = (ΔI _max / I ₀ ) · b is obtained. If DAPA binds T and T · TM with equal affinity, TM binds T and T · D with equal affinity.

_Thus, thrombin _{K TM} - Defining a dissociation constant for thrombomodulin _{interaction, [T] [TM] =} K TM [T · TM]; [T · D] [TM] = K TM [T · TM · D ]; And ([T] + [T · D]) · [TM] = K _TM ([T · TM] + [T · TM · D]). The immediately preceding equation is the same as f · [TM] = K _TM · b. Since f = 1−b and [TM] = [TM] ₀ −b · [T] ₀ , where [TM] ₀ is the total concentration of thrombomodulin, the following equation is obtained: (1-b ) ([TM] ₀ −b · [T] ₀ ) = K _TM · b. This is a quadratic equation for b. Solving b in the above equation and substituting (ΔI / I ₀ ) for b gives the following equation: (ΔI / I ₀ ) = (ΔI _max / I ₀ ) · 0.5 · (K _TM + [T] ₀ + [TM] ₀ − ((K _TM + [T] ₀ + [TM] ₀ ) ² −4 · [T] ₀ · [TM] ₀ ) ^1/2 ). This immediately preceding equation gives the relationship between the fluorescence intensity value, the set concentration of thrombomodulin and thrombin, the dissociation constant for the thrombin-thrombomodulin interaction, and the fluorescence intensity increment that is a manifestation of the interaction between thrombomodulin and thrombin-DAPA. Represent. [TM] ₀ and the independent variables, with K _TM and [Delta] I _max as the best fit parameters were fitted intensity data in the above equation by nonlinear regression analysis.

3. Results Thrombin binds to soluble thrombomodulin with an affinity of K _D = 23 ± 14 nM.

Binding of thrombin to soluble thrombomodulin was measured by perturbation of DAPA fluorescence. As shown in FIG. 6, the titration curve showed an increase in relative fluorescence for a concentration range of soluble thrombomodulin between 0 nM and 75 nM. Data analysis revealed that thrombin binding to soluble thrombomodulin was characterized by K _D = 23 ± 14 nM.

Example III
Using analytical fluorescence kinetics assay cofactor activity for mutations thrombomodulin analogs were affinity, expressed as K _D values determined for binding between thrombin and thrombomodulin analogs.

1. Experimental procedure Materials and methods.

The ability of the TM mutant to act as a cofactor for thrombin-mediated activation of protein C was directly assayed in shockate. Recombinant human protein C is available from Dr. From John McPherson (Genzyme Corp., Framingham, Mass.) And purified as described (BioTechnology 1990; 8: 655-661). 25 [mu] L of each schokate and the same amount of recombinant human protein C (0.3 [mu] M final concentration) and human alpha thrombin (Sigma Chemicals, St. Louis, MO) were mixed in a microtiter plate at a final concentration of 1 nM. All reagents used were diluted with 20 mM Tris, pH 7.4 / 100 mM NaCl / 3.75 mM CaCl ₂ /0.1% NaN ₃ (w / V) containing 5 mg / mL bovine serum albumin. The mixture was incubated for 1 hour at 37 ° C. and the reaction was stopped by the addition of 25 μL hirudin (Sigma Chemicals, St. Louis, MO) at 800 units / mL. The amount of protein C activated was determined by the addition of 100 μL of the chromogenic substrate D-valyl-L-leucyl-L-arginine-p-nitroanilide (S-2266) (1 mM). The change is measured by absorbance at 405 nm over time using a plate reader. The data for each sample is recorded and determined as milliOD units / minute by measuring absorbance every 10 seconds for 15 minutes using a Molecular Devices plate reader. All assays were transfected with either triplicate shot samples, ie, pSELECT-1 vector (no TM), pTHR211 (wild type) or pMJM57 (pTHR211 where 388 methionine was altered to leucine). Each DH5 alpha cell was included as an internal control. The cofactor activity of TM mutants was expressed as the average of those obtained for pMJM57.

  Statistical analysis.

  Each mutant was assayed for activity at least twice (three for mutants that isolated only two positive clones) and all data were included in determining the significance of differences using the Student t test. The variation rate between the plates was 16.7% (n = 18).

Western Blot Analysis E. coli syogate was run under reducing conditions in 10% Tris-tricine SDS PAGE according to manufacturer's specifications (Novex Inc. San Diego, CA). Boil the silicate for 10 minutes in sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.0025% bromophenol blue) containing 10 mM dithiothreitol, followed by 50 mM iodoacetamide. Reduced and alkylated samples were prepared by incubating with.

Proteins were transferred to nitrocellulose filters in transcription buffer (192 mM glycine, 25 mM Tris, pH 8.3, 20% methanol) at 4 ° C. The nitrocellulose filter was blocked with blocking buffer (1% bovine serum albumin, 0.9% NaCl, 0.05% NaN _{3 in} 10 mM Tris, pH 7.5) and then mouse polyclonal anti-antigen in the blocking buffer. Incubated with serum (produced against the reduced and alkylated EGF domain of human thrombomodulin). After washing with wash buffer (10 mM Tris, pH 7.5, 0.9% NaCl, 0.05% NaN ₃ , 0.05% Tween 20), the filter was washed with blocking buffer containing 0.05% Tween 20 Incubated with biotinylated goat anti-mouse IgG antibody in. Proteins were detected using Vectastein ABC solution (Vector Laboratories, Burlingame, Calif.) And ECL detection system (Amersham Corporation, Arlington Heights, Ill.) According to the manufacturer's specifications.

Example IV
Using analytical fluorescence kinetics assay of thrombomodulin analogs of TAFI and protein C activation, has an affinity, expressed as K _D values determined for binding of thrombin and thrombomodulin analogs.

1. Experimental Procedure Proteins and Reagents Solulin (residues 4-490), TM _E (residues 227-462), TM _E c-loop 3-6 (residues 333-462) and TM _E i4-6 (residues 345-362) Truncated thrombomodulin was prepared as described by Parkinson et al (Biochem. Biophys. Res. Commun. 1992; 185: 567-576). Sf9 cells were transfected with the TM construct and the protein was isolated from the medium by a combination of anion exchange, gel filtration and chromatographic procedures utilizing thrombin affinity. Purity was greater than 95% as assessed by SDS-polyacrylamide gel electrophoresis and silver staining. Human plasma TAFI was isolated as described by Bajzar et al. (J. Biol. Chem. 1995: 270: 14477-14484). Human protein C and thrombin were prepared as described by Bajzar and Nesheim (J. Biol. Chem. 1993; 268: 8608-8616). The thrombin inhibitor dansyl arginine N- (3-ethyl-1,5-pentanediyl) amide (DAPA) was synthesized as described by Nesheim et al. (Biochemistry 1979; 18: 996-1003). Point mutations resulting from alanine scanning were generated from the TM _E M388L construct. The protein was expressed in Escherichia coli. The procedure and preparation of periplasmic extracts is described by Nagashima et al. (J. Biol. Chem. 1993; 268: 8608-8616). HEPES, the basic carboxypeptidase substrate hipryl arginine, cyanuric chloride, and 1,4-dioxane were obtained from Sigma. All other reagents were of analytical quality.

  Measurement of protein C and TAFI activation rates at point mutations of thrombomodulin analogs.

For activation of TAFI, 20 μL aliquots of each periplasmic extract were preincubated with thrombin (final 13 nM) at room temperature in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl ₂ for 5 minutes. The mixture was then incubated with purified recombinant TAFI (final 18 nM) and substrate, Hipril-Arginine (final 1.0 mM) in a final volume of 60 μL for 60 minutes. Hydrolysis of hipril-arginine to hippuric acid, followed by hippuric acid chromogen with 80 μL phosphate buffer (0.2 M, pH 8.3) and 60 μL 3% cyanuric acid (w / v) in dioxane The amount of activated TAFI was quantified by measuring the conversion of. After careful mixing, the absorbance of the clear supernatant was measured at 382 nm. The amount of thrombin-dependent activation of TAFI was calculated by subtracting the background absorbance generated in the absence of thrombin for each mutant. Activation of protein C by the TM _E M388L-alanine mutant was assayed as follows.

All samples and reagents APC assay diluent (20mM Tris-HCl, pH7.4,100mM NaCl , 2.5mM CaCl 2, 0.5% BSA) was diluted with. Samples and TM standards (0-1 nM) were incubated with 0.5 μM protein C and 1 nM thrombin in a 96 well plate at 37 ° C. in a total volume of 60 μL for 60 minutes to give APC, followed by 20 μL hirudin (0 .16 U / μL, 570 nM). The amount of APC formed was determined by monitoring the hydrolysis of S-2266 (100 μL of 1 mM) at 1 minute intervals at 405 nm using a plate reader (Molecular Devices Corp., Menlo Park, Calif.). Were determined. 1 U of activity gives 1 pmol of activated protein C / min (37 ° C.).

All assays, pSelect-1 vector (no _TM E), contained wild-type _TM E (M388) or _TM E (M388L) extracts of transfected DH5α cells either as an internal control. The cofactor activity of the TM _E (M388L) alanine mutant was expressed as a percentage of the activity of TM _E (M388L). Each TM mutant was assayed in duplicate for both protein C and TAFI activation using three independent preparations of extracts.

2. Results The results obtained with the TM mutant (FIG. 7) show that 5 out of 8 mutants have substantially reduced cofactor activity. Four of these five mutants also show a concomitant reduction in TAFI activation activity. Only the mutation at F376A resulted in an enormous loss of protein C activation, but only a modest reduction with respect to TAFI activation. Interestingly, the difference in the importance of Phe ³⁷⁶ for TAFI and protein C activation suggests that the requirements for thrombomodulin structure are more constrained when protein C is a substrate for the thrombin-thrombomodulin complex. Yes.

(Example V)
Analysis on oxidation of Met-specific TM mutants for protein C activation Using a specific methionine mutant of a thrombomodulin analog, a protein C activation assay was used to complement these residues. The role for factor activation was also analyzed for protein oxidation.

1. Experimental procedure Proteins and reagents.

  Human recombinant protein C is available from Genzyme Corp. (Boston, Massachusetts). Bovine thrombin is available from Miles Laboratories Inc. (Dallas, Texas). D-Val-Leu-L-Arg-p-nitroanilide was prepared as described by Glaser et al. (Prep. Biochem. 111975; 5: 333-348). Human alpha-thrombin (4,000 NIH U / mg), bovine serum albumin (factor V) and chloramine T are available from Sigma Chemical Co. (From St. Louis, Missouri).

Expression of TM _E (Sf9).

  All procedures were performed at 4 ° C. The DNA sequence encoding TM's 6th EGF-like repeat (amino acids 227-462) was ligated to the insect protease, hypodermin A, signal sequence and the hybrid gene was under the control of the polyhedron gene promoter in the baculovirus shuttle vector pTMHY101. placed. Recombinant virus was produced using standard techniques. The described mutant analogs are prepared by use of a mutator site-directed mutagenesis kit (Stratagene, Inc., La Jolla, Calif.), Which allows the virus to be expressed for expression in a baculovirus system. Prepared.

Purification and Oxidation with Chloramine T Growth media containing a secreted mutant of TME (Sf9) was clarified by centrifugation, lyophilized, and 1:10 volumes of 0.2% NEM-Ac, pH 7 and 0.008. Redissolved in% Tween 80. Aliquots are treated with either 5 μL H ₂ O or 5 μL 100 mM chloramine T, incubated for 20 minutes at room temperature, diluted to remove oxidant, and NAP-5 column (20 mM Tris-HCl, 0.1 M NaCl). , 2.5 mM CaCl ₂ , 5 mg / mL BSA, pH 7.4; Pharmacia Inc.) and assayed for protein C activation as follows.

Measurement of TM cofactor activity (APC assay)
All samples and reagents APC assay diluent (20mM Tris-HCl, pH7.4,100mM NaCl , 2.5mM CaCl 2, 0.5% BSA) was diluted with. Samples and TM standards (0-1 nM) were incubated with 0.5 μM protein C and 1 nM thrombin in a 96 well plate at 37 ° C. in a total volume of 60 μL for 60 minutes to give APC, followed by 20 μL hirudin (0 .16 U / μL, 570 nM). Determine the amount of APC formed by monitoring the hydrolysis of S-2266 (100 μL of 1 mM) at 1 minute intervals at 405 nm using a plate reader (Molecular Devices Corp., Menlo Park, Calif.) did. 1 U of activity gives 1 pmol of activated protein C / min (37 ° C.).

2. Results Reduction of TM cofactor activity by oxidation of Met388 Mutants and wild type TME (Sf9) were expressed in insect cells, treated with chloramine T, assayed for cofactor activity, and the results compared (Table 2). When the TM _E is treated with an oxidizing agent such as chloramine T, TM _E loses approximately 85% of its cofactor activity (see Table 2). Site-directed mutations of Met ²⁹¹ and Met ³⁸⁸ demonstrate that inactivation of TME (Sf9) is due to single methionine oxidation. The derivative carrying Met ³⁸⁸ was inactivated to a similar extent (> 80%) by chloramine T, whereas the Met ³⁸⁸ Leu mutant was resistant. Mutants in which Met ²⁹¹ was substituted were active but not resistant to oxidative inactivation.

Example VI
Analysis of TM analogs with mutations in the interdomain loop between EGF4 and EGF5 (Gln387, Met388, Phe389) Using specific mutants of thrombomodulin analogs, the role of these residues and their oxidation Were analyzed using a protein C activation assay.

1. Experimental procedure Plasmid construction. A thrombomodulin fragment consisting only of the EGF-like domain (TM _E ) was expressed in E. coli as follows: Primer 5′-CCGGGATCCTCAACAGTCGGTGCCCAATGTGGCG-3 ′ and 5′-CCGGGATCCTGCAGCGTGGAGAACGGCGGCGCGC-3 ′ from the genomic DNA reaction using the human genome chain reaction A DNA fragment encoding the full length TM _E (residues 227-462) was obtained. This fragment was placed under the control of the β-lactamase promoter and the signal sequence in pKT279. The resulting plasmid EcoRV-BgIII fragment and the pGEM3zf-ScaI-SacI fragment containing the f1 origin of replication were then inserted into the EcoRV-BamHI and ScaI-SacI sites of the pSelect-1 vector, respectively. An E. coli expression plasmid pTHR211 was constructed. Use the site-directed mutagenesis procedure described for the modified site in vitro mutagenesis kit to encode a TM mutant at position 387, 388 or 389 using a single-stranded pTHR211 DNA template A plasmid was constructed. Each primer for the site-specific mutation was confirmed by restriction analysis.

To determine the cofactor activity of the mutant, individual E. coli cultures expressing the mutant protein were centrifuged, washed, 20% sucrose, 300 mM Tris-HCl, pH 8.0, 1 mM EDTA. The cell pellet was incubated in 0.5 mM MgCl ₂ (10 min, 4 ° C.). Shots were prepared by centrifugation (10 min, 4 ° C.) of cell pellets treated with 0.5 mM MgCl ₂ and assayed in the APC assay. Data is the average of results from each of three independent clones.

Measurement of TM cofactor activity (APC assay)
All samples and reagents APC assay diluent (20mM Tris-HCl, pH7.4,100mM NaCl , 2.5mM CaCl 2, 0.5% BSA) was diluted with. Samples and TM standards (0-1 nM) were incubated with 0.5 μM protein C and 1 nM thrombin in a 96 well plate at 37 ° C. in a total volume of 60 μL for 60 minutes to give APC, followed by 20 μL hirudin (0 .16 U / μL, 570 nM). Determine the amount of APC formed by monitoring the hydrolysis of S-2266 (100 μL of 1 mM) at 1 minute intervals at 405 nm using a plate reader (Molecular Devices Corp., Menlo Park, Calif.) did. 1 U of activity gives 1 pmol of activated protein C / min (37 ° C.).

2. Results Reduction of the cofactor activity of TM by mutation of the inter-loop domain Using site-directed mutagenesis, expressing TM mutants with amino acid modifications, either deletions or insertions at positions 387, 388 or 389 (FIG. 8). Mutant TM cofactor activity is the mean value obtained from three independent clones and is expressed as a percentage of the known activity for TME (Sf9) WT. Gel scans on Western blots were performed using polyclonal antibodies against TM for all novel mutants at position 388 and for selected mutants at position 387. These scans yielded approximately equal amounts of TM. This indicates that differences in expression cannot cause observed differences in activity. In addition, independent substitutions at positions 387 (FIG. 8A), 388 (FIG. 8B), 389 (FIG. 8C), or insertions and deletions anywhere within the interdomain loop (FIG. 8D) generally lead to APC assays. resulting in causing analogue is defective cofactor than the wild-type TM _E in. Analogs in which Gln ³⁸⁷ is replaced by Thr, Met or Ala retain> 70% cofactor activity, but substitution with Glu residues reduces this to 58% of the control and all other Of amino acids results in> 50% loss. Only substitution of Met ³⁸⁸ with Leu results in cofactor activity (1.8-fold) that is substantially higher than wild-type. All other substitutions of Met ³⁸⁸ except Gln and Tyr result in a> 50% loss of cofactor activity. TM cofactor activity is less sensitive to Phe ³⁸⁹ amino acid substitutions, and none of the point mutations at this position retain> 70% of the activity found in controls. With the exception of Met388Pro, which retained 30% activity, substitution of Pro or Cys at any position reduced its activity to> 10%. By varying either the length of the inter-domain loop between EGF4 and EGF5 insertion Ala to deletion or four possible positions each individual amino acid, as a result, the activity of the wild-type TM _E Mutants with less than 10% are obtained.

(Example VII)
Analysis of fibrinolysis in canine hemophilia plasma Soluble thrombomodulin that reduces or increases clot lysis time in whole blood or plasma of dogs with hemophilia A using an in vitro clot lysis model and thromboelastography ( The ability of Solulin) was tested.

Materials and Methods Clot dissolution assay The clot dissolution assay was performed as described in Example I of the present invention.

Thromboelastography Thromboelastography was performed as described in Prasad et al., 2008; Blood 111: 672-679. Canine haemophilia whole blood (± factor VIII neutralizing antibody) (320 μL) was collected from Haemoscope TEG® 5000 (Haemonetics Corp., Mass.) Containing 90 nM thrombin, 9 nM tPA and 0-390 nM sTM. , Braintree). After careful mixing, the pins were sheeted and clotting and fibrinolysis were continuously monitored. The Haemoscope TEG® 5000 allows the measurement of clot time, clot dynamics, clot strength and clot stability (fibrinolysis) by measuring the torque of the wire connected to the clot by pins. As the clot forms, the pin torque increases, and this torque is represented by an increase in amplitude (power). Similarly, clot decomposition during fibrinolysis results in a decrease in torque and a decrease in amplitude.

  Inhibitory antibodies (inhibitors) generated by some dogs in Queen's University haemophilic dog colonies are described by Giles et al., 1984; Blood 63: 451-456 and by Tinlin et al., 1993; Thromb Haemost 69: 21-24. It has been previously described.

  Hemophilia dog plasma with inhibitors was drawn from dogs with inhibitor titers of> 150 Bethesda units (> 5 BU is considered untreatable with Factor VIII replacement therapy).

Results Solulin prolonged clot lysis even at a concentration of 500 nM.

  In canine hemophilia plasma, Solulin increases clot lysis time in a dose-dependent manner, which is approximately doubled in the presence of 25 nM Solulin and corresponds to the highest dose tested, 200-500 nM. A plateau of approximately 9 times extension was reached with Solulin (see FIG. 9).

  Conclusion: Even at very high concentrations in hemophilia plasma, the remarkable antifibrinolytic effect of Solulin strongly demonstrates the effective and safe use of thrombomodulin analogs in hemophilia.

  Solulin prolongs clot lysis in whole blood from canines with hemophilia A.

  In the thromboelastogram, the maximum amplitude of the torsion wire is a measure of the maximum clot strength. In these experiments (see FIG. 11A), sequential clot formation and lysis were induced by transferring canine hemophilia whole blood into cuvettes containing 90 nM thrombin and 9 nM rt-PA. Solulin was present at a concentration of 0-390 nM. An amplitude of approximately ± 85 mm was observed in the absence of Solulin. Solulin at concentrations of 100 and 250 nM completely blocked clot lysis: the amplitude was larger and did not change from 80 to 90 minutes (end of experiment). Further increase in Solulin concentration to 390 nM was still accompanied by greater amplitude and slower clot lysis than control experiments (No Solulin) (FIG. 11A).

  In this experiment, the area under the clot dissolution curve (AUCL) was used for quantification of clot hardness, whereby Solulin at 100 and 250 nM increased this parameter by more than 5 times (FIG. 11B). The extension of clot dissolution time in the same experiment is plotted in FIG. 11C. The dissolution time was increased from 20 minutes in the absence of Solulin to 3 hours at 100 and 250 nM Solulin.

  Conclusion: The anti-fibrinolytic effect of Solulin demonstrated by thromboelastography is consistent with the results of the above clot lysis assay. Furthermore, Solulin appears to increase clot hardness.

  Solulin prolongs clot lysis in whole blood from canines with hemophilia A in the presence of anti-factor VIII antibodies.

  These thromboelastography experiments were performed as described above with blood known to contain high titers of anti-factor VIII antibodies (> 150 Bethesda units): 90 nM thrombin and 9 nM rt Sequential clot formation and lysis was induced by transferring canine hemophilia whole blood into cuvettes containing PA. Solulin was present at a concentration of 0-3510 nM. An amplitude of approximately ± 20 mm was observed without Solulin (see FIG. 12A). The Solulin dose increased the amplitude to approximately ± 60 mm in a dose-dependent manner at concentrations of 100 and 250 nM, reaching an amplitude of approximately ± 90 mm at 390 nM.

  In addition, Solulin extended the clot lysis time in a dose-dependent manner from approximately 18 minutes in the control to approximately 46 minutes in 390 nM Solulin (see FIG. 12A). An increase in clot hardness is also indicated by an enhancement greater than 10 times the area under the elastic curve (compare 0 nM Solulin with 390 nM; see FIG. 12B).

  Conclusion: Although anti-factor VIII antibodies reduced virtually complete inhibition of factor VIII activity, Solulin's ability to prolong clot lysis could not be suppressed. Viewed from another angle, this experiment is sufficient for the presence of residual factor VIII activity in hemophilic whole blood (absence of antibody, see FIG. 11A) to increase the efficacy of Solulin to prolong clot lysis. It is shown that.

  This supports the use of the claimed thrombomodulin analogs in hemophilia patients treated with Factor VIII. In addition, patients suffering from hemophilia and still having functional factor VIII concentrations could also be treated with the claimed thrombomodulin analogs. Finally, patients lacking factor VIII or exhibiting high titers of anti-factor VIII antibodies are also subject to treatment with the claimed thrombomodulin analogs.

Example VIII
Ratio of TAFI activation to protein C activation for various TM analogs Several TM analogs were generated as shown in FIG. 19 and given in SEQ ID NOs: 5-11. Solulin analogs with Phe376Ala exchange were tested in vitro for protein C and TAFI activation in comparison to Solulin.

Materials and Methods Measurement of TM cofactor activity (APC assay)
A DNA fragment encoding Solulin with the selected mutation was synthesized and cloned into the pGAEx vector (a standard HEK293-derived vector). The pGAEx vector contains the standard promoter, leader and excretion sequences for HEK293 cells, and a His-6 tag at the C-terminus of the protein. HEK293 cells were transfected with the pGAEx-Solulin vector (transient transfection) and cultured in suspension on a 1 liter scale. Six days after transfection, the protein was recovered by Ni-HiTrap column (linear gradient of 20-500 mM imidazole in PBS, 500 mM NaCl). The purification target was a His-6 tag. Proteins were analyzed after dialysis (16 hours against PBS). Protein was quantified by Coomassie Blue staining of SDS-PAGE. Thereafter, protein identity was verified by Western blot (labeled anti-His-6 antibody).

TM cofactor (APC assay) and TAFI activity measurements TM similar in assay buffer containing 0 to 2 μM TAFI or protein C, 0.5 nM thrombin, 5 mM calcium chloride and 25 nM Solulin or Solulin Phe376Ala analog, respectively The body was tested.

  The reaction was quenched with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) for TAFI experiments or hirudin for protein C.

  TAFIa and activated protein C activity were determined using N- (4-methoxyphenylazoformyl) -Arg-OH (AAFR) and pyroGlu-Pro-Arg-pNA.HCl (S-2366), respectively. . The amount of TAFIa or aPC produced in 10 minutes was determined by comparing the substrate hydrolysis rate to a set of standards. Using standards, we determined the kinetics of TAFI and protein C activation by IIa-sTM.

Results The exchange of Phe376 for Ala results in an increased ratio of TAFI to protein C activation rates, with the increase being more pronounced at lower substrate concentrations. Michaelis-Menten constants could be determined for TAFI activation (FIG. 15, upper panel), but such constants could not be derived from protein C activation (FIG. 15, lower panel).

  The ratio of activation rates of TAFI and protein C (TAFI / protein C) by Solulin and Phe376Ala at various substrate concentrations is illustrated in FIG. Solulin's high TAFI / PC ratio could be further improved by amino acid exchange from Phe376 to Ala.

Conclusion:
An increase in the TAFI / protein C ratio, which can be used as a measure of TAFI preference, occurred at reasonable substrate (TAFI or protein C) concentrations. This is most noticeable at the lowest concentration tested (250 nm), close to physiological concentrations of both TAFI (about 75-220 nM) and protein C (about 70 nM), and both Solulin and its variants have preferential TAFI activity. Confirm the claim that it is suitable for On the other hand, in particular, protein C activation by Phe376Ala is so slow that it must be considered unlikely that it can enhance protein C activation to any appreciable extent at physiological protein C concentrations (about 70 nM). It has been found. Therefore, a significant reduction in both TAFI and protein C activation capacity that still allows TAFI activation but actually interferes with protein C activation under physiological conditions has led to an additional sudden increase in Phe376Ala. You can see the related effects of the mutation.

  Furthermore, these results support the overall importance of single amino acid mutations and in particular Phe376Ala. The beneficial effect of this mutation is not only observed for Solulin fragments ranging from EGF1 to EGF6, but also for full-length soluble thrombomodulin containing an N-terminal lectin-like domain, six EGF domains and an O-linked glycosylation domain. This is because that.

Example IX
In Vivo Analysis of Fibrinolysis in a Canine Model of Hemophilia Study Objectives Recovery of Factor VIII (fVIII) is a recourse for treatment of patients with hemophilia A. While a significant number of patients develop neutralizing anti-fVIII antibodies while undergoing effective treatment in most cases, so far, this problem cannot be overcome with the available adjuvant treatment options. Other disadvantages of fVIII therapy include the need for complex dose determination and intravenous administration and periodic monitoring.

  After all, it is best not to produce sufficient amounts of fVIII, but in addition there is increasing evidence that the uncontrolled activity of tissue plasminogen activator (tPA) also contributes to this pathology. . Normally, clot growth and maturation is protected from tPA by activation of thrombin-activated fibrinolysis inhibitor (TAFI). It is activated by a complex formed by thrombin generated in the process of vascular injury closure and thrombomodulin, a protein important in the endothelial cell membrane. The thrombin / thrombomodulin complex binds the plasminogen of tPA by converting the plasma zymogen TAFI to its active form (TAFIa), which removes the carboxy-terminal lysine and arginine from the clot fibrin. It acts to impair its ability to activate plasminogen in a higher order structure. This control of tPA potency is incomplete in hemophilia A, due to insufficient thrombin generation and possibly due to reduced utilization of zymogen, TAFI. Therefore, one of the hallmarks of hemophilia A is premature clot lysis with tPA.

  In vitro studies have shown that Solulin can partially or completely correct premature clot lysis in plasma from hemophilia patients, depending on tPA concentration. The aim of this study is to test whether this in vitro efficacy can be confirmed in vivo using a well-established hemophilia canine model. In order to justify the use of this animal model for this purpose, preliminary experiments confirmed that hemophiliac dog plasma, like human plasma, caused Solulin to correct premature clot lysis.

Research substance identification: Solulin
Physical state: Clear colorless solution, pH 7.0
Dissociation buffer: sodium phosphate, 13.4; potassium chloride, 2.7; sodium chloride, 137 (all in mmol / L); mannitol, 5%
Contents of vial: 3 mg Solulin in 1 mL (3 mg / mL)
Vial handling: Use ethanol to wipe the vial septum, then insert a sterile needle through the septum to withdraw the required volume. Store the vial containing the remaining amount in the refrigerator.
Storage conditions and time: Store in a refrigerator at 2-6 ° C. The remaining amount can be stored in the refrigerator for 28 days. Within this period, they can be used for further experiments, but it is recommended to use separate vials for each dog.

Preparation of Dosage Formulation Take an appropriate amount from the original Solulin vial (s) and supplement with 0.9% NaCl to a 3 mL dose.
The final dosage formulation will be freshly prepared for each experimental day.

Animals: Species and Management This study was conducted in dogs with hemophilia A (hemophilia A dogs) characterized by “cured” FVIII gene therapy dogs. After canine FVIII gene therapy, these dogs had undetectable plasma FVIII levels (<1%) but showed a reduction in their whole blood clot time and further idiopathic bleeding for at least 3 years after treatment. Never experienced an episode (standard frequency approximately 5 / year). These dogs can be considered to show partial phenotypic correction after gene transfer. They best reproduce the clinical picture demonstrated in approximately 10% of severe hemophilia A patients who rarely bleed (plasma FVIII <1%). Such a profile also reflects the outcome of the low dose FVIII prevention protocol. The age and weight of these animals were approximately 3 years and 10-15 kg, respectively. Only healthy animals with the exception of haemophilia were included in this study.

Experimental procedure Preliminary study (pharmacokinetics and in vitro efficacy)
As a first step, the pharmacokinetics of Solulin together with pharmacodynamic effects on coagulation / fibrinolysis were investigated in hemophilia dogs. Based on in vitro potency data available for canine plasma and pharmacokinetic modeling, Solulin was administered by intravenous bolus injection at a dose of 0.5 mg / kg in a volume of 3 mL.

  Depending on the results obtained in the first dog, the second dog was treated in the same way or with a matched dose of Solulin. Subsequently, the start of the main study or further investigation of pharmacokinetics was decided.

Blood sample preparation Two different sets of blood samples were taken from the contralateral leg (see table below).

  Whole blood was collected using citrate buffer (final volume: 1 part 3.13% sodium citrate mixed with 9 parts blood) and processed immediately (see preparation below). Unless they were assayed in close time relationship according to the instructions for the individual splits, they were appropriately labeled and stored at −80 ° C. until further use.

Sample 1
The first milliliter to be discarded was taken at the time indicated in the table above, citrated and divided into five parts.

  14 mL of citrated whole blood was distributed into aliquot 1 (thromboelastography x 5 mL whole blood) and aliquot 2 (whole blood clotting: 2 mL whole blood).

  The remaining amount was centrifuged at 1500 g for 15 minutes. Divide the resulting supernatant into 3 parts (split part 3, Solulin concentration, 0.5 mL plasma; split part 4, activated TAFI, 0.5 mL plasma; split 5, PT / aPPT, 0.5 mL plasma) And stored at −80 ° C. until further use.

Sample 2:
Sample 2 (2 mL; discard the first 1 mL) was taken from the opposite leg of Sample 1 and citrated. Sampling times are shown in the table above.

  This sample is for determining the thrombin generation potential and requires special prior knowledge. It is important to avoid contact activation, which means that the sample should be drawn with the needle as thick as possible (preferably not from the butterfly needle).

The required plasma volume is 0.5 mL. The plasma preparation for thrombin generation potential is described in detail:
First centrifugation stage:
○ Enter the following centrifugation parameters:
-Decomposition power: 9
-Rotor speed 3790 at r = 156mm
-Duration: 5 minutes.
O Centrifuge the sample for 5 minutes at 2500 g (for the Hetrich Rotina 35R this is reached at a setting of 3790 rpm at r = 156 mm)
○ Using a plastic disposable Pasteur pipette, transfer the plasma supernatant to a labeled plastic centrifuge tube. Care is taken to leave 0.5 cm plasma on the buffy coat and not to draw any cells from the buffy coat.

Second centrifugation stage:
○ Apply the following centrifugation parameters:
-Temperature: 18 ° C
-Centrifugation speed: 11000 RPM at r = 156 mm
-Duration: 10 minutes o Centrifuge plasma at 10000 g for 10 minutes.

(For Universal 30RF, this is reached at r = 156 mm and 11000 rpm)
O Inject or pipette the contents of the centrifuge tube into the labeled tube in one move (do not mix the precipitate and supernatant).
O Where applicable, combine several identical plasma contents into one large plastic tube.

Pharmacokinetic and pharmacodynamic assays Thromboelastography: Thromboelastography was performed on citrated whole blood. 340 μL of whole blood was added to a Haemoscope TEG® 5000 (Haemonetics Corp., Braintree, Mass.) Channel containing a 20 μL solution of Innovin 1/15000 dilution (18 ×), 270 mM CaCl ₂ and 18 nM tPA. Add to. After thorough mixing, the pins were sealed and solidification and fibrinolysis were continuously monitored. The Haemoscope TEG® 5000 allows the measurement of clot time, clot dynamics, clot strength and clot stability (fibrinolysis) by measuring the torque of the wire connected to the clot by pins.

  Whole blood clotting time: Whole blood clotting time is determined by incubating citrated whole blood in a glass test tube at 37 ° C. and continuously monitoring clot formation.

  Pharmacokinetics. Plasma concentrations were determined using an ELISA validated for canine plasma. This research will be under the responsibility of the sponsor. Summarize results in final report.

  Activated TAFI. TAFIa was determined using the method of Kim et al. (Anal Biochem. (2008), 372 (1): 32-40). Briefly, fibrin degradation products containing a covalently linked quencher moiety (QSY-FDP) bind to fluorescently labeled plasminogen (F-Pg). When F-Pg is bound to QSY-FDP, the fluorescence is quenched. Addition of TAFIa removes the F-Pg binding site and releases F-Pg from QSY-FDP, resulting in increased fluorescence. The rate of this fluorescence increase is proportional to the TAFIa concentration. Plasma spiked with known concentrations of TAFIa was used to generate a standard curve that was used to determine the TAFIa concentration in plasma samples.

  Coagulation assay. Diluted PT (dPT) and aPTT assays were performed. The diluted PT assay requires 1/5 dilution of the test platelet poor plasma. TriniCLOT PT HTF thromboplastin reagent is added to the plasma, after which the sample is remineralized after a 180 second incubation. aPTT was performed with TriniCLOT Automated APTT reagent. Again, a 180 second incubation is performed, after which the sample is remineralized. All studies were performed on a Coag-A-Mate MAX (bioMerieux) automatic coagulometer.

  Endogenous thrombin potential. Endogenous thrombin potential was determined with a 96-well plate fluorometer using a calibrated automated thrombogram (CAT) assay. The CAT assay was performed using CoagScope B. V. (Netherlands, Maastricht) with intellectual property rights.

  Briefly, the CAT assay measures the concentration of thrombin in clotting plasma over time. Citrated plasma is induced with a PPP reagent containing tissue factor and phospholipid. The plasma is remineralized with another reagent (FluCa) containing calcium chloride and a fluorogenic substrate. This substrate is converted to a fluorophore by thrombin and the fluorescence is followed with a fluorometer. At the same time, another sample of the same plasma is measured in the presence of a thrombin calibrator (calibrated thrombin activity). By comparing the results of these parallel measurements, it is possible to calculate the thrombin concentration over time.

  Main research (cuticle bleeding assay)

Number of animals, study drug and dose level Three hemophilia dogs were studied to test the effect of Solulin on blood clotting and fibrinolysis. Solulin was administered by intravenous bolus injection at a dose selected according to the preliminary study described in 8.1.

Anesthesia and supportive dosing Premedication: Hydrocortisone 100 mg and Benadoryl 50 mg IV
Maintenance: 1-2% isoflurane Pain management after surgery: Buprenorphine

Time course and procedure The course of the experiment can be seen in the table below. After 15 minutes of anesthesia, a baseline epidermal test is performed and then observed for 15 minutes. Thereafter, nominally 30 minutes after anesthesia, the dog is given an intravenous bolus injection of Solulin based on its actual body weight. After 30 minutes exposure to Solulin, a second epidermal assay is performed on the contralateral paw. Thereafter, since observation is performed for 15 minutes, the total anesthetic time is approximately 75 minutes.

  For the epidermal bleeding test, the dog is placed in a recumbent position and all hair around the nail bed is removed by cutting around the base of the claws that will be used in the epidermal bleeding time assay. Apply silicone grease to the claws to prevent blood from returning under the claws. Cut the nail proximal to the dorsal nail groove using a guillotine clipper that allows the apex of the epidermis to be seen and the spring-loaded blade slides. The animal's paw is then placed out of the end of the operating table and blood is allowed to fall freely from the cut epidermis. Record the number of blood drops for each 15-minute period thereafter and convert to the 15-minute cumulative score. After observing for 15 minutes, if the epidermis is still bleeding, the injured site is cauterized by topical application of silver nitrate.

  The same experienced veterinarian nurse performs all assays.

Results and Discussion Dogs with severe hemophilia A were studied in vivo. The first dog was injected with a dose of 500 μg / kg, derived by a concentration of several hundred nM Solulin found to be effective in the in vitro experiments presented in Example VII, observed after the in vitro addition of Solulin. It was seen whether an improvement in thromboelastogram could be confirmed in vivo. Contrary to expectations, this dose was not only ineffective, but also severely inhibited clot formation and intensity (Figure 17). This effect was manifested 30 minutes after dosing and was maintained at 2 hours.

  At the end of the day, the decrease in clot formation was still evident, but the inhibition seemed to subside slowly. This experiment could be considered a failure because there was a technical defect in the 24-hour test. Even so, it was decided to draw a final sample at 48 hours to see if the animal recovered. Although this seemed to be the case, it seemed somewhat surprising that there was still a slight improvement trend in the baseline thromboelastogram. Encouraged by this idea, samples were examined 72 hours after Solulin, and quite unexpectedly a clear improvement in clot formation and strength was observed. This is because a much lower dose is effective than would be expected from in vitro studies, and the higher the dose, the higher the plasma concentration will be established and this higher plasma concentration will degrade the characteristics of the clot. This hypothesis was tested in further severe hemophilia dogs (Figure 18).

  The second dog received a much lower dose of Solulin (10 μg / kg) than the first. This experiment supported the hypothesis that higher plasma concentrations have no effect on improving clot properties, but can be improved at concentrations as low as 0.2 nM.

Description of Tables Table 1: List of data used to generate FIG. 4, including absolute dissolution time in the presence of PTCI to allow determination of dissolution time under each condition. In all cases, the dissolution times for those obtained in the presence of the TAFIa inhibitor, PTCI, are displayed. TAFI, thrombin activated fibrinolysis inhibitor; PTCI, potato tuber carboxypeptidase inhibitor.

Table 2: Results after treatment of TM _E (Sf9) site-specific mutant analogs with chloramine T oxidized chloramine T were expressed as a percentage of activity after control treatment. ^* Average value of duplicate determination and deviation from the average.

Claims (34)

A thrombomodulin analog having reduced cofactor activity when bound to thrombin compared to TM _E M338L;
a. B) the amino acid sequence described in SEQ ID NO: 3, or c) the amino acid sequence described in SEQ ID NO: 4, or d) the amino acid of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. An amino acid sequence having at least 90%, more preferably at least 95%, most preferably at least 98% identity to the sequence, or e) six EGF-like repeats of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 Domain (amino acid positions 227 to 462 when numbered in SEQ ID NO: 1), EGF-like repeat domain 3 to EGF-like repeat domain 6 of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 (when numbered in SEQ ID NO: 1) Amino acid positions 307 to 462) or SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: Having a thrombomodulin fragment consisting essentially of the EGF-like repeat domain 6 (amino acid positions 333 to 462 as numbered in SEQ ID NO: 1) from the c-loop of 4 EGF-like repeat domains 3;
Phenylalanine at position 376 (when numbered according to SEQ ID NO: 1) has been deleted or replaced by glycine, alanine, leucine, isoleucine,
Thrombomodulin analog. Further comprising a deletion or substitution of a glutamine residue at position 387 (when numbered in SEQ ID NO: 1), wherein the substitution is preferably Met, Thr, Ala, Glu, His, Arg, Ser, Val, Lys 2. Thrombomodulin according to claim 1, substituted with Gly, Ile, Tr, Tyr, Leu, Asn, Phe, Asp, Cys. Further comprising a deletion or substitution of a methionine residue at position 388 (when numbered in SEQ ID NO), wherein the methionine residue is preferably Gln, Tyr, Ile, Phe, His, Arg, Pro, Val, The thrombomodulin according to claim 1 or 2, which is substituted with Thr, Ser, Ala, Trp, Asn, Lys, Gly, Glu, Asp, Cys. Further comprising a deletion or substitution of a phenylalanine residue at position 389 (when numbered in SEQ ID NO: 1), wherein the phenylalanine is preferably Val, Glu, Thr, Ala, His, Trp, Asp, Gln, Leu A thrombomodulin according to any of the preceding claims, which is substituted with Ile, Asn, Asn, Ser, Arg, Lys, Met, Tyr, Gly, Cys, Pro. 9. Thrombomodulin according to any of the preceding claims comprising one or more first and second amino acid modifications as shown in Table 4. 9. Thrombomodulin according to any of the preceding claims comprising one or more first, second and third amino acid modifications as shown in Table 5. a) a TM analog is defined as described in one of the above claims, or b) said TM analog exhibits an antifibrinolytic effect at a therapeutically effective dosage,
Use of a thrombomodulin analogue for the manufacture of a medicament for the treatment of coagulopathy with hyperfibrinolysis. Said thrombomodulin analogue has the following characteristics:
(I) is reduced as compared to the rabbit lung thrombomodulin, binding affinity to thrombin, and / or binding affinity to thrombin with 0.2nM greater than k _D values, and / or (ii) TM analog Reduced cofactor activity compared to TMEM388L cofactor activity,
(Iii) increased ratio of TAFI activation activity to cofactor activity compared to TM analog TM _E M388L;
8. Use according to claim 7, presenting one or more of: The coagulation disorder with increased fibrinolysis is a group of diseases as follows: hemophilia A, hemophilia B, hemophilia C, von Willebrand disease (vWD), acquired von Willebrand disease, Selected from factor X deficiency, parahaemophilia, inherited disorder of factor I, II, V or VII clotting factor, hemorrhagic disorder with circulating anticoagulants or acquired coagulopathy 8. Use according to 8. The thrombomodulin analog is one of a bleeding event selected from the group consisting of intracranial or other CNS bleeding, bleeding in joints, microcapillaries, muscles, gastrointestinal tract, respiratory tract, retroperitoneal cavity or soft tissue Use according to any of the preceding claims, used to treat the above. Use according to any of the preceding claims, wherein the patient has an anti-factor VIII antibody. 8. A patient according to any of the preceding claims, wherein the patient is treated with factor VIII, preferably recombinant factor VIII or a recombinant B domain deleted factor VIII molecule, more preferably octocog alpha or moloccog alpha. Use of. Said thrombomodulin analogue is provided in combination with factor VIII, preferably recombinant factor VIII, recombinant B domain deleted factor VIII molecule, more preferably octocog alpha or moloccog alpha Use of a thrombomodulin analogue according to any of the preceding claims for the treatment of coagulopathy with hyperfibrinolysis. Use according to any of the preceding claims, wherein the patient is treated with factor VIII, preferably recombinant factor VIII, more preferably Octocog alpha. Use according to any of the preceding claims, wherein the thrombomodulin analogue is administered at the time of a bleeding episode. Use according to any of the preceding claims, wherein the thrombomodulin analogue is administered prior to increased risk of bleeding (eg surgery or tooth extraction). Use according to any of the preceding claims, wherein the thrombomodulin analogue is administered to a patient who is refractory to transfusion / plasma transfusion or coagulation factor replacement therapy. The thrombomodulin analog is administered in multiple doses, preferably once a day, once every two days, or every 3, 4, 5, 6, or 7 days over a total period of less than 1 week to 4 weeks Furthermore, the use according to any of the preceding claims, more preferably administered as long-term administration. Use according to any of the preceding claims, wherein the thrombomodulin analogue is given as a parenteral application, preferably as an intravenous or subcutaneous application. Use according to any of the preceding claims, wherein the thrombomodulin analogue is a soluble TM analogue. 21. Use according to claim 20, wherein the thrombomodulin analog is a human soluble TM analog. Said thrombomodulin analogue comprises at least one structural domain selected from the group comprising EGF3, EGF4, EGF5, EGF6, preferably comprising fragments EGF3-EGF6 and more preferably comprising EGF domains 1-6 Use according to any of the preceding claims. Use according to any of the preceding claims, wherein the thrombomodulin analogue consists of EGF domains EGF1 to EGF6, and more preferably consists of EGF domains EGF3 to EGF6. The thrombomodulin analog has an amino acid sequence corresponding to the amino acid sequence of mature thrombomodulin (shown in SEQ ID NO: 1 or SEQ ID NO: 3), as well as the following modifications:
a) Removal of amino acids 1 to 3 b) M388L
c) R456G
d) H457Q
e) Use according to any of the preceding claims, comprising one or more of S474A and termination at P490. Use according to any of the preceding claims, wherein the thrombomodulin analogue has an amino acid sequence comprising a sequence of at least 85%, or at least 90% or 95% sequence identity with SEQ ID NO: 2. Said thrombomodulin analogue is a native sequence (according to SEQ ID NO: 1 or SEQ ID NO: 3)
aa) ³⁴⁹ Asp;
bb) ³⁵⁵ Asn;
ac) ³⁵⁷ Glu;
ad) ³⁵⁸ Tyr;
ae) ³⁵⁹ Gln;
af) ³⁶¹ Gln;
ag) ³⁶³ Leu;
ah) ³⁶⁴ Asn;
ai) ³⁶⁸ Tyr;
aj) ³⁷¹ Val;
ak) ³⁷⁴ Glu;
al) ³⁷⁶ Phe;
am) ³⁸⁴ His;
an) ³⁸⁵ Arg;
ba) ³⁸⁷ Gln;
bb) ³⁸⁹ Phe;
bc) ³⁹⁸ Asp;
bd) ⁴⁰⁰ Asp;
be) ⁴⁰² Asn;
bf) ⁴⁰³ Thr;
bg) ⁴⁰⁸ Glu;
bh) ⁴¹¹ Glu;
bi) ⁴¹³ Tyr;
bj) ⁴¹⁴ Ile;
bk) ⁴¹⁵ Leu;
bl) ⁴¹⁶ Asp;
bm) ⁴¹⁷ Asp;
bn) ⁴²⁰ Ile;
bo) ⁴²³ Asp;
bp) ⁴²⁴ Ile;
bq) ⁴²⁵ Asp;
br) ⁴²⁶ Glu;
ca) ⁴²⁸ Glu;
cb) ⁴²⁹ Asp;
cc) ⁴³² Phe;
cd) ⁴³⁴ Ser;
ce) ⁴³⁶ Val;
cf) ⁴³⁸ His;
cg) ⁴³⁹ Asp;
ch) ⁴⁴⁰ Leu;
ci) ⁴⁴³ Thr;
cj) ⁴⁴⁴ Phe;
ck) ⁴⁴⁵ Glu;
cl) ⁴⁵⁶ Arg;
cm) ⁴⁵⁸ Ile; or cn) ⁴⁶¹ Asp
Use according to any of the preceding claims, having amino acid modifications at one or more positions corresponding to. The thrombomodulin analog has a phenylalanine modification at position 376 according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably substituted with an aliphatic amino acid, more preferably substituted with glycine, alanine, valine, leucine, or isoleucine. The use according to any of the preceding claims, which is most preferably substituted with alanine. Said thrombomodulin analogue has the following amino acids according to SEQ ID NO: 1 or SEQ ID NO: 3:
a) ³⁸⁷ Gln;
b) ³⁸⁸ Met;
b) ³⁸⁹ Phe;
The amino acid is deleted, the amino acid is inserted with one or more additional amino acids, or the amino acid is preferably substituted, Use according to any of the preceding claims. Use according to any of the preceding claims, wherein the thrombomodulin analogue is used in its oxidized form, preferably oxidized with chloramine T, hydrogen peroxide or sodium periodate. 20. The methionine residue at position 388 (according to SEQ ID NO: 1 or SEQ ID NO: 3) is oxidized, preferably one or more of the methionine residues in the TM analogue is oxidized. Use of. A method of screening for analogs of thrombomodulin suitable for the treatment of coagulation disorders with increased fibrinolysis, comprising:
The thrombomodulin has the following characteristics:
(I) reduced binding affinity to thrombin,
(Ii) reduced cofactor activity,
(Iii) exhibit one or more of increased TAFI activation activities, the method comprising:
a) making one or more amino acid substitutions of the thrombomodulin sequence (SEQ ID NO: 1 or SEQ ID NO: 3), preferably at the amino acid position according to claim 15;
b) The modified analog and a control molecule, preferably rabbit lung TM or soluble human TM analog, with the following properties:
ba) Binding affinity to thrombin (KD value);
bb) cofactor activity;
bc) TAFI activation activity or TAFIa potential;
bd) ratio of TAFI activating activity to cofactor activity;
be) the effect of protein oxidation;
bf) comparing to one or more of the effects on clot lysis over time in an in vitro assay; or bg) in a coagulation related animal model. 21. A method of treating a coagulation disorder associated with hyperfibrinolysis, comprising administering a therapeutically effective amount of a thrombomodulin analog according to any of claims 1-20. 21. Treatment of coagulopathy with hyperfibrinolysis comprising administering a thrombomodulin analogue according to any of claims 1 to 20 at a dose between 0.75 and 140 μg / kg body weight of the subject to be treated Treatment method. 21. A medicament comprising a thrombomodulin analogue according to any of claims 1 to 20 for treating a coagulation disorder with hyperfibrinolysis at a dose between 0.75 and 140 pg / kg body weight of the subject to be treated. Composition.