Intravascular thrombolytic agents

Abstract

The present invention relates to a dissolving agent for 'intravascular thrombus', and more specifically, to a polypeptide, gene and pharmaceutical composition containing the same, which is composed of a thrombus recognition domain and a thrombus dissolving domain for 'intravascular thrombus' and has the function of dissolving 'intravascular thrombus'. The polypeptide of the present invention that recognizes 'intravascular thrombus' and dissolves 'intravascular thrombus' is characterized in that it is composed of a thrombus dissolving domain including an amino acid sequence recorded as sequence number 1 or sequence number 2 and a thrombus recognition domain including an amino acid sequence recorded as sequence number 3 or sequence number 4. The polypeptide that recognizes 'intravascular thrombus' and dissolves thrombus according to the present invention has the effect of preventing and treating thrombosis by dissolving thrombus in the blood of mammals without severe bleeding side effects, and therefore can be widely used as a preventive or therapeutic agent for thrombosis and related diseases.

Description

Technical Field

[0001] The present invention relates to a dissolving agent for 'intravascular thrombus', and more specifically, to a polypeptide, gene and pharmaceutical composition containing the same, which is composed of a thrombus recognition domain and a thrombolytic domain for 'intravascular thrombus' and has the function of dissolving 'intravascular thrombus'. Background Art

[0002] When blood vessel tissue is injured, blood will flow out of the blood vessel, so blood clots will form around the wound to prevent bleeding. Tissue thrombosis that is limited to the wound site to prevent bleeding is a normal wound healing process and is a necessary process for the survival of animals including humans. In contrast, the presence of abnormal blood clots in blood vessels through which blood flows is called "intravascular thrombus". If the intravascular thrombus is not removed, it will block the flow of blood and cause thrombosis. When the flow of blood is blocked, it will cause fatal thrombosis diseases such as cerebral stroke, pulmonary infarction and myocardial infarction, which cause tissue necrosis due to hypoxia. Therefore, if thrombosis occurs, immediate treatment is urgently needed.

[0003] Thrombosis is a very serious disease with a high incidence rate, and various therapeutic agents have been developed. Thrombolytics are direct therapeutic agents for dissolving blood clots, and representative examples include tissue-type plasminogen activator (tPA) (US4766075, US5185259, US5587159, US5869314, US6274335), urokinase (US4259447, US4851345, US5055295, US6759042), and streptokinase (US3855065, US5011686, US7105327). These thrombolytic agents, i.e., tPA (alteplase, reteplase), urokinase, streptokinase, etc., all have the effect of cutting off the plasminogen in the body and activating it into plasmin, and the activated plasmin breaks down the thrombus and treats thrombosis. However, the activated plasmin also breaks down the thrombus used to prevent bleeding from damaged blood vessels, thereby inducing severe bleeding at the wound site. This is because plasmin is a nonspecific thrombolytic agent that does not have specificity for 'intravascular thrombus'. Due to the severe bleeding side effects of plasmin, the thrombolytic agents that produce plasmin, i.e., tPA, urokinase, and streptokinase, are very limited and are only used in certain cases.

[0004] Due to the fatal side effects of thrombolytic agents, in fact, in hospitals, anticoagulants such as heparin, warfarin, davigatran or antiplatelets such as aspirin are used to replace thrombolytic agents for patients with thrombosis. Anticoagulants and antiplatelets will not decompose the thrombus that has been formed, but will prevent the formation of extra thrombus in the blood vessel. That is, anticoagulants and antiplatelet drugs do not have thrombus decomposition ability, so fatal bleeding is less, but there is no large effect on the treatment of thrombosis. In addition, because anticoagulants and antiplatelets can interfere with thrombosis, wound healing is hindered, so that the bleeding side effect is also very serious.

[0005] Therefore, in order to treat thrombosis, it is urgently necessary to develop a dissolving agent that is specific to 'intravascular thrombus', which can accurately identify and decompose only 'intravascular thrombus' and minimize bleeding side effects without interfering with normal wound healing. Summary of the invention

[0006] Technical issues to be solved

[0007] The inventors of the present invention have been developing an "intravascular thrombus" dissolving agent after realizing that an "intravascular thrombus" dissolving agent that accurately dissolves only the "intravascular thrombus" that causes thrombosis without interfering with the normal blood coagulation process and wound healing in vascular tissue is the most ideal thrombosis therapeutic agent. Therefore, the purpose of the present invention is to provide an "intravascular thrombus" dissolving agent of an innovative concept as a thrombosis therapeutic agent, which "intravascular thrombus" dissolving agent accurately dissolves only the "intravascular thrombus" that causes thrombosis without any serious bleeding side effects such as systemic bleeding.

[0008] Solutions to the problem

[0009] In order to achieve the above-mentioned purpose, the present invention provides a polypeptide, which recognizes 'intravascular thrombus' and dissolves 'intravascular thrombus', and is characterized in that it is composed of a thrombus-dissolving domain including an amino acid sequence recorded as sequence number 1 or sequence number 2 and a thrombus-recognition domain including an amino acid sequence recorded as sequence number 3 or sequence number 4.

[0010] The present invention also provides a thrombolytic domain gene, which encodes a thrombolytic domain having an amino acid sequence recorded as sequence number 1 or sequence number 2, and is characterized in that it has a base sequence recorded as sequence number 5 or sequence number 6.

[0011] The present invention also provides a thrombus recognition domain gene, which encodes a thrombus recognition domain having an amino acid sequence recorded as sequence number 3 or sequence number 4, and is characterized in that it has a base sequence recorded as sequence number 7 or sequence number 8.

[0012] The present invention also provides a pharmaceutical composition for treating or preventing thrombosis and related diseases, characterized in that it comprises a polypeptide or a gene encoding the polypeptide as an effective ingredient that recognizes 'intravascular thrombus' and dissolves the thrombus.

[0013] Effects of the Invention

[0014] The polypeptide that recognizes 'intravascular thrombus' and dissolves the thrombus according to the present invention has the effect of preventing and treating thrombosis by dissolving the thrombus in the blood of mammals without severe bleeding side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The results of confirming the thrombus-dissolving ability after treating the isolated thrombus with the thrombolytic enzyme SK or HtrA1 according to Experimental Example 1 of the invention (a; image of the thrombus dissolved (HA1) and the thrombus clot image (SK) without thrombus dissolution, b; thrombus clot solubility expressed in % before treatment, c; thrombus degradation product FDP (fibrin degradation products), d; amount of thrombus degradation product D-dimer (Ctrl: control group, SK: streptokinase, HA1; polypeptide HtrA1 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0016] Figure 2 The results of confirming the thrombus-dissolving ability after treating the isolated thrombus with the thrombolytic enzyme SK or HtrA2 according to Experimental Example 1 of the invention (a; image of dissolved thrombus (HA2) and image of undissolved thrombus (SK); b; solubility of thrombus expressed in % before treatment; c; thrombus degradation product FDP (fibrin degradation products); d; amount of thrombus degradation product D-dimer (Ctrl: control group; SK: streptokinase; HA2; polypeptide HtrA2 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0017] Figure 3 These are the results of confirming the plasminogen activity performance of the 'intravascular thrombus' dissolving polypeptide HtrA1 according to Experimental Example 2 of the invention (a; fluorescence intensity measurement value of the matrix degraded by plasmin generated by the activation of plasminogen, b; SDS-PAGE image confirming the plasmin band generated by the activation of plasminogen (Ctrl: control group, PL: plasmin, SK: streptokinase, UK: urokinase, HA1; polypeptide HtrA1 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0018] Figure 4 These are the results of confirming the plasminogen activity performance of the 'intravascular thrombus' dissolving polypeptide HtrA2 according to Experimental Example 2 of the invention (a; fluorescence intensity measurement value of the matrix degraded by plasmin generated by the activation of plasminogen, b; SDS-PAGE image confirming the plasmin band generated by the activation of plasminogen (Ctrl: control group, PL: plasmin, SK: streptokinase, UK: urokinase, HA2; polypeptide HtrA2 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0019] Figure 5The results of confirming whether the fibrinolysis components appearing in the wound healing process are active in order to evaluate whether the intravascular thrombolytic polypeptide of the present invention has thrombus specificity according to Experimental Example 3 of the present invention (a; an image of SDS-PAGE confirming whether fibrinogen is decomposed into fibrin after fibrinogen is treated with each thrombolytic enzyme, b; an image of immunoblot confirming whether fibronectin is decomposed after cellular fibronectin is treated with each thrombolytic enzyme, c; an image of immunoblot confirming whether fibronectin is decomposed after plasma fibronectin is treated with each thrombolytic enzyme (Ctrl: control group, PL: plasmin, SK: streptokinase, UK: urokinase, HA1; the polypeptide HtrA1 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0020] Figure 6 The thrombotic tail image results are obtained by confirming the therapeutic effect of the 'intravascular thrombus' dissolving polypeptide of the present invention on thrombosis in tail thrombosis mice using an animal model according to Example 1 of the present invention (Ctrl: control group, PL: plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; polypeptides HtrA1 and HA2 of the present invention that recognize 'intravascular thrombus' and dissolve thrombus; polypeptide HtrA2 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0021] Figure 7 The H&E staining image results of the thrombotic tail tissue were obtained by confirming the therapeutic effect of the 'intravascular thrombus' dissolving polypeptide of the present invention on thrombosis in the tail thrombosis mice using the animal model according to Example 1 of the present invention (Ctrl: control group, PL: plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; polypeptides HtrA1 and HA2 of the present invention that recognize 'intravascular thrombus' and dissolve thrombus; polypeptide HtrA2 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus)

[0022] Figure 8 This is the bleeding image result of confirming the effect of the 'intravascular thrombus' dissolving polypeptide of the present invention on wound healing using a trauma animal model according to Example 3 of the present invention (Ctrl: control group, PL: plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; polypeptides HtrA1 and HA2 of the present invention that recognize 'intravascular thrombus' and dissolve thrombus; polypeptide HtrA2 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0023] Fig. 9 The results of the bleeding test using the trauma animal model to confirm the effect of the 'intravascular thrombus' dissolving polypeptide HtrA1 of the present invention on wound healing according to Example 3 of the present invention. a; bleeding time measurement value, b; bleeding volume measurement value, c; hemoglobin content measurement value in bleeding blood, d; wound healing and blood coagulation time measurement value (Ctrl: control group, PL: plasmin, SK: streptokinase, UK: urokinase, HA1; polypeptide HtrA1 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0024] Fig.10 The results of the bleeding test using the trauma animal model to confirm the effect of the 'intravascular thrombus' dissolving polypeptide HtrA2 of the present invention on wound healing according to Example 3 of the present invention. a; bleeding time measurement value, b; bleeding volume measurement value, c; hemoglobin content measurement value in bleeding blood, d; wound healing and blood coagulation time measurement value (Ctrl: control group, PL: plasmin, SK: streptokinase, tPA; tissue plasminogen activator, HA2; polypeptide HtrA2 of the present invention that recognizes 'intravascular thrombus' and dissolves thrombus).

[0025] Best Mode for Carrying Out the Invention

[0026] Various drugs have been developed to remove blood clots, but existing thrombolytic agents have serious bleeding side effects and are difficult to effectively treat thrombosis and related diseases. In addition, these treatments cannot prevent thrombosis in advance.

[0027] The present inventors focused on the fact that 'intravascular thrombus' is a protein aggregate and that quality control protein that degrades aggregated protein is essential for biological survival, and attempted to confirm the existence of an endogenous protease (endogenous proteinase) in the body that has a domain that recognizes misfolded / aggregated proteins and a domain that decomposes and removes misfolded / aggregated proteins, and that the endogenous protease can degrade intravascular thrombus.

[0028] Therefore, in the present invention, domains that recognize misfolded / aggregated proteins, domains that degrade and remove misfolded / aggregated proteins, and quality control proteins containing these domains were explored. As a result, it was confirmed that a polypeptide containing a thrombus recognition domain and a thrombolytic domain can treat thrombosis without severe bleeding side effects by specifically recognizing and degrading "intravascular thrombus" and removing it, thereby recognizing "intravascular thrombus" and dissolving thrombus.

[0029] That is, in the present invention, it can be confirmed that the polypeptide that dissolves thrombus by recognizing "intravascular thrombus" including an intravascular thrombus recognition domain and a thrombolytic domain (1) has excellent thrombolytic ability, (2) unlike existing thrombolytic agents, does not activate plasminogen to plasmin, and therefore does not have the risk of bleeding due to the production of plasmin, (3) unlike existing thrombolytic agents, does not decompose fibrinogen, c-fibronectin and p-fibronectin, which are important for wound healing, and therefore does not interfere with wound healing, (4) effectively treats thrombosis in an in vivo animal model, (5) unlike existing thrombolytic agents, the post-treatment recovery survival rate of thromboembolism in an in vivo animal model reaches 100%, and (6) unlike existing thrombolytic agents, does not interfere with blood coagulation and wound healing in an in vivo bleeding animal experiment, and therefore has no bleeding side effects.

[0030] That is, in one embodiment of the present invention, the results of confirming the therapeutic effect in a mouse tail thrombosis model confirmed that, unlike conventional thrombolytic agents, the polypeptide of the present invention that recognizes and dissolves "intravascular thrombus" has a healing effect on thrombosis.

[0031] In the mouse model of pulmonary thromboembolism, which is another embodiment of the present invention, although individual deaths were confirmed in the group treated with existing thrombolytic agents, the survival rate was 100% in the group treated with the polypeptide that recognizes 'intravascular thrombus' and dissolves the thrombus, thereby confirming that the polypeptide that recognizes 'intravascular thrombus' and dissolves the thrombus of the present invention perfectly treats fatal thromboembolism.

[0032] In addition, the results of confirming the wound healing ability in the mouse tail bleeding experiment confirmed that the wound site bleeding time, bleeding volume, amount of hemoglobin loss, blood clotting time, etc. of the polypeptide treatment group of the present invention that recognizes "intravascular thrombus" and dissolves thrombus are excellent to the level of the untreated control group. Therefore, unlike existing thrombolytic agents, it has a perfect wound healing effect without any bleeding side effects.

[0033] Therefore, on the one hand, the present invention relates to a polypeptide that recognizes 'intravascular thrombus' and dissolves the thrombus, characterized in that it is composed of a thrombus-dissolving domain including an amino acid sequence recorded as sequence number 1 or sequence number 2 and a thrombus-recognition domain including an amino acid sequence recorded as sequence number 3 or sequence number 4.

[0034] In the present invention, the polypeptide may be composed of a thrombolytic domain including an amino acid sequence recorded as sequence number 1 and a thrombus recognition domain including an amino acid sequence recorded as sequence number 3, or may be composed of a thrombolytic domain including an amino acid sequence recorded as sequence number 2 and a thrombus recognition domain including an amino acid sequence recorded as sequence number 4.

[0035] In the present invention, the polypeptide that recognizes 'intravascular thrombus' and dissolves the thrombus is characterized in that it preferably belongs to the High Temperature Requirement (Htr) family of serine proteases, which are trypsin-like polypeptides, and more preferably includes HtrA1 and HtrA2.

[0036] The thrombolytic domain is characterized in that it can be a domain having a similarity (homology) of more than 50%, preferably more than 80%, and more preferably more than 90% with the amino acid sequence of sequence number 1 or sequence number 2. The thrombolytic domain is characterized in that it includes a GNSGGPL peptide or a similar peptide that confers serine protease activity.

[0037] In addition, the thrombus recognition domain is characterized in that it can be a domain having a homology of more than 50%, preferably more than 80%, and more preferably more than 90% with the amino acid sequence of sequence number 3 or sequence number 4. The thrombus recognition domain is characterized in that it has a topology structure composed of multiple combinations of β-folded chains (beta-strand) and α-helix (alpha-helix). That is, the thrombus recognition domain can be a PDZ domain or a PDZ-like domain that has the function of recognizing "intravascular thrombus" and regulating the activity of lytic enzymes.

[0038] On the other hand, the present invention relates to a thrombolytic domain gene, which encodes a thrombolytic domain having an amino acid sequence recorded as sequence number 1 or sequence number 2, and is characterized in that it has a base sequence recorded as sequence number 5 or sequence number 6.

[0039] The thrombolytic domain gene is characterized in that it has a homology of 50% or more, preferably 80% or more, and more preferably 90% or more to the base sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0040] On the other hand, the present invention relates to a thrombus recognition domain gene, which encodes a thrombus recognition domain having an amino acid sequence recorded as sequence number 3 or sequence number 4, and is characterized in that it has a base sequence recorded as sequence number 7 or sequence number 8.

[0041] The thrombus recognition domain gene is characterized in that it has a homology of 50% or more, preferably 80% or more, and more preferably 90% or more to the base sequence of sequence number 7 or sequence number 8.

[0042] In another aspect, the present invention relates to a pharmaceutical composition for treating or preventing thrombosis and related diseases, characterized in that it comprises a polypeptide or a gene encoding the polypeptide that recognizes 'intravascular thrombus' and dissolves the thrombus as an effective ingredient.

[0043] The pharmaceutical composition of the present invention may include pharmaceutically acceptable carriers commonly used in preparations, such as lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but are not limited thereto.

[0044] In addition to the above ingredients, the pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0045] The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally. In the case of parenterally, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc.

[0046] The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways according to factors such as the formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity. On the other hand, the preferred dosage of the pharmaceutical composition of the present invention is 0.0001-1000 μg per day.

[0047] The pharmaceutical composition of the present invention is formulated by using a pharmaceutically acceptable carrier and / or excipient according to a method that is easily implemented by a person of ordinary skill in the art, so that it can be prepared in a unit dose form or prepared by packaging in a multi-dose container. In this case, the preparation can be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or can be in the form of an extract, powder, granule, tablet or capsule, and can also include a dispersant or stabilizer.

[0048] The pharmaceutical composition for treating or preventing thrombosis and related diseases according to the present invention has the effect of dissolving 'intravascular thrombus' and minimizing bleeding side effects when administered to mammals.

[0049] In the present invention, the thrombosis and related diseases are preferably selected from any one of the group consisting of diseases caused by 'intravascular thrombosis' such as thrombosis, embolism, thromboembolism, arterial thromboembolism, venous thromboembolism (VTE), cardiovascular disease, cerebrovascular disease, ischemic disease, etc., but not limited thereto. As a specific example, it is preferably selected from any one of the group consisting of deep vein thrombosis (DVT), pulmonary embolism (PE), ischemic stroke, stroke, cerebral hemorrhage, cerebral infarction, myocardial infarction, cardioplegia and unstable angina, but not limited thereto. DETAILED DESCRIPTION

[0050] Hereinafter, the present invention will be described in more detail by specific examples. However, these examples are only used to describe the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0051] Experimental Example 1: Evaluation of in vitro thrombolytic ability

[0052] HtrA1 and HtrA2, which are polypeptides that recognize 'intravascular thrombi' and dissolve thrombi, were prepared as recombinant proteins. For this purpose, cDNA corresponding to 157-480 of the HtrA1 amino acid sequence (SEQ ID NO. 9) including both the intravascular thrombotic domain and the thrombolytic domain was amplified by PCR using a forward primer (5'-AATTCATATGCAAGGGCAGGAAGATCCCA-3') (SEQ ID NO. 11) and a reverse primer (5'-TATCTCGAGCTATGGGTCAATTTCTTCGGG-3') (SEQ ID NO. 12). The PCR conditions were initial denaturation at 95°C for 5 minutes, followed by amplification (at 95°C for 30 seconds, at 63°C for 1 minute, at 72°C for 2 minutes) repeated 34 times, and then at 72°C for 10 minutes. It was subcloned into the NdeI / XhoI site of the pET28a+ expression vector (Novagen, USA) as an expression vector. The obtained HtrA1 recombinant plasmid was electroporated in E. coli BL21 (DE3) pLysS (Stratagene, USA), and IPTG was added during the culture to express HtrA1. The culture solution of the recombinant E. coli was treated with ultrasound to prepare a cell lysate, which was separated by an econo-pac column (Bio-Rad), and then purified by a PD-10 column (Amersham, US) to obtain the recombinant protein HtrA1. In the case of HtrA2, cDNA corresponding to 134-458 of the amino acid sequence of HtrA2 (SEQ ID NO: 10) was amplified by PCR using a forward primer (5'-GTCCTCGCCCATATGGCCGTCCCTAGCC-3') (SEQ ID NO: 13) and a reverse primer (5'-GGCTCTCTCGAGTCATTCTGTGACCTCAGGG-3') (SEQ ID NO: 14). The PCR conditions were initial denaturation at 95°C for 5 minutes, followed by amplification (at 95°C for 30 seconds, at 65°C for 45 seconds, at 72°C for 1 minute) repeated 34 times and then at 72°C for 10 minutes. After that, the recombinant protein HtrA2 was obtained by subcloning into the pET28a+ expression vector (Novagen, USA) in the same manner as HtrA1 and then expressing.

[0053] The thrombolytic activity was confirmed when the prepared recombinant proteins HtrA1 and HtrA2 were treated with isolated thrombi. Thrombi were formed using platelet-rich blood, and the control group or each thrombolytic enzyme (2 mg / ml) was treated with 50 mM Tris-HCl at 37°C for 24 hours. The weight of the thrombus was measured after treatment with each enzyme and expressed as % of the weight before treatment. In addition, the amount of fibrin degradation products (FDP) and D-dimer generated by the decomposition of fibrin polymers constituting the thrombus was quantified.

[0054] exist Figure 1 In the study, HtrA1 showed a significantly higher thrombolytic capacity ( Figure 1 a, b) and fibrin decomposition capacity ( Figure 1 c, d) are the best. Figure 2 In the case of HtrA2, it still showed superior thrombolytic ability than existing thrombolytic enzymes ( Figure 2 a, b) and fibrin decomposition capacity ( Figure 2 c, d).

[0055] Experimental Example 2: Evaluation of plasmin production ability (thrombolytic mechanism)

[0056] The thrombolytic mechanism of current thrombolytic agents is a mechanism of action in which plasmin-dependent fibrinolysis occurs by activating plasminogen to plasmin. In order to confirm the mechanism of action compared with existing thrombolytic enzymes, each thrombolytic enzyme (0.1 mg / ml) was first added to plasminogen (1.23 μM) and plasmin-specific fluorescent substrate Boc-Glu-Lys-Lys-MCA (100 μM), and then cultured. The activation of plasmin by plasminogen was confirmed by measuring the fluorescence intensity of the substrate dissolved by plasmin. After each enzyme (0.15 mg / ml) was treated with plasminogen (5.14 μM), the plasmin band actually generated by the activation of plasminogen was confirmed by SDS-PAGE.

[0057] When compared to existing thrombolytic enzymes, HtrA1 has no plasminogen activation effect at all ( Figure 3 a), and of course no plasmin was produced ( Figure 3 b). Similarly, HtrA2 had no plasminogen activation effect ( Figure 4 As a result, no plasmin is produced ( Figure 4 b).

[0058] Experimental Example 3: Thrombus-specific evaluation

[0059] In order to evaluate the thrombotic specificity of HtrA1, it was confirmed whether it has activity on the fibrinolytic components that appear during wound healing. To this end, the fibrin clot obtained by reacting fibrinogen with thrombin was incubated with a 50mM Tris-HCl control group or a thrombolytic enzyme (2mg / ml) at 37°C for 24 hours. The dissolution of the fibrin clot was measured using a spectrophotometer at a wavelength of 415nm. In order to observe the activity of HtrA1 on fibrinogen, fibrinogen (5μM) was incubated with a 50mM Tris-HCl control group or each thrombolytic enzyme (0.15mg / ml) at 37°C for 3 hours, and then SDS-PAGE was used to confirm whether the fibrinogen was decomposed. To observe the activity of HtrA1 on fibronectin, fibronectin (1.52 μM) was incubated with each thrombolytic enzyme (0.15 mg / ml) at 37°C for 3 hours, then separated on 4-12% SDS gel and immunostained with anti-cFN (anti-cFN) or anti-pFN (anti-pFN) antibodies.

[0060] As shown in Table 1, in the case of HtrA1 and HtrA2, when compared with the untreated control group, not only did they show statistically significant differences, but also the absorbance of streptokinase, which is an existing thrombolytic enzyme, showed statistically significant differences. In terms of fibrin clot dissolving performance, HtrA1 was the most excellent, followed by HtrA2.

[0061] [Table 1]

[0062] Group A415 Comparison 1.870±0.05 Streptokinase <![CDATA[1.335±0.07 a ]]> Urokinase <![CDATA[1.047±0.06 a ]]> HtrA1 <![CDATA[1.055±0.10 a,b ]]> HtrA2 <![CDATA[1.002±0.09 a,b ]]>

[0063] ap<0.05significant difference from the control group

[0064] bp<0.01Significant difference with streptokinase group

[0065] However, unlike existing thrombolytic enzymes, HtrA1 was shown not to break down fibrinogen, which is important for hemostasis at the wound site. Figure 5 a), nor does it break down cellular fibronectin, which is important for wound healing ( Figure 5 b) and plasma fibronectin (plasma fibr onectin) ( Figure 5 c) and save.

[0066] Finally, to observe the activity of HtrA1 on the wound healing process, wounds (~30 mm) were made by externally incising the tail skin of BALB / c mice. 2). The excised wound fragments were incubated with 50 mM Tris-HCl of the control group or each thrombolytic enzyme (2 mg / ml) at 37°C for 72 hours. Whether the wound has healed was determined by observing the liquid containing the wound fragments and measuring the absorbance at 550 nm. As described above, in the wound tissue experiment using actual animals, since the wound tissue wound did not heal when treated with the existing thrombolytic enzyme, the absorbance value caused by bleeding could be confirmed, but in the case of HtrA1 or HtrA2 treatment, since the absorbance was low to a level similar to that of the control group, it was confirmed that normal wound healing had occurred (Table 2).

[0067] [Table 2]

[0068] Group A550 Comparison 0.235±0.031 Streptokinase <![CDATA[0.471±0.018 a ]]> Urokinase <![CDATA[0.404±0.037 a ]]> HtrA1 <![CDATA[0.203±0.028 a,b ]]> HtrA2 <![CDATA[0.247±0.033 a,b ]]>

[0069] ap<0.05significant difference from the control group

[0070] bp<0.01Significant difference with streptokinase group

[0071] Example 1: Therapeutic effect of thrombosis

[0072] In order to evaluate the therapeutic effect of the product of the present invention on thrombosis, an animal experiment using a tail thrombosis model was conducted. A tail thrombosis model induced by κ-carrageenan was established in 15-week-old female BALB / c mice. Each group (n=8) with tail thrombosis was injected with physiological saline (control group) or each thrombolytic enzyme into the abdominal cavity, and the length and ratio of the thrombotic site were measured 24 hours later. The results are shown in Tables 3 and 4.

[0073] [Table 3]

[0074]

[0075] ap<0.001 and HtrA1(40mg / kg) group

[0076] bp<0.01Significant difference with HtrA1(40mg / kg) group

[0077] As shown in Table 3, in the mouse tail thrombosis model, in the case of the HtrA1-treated group, when compared with the existing thrombolytic enzymes, it was confirmed that the tail length and frequency of the tail thrombosis were significantly reduced, and this thrombolytic effect increased in proportion to the volume. In other words, in the case of HtrA1, it was confirmed that there was a statistically significant level of thrombosis therapeutic effect even compared with the existing thrombolytic enzymes.

[0078] [Table 4]

[0079]

[0080] ap<0.001 and HtrA2 (40 mg / kg) group

[0081] bp<0.01Significant difference with HtrA2(40mg / kg) group

[0082] Furthermore, as shown in Table 4, in the mouse tail thrombosis model, in the case of the HtrA2-treated group, when compared with the existing thrombolytic enzyme, it was confirmed that the tail length and frequency of the tail thrombosis were significantly reduced, and this thrombolytic effect increased in proportion to the volume. In the case of HtrA2, not only compared with the untreated group but also compared with the existing thrombolytic enzyme, there was a statistically significant level of thrombosis therapeutic effect.

[0083] exist Figure 7 In the experiment, the results of H&E staining of the thrombotic tail tissue were observed. As with the thrombotic tail results described above, the results directly confirmed the thrombus clot and the degree of thrombus dissolution. In the case of the HtrA1 and HtrA2 treatment groups, no thrombus clot was found at all, and when compared with existing thrombolytic enzymes, excellent thrombosis treatment was also demonstrated.

[0084] Example 2: Therapeutic Effects of Pulmonary Thromboembolism

[0085] Next, in order to evaluate the preventive and therapeutic effects of thromboembolism, an animal experiment using a pulmonary thromboembolism model was conducted. A pulmonary thromboembolism model induced by adenosine 5'-diphosphate (ADP) was established in 15-week-old female C57BL / 6 mice. Each group of mice with pulmonary thromboembolism (n=8) was fasted for more than 12 hours, and then physiological saline (control group) or thrombolytic enzyme was intravenously injected at a dose of 40 mg / kg. 30 minutes later, ADP (150 mg / kg) was injected into the mice to induce pulmonary thromboembolism, and then death was confirmed. The results are shown in Tables 5 and 6.

[0086] [Table 5]

[0087] Group Dosage (mg / kg) Deaths / Total Protection rate (%) Comparison - 6 / 6 0 Plasmin 40 3 / 6 50 Streptokinase 40 4 / 6 33 Urokinase 40 2 / 6 67 HtrA1 40 0 / 6 100

[0088] From Table 5, in the mouse model of pulmonary thromboembolism, the survival rates of the plasmin, streptokinase and urokinase treatment groups were 50%, 33% and 66% respectively, while the survival rate of the HtrA1 treatment group was 100%. It can be seen that the HtrA1 of the present invention has a perfect therapeutic and preventive effect on fatal pulmonary thromboembolism.

[0089] [Table 6]

[0090] Group Dosage (mg / kg) Deaths / Total Protection rate (%) Comparison - 5 / 5 0 Plasmin 40 2 / 5 60 Streptokinase 40 4 / 5 20 tPA 40 3 / 5 40 HtrA2 40 0 / 5 100

[0091] From Table 6, in the mouse model of pulmonary thromboembolism, the survival rates of the plasmin, streptokinase and tPA treatment groups were 60%, 20% and 40% respectively, while the survival rate of the HtrA2 treatment group was 100%. Based on this, it can be seen that the HtrA2 of the present invention also has a perfect therapeutic and preventive effect on fatal pulmonary thromboembolism.

[0092] Example 3: Effect of Eliminating Risk of In Vivo Bleeding

[0093] When the thrombolytic protein HtrA1 is administered, in order to determine whether there is a risk of in vivo bleeding during wound healing, a tail bleeding examination was performed using 15-week-old C57BL / 6 female mice. Physiological saline (control group) or each thrombolytic enzyme was intravenously injected into the mouse group (n=5) at a dose of 40 mg / kg. After 30 minutes, the tail was cut off from the anesthetized mouse, and blood was collected until the bleeding stopped. The bleeding time and amount were recorded, and the hemoglobin content in the collected blood was measured. In addition, the time required for blood clotting in the cut tail vein of the mouse was recorded.

[0094] like Figure 8 As shown, in the mouse tail bleeding experiment, it was confirmed that HtrA1 and HtrA2 significantly reduced bleeding compared with other thrombolytic enzymes. In the case of the HtrA1-treated group, the actual bleeding time ( Fig. 9 a) Amount of bleeding ( Fig. 9 b) The amount of hemoglobin lost ( Fig. 9 c) Time taken to stop bleeding ( Fig. 9 The results of d) confirmed that the bleeding side effect could not be reduced as compared with the existing thrombolytic enzymes and was at a level similar to that of the control group.

[0095] In the case of the HtrA2-treated group, actual bleeding time was also measured ( Fig.10 a) Amount of bleeding ( Fig.10 b) The amount of hemoglobin lost ( Fig.10 c) Time taken to stop bleeding ( Fig.10 The results of d) confirmed that the bleeding side effect could not be reduced compared with the existing thrombolytic enzymes and was at a level similar to the control group. HtrA1 and HtrA2 do not interfere with the hemostasis process as part of the wound healing process, thus perfectly reducing the risk of bleeding, and therefore it is known that they can be used as a thrombosis therapeutic agent with significantly reduced bleeding side effects.

[0096] Possible industrial applications

[0097] The polypeptide that recognizes and dissolves intravascular thrombi according to the present invention has the effect of preventing and treating thrombosis by dissolving thrombi in mammalian blood without severe bleeding side effects, and can therefore be widely used as a preventive or therapeutic agent for thrombosis and related diseases. <110> JINIS Co., Ltd. <120> Intravascular thrombolytic agents <130> OPP20020-PCT <150> KR 2019-0131585 <151> 2019-10-22 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 161 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the thrombolytic domain of HtrA1 (HtrA1 as1) <400> 1 Gly Ser Gly Phe Ile Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala 1 5 10 15 His Val Val Thr Asn Lys His Arg Val Lys Val Glu Leu Lys Asn Gly 20 25 30 Ala Thr Tyr Glu Ala Lys Ile Lys Asp Val Asp Glu Lys Ala Asp Ile 35 40 45 Ala Leu Ile Lys Ile Asp His Gln Gly Lys Leu Pro Val Leu Leu Leu 50 55 60 Gly Arg Ser Ser Glu Leu Arg Pro Gly Glu Phe Val Val Ala Ile Gly 65 70 75 80 Ser Pro Phe Ser Leu Gln Asn Thr Val Thr Thr Gly Ile Val Ser Thr 85 90 95 Thr Gln Arg Gly Gly Lys Glu Leu Gly Leu Arg Asn Ser Asp Met Asp 100 105 110 Tyr Ile Gln Thr Asp Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro 115 120 125 Leu Val Asn Leu Asp Gly Glu Val Ile Gly Ile Asn Thr Leu Lys Val 130 135 140 Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp Lys Ile Lys Lys Phe 145 150 155 160 Leu <210> 2 <211> 177 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the thrombolytic domain of HtrA2 (HtrA2 as1) <400> 2 Ile Leu Asp Arg His Pro Phe Leu Gly Arg Glu Val Pro Ile Ser Asn 1 5 10 15 Gly Ser Gly Phe Val Val Ala Ala Asp Gly Leu Ile Val Thr Asn Ala 20 25 30 His Val Val Ala Asp Arg Arg Arg Val Arg Val Arg Leu Leu Ser Gly 35 40 45 Asp Thr Tyr Glu Ala Val Val Thr Ala Val Asp Pro Val Ala Asp Ile 50 55 60 Ala Thr Leu Arg Ile Gln Thr Lys Glu Pro Leu Pro Thr Leu Pro Leu 65 70 75 80 Gly Arg Ser Ala Asp Val Arg Gln Gly Glu Phe Val Val Ala Met Gly 85 90 95 Ser Pro Phe Ala Leu Gln Asn Thr Ile Thr Ser Gly Ile Val Ser Ser 100 105 110 Ala Gln Arg Pro Ala Arg Asp Leu Gly Leu Pro Gln Thr Asn Val Glu 115 120 125 Tyr Ile Gln Thr Asp Ala Ala Ile Asp Phe Gly Asn Ser Gly Gly Pro 130 135 140 Leu Val Asn Leu Asp Gly Glu Val Ile Gly Val Asn Thr Met Lys Val 145 150 155 160 Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp Arg Leu Arg Glu Phe 165 170 175 Leu <210> 3 <211> 103 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the thrombus recognition domain of HtrA1 (HtrA1 as2) <400> 3 Thr Glu Ser His Asp Arg Gln Ala Lys Gly Lys Ala Ile Thr Lys Lys 1 5 10 15 Lys Tyr Ile Gly Ile Arg Met Met Ser Leu Thr Ser Ser Lys Ala Lys 20 25 30 Glu Leu Lys Asp Arg His Arg Asp Phe Pro Asp Val Ile Ser Gly Ala 35 40 45 Tyr Ile Ile Glu Val Ile Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu 50 55 60 Lys Glu Asn Asp Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Ser 65 70 75 80 Ala Asn Asp Val Ser Asp Val Ile Lys Arg Glu Ser Thr Leu Asn Met 85 90 95 Val Val Arg Arg Gly Asn Glu 100 <210> 4 <211> 82 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the thrombus recognition domain of HtrA2 (HtrA2 as2) <400> 4 Val Met Met Leu Thr Leu Ser Pro Ser Ile Leu Ala Glu Leu Gln Leu 1 5 10 15 Arg Glu Pro Ser Phe Pro Asp Val Gln His Gly Val Leu Ile His Lys 20 25 30 Val Ile Leu Gly Ser Pro Ala His Arg Ala Gly Leu Arg Pro Gly Asp 35 40 45 Val Ile Leu Ala Ile Gly Glu Gln Met Val Gln Asn Ala Glu Asp Val 50 55 60 White Glue Ala Val Arg Thr Gln Ser Gln Leu Ala Val Gln Ile Arg Arg 65 70 75 80 Gly Arg <210> 5 <211> 483 <212> DNA <213> The snowstorm <220> <223> HtrA1-binding transcription factor (HtrA1 bs1) <400> 5 gggtctgggt ttattgtgtc ggagatgga ctgatcgtga caaatgccca cgtggtgacc 120. aacaagcacc gggtcaaagt tgagctgaag aacggtgcca cttacgaagc caaaatcaag gatgtggatg agaaagcaga catcgcactc atcaaaattg accaccaggg caagctgcct gtcctgctgc ttggccgctc ctcagagctg cggccggggag agttcgtggt cgccatcgga 240 agcccgtttt cccttcaaaa cacagtcacc accgggatcg tgagcaccac ccagcgaggc 300 ggcaaagagc tggggctccg caactcagac atggactaca tccagaccga cgccatcatc aactatggaa actcgggagg cccgttagta aacctggacg gtgaagtgat tggaattaac 420 actttgaaag tgacagctgg aatctccttt gcaatcccat ctgataagat taaaaagttc 480 ctc 483 <210> 6 <211> 531 <212> DNA <213> Artificial sequence <220> <223> Base sequence of the thrombolytic domain of HtrA2 (HtrA2 bs1) <400> 6 atcctggacc ggcacccttt cttgggccgc gaggtcccta tctcgaacgg ctcaggattc 60 gtggtggctg ccgatgggct cattgtcacc aacgcccatg tggtggctga tcggcgcaga 120 gtccgtgtga gactgctaag cggcgacacg tatgaggccg tggtcacagc tgtggatccc 180 gtggcagaca tcgcaacgct gaggattcag actaaggagc ctctccccac gctgcctctg 240 ggacgctcag ctgatgtccg gcaaggggag tttgttgttg ccatgggaag tccctttgca 300 ctgcagaaca cgatcacatc cggcattgtt agctctgctc agcgtccagc cagagacctg 360 ggactccccc aaaccaatgt ggaatacatt caaactgatg cagctattga ttttggaaac 420 tctggaggtc ccctggttaa cctggatggg gaggtgattg gagtgaacac catgaaggtc 480 acagctggaa tctcctttgc catcccttct gatcgtcttc gagagtttct g 531 <210> 7 <211> 309 <212> DNA <213> Artificial sequence <220> <223> HtrA1 thrombus recognition domain base sequence (HtrA1 bs2) <400> 7 acggagtccc atgaccgaca ggccaaagga aaagccatca ccaagaagaa gtatattggt 60 atccgaatga tgtcactcac gtccagcaaa gccaaagagc tgaaggaccg gcaccgggac 120 ttccccagacg tgatctcagg agcgtatata attgaagtaa ttcctgatac cccagcagaa 180 gctggtggtc tcaaggaaaa cgacgtcata atcagcatca atggacagtc cgtggtctcc 240 gccaatgatg tcagcgacgt cattaaaagg gaaagcaccc tgaacatggt ggtccgcagg 300 ggtaatgaa 309 <210> 8 <211> 246 <212> DNA <213> Artificial sequence <220> <223> HtrA2 bs2 thrombus recognition domain base sequence <400> 8 gtgatgatgc tgaccctgag tcccagcatc cttgctgaac tacagcttcg agaaccaagc 60 tttcccgatg ttcagcatgg tgtactcatc cataaagtca tcctgggctc ccctgcacac 120 cgggctggtc tgcggcctgg tgatgtgatt ttggccattg gggagcagat ggtacaaaat 180 gctgaagatg tttatgaagc tgttcgaacc caatcccagt tggcagtgca gatccggcgg 240 ggacga 246 <210> 9 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HtrA1 (HtrA1) <400> 9 Met Gln Ile Pro Arg Ala Ala Leu Leu Pro Leu Leu Leu Leu Leu Leu 1 5 10 15 Ala Ala Pro Ala Ser Ala Gln Leu Ser Arg Ala Gly Arg Ser Ala Pro 20 25 30 Leu Ala Ala Gly Cys Pro Asp Arg Cys Glu Pro Ala Arg Cys Pro Pro 35 40 45 Gln Pro Glu His Cys Glu Gly Gly Arg Ala Arg Asp Ala Cys Gly Cys 50 55 60 Cys Glu Val Cys Gly Ala Pro Glu Gly Ala Ala Cys Gly Leu Gln Glu 65 70 75 80 Gly Pro Cys Gly Glu Gly Leu Gln Cys Val Val Pro Phe Gly Val Pro 85 90 95 Ala Ser Ala Thr Val Arg Arg Arg Ala Gln Ala Gly Leu Cys Val Cys 100 105 110 Ala Ser Ser Glu Pro Val Cys Gly Ser Asp Ala Asn Thr Tyr Ala Asn 115 120 125 Leu Cys Gln Leu Arg Ala Ala Ser Arg Arg Ser Glu Arg Leu His Arg 130 135 140 Pro Pro Val Ile Val Leu Gln Arg Gly Ala Cys Gly Gln Gly Gln Glu 145 150 155 160 Asp Pro Asn Ser Leu Arg His Lys Tyr Asn Phe Ile Ala Asp Val Val 165 170 175 Glu Lys Ile Ala Pro Ala Val Val His Ile Glu Leu Phe Arg Lys Leu 180 185 190 Pro Phe Ser Lys Arg Glu Val Pro Val Ala Ser Gly Ser Gly Phe Ile 195 200 205 Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala His Val Val Thr Asn 210 215 220 Lys His Arg Val Lys Val Glu Leu Lys Asn Gly Ala Thr Tyr Glu Ala 225 230 235 240 Lys Ile Lys Asp Val Asp Glu Lys Ala Asp Ile Ala Leu Ile Lys Ile 245 250 255 Asp His Gln Gly Lys Leu Pro Val Leu Leu Leu Gly Arg Ser Ser Glu 260 265 270 Leu Arg Pro Gly Glu Phe Val Val Ala Ile Gly Ser Pro Phe Ser Leu 275 280 285 Gln Asn Thr Val Thr Thr Gly Ile Val Ser Thr Thr Gln Arg Gly Gly 290 295 300 Lys Glu Leu Gly Leu Arg Asn Ser Asp Met Asp Tyr Ile Gln Thr Asp 305 310 315 320 Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu Val Asn Leu Asp 325 330 335 Gly Glu Val Ile Gly Ile Asn Thr Leu Lys Val Thr Ala Gly Ile Ser 340 345 350 Phe Ala Ile Pro Ser Asp Lys Ile Lys Lys Phe Leu Thr Glu Ser His 355 360 365 Asp Arg Gln Ala Lys Gly Lys Ala Ile Thr Lys Lys Lys Tyr Ile Gly 370 375 380 Ile Arg Met Met Ser Leu Thr Ser Ser Lys Ala Lys Glu Leu Lys Asp 385 390 395 400 Arg His Arg Asp Phe Pro Asp Val Ile Ser Gly Ala Tyr Ile Ile Glu 405 410 415 Val Ile Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp 420 425 430 Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Ser Ala Asn Asp Val 435 440 445 Ser Asp Val Ile Lys Arg Glu Ser Thr Leu Asn Met Val Val Arg Arg 450 455 460 Gly Asn Glu Asp Ile Met Ile Thr Val Ile Pro Glu Glu Ile Asp Pro 465 470 475 480 <210> 10 <211> 458 <212> PRT <213> artificial sequence <220> <223> HtrA2's amino acid sequence (HtrA2) <400> 10 Met Ala Ala Pro Arg Ala Gly Arg Gly Ala Gly Trp Ser Leu Arg Ala 1 5 10 15 Trp Arg Ala Leu Gly Gly Ile Arg Trp Gly Arg Arg Pro Arg Leu Thr 20 25 30 Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Ser Asp Pro Arg Ala 35 40 45 Arg Val Thr Tyr Gly Thr Pro Ser Leu Trp Ala Arg Leu Ser Val Gly 50 55 60 Val Thr Glu Pro Arg Ala Cys Leu Thr Ser Gly Thr Pro Gly Pro Arg 65 70 75 80 Ala Gln Leu Thr Ala Val Thr Pro Asp Thr Arg Thr Arg Glu Ala Ser 85 90 95 Glu Asn Ser Gly Thr Arg Ser Arg Ala Trp Leu Ala Val Ala Leu Gly 100 105 110 Ala Gly Gly Ala Val Leu Leu Leu Leu Trp Gly Gly Gly Arg Gly Pro 115 120 125 Pro Ala Val Leu Ala Ala Val Pro Ser Pro Pro Pro Ala Ser Pro Arg 130 135 140 Ser Gln Tyr Asn Phe Ile Ala Asp Val Val Glu Lys Thr Ala Pro Ala 145 150 155 160 Val Val Tyr Ile Glu Ile Leu Asp Arg His Pro Phe Leu Gly Arg Glu 165 170 175 Val Pro Ile Ser Asn Gly Ser Gly Phe Val Val Ala Ala Asp Gly Leu 180 185 190 Ile Val Thr Asn Ala His Val Val Ala Asp Arg Arg Arg Val Arg Val 195 200 205 Arg Leu Leu Ser Gly Asp Thr Tyr Glu Ala Val Val Thr Ala Val Asp 210 215 220 Pro Val Ala Asp Ile Ala Thr Leu Arg Ile Gln Thr Lys Glu Pro Leu 225 230 235 240 Pro Thr Leu Pro Leu Gly Arg Ser Ala Asp Val Arg Gln Gly Glu Phe 245 250 255 Val Val Ala Met Gly Ser Pro Phe Ala Leu Gln Asn Thr Ile Thr Ser 260 265 270 Gly Ile Val Ser Ser Ala Gln Arg Pro Ala Arg Asp Leu Gly Leu Pro 275 280 285 Gln Thr Asn Val Glu Tyr Ile Gln Thr Asp Ala Ala Ile Asp Phe Gly 290 295 300 Asn Ser Gly Gly Pro Leu Val Asn Leu Asp Gly Glu Val Ile Gly Val 305 310 315 320 Asn Thr Met Lys Val Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp 325 330 335 Arg Leu Arg Glu Phe Leu His Arg Gly Glu Lys Lys Asn Ser Ser Ser 340 345 350 Gly Ile Ser Gly Ser Gln Arg Arg Tyr Ile Gly Val Met Met Leu Thr 355 360 365 Leu Ser Pro Ser Ile Leu Ala Glu Leu Gln Leu Arg Glu Pro Ser Phe 370 375 380 Pro Asp Val Gln His Gly Val Leu Ile His Lys Val Ile Leu Gly Ser 385 390 395 400 Pro Ala His Arg Ala Gly Leu Arg Pro Gly Asp Val Ile Leu Ala Ile 405 410 415 Gly Glu Gln Met Val Gln Asn Ala Glu Asp Val Tyr Glu Ala Val Arg 420 425 430 Thr Gln Ser Gln Leu Ala Val Gln Ile Arg Arg Gly Arg Glu Thr Leu 435 440 445 Thr Leu Tyr Val Thr Pro Glu Val Thr Glu 450 455 <210> 11 <211> 29 <212> DNA <213> Artificial sequence <220> <223> HtrA1 forward primer <400> 11 aattcatatg caagggcagg aagatccca 29 <210> 12 <211> 30 <212> DNA <213> Artificial sequence <220> <223> HtrA1 reverse primer <400> 12 tatctcgagc tatgggtcaa tttcttcggg 30 <210> 13 <211> 28 <212> DNA <213> Artificial sequence <220> <223> HtrA2 forward primer <400> 13 gtcctcgccc atatggccgt ccctagcc 28 <210> 14 <211> 29 <212> DNA <213> Artificial sequence <220> <223> HtrA2 reverse primer <400> 14 ggctctcgag tcattctgtg acctcaggg 29

Claims

1. Use of a polypeptide consisting of the amino acid sequence 157-480 of the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence 134-458 of the amino acid sequence of SEQ ID NO: 10, in the preparation of a medicament for treating or preventing thrombosis.

Images