KR20210047828A - Thrombolytic agents for Intravascular clots - Google Patents

Abstract

The present invention relates to an "intravascular thrombus" lytic agent and, more particularly, to a polypeptide, a gene, and a pharmaceutical composition comprising the same, wherein the polypeptide is composed of a thrombo-recognition domain and a thrombolytic domain for "intravascular thrombus" and functions to lyse an "intravascular thrombus". The polypeptide recognizing and lysing an "intravascular thrombus" of the present invention is composed of a thrombolytic domain including the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and a thrombo-recognition domain including the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. The polypeptide that recognizes and lyses an "intravascular thrombus" according to the present invention lyses intravascular thrombi in mammals without significant hemorrhage side effects and has a prophylactic and therapeutic effect on thrombosis, thus finding wide applications as a prophylactic or therapeutic agent for thrombosis and related diseases.

Description

'Thrombolytic agents for Intravascular clots}

The present invention relates to an agent for dissolving'blood clots in blood vessels', and more particularly, consisting of a thrombo-recognition domain and a thrombolytic domain for'blood clots in blood vessels', and'blood clots in blood vessels' It relates to a polypeptide, a gene, and a pharmaceutical composition containing the same.

When a vascular tissue is damaged, blood comes out of the blood vessel, so a blood clot is formed in the vascular tissue around the wound to prevent bleeding. A blood clot, which is limited to the wound site and prevents bleeding, is a normal wound healing process and is an essential process for the survival of animals including humans. In contrast, a case where an abnormal blood clot is present in a blood vessel through which blood flows is called an'intravascular thrombus'. If the blood clots in the blood vessels are not removed, they block the flow of blood and cause thrombosis. Blocked blood flow can lead to fatal thrombosis, such as stroke, menopause, and myocardial infarction, which causes tissue necrosis due to hypoxia. Therefore, when thrombosis occurs, immediate treatment is urgent.

Thrombosis is a very serious disease and its incidence is high, and various treatments have been developed. Thrombolytics are direct treatments that dissolve blood clots, tissue-type plasminogen activator (tPA) (US4766075, US5185259, US5587159, US5869314, US6274335), urokinase (US4259447, US4851345, US5055295, US6759042), streptokinase (US3855065, US5011686, US5011686) Is representative. All of these thrombolytic agents, such as tPA (alteplase, reteplase), urokinase, and streptokinase, cleave plasminogen in the body and activate it with plasmin, and the activated plasmin decomposes blood clots and has a therapeutic effect on thrombosis. However, activated plasmin degrades blood clots to prevent bleeding of damaged blood vessels, causing severe bleeding from the wound site. This is because plasmin is a non-specific thrombolytic agent that has no specificity for'blood clots in blood vessels'. Because of the severe bleeding side effects of plasmin, the thrombolytic agents that produce plasmin, such as tPA, urokinase, and streptokinase, are all very limited and used in only a few cases.

Because of the fatal side effects of thrombolytic drugs, in actual hospitals, anticoagulants such as heparin, warfarin, and davigatran or antiplatelet drugs such as aspirin are used instead of thrombolytics for patients with thrombosis. Anticoagulants and antiplatelet agents do not break down blood clots that have already been formed, but prevent the formation of additional clots in the blood vessels. In other words, anticoagulants and antiplatelet drugs do not have thrombolytic ability, so fatal bleeding is small, but they are not very effective in treating thrombosis. In addition, anticoagulants and antiplatelet agents interfere with the formation of blood clots, thus hindering wound healing, and bleeding side effects are also serious.

Therefore, in order to treat thrombosis, it is urgently needed to develop a dissolving agent having specificity for'blood clots' that accurately recognizes and decomposes only'intravascular thrombi' and minimizes bleeding side effects by not interfering with normal wound healing.

The inventors of the present invention recognized the fact that the'intravascular thrombus' dissolving agent that accurately decomposes only the'intravascular thrombus' that causes thrombosis without interfering with the blood clotting process and wound healing normally occurring in the vascular tissue, is the most ideal thrombosis treatment, ' Efforts have been made to develop an intravascular thrombus dissolving agent. Accordingly, an object of the present invention is to provide an innovative concept of a'intravascular thrombus' dissolving agent as a therapeutic agent for thrombosis, which does not have any serious bleeding side effects such as systemic bleeding by accurately dissolving only the'intravascular thrombus' causing thrombosis.

In order to achieve the above object, the present invention is composed of a thrombolytic domain comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and a thrombus recognition domain comprising an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 It provides a polypeptide that recognizes'intravascular thrombus' and dissolves'intravascular thrombus'.

The present invention also provides a thrombolytic domain gene, characterized in that it has a nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, which encodes a thrombolytic domain having an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. to provide.

The present invention further comprises a thrombosis recognition domain gene, characterized in that it has a nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8, encoding a thrombus recognition domain having an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 to provide.

The present invention also provides a pharmaceutical composition for the treatment or prevention of thrombosis and related diseases, characterized in that it comprises a polypeptide that dissolves a blood clot by recognizing a'blood clot' or a gene encoding the same as an active ingredient.

According to the present invention, the polypeptide that dissolves blood clots by recognizing'thrombosis in blood vessels' has the effect of preventing and treating thrombosis by dissolving blood clots in the blood of mammals without serious bleeding side effects, thus preventing thrombosis and related diseases. Or it can be widely used as a therapeutic agent.

1 is a result of confirming the thrombolytic ability after treatment with the thrombolytic enzyme SK or HtrA1 on the ex vivo thrombus according to Experimental Example 1 of the present invention (a; the image in which the thrombus is dissolved (HA1) and the undissolved thrombus clump Image (SK) b; blood clot solubility in% before treatment, c; fibrin degradation products (FDP), d; amount of D-dimer, a decomposition product of blood clots. (Ctrl: control, SK: streptokinase, HA1; polypeptide HtrA1 that dissolves blood clots by recognizing the'thrombosis in blood vessels' of the present invention).
Figure 2 is a result of confirming the thrombolytic ability after treatment with the thrombolytic enzyme SK or HtrA2 on the ex vivo thrombus according to Experimental Example 1 of the present invention (a; the image in which the thrombi are dissolved (HA2) and the undissolved thrombus clump Image (SK), b; blood clot solubility in% before treatment, c; fibrin degradation products (FDP), d; amount of D-dimer, a decomposition product of blood clots. (Ctrl: control, SK: streptokinase, HA2; polypeptide HtrA2 that dissolves blood clots by recognizing the'thrombosis in blood vessels' of the present invention).
3 is a result of confirming the plasminogen activity of the'blood clot' lysis polypeptide HtrA1 according to Experimental Example 2 of the present invention (a; fluorescence intensity measurement of a substrate degraded by plasmin generated by activation of plasminogen, b; SDS-PAGE image confirming the plasmin band generated by activation of plasminogen). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA1; polypeptide HtrA1 that recognizes the'intravascular thrombus' of the present invention and dissolves thrombi).
4 is a result of confirming the plasminogen activity of HtrA2, the polypeptide for lysis of'blood clots' according to Experimental Example 2 of the present invention (a; fluorescence intensity measurement of a substrate degraded by plasmin generated by activation of plasminogen, b; SDS-PAGE image confirming the plasmin band generated by activation of plasminogen). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA2; polypeptide HtrA2 that dissolves thrombi by recognizing the'thrombosis in blood vessels' of the present invention).
5 is a result of confirming the activity of fibrinolysis components that appear in the wound healing process in order to evaluate whether the intravascular thrombolytic polypeptide of the present invention has thrombogenicity according to Experimental Example 3 of the present invention (a; fibrinogen SDS-PAGE confirms whether fibrinogen is degraded into fibrin after treatment with each thrombolytic enzyme in the b; image confirming whether fibronectin is degraded by immuno-blot after treating each thrombolytic enzyme in cellular fibronectin, c ; After treatment with each thrombolytic enzyme in plasma fibronectin, the image confirmed by immuno-blot whether fibronectin was degraded). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA1; polypeptide HtrA1 that recognizes the'intravascular thrombus' of the present invention and dissolves thrombi).
6 is a thrombosis tail image result confirming the therapeutic efficacy of the'intravascular thrombus' lytic polypeptide of the present invention for thrombosis in tail thrombosis mice using an animal model according to Example 1 of the present invention (Ctrl: control, PL: Plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; Polypeptides HtrA1, HA2 that dissolve thrombi by recognizing the'intravascular thrombus' of the present invention; the'intravascular thrombus' of the present invention Polypeptides HtrA2 that recognizes and dissolves blood clots).
7 is an H & E staining image result of thrombosis tail tissue confirming the therapeutic efficacy of the'intravascular thrombus' lysis polypeptide of the present invention for thrombosis in tail thrombosis mice using an animal model according to Example 1 of the present invention ( Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; Polypeptides HtrA1, HA2 that dissolve thrombi by recognizing the'intravascular thrombus' of the present invention; of the present invention Polypeptide HtrA2 that recognizes'thrombosis in blood vessels' and dissolves blood clots.
8 is a bleeding image result confirming the effect of the'intravascular thrombus' lysis polypeptide of the present invention on wound healing using a wound animal model according to Example 3 of the present invention (Ctrl: control, PL: plasmin, SK : Streptokinase, UK; Urokinase, tPA; tissue plasminogen activator, HA1; Polypeptides HtrA1, HA2 that dissolves blood clots by recognizing the'thrombosis in blood vessels' of the present invention; Solubilizing polypeptide HtrA2).
9 is a bleeding test result confirming the effect of the'intravascular thrombus' lysis polypeptide HtrA1 of the present invention on wound healing using a wound animal model according to Example 3 of the present invention (a; measured bleeding time, b; amount of bleeding) Measurement, c; measurement of hemoglobin content in bleeding fluid, d; measurement of time taken to wound healing and blood clotting). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA1; polypeptide HtrA1 that recognizes the'intravascular thrombus' of the present invention and dissolves thrombi).
10 is a bleeding test result confirming the effect of the'intravascular thrombus' lysis polypeptide HtrA2 of the present invention on wound healing using a wound animal model according to Example 3 of the present invention (a; measured bleeding time, b; amount of bleeding) Measurement, c; measurement of hemoglobin content in bleeding fluid, d; measurement of time taken to wound healing and blood clotting). (Ctrl: control, PL: plasmin, SK: streptokinase, tPA; tissue plasminogen activator, HA2; polypeptide HtrA2 that dissolves blood clots by recognizing the'thrombosis in blood vessels' of the present invention).

Various drugs have been developed to remove blood clots, but all of the existing thrombolytic agents have serious bleeding side effects, making it difficult to effectively treat thrombosis and related diseases. In addition, these treatments cannot prevent thrombosis in advance.

The present inventors focused on the fact that'blood clots in blood vessels' are a kind of protein aggregate, and that quality control proteins that degrade aggregated proteins are essential for the survival of living organisms, so that domains that recognize misfolded/aggregated proteins and misfolded/aggregated proteins are identified. We tried to confirm that endogenous proteinase, which has a domain that is decomposed and removed, exists in the body, and that this endogenous proteinase can degrade blood clots in blood vessels.

Accordingly, 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 searched. As a result, they included a thrombosis recognition domain and a thrombolytic domain. By specifically recognizing and decomposing and removing the'intravascular thrombus', it was confirmed that the polypeptide that recognizes the'intravascular thrombus' and dissolves the thrombus can treat thrombosis without serious bleeding side effects.

That is, in the present invention, the polypeptide that dissolves a blood clot by recognizing'intravascular thrombus' including an intravascular thrombus recognition domain and a thrombolytic domain (1) has excellent thrombolytic ability, and (2) unlike existing thrombolytic agents. , As it does not activate plasminogen with plasmin, there is no risk of bleeding due to plasmin production.(3) Unlike conventional thrombolytic agents, it does not decompose fibrinogen, c-fibronectin, and p-fibronectin, which are important for wound healing. (4) effectively treats thrombosis in an in vivo animal model, (5), unlike conventional thrombolytic drugs, cures the survival rate after treatment of thromboembolism in an in vivo animal model to 100%, and (6) Unlike conventional thrombolytic drugs, in vivo bleeding animal experiments did not interfere with blood clotting and wound healing, so it was confirmed that there were no bleeding side effects.

That is, in one embodiment of the present invention, as a result of confirming the treatment efficacy in a mouse tail thrombosis model, it was confirmed that the polypeptide that recognizes and dissolves the'intravascular thrombus' of the present invention has a cure effect on thrombosis unlike the existing thrombolytic agents. I did.

In another embodiment of the present invention, in the pulmonary thromboembolic mouse model, individual death can be confirmed in the group treated with existing thrombolytic agents, but the survival rate 100% in the polypeptide treatment group that dissolves the thrombus by recognizing the'intravascular thrombus' of the present invention. As a result, it was confirmed that the polypeptide that dissolves the blood clot by recognizing the'intravascular thrombus' of the present invention completely cures the lethal thromboembolism.

In addition, as a result of confirming the wound healing ability in the mouse tail bleeding experiment, the polypeptide-treated group that recognizes the'intravascular thrombus' of the present invention and dissolves the thrombus is the bleeding time at the wound site, the amount of hemorrhage, the amount of hemoglobin lost, the blood clotting time It was excellent at the level of this untreated control group, and unlike existing thrombolytic agents, it was confirmed that it had a perfect wound healing effect without any bleeding side effects.

Therefore, in one aspect, the present invention is characterized by consisting of a thrombolytic domain comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and a thrombus recognition domain comprising an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 As described above, it relates to a polypeptide that dissolves a blood clot by recognizing an'intravascular thrombus'.

In the present invention, the polypeptide is composed of a thrombolytic domain comprising an amino acid sequence represented by SEQ ID NO: 1 and a thrombus recognition domain comprising an amino acid sequence represented by SEQ ID NO: 3, or the polypeptide is represented by SEQ ID NO: 2. It may consist of a thrombolytic domain including an amino acid sequence and a thrombosis recognition domain including an amino acid sequence represented by SEQ ID NO: 4.

In the present invention, the polypeptide that dissolves the blood clot by recognizing the'blood clot' is preferably a trypsin-like polypeptide, characterized in that it belongs to the High Temperature Requirement (Htr) family of serine protease, and more preferably It is characterized by including HtrA1 and HtrA2.

The thrombolytic domain may be a domain having 50% or more, preferably 80% or more, more preferably 90% or more similarity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the thrombolytic domain is a GNSGGPL peptide or It is characterized in that it is a domain that confers serine protease activity, including similar peptides.

In addition, the thrombus recognition domain may be a domain having 50% or more, preferably 80% or more, more preferably 90% or more similarity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and the thrombus recognition domain, It is characterized by having a topology structure composed of a plurality of combinations of beta-strand and alpha-helix. That is, the thrombus recognition domain may be a PDZ domain or a PDZ-like domain having a function of recognizing'blood clots in blood vessels' and regulating the activity of lytic enzymes.

In another aspect, the present invention encodes a thrombolytic domain having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, characterized in that it has a nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, thrombolytic domain It's about genes.

The thrombolytic domain gene is characterized by having 70% or more, preferably 80% or more, more preferably 90% or more similarity to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

In another aspect, the present invention encodes a thrombus recognition domain having an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, characterized in that it has a nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8, a thrombus recognition domain It's about genes.

The thrombus recognition domain gene is characterized by having 70% or more, preferably 80% or more, more preferably 90% or more similarity to the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

In another aspect, the present invention relates to a pharmaceutical composition for treating or preventing thrombosis and related diseases, characterized in that it comprises as an active ingredient a polypeptide that dissolves a blood clot by recognizing a'blood clot' or a gene encoding the same. will be.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. may be exemplified, It is not limited thereto.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc. I can.

The appropriate dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. Meanwhile, the preferred dosage of the pharmaceutical composition of the present invention is 0.0001-1000 μg per day.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Alternatively, it may be prepared by enclosing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The pharmaceutical composition for the treatment or prevention of thrombosis and related diseases according to the present invention has an effect of dissolving'intravascular thrombi' and minimizing bleeding side effects when administered to a mammal.

In the present invention, the thrombosis and related diseases are thrombosis, embolism, thromboembolism, arterial thromboembolism, venous thromboembolism (VTE), cardiovascular disease. ), cerebrovascular disease, ischemic disease, etc. It is preferably any one selected from the group consisting of diseases caused by'intravascular thrombi', but is not limited thereto. Specific examples include Deep Vein Thrombosis (DVT), Pulmonary Embolism (PE), ischemic stroke, stroke, cerebral hemorrhage, cerebral infarction, myocardial infarction, heart attack, and unstable angina. Any one selected is preferred, but is not limited thereto.

[Example]

Hereinafter, the present invention will be described in more detail through specific examples. However, these examples are only for describing the present invention in more detail, and the scope of the present invention is not limited by these examples.

Experimental Example 1: Evaluation of ex vivo thrombolytic ability

HtrA1 and HtrA2, the polypeptides that recognize'blood clots' and dissolve blood clots, were prepared as recombinant proteins. For this, cDNA corresponding to 157-480 of the HtrA1 amino acid sequence (SEQ ID NO: 9), which includes both the intravascular thrombus domain and the thrombolytic domain, was used as a forward primer (5'-AATTCATATGCAAGGGCAGGAAGATCCCA-3') and reverse primer (5'-TATCTCGAGCTATGGGTCAATTTCTTCGGG -3') was amplified by PCR. PCR conditions were initial denaturation at 95°C for 5 minutes, amplification (95°C for 30 seconds, 63°C for 1 minute, 72°C for 2 minutes) repeated 34 times and then 72°C for 10 minutes. This was subcloned into the NdeI/XhoI site of the expression vector pET28a+ expression vector (Novagen, USA). The obtained HtrA1 recombinant plasmid was electroporated in E. coli BL21 (DE3) pLysS (Stratagene, USA) and then incubated with IPTG to express HtrA1. Recombinant E. coli culture solution was sonicated to make cell lysate, and it was separated by passing through an econo-pac chromatography column (Bio-Rad), and then purified by PD-10 column (Amersham, US) to obtain recombinant protein HtrA1. In the case of HtrA2, cDNA corresponding to 134-458 of the HtrA2 amino acid sequence (SEQ ID NO: 10) was PCR by using forward primer (5'-GTCCTCGCCCATATGGCCGTCCCTAGCC-3') and reverse primer (5'-GGCTCTCGAGTCATTCTGTGACCTCAGGG-3'). Amplified. PCR conditions were initial denaturation at 95°C for 5 minutes, then amplification (95°C for 30 seconds, 65°C for 45 seconds, 72°C for 1 minute) was repeated 34 times and then 72°C for 10 minutes. After obtaining this, the recombinant protein HtrA2 was obtained by subcloning and expressing it in a pET28a+ expression vector (Novagen, USA) in the same manner as HtrA1.

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

In FIG. 1, compared with conventional thrombolytic enzymes, the thrombolytic ability of HtrA1 (a and b of FIG. 1) and fibrin resolution (c and d of FIG. 1) were the most excellent. In FIG. 2, HtrA2 also showed superior thrombolytic ability (a, b in FIG. 2) and fibrin resolution (c, d in FIG. 2) than conventional thrombolytic enzymes.

Experimental Example 2: Evaluation of plasmin production ability (before thrombosis)

The current mechanism of thrombolysis of thrombolytic drugs is the mechanism of action of plasmin-dependent fibrinolysis by activating plasmininogen into plasmin. In order to confirm the mechanism of action compared with existing thrombolytic enzymes, first, each thrombolytic enzyme (0.1) was added to plasminogen (1.23 μM) and plasmin-specific fluorescent substrate Boc-Glu-Lys-Lys-MCA (100 μM). mg/ml) was added and then cultured. The activation of plasminogen on plasmin was confirmed by measuring the fluorescence intensity of the substrate dissolved by plasmin. The plasmin band generated by the activation of the actual plasminogen was confirmed by SDS-PAGE after each enzyme (0.15 mg/ml) was treated with plasminogen (5.14 μM).

Compared with the existing thrombolytic enzyme, HtrA1 had no plasminogen activation effect at all (Fig. 3a), and of course, no plasmin was produced (Fig. 3b). Likewise, HtrA2 also had no plasminogen activation efficacy (FIG. 4A), and as a result, there was no plasmin production (FIG. 4B).

Experimental Example 3: Thrombosis specificity evaluation

In order to evaluate the thrombotic specificity of HtrA1, the activity of fibrinolysis components that appeared in the wound healing process was confirmed. For this, fibrin clot obtained by reacting fibrinogen with thrombin was incubated with 50 mM Tris-HCl control or thrombolytic enzyme (2 mg/ml) at 37°C for 24 hours. The dissolution of fibrin clot was measured at a wavelength of 415 nm using a spectrophotometer. In order to see the activity of HtrA1 against fibrinogen, fibrinogen (5 μM) was incubated with 50 mM Tris-HCl control or each thrombolytic enzyme (0.15 mg/ml) for 3 hours at 37°C, and then the decomposition of fibrinogen was determined by SDS-PAGE. Confirmed as. In order to see the activity of HtrA1 on fibrinectin, fibronectin (1.52 μM) was incubated with each thrombolytic enzyme (0.15 mg/ml) at 37°C for 3 hours, and then separated on a 4-12% SDS gel. Immunostaining was performed with cFN or anti-pFN antibody.

As shown in Table 1, HtrA1 and HtrA2 showed a statistically significant difference in absorbance when compared to the untreated control group, as well as a statistically significant difference in absorbance for streptokinase, an existing thrombolytic enzyme. HtrA1 was the most excellent in solubility for fibrin clot, followed by HtrA2.

Groups A415 Control 1.870 ± 0.05 Streptokinase 1.335 ± 0.07 a Urokinase 1.047 ± 0.06 a HtrA1 1.055 ± 0.10 a,b HtrA2 1.002 ± 0.09 a,b

a p<0.05 significance difference from control group

b p<0.01 significance difference from Streptokinase group

Nevertheless, unlike existing thrombolytic enzymes, HtrA1 did not decompose fibrinogen, which is important for hemostasis at the wound site (Fig. 5a), and cellular fibronectin (Fig. 5b) and plasma fibronectin (Fig. 5c), which are important for wound healing. It has been shown to preserve without disassembling.

Finally, in order to see the activity of HtrA1 on the wound healing process, a wound (~30 mm ² ) in which the tail skin of BALB/c mice was excised from the outside was made. The excised wound pieces were incubated with 50 mM Tris-HCl or each thrombolytic enzyme (2 mg/ml) at 37° C. for 72 hours. Whether the wound healed was determined by observing the liquid containing the wound fragment and measuring the absorbance at 550 nm. As described above, in a wound tissue experiment using an actual animal, the absorbance level due to bleeding can be confirmed because the wound tissue is not healed during the conventional thrombolytic enzyme treatment. It was confirmed that normal wound healing occurred (Table 2).

Groups A550 Control 0.235 ± 0.031 Streptokinase 0.471 ± 0.018 a Urokinase 0.404 ± 0.037 a HtrA1 0.203 ± 0.028 a,b HtrA2 0.247 ± 0.033 a,b

a p<0.05 significance difference from control group

b p<0.01 significance difference from Streptokinase group

Example 1: Therapeutic effect of thrombosis

Animal experiments using a tail thrombosis model were performed to evaluate the efficacy of thrombosis treatment by the outcome of the present invention. A κ-carrageenan induced tail thrombosis model was established in 15 week old female BALB/c mice. Saline (control) or each thrombolytic enzyme was injected intraperitoneally into each group (n = 8) of mice with tail thrombosis, and the length and ratio of the thrombosis site were measured after 24 hours, and the results are shown in Tables 3 and 4. I got it.

Groups Dose (mg/kg) Tail Length (cm) Thrombosis Length (cm) Thrombosis Ratio (%) Control - 9.21±0.08 9.07±0.11 a 98.50±0.80 a Plasmin 40 9.02±0.07 3.37±0.45 b 37.39±5.10 b Streptokinase 40 9.10±0.10 5.11±1.22 a 56.16±13.4 a Urokinase 40 9.07±0.10 4.27±0.60 a 47.08±6.51 a HtrA1 (10mg/kg) 10 9.18±0.16 4.72±1.06 51.33±10.9 HtrA1 (20mg/kg) 20 9.12±0.10 3.41±0.57 37.41±6.43 HtrA1 (40mg/kg) 40 9.17±0.12 1.61±0.49 17.56±5.47

a p<0.001 significance difference from HtrA1 (40mg/kg) group

b p<0.01 Significance difference from HtrA1 (40mg/kg) group

As shown in Table 3, in the mouse tail thrombosis model, compared with conventional thrombolytic enzymes, in the case of the HtrA1 treatment group, the length and frequency of thrombosis tails in the tail are significantly reduced, and this thrombolytic effect increases in proportion to the dose. Confirmed that. In other words, it was confirmed that HtrA1 had a statistically significant thrombosis treatment effect even when compared with existing thrombolytic enzymes.

Groups Dose (mg/kg) Tail Length (cm) Thrombosis Length (cm) Thrombosis Ratio (%) Control - 9.19±0.09 9.03±0.14 a 98.25±1.16 a Plasmin 40 9.03±0.06 3.40±0.41 b 37.65±4.62 b Streptokinase 40 9.09±0.09 4.97±1.16 a 54.66±12.7 a tPA 40 9.06±0.08 3.49±0.53 b 38.53±6.06 b HtrA2 (10mg/kg) 10 9.15±0.12 4.55±1.11 49.62±11.5 HtrA2 (20mg/kg) 20 9.11±0.11 4.01±0.83 44.01±9.18 HtrA2 (40mg/kg) 40 9.14±0.08 2.19±0.75 23.93±8.20

a p<0.001 significance difference from HtrA2 (40mg/kg) group

b p<0.01 Significance difference from HtrA2 (40mg/kg) group

In addition, as shown in Table 4, it can be seen that in the case of the HtrA2 treatment group, the tail length and frequency of thrombosis in the tail are also significantly reduced when compared with the existing thrombolytic enzymes in the mouse tail thrombosis model, and this thrombosis dissolution effect is proportional to the dose. It was confirmed that it increased. In the case of HtrA2, it was confirmed that not only the untreated group but also had a statistically significant thrombosis treatment effect when compared with the existing thrombolytic enzymes.

In FIG. 7, as a result of observing the thrombosis tail tissue by H & E staining, as a result of confirming the degree of direct thrombosis and thrombosis dissolution as in the gastric thrombosis tail result, in the case of the HtrA1 and HtrA2 treatment groups, no thrombosis was found, Compared with existing thrombolytic enzymes, it showed excellent thrombosis treatment.

Example 2: Therapeutic effect of pulmonary thromboembolism

Next, an animal experiment using a pulmonary thromboembolic model was performed to evaluate the prevention and treatment efficacy of thromboembolism. A pulmonary thromboembolic model induced by Adenosine 5'-diphosphate (ADP) was established in 15 week old female C57BL/6 mice. Each group (n = 8) of pulmonary thromboembolic mice was fasted for 12 hours or more, and then saline (control) or thrombolytic enzyme was injected intravenously at a dose of 40 mg/kg. After 30 minutes, the mice were injected with ADP (150 mg/kg) to induce pulmonary thromboembolism, and then death was confirmed, and the results are shown in Tables 5 and 6.

Groups Dose (mg/kg) Lethal number/total Protection rate (%) Control - 6/6 0 Plasmin 40 3/6 50 Streptokinase 40 4/6 33 Urokinase 40 2/6 67 HtrA1 40 0/6 100

From Table 5, in the pulmonary thromboembolic mouse model, the survival rates of the plasmin, streptokinase, and urokinase-treated groups were 50%, 33%, and 66%, respectively, but the survival rate of the HtrA1-treated group was 100%, from which the present invention It was found that HtrA1 has a complete therapeutic and preventive effect on fatal pulmonary thromboembolism.

Groups Dose(mg/kg) Lethal number/total Protection rate (%) Control - 5/5 0 Plasmin 40 2/5 60 Streptokinase 40 4/5 20 tPA 40 3/5 40 HtrA2 40 0/5 100

From Table 6, in the pulmonary thromboembolic mouse model, in the case of the plasmin, streptokinase, and tPA-treated group, the survival rates were 60%, 20%, and 40%, respectively, but the survival rate of the HtrA2-treated group was 100%. HtrA2 was also found to have a perfect treatment and prevention effect for pulmonary thromboembolism.

Example 3: Elimination of risk of bleeding in the body

When the thrombolytic protein HtrA1 was administered, a tail bleeding test was performed using a 15-week-old C57BL/6 female mouse to determine whether there was a risk of bleeding in the body during the wound healing process. Saline (control) or each thrombolytic enzyme was injected intravenously into a group of mice (n = 5) at a dose of 40 mg/kg. After 30 minutes, the tail was cut from the anesthetized mouse and blood was collected until the bleeding stopped, and the bleeding time and the amount of bleeding were recorded, and the collected blood was measured for the hemoglobin content. In addition, the time required for blood to coagulate in the rat's cut tail vein was recorded.

As shown in FIG. 8, it was confirmed that the bleeding of HtrA1 and HtrA2 was significantly less compared to other thrombolytic enzymes in the mouse tail bleeding experiment. In the case of the HtrA1 treatment group, as a result of measuring the actual bleeding time (Fig. 9 a), the amount of bleeding (Fig. 9 b), the amount of hemoglobin lost (Fig. 9 c), and the time taken to hemostasis (Fig. 9 d), It was confirmed that the bleeding side effects were reduced, which could not be compared with the existing thrombolytic enzymes, and the level was similar to that of the control group.

In the case of the HtrA2 treatment group, the bleeding time (FIG. 10 a ), the amount of bleeding (FIG. 10 b ), the amount of hemoglobin lost (FIG. 10 c ), and the time taken to hemostasis (FIG. 10 d) were measured. It was confirmed that the bleeding side effects were reduced, which could not be compared with the thrombolytic enzymes of, and was similar to that of the control group. HtrA1 and HtrA2 do not interfere with the hemostatic process, which is part of the wound healing process, and thus completely reduce the risk of bleeding, so it can be seen that they can be used as thrombosis treatments that dramatically reduce bleeding side effects.

Example 4: Evaluation of similarity according to species

The similarity between the amino acid sequences of SEQ ID NOs: 1 to 4 and SEQ ID NOs: 9 and 10 of the present invention and the amino acid sequences of mice, rats and bovines was evaluated at, and the results are shown in Tables 7 and 8 below. Indicated.

The species sequence of the serine protease domain-HtrA1 corresponding to SEQ ID NO: 1 is as follows.

Mouse amino acid sequence (SEQ ID NO: 15): GSGFIVSEDGLIVTNAHVVTNKNRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGKSDLK

Rat amino acid sequence (SEQ ID NO: 16): GSGFIVSEDGLIVTNAHVVTNKNRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGKISDK

Bovine amino acid sequence (SEQ ID NO: 17): GSGFIVSEDGLIVTNAHVVTNKHRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLGISFAIPSDKIKFLVNLDGEVIGINTLK

The species sequence of the serine protease domain-HtrA2 corresponding to SEQ ID NO: 2 is as follows.

Mouse amino acid sequence (SEQ ID NO: 18): ILDRHPFSGREVPISNGSGFVVASDGLIVTNAHVVADRRRVRVRLPSGDTYEAMVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPARDLGLPQNNVEYIQTDAAIDFFAGNSGGSDRLREF

Rat amino acid sequence (SEQ ID NO: 19): ILDRHPFSGREVPISNGSGFIVASDGLIVTNAHVVADRRRVRVRLPSGDTYEAMVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPARDLGLPQTNVEYIQPLVDAAIGDFGNSDRLREF

Bovine amino acid sequence (SEQ ID NO: 20): ILGRHPFSGREVPISNGSGFVVAADGLIVTNAHVVADRRRVRVRLPSGDTYEAVVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPAKDLGLPQTNVEYIQVNLDGEFAGNVEYIQTNLDAIDFGNSREF

The species sequence of the thrombus recognition domain PDZ-HtrA1 corresponding to SEQ ID NO: 3 is as follows.

Mouse amino acid sequence (SEQ ID NO: 21): TESHDRQAKGKAVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVTANDVSDVIKKENTLNMVVRRGNE

Rat amino acid sequence (SEQ ID NO: 22): TESHDRQAKGKTVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVTANDVSDVIKKENTLNMVVRRGNE

Bovine amino acid sequence (SEQ ID NO: 23): TESHDRQAKGKAITKKKYIGIRMMSLTPSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVSDVIKKESTLNMVVRRGNE

The species sequence of the thrombus recognition domain PDZ-HtrA2 corresponding to SEQ ID NO: 4 is as follows.

Mouse amino acid sequence (SEQ ID NO: 24): VMMLTLTPSILIELQLREPSFPDVQHGVLIHKVILGSPAHRAGLRPGDVILAIGEKLAQNAEDVYEAVRTQSQLAVRIRRGSE

Rat amino acid sequence (SEQ ID NO: 25): VMMLTLTPSILAELQLREPSFPDVQHGVLIHKVILGSPAHRAGLRPADVILAIGEKMIQNAEDVYEAVRTQSQLAVRIRRGP

Bovine amino acid sequence (SEQ ID NO: 26): VMMLTLTPSILAELQLREPSFPDVQHGVLIHKVILDSPAHRAGLRPGDVILAIGEQLVQNAEDIYEAVRTQSQLAVRIRRGQ

Sequence number Domain Similarity (%) Human Mouse Rat Bovine Seq.1 Serine protease domain -HtrA1 100 99.4 99.4 92.5 Seq.2 Serine protease domain -HtrA2 100 97.2 97.2 97.2 Seq.3 Blood clot recognition main PDZ-HtrA1 100 95.1 95.1 97.1 Seq.4 Recognition of blood clotsmain
PDZ -HtrA2 100 92.6 93.8 92.7

The species sequence of the full length sequence-HtrA1 corresponding to SEQ ID NO: 9 is as follows.

Mouse amino acid sequence (SEQ ID NO: 27): MQSLRTTLLSLLLLLLAAPSLALPSGTGRSAPAATVCPEHCDPTRCAPPPTDCEGGRVRDACGCCEVCGALEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDAKTYTNLCQLRAASRRSEKLRQPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELYRKLPFSKREVPVASGSGFIVSEDGLIVTNAHVVTNKNRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKAVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVTANDVSDVIKKENTLNMVVRRGNEDIVITVIPEEIDP

Rat amino acid sequence (SEQ ID NO: 28): MQFLRTALLSLLLLLLAAPSLALPSGISRSAPAATVCPEHCDPTRCAPPPTDCEGGRVRDACGCCEVCGALEGAVCGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDAKTYTNLCQLRAASRRSEKLRQPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELYRKLPFSKREVPVASGSGFIVSEDGLIVTNAHVVTNKNRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKTVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVTANDVSDVIKKENTLNMVVRRGNEDIVITVVPEEIDP

Bovine amino acid sequence (SEQ ID NO: 29): MQPPRAPFLPPPTPPLLLLLLLLAAPASAQPARAGRSAPGAAGCPERCDPARCAPPPGSCEGGRVRDACGCCEVCGAPEGAECGLQEGPCGEGLQCVVPFGVPASATVRRRAQSGLCVCASNEPVCGSDAKTYTNLCQLRAASRRSERLHQPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELFRKLPFSKREVPVASGSGFIVSEDGLIVTNAHVVTNKHRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTPSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVSDVIKKESTLNMVVRRGNEDIMITVIPEEIDP

The species sequence of the full length sequence-HtrA2 corresponding to SEQ ID NO: 10 is as follows.

Mouse amino acid sequence (SEQ ID NO: 30): MAALKAGRGANWSLRAWRALGGIFWRKPPLLAPDLRALLTSGTPDSQIWMTYGTPSLPAQVPEGFLASRADLTSRTPDLWARLNVGTSGSSDQEARRSPGSRRREWLAVAVGAGGAVVLLLWGWGRGLSTVLAAVPAPPPTSPRSQYNFIADVVEKTAPAVVYIEILDRHPFSGREVPISNGSGFVVASDGLIVTNAHVVADRRRVRVRLPSGDTYEAMVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPARDLGLPQNNVEYIQTDAAIDFGNSGGPLVNLDGEVIGVNTMKVTAGISFAIPSDRLREFLHRGEKKNSWFGTSGSQRRYIGVMMLTLTPSILIELQLREPSFPDVQHGVLIHKVILGSPAHRAGLRPGDVILAIGEKLAQNAEDVYEAVRTQSQLAVRIRRGSETLTLYVTPEVTE

Rat amino acid sequence (SEQ ID NO: 31): MAALKAGRGANWSLRAWRALGGIFWRKGPLLAPDLRALLTSGTPDPQARMTYGTPSLPARVPLGVLASRANLTSGTPDLWVRLTVGTPGSSDQEDCGSPGSRRREWLAVALGAGGAVLLLLWGWGRGPSTVLAAVPAPPPTSPRSQYNFIADVVEKTAPAVVYIEILDRHPFSGREVPISNGSGFIVASDGLIVTNAHVVADRRRVRVRLPSGDTYEAMVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPARDLGLPQTNVEYIQTDAAIDFGNSGGPLVNLDGEVIGVNTMKVTAGISFAIPSDRLREFLHRGEKKNSWFGISGSQRRYIGVMMLTLTPSILAELQLREPSFPDVQHGVLIHKVILGSPAHRAGLRPADVILAIGEKMIQNAEDVYEAVRTQSQLAVRIRRGPETLTLYVTPEVTE

Bovine amino acid sequence (SEQ ID NO: 32): MAALRAGRGAGWSLRGWRALWGGRWGKGPLLTPDLRALLTSGTPDPRTRVTYGTPSFRARLSVGVPEPRTCLRSRTSDLRARLIAGTPDPRTPEDSGTPGTRLRVWLAVALGAGGAVLLLFWGGGRGPPAVLASVLGSPPTSPRSQYNFIADVVEKTAPAVVYIEILGRHPFSGREVPISNGSGFVVAADGLIVTNAHVVADRRRVRVRLPSGDTYEAVVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPAKDLGLPQTNVEYIQTDAAIDFGNSGGPLVNLDGEVIGVNTMKVTSGISFAIPSDRLREFLHRGEKKNSWFGISGSQRRYIGVMMLTLTPSILAELQLREPSFPDVQHGVLIHKVILDSPAHRAGLRPGDVILAIGEQLVQNAEDIYEAVRTQSQLAVRIRRGQETLTLYVTPEVTE

Sequence number Full length HtrA Similarity (%) Human Mouse Rat Bovine Seq.9 Full length sequence-HtrA1 100 92.3 91.9 94.0 Seq.10 Full length sequence-HtrA2 100 84.9 87.3 89.5

Example 5: Evaluation of thrombolytic activity of thrombolytic protein having homology with thrombolytic domain or whether it is a thrombosis of thrombolytics

Mouse thrombolytic protein HtrA1 (MHT1) (SEQ ID NO: 27), a homologous enzyme for human thrombolytic proteins HtrA1 (HHT1) (SEQ ID NO: 9) and HtrA2 (HHT2) (SEQ ID NO: 10), rat thrombolytic protein HtrA1 (RHT1) (SEQ ID NO: 28) and bovine thrombolytic protein HtrA1 (BHT1) (SEQ ID NO: 29) gene clones were prepared and expressed, each thrombolytic protein was separated and purified, and the following SATA (spectrophotometric analysis of thrombolytic activity) thrombolytic analysis method The blood clot solubility of each thrombolytic protein was measured.

1. To 300 μL of whole blood treated with sodium citrate, an anticoagulant, _{30 μL of 250 mM CaCl 2} and 200 μL of a partial thromboplastin time (PTT) test reagent were added, and then allowed to stand at room temperature for 10 minutes to form a blood clot.

2. After each 100 μL of the thrombolytic agent to be tested was added to this blood clot solution, it was allowed to stand at 37° C. for 20 minutes to induce a thrombolytic reaction.

3. After the thrombolytic reaction, 1 mL of saline solution was added to stop the thrombolytic reaction.

4. This solution was diluted 25 times with saline solution, absorbance was measured at 405 nm, and the thrombolytic activity of HtrA1, HtrA2, and each homologous enzyme was measured by comparing with the blood standard curve. Table 9 shows the results.

Modified thrombolytic Thrombolytic activity (μg/mL) Control 0 HHT1 32.6 ± 5.1 HHT2 15.3 ± 4.8 MHT1 38.9 ± 7.3 RHT1 35.6 ± 4.2 BHT1 31.2 ± 3.9

From Table 9, as a result of comparative analysis of human thrombolytic protein and its homologous enzyme, mouse, rat, and bovine thrombolytic protein, it was confirmed that they all effectively dissolve blood clots in common. These results show that the thrombolytic protein, which dissolves blood clots, which is fatal to survival, has its evolutionary function, so that the homologous thrombolytic protein of animals having homology with human thrombolytic protein can be used for human thrombolysis. Indicates that there is. Accordingly, in the present invention, not only human thrombolytic proteins but also animal thrombolytic proteins having similarity thereto may be included as therapeutic agents for the purpose of human thrombolysis.

<110> JINIS CO., LTD. <120> Thrombolytic agents for Intravascular clots <130> P19135 <150> KR 2019-0131585 <151> 2019-10-22 <160> 32 <170> KoPatentIn 3.0 <210> 1 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of 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 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 thrombus cognitive 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 thrombus cognitive 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 Tyr Glu 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> Artificial Sequence <220> <223> base sequence of thrombolytic domain of HtrA1 (HtrA1 bs1) <400> 5 gggtctgggt ttattgtgtc ggaagatgga ctgatcgtga caaatgccca cgtggtgacc 60 aacaagcacc gggtcaaagt tgagctgaag aacggtgcca cttacgaagc caaaatcaag 120 gatgtggatg agaaagcaga catcgcactc atcaaaattg accaccaggg caagctgcct 180 gtcctgctgc ttggccgctc ctcagagctg cggccgggag agttcgtggt cgccatcgga 240 agcccgtttt cccttcaaaa cacagtcacc accgggatcg tgagcaccac ccagcgaggc 300 ggcaaagagc tggggctccg caactcagac atggactaca tccagaccga cgccatcatc 360 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 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> base sequence of thrombus cognitive domain of HtrA1 (HtrA1 bs2) <400> 7 acggagtccc atgaccgaca ggccaaagga aaagccatca ccaagaagaa gtatattggt 60 atccgaatga tgtcactcac gtccagcaaa gccaaagagc tgaaggaccg gcaccgggac 120 ttcccagacg 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> base sequence of thrombus cognitive domain of HtrA2 (HtrA2 bs2) <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> amino acid sequence of HtrA2 (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 <210> 15 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> serine protease domain HtrA1 of Mouse <400> 15 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 Asn 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> 16 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> serine protease domain HtrA1 of Rat <400> 16 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 Asn 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> 17 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> serine protease domain HtrA1 of Bovine <400> 17 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> 18 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> serine protease domain HtrA2 of Mouse <400> 18 Ile Leu Asp Arg His Pro Phe Ser Gly Arg Glu Val Pro Ile Ser Asn 1 5 10 15 Gly Ser Gly Phe Val Val Ala Ser Asp Gly Leu Ile Val Thr Asn Ala 20 25 30 His Val Val Ala Asp Arg Arg Arg Val Arg Val Arg Leu Pro Ser Gly 35 40 45 Asp Thr Tyr Glu Ala Met 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 Asn 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> 19 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> serine protease domain HtrA2 of Rat <400> 19 Ile Leu Asp Arg His Pro Phe Ser Gly Arg Glu Val Pro Ile Ser Asn 1 5 10 15 Gly Ser Gly Phe Ile Val Ala Ser Asp Gly Leu Ile Val Thr Asn Ala 20 25 30 His Val Val Ala Asp Arg Arg Arg Val Arg Val Arg Leu Pro Ser Gly 35 40 45 Asp Thr Tyr Glu Ala Met 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> 20 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> serine protease domain HtrA2 of Bovine <400> 20 Ile Leu Gly Arg His Pro Phe Ser 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 Pro 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 Lys 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 Ser Gly Ile Ser Phe Ala Ile Pro Ser Asp Arg Leu Arg Glu Phe 165 170 175 Leu <210> 21 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> PDZ-HtrA1 of Mouse <400> 21 Thr Glu Ser His Asp Arg Gln Ala Lys Gly Lys Ala Val 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 Leu 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 Thr 65 70 75 80 Ala Asn Asp Val Ser Asp Val Ile Lys Lys Glu Asn Thr Leu Asn Met 85 90 95 Val Val Arg Arg Gly Asn Glu 100 <210> 22 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> PDZ-HtrA1 of Rat <400> 22 Thr Glu Ser His Asp Arg Gln Ala Lys Gly Lys Thr Val 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 Thr 65 70 75 80 Ala Asn Asp Val Ser Asp Val Ile Lys Lys Glu Asn Thr Leu Asn Met 85 90 95 Val Val Arg Arg Gly Asn Glu 100 <210> 23 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> PDZ-HtrA1 of Bovine <400> 23 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 Pro Ser Lys Ala Lys 20 25 30 Glu Leu Lys Asp Arg His Arg Asp Phe Pro Asp Val Leu 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 Lys Glu Ser Thr Leu Asn Met 85 90 95 Val Val Arg Arg Gly Asn Glu 100 <210> 24 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> PDZ-HtrA2 of Mouse <400> 24 Val Met Met Leu Thr Leu Thr Pro Ser Ile Leu Ile 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 Lys Leu Ala Gln Asn Ala Glu Asp Val 50 55 60 Tyr Glu Ala Val Arg Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg 65 70 75 80 Gly Ser Glu <210> 25 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> PDZ-HtrA2 of Rat <400> 25 Val Met Met Leu Thr Leu Thr 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 Ala Asp 35 40 45 Val Ile Leu Ala Ile Gly Glu Lys Met Ile Gln Asn Ala Glu Asp Val 50 55 60 Tyr Glu Ala Val Arg Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg 65 70 75 80 Gly Pro <210> 26 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> PDZ-HtrA2 of Bovine <400> 26 Val Met Met Leu Thr Leu Thr 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 Asp Ser Pro Ala His Arg Ala Gly Leu Arg Pro Gly Asp 35 40 45 Val Ile Leu Ala Ile Gly Glu Gln Leu Val Gln Asn Ala Glu Asp Ile 50 55 60 Tyr Glu Ala Val Arg Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg 65 70 75 80 Gly Gln <210> 27 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Full length sequence-HtrA1 of Mouse <400> 27 Met Gln Ser Leu Arg Thr Thr Leu Leu Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Ala Ala Pro Ser Leu Ala Leu Pro Ser Gly Thr Gly Arg Ser Ala Pro 20 25 30 Ala Ala Thr Val Cys Pro Glu His Cys Asp Pro Thr Arg Cys Ala Pro 35 40 45 Pro Pro Thr Asp Cys Glu Gly Gly Arg Val Arg Asp Ala Cys Gly Cys 50 55 60 Cys Glu Val Cys Gly Ala Leu 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 Lys Thr Tyr Thr Asn 115 120 125 Leu Cys Gln Leu Arg Ala Ala Ser Arg Arg Ser Glu Lys Leu Arg Gln 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 Tyr 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 Asn 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 Val 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 Leu 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 Thr Ala Asn Asp Val 435 440 445 Ser Asp Val Ile Lys Lys Glu Asn Thr Leu Asn Met Val Val Arg Arg 450 455 460 Gly Asn Glu Asp Ile Val Ile Thr Val Ile Pro Glu Glu Ile Asp Pro 465 470 475 480 <210> 28 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> Full length sequence-HtrA1 of Rat <400> 28 Met Gln Phe Leu Arg Thr Ala Leu Leu Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Ala Ala Pro Ser Leu Ala Leu Pro Ser Gly Ile Ser Arg Ser Ala Pro 20 25 30 Ala Ala Thr Val Cys Pro Glu His Cys Asp Pro Thr Arg Cys Ala Pro 35 40 45 Pro Pro Thr Asp Cys Glu Gly Gly Arg Val Arg Asp Ala Cys Gly Cys 50 55 60 Cys Glu Val Cys Gly Ala Leu Glu Gly Ala Val 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 Lys Thr Tyr Thr Asn 115 120 125 Leu Cys Gln Leu Arg Ala Ala Ser Arg Arg Ser Glu Lys Leu Arg Gln 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 Tyr 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 Asn 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 Thr Val 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 Thr Ala Asn Asp Val 435 440 445 Ser Asp Val Ile Lys Lys Glu Asn Thr Leu Asn Met Val Val Arg Arg 450 455 460 Gly Asn Glu Asp Ile Val Ile Thr Val Val Pro Glu Glu Ile Asp Pro 465 470 475 480 <210> 29 <211> 487 <212> PRT <213> Artificial Sequence <220> <223> Full length sequence-HtrA1 of Bovine <400> 29 Met Gln Pro Pro Arg Ala Pro Phe Leu Pro Pro Pro Thr Pro Pro Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Ala Ala Pro Ala Ser Ala Gln Pro Ala 20 25 30 Arg Ala Gly Arg Ser Ala Pro Gly Ala Ala Gly Cys Pro Glu Arg Cys 35 40 45 Asp Pro Ala Arg Cys Ala Pro Pro Pro Gly Ser Cys Glu Gly Gly Arg 50 55 60 Val Arg Asp Ala Cys Gly Cys Cys Glu Val Cys Gly Ala Pro Glu Gly 65 70 75 80 Ala Glu Cys Gly Leu Gln Glu Gly Pro Cys Gly Glu Gly Leu Gln Cys 85 90 95 Val Val Pro Phe Gly Val Pro Ala Ser Ala Thr Val Arg Arg Arg Ala 100 105 110 Gln Ser Gly Leu Cys Val Cys Ala Ser Asn Glu Pro Val Cys Gly Ser 115 120 125 Asp Ala Lys Thr Tyr Thr Asn Leu Cys Gln Leu Arg Ala Ala Ser Arg 130 135 140 Arg Ser Glu Arg Leu His Gln Pro Pro Val Ile Val Leu Gln Arg Gly 145 150 155 160 Ala Cys Gly Gln Gly Gln Glu Asp Pro Asn Ser Leu Arg His Lys Tyr 165 170 175 Asn Phe Ile Ala Asp Val Val Glu Lys Ile Ala Pro Ala Val Val His 180 185 190 Ile Glu Leu Phe Arg Lys Leu Pro Phe Ser Lys Arg Glu Val Pro Val 195 200 205 Ala Ser Gly Ser Gly Phe Ile Val Ser Glu Asp Gly Leu Ile Val Thr 210 215 220 Asn Ala His Val Val Thr Asn Lys His Arg Val Lys Val Glu Leu Lys 225 230 235 240 Asn Gly Ala Thr Tyr Glu Ala Lys Ile Lys Asp Val Asp Glu Lys Ala 245 250 255 Asp Ile Ala Leu Ile Lys Ile Asp His Gln Gly Lys Leu Pro Val Leu 260 265 270 Leu Leu Gly Arg Ser Ser Glu Leu Arg Pro Gly Glu Phe Val Val Ala 275 280 285 Ile Gly Ser Pro Phe Ser Leu Gln Asn Thr Val Thr Thr Gly Ile Val 290 295 300 Ser Thr Thr Gln Arg Gly Gly Lys Glu Leu Gly Leu Arg Asn Ser Asp 305 310 315 320 Met Asp Tyr Ile Gln Thr Asp Ala Ile Ile Asn Tyr Gly Asn Ser Gly 325 330 335 Gly Pro Leu Val Asn Leu Asp Gly Glu Val Ile Gly Ile Asn Thr Leu 340 345 350 Lys Val Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp Lys Ile Lys 355 360 365 Lys Phe Leu Thr Glu Ser His Asp Arg Gln Ala Lys Gly Lys Ala Ile 370 375 380 Thr Lys Lys Lys Tyr Ile Gly Ile Arg Met Met Ser Leu Thr Pro Ser 385 390 395 400 Lys Ala Lys Glu Leu Lys Asp Arg His Arg Asp Phe Pro Asp Val Leu 405 410 415 Ser Gly Ala Tyr Ile Ile Glu Val Ile Pro Asp Thr Pro Ala Glu Ala 420 425 430 Gly Gly Leu Lys Glu Asn Asp Val Ile Ile Ser Ile Asn Gly Gln Ser 435 440 445 Val Val Ser Ala Asn Asp Val Ser Asp Val Ile Lys Lys Glu Ser Thr 450 455 460 Leu Asn Met Val Val Arg Arg Gly Asn Glu Asp Ile Met Ile Thr Val 465 470 475 480 Ile Pro Glu Glu Ile Asp Pro 485 <210> 30 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Full length sequence-HtrA2 of Mouse <400> 30 Met Ala Ala Leu Lys Ala Gly Arg Gly Ala Asn Trp Ser Leu Arg Ala 1 5 10 15 Trp Arg Ala Leu Gly Gly Ile Phe Trp Arg Lys Pro Pro Leu Leu Ala 20 25 30 Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Pro Asp Ser Gln Ile 35 40 45 Trp Met Thr Tyr Gly Thr Pro Ser Leu Pro Ala Gln Val Pro Glu Gly 50 55 60 Phe Leu Ala Ser Arg Ala Asp Leu Thr Ser Arg Thr Pro Asp Leu Trp 65 70 75 80 Ala Arg Leu Asn Val Gly Thr Ser Gly Ser Ser Asp Gln Glu Ala Arg 85 90 95 Arg Ser Pro Gly Ser Arg Arg Arg Glu Trp Leu Ala Val Ala Val Gly 100 105 110 Ala Gly Gly Ala Val Val Leu Leu Leu Trp Gly Trp Gly Arg Gly Leu 115 120 125 Ser Thr Val Leu Ala Ala Val Pro Ala Pro Pro Pro Thr 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 Ser Gly Arg Glu 165 170 175 Val Pro Ile Ser Asn Gly Ser Gly Phe Val Val Ala Ser 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 Pro Ser Gly Asp Thr Tyr Glu Ala Met 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 Asn 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 Trp Phe 340 345 350 Gly Thr Ser Gly Ser Gln Arg Arg Tyr Ile Gly Val Met Met Leu Thr 355 360 365 Leu Thr Pro Ser Ile Leu Ile 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 Lys Leu Ala Gln Asn Ala Glu Asp Val Tyr Glu Ala Val Arg 420 425 430 Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg Gly Ser Glu Thr Leu 435 440 445 Thr Leu Tyr Val Thr Pro Glu Val Thr Glu 450 455 <210> 31 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Full length sequence-HtrA2 of Rat <400> 31 Met Ala Ala Leu Lys Ala Gly Arg Gly Ala Asn Trp Ser Leu Arg Ala 1 5 10 15 Trp Arg Ala Leu Gly Gly Ile Phe Trp Arg Lys Gly Pro Leu Leu Ala 20 25 30 Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Pro Asp Pro Gln Ala 35 40 45 Arg Met Thr Tyr Gly Thr Pro Ser Leu Pro Ala Arg Val Pro Leu Gly 50 55 60 Val Leu Ala Ser Arg Ala Asn Leu Thr Ser Gly Thr Pro Asp Leu Trp 65 70 75 80 Val Arg Leu Thr Val Gly Thr Pro Gly Ser Ser Asp Gln Glu Asp Cys 85 90 95 Gly Ser Pro Gly Ser Arg Arg Arg Glu Trp Leu Ala Val Ala Leu Gly 100 105 110 Ala Gly Gly Ala Val Leu Leu Leu Leu Trp Gly Trp Gly Arg Gly Pro 115 120 125 Ser Thr Val Leu Ala Ala Val Pro Ala Pro Pro Pro Thr 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 Ser Gly Arg Glu 165 170 175 Val Pro Ile Ser Asn Gly Ser Gly Phe Ile Val Ala Ser 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 Pro Ser Gly Asp Thr Tyr Glu Ala Met 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 Trp Phe 340 345 350 Gly Ile Ser Gly Ser Gln Arg Arg Tyr Ile Gly Val Met Met Leu Thr 355 360 365 Leu Thr 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 Ala Asp Val Ile Leu Ala Ile 405 410 415 Gly Glu Lys Met Ile Gln Asn Ala Glu Asp Val Tyr Glu Ala Val Arg 420 425 430 Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg Gly Pro Glu Thr Leu 435 440 445 Thr Leu Tyr Val Thr Pro Glu Val Thr Glu 450 455 <210> 32 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Full length sequence-HtrA2 of Bovine <400> 32 Met Ala Ala Leu Arg Ala Gly Arg Gly Ala Gly Trp Ser Leu Arg Gly 1 5 10 15 Trp Arg Ala Leu Trp Gly Gly Arg Trp Gly Lys Gly Pro Leu Leu Thr 20 25 30 Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Pro Asp Pro Arg Thr 35 40 45 Arg Val Thr Tyr Gly Thr Pro Ser Phe Arg Ala Arg Leu Ser Val Gly 50 55 60 Val Pro Glu Pro Arg Thr Cys Leu Arg Ser Arg Thr Ser Asp Leu Arg 65 70 75 80 Ala Arg Leu Ile Ala Gly Thr Pro Asp Pro Arg Thr Pro Glu Asp Ser 85 90 95 Gly Thr Pro Gly Thr Arg Leu Arg Val Trp Leu Ala Val Ala Leu Gly 100 105 110 Ala Gly Gly Ala Val Leu Leu Leu Phe Trp Gly Gly Gly Arg Gly Pro 115 120 125 Pro Ala Val Leu Ala Ser Val Leu Gly Ser Pro Pro Thr 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 Gly Arg His Pro Phe Ser 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 Pro 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 Lys 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 Ser 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 Trp Phe 340 345 350 Gly Ile Ser Gly Ser Gln Arg Arg Tyr Ile Gly Val Met Met Leu Thr 355 360 365 Leu Thr 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 Asp 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 Leu Val Gln Asn Ala Glu Asp Ile Tyr Glu Ala Val Arg 420 425 430 Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg Gly Gln Glu Thr Leu 435 440 445 Thr Leu Tyr Val Thr Pro Glu Val Thr Glu 450 455

Claims (6)

(a) consisting of a thrombolytic domain having 80% or more similarity to the amino acid sequence of SEQ ID NO: 1 and a thrombus recognition domain having 80% or more similarity to the amino acid sequence of SEQ ID NO: 3, or (b) with the amino acid sequence of SEQ ID NO: 2 A polypeptide that recognizes'intravascular thrombi' and dissolves thrombi, characterized in that it consists of a thrombolytic domain having 80% or more similarity and a thrombus recognition domain having 80% or more similarity to the amino acid sequence of SEQ ID NO: 4.
A polypeptide that dissolves a blood clot by recognizing a'blood clot' consisting of the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10.
The polypeptide according to claim 2, wherein the polypeptide has 80% or more similarity to the amino acid sequence of SEQ ID NO: 9 or 10, which recognizes'blood clots in blood vessels' and dissolves blood clots.
A thrombolytic domain gene, characterized in that it has 80% or more similarity to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, which encodes the thrombolytic domain having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
A thrombus recognition domain gene, characterized in that it has 80% or more similarity to the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8, which encodes a thrombus recognition domain having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
A pharmaceutical composition for treating or preventing thrombosis and related diseases, characterized in that it comprises a polypeptide that dissolves a blood clot by recognizing the'thrombosis in blood vessels' of any one of claims 1 to 3 or a gene encoding the same as an active ingredient .

Images