KR102362115B1 - Thrombolytic agents for Intravascular clots - Google Patents

Abstract

The present invention relates to an 'intravascular thrombus' dissolving agent, and more particularly, it is composed of a thrombo-recognition domain and a thrombolytic domain for 'intravascular thrombus'. It relates to a polypeptide, a gene, and a pharmaceutical composition containing the same.
The polypeptide for recognizing 'intravascular thrombus' of the present invention and dissolving thrombi includes a thrombolytic domain comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 It is characterized in that it consists of a thrombus recognition domain.
According to the present invention, the polypeptide for dissolving thrombus by recognizing 'intravascular thrombus' dissolves thrombus in the blood of a mammal without serious bleeding side effects and has a preventive and therapeutic effect on thrombosis, prevention of thrombosis and related diseases Or it can be widely used as a therapeutic agent.

Description

'Thrombolytic agents for Intravascular clots'

The present invention relates to a 'intravascular thrombus' dissolving agent, and more particularly, it is composed of a thrombo-recognition domain and a thrombolytic domain for 'intravascular thrombus'. It relates to a polypeptide, a gene, and a pharmaceutical composition containing the same.

When a vascular tissue is injured, blood flows out of the blood vessel, so a thrombus is formed in the vascular tissue around the wound to prevent bleeding. A blood clot that is confined 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. On the other hand, a case in which there is an abnormal blood clot in a blood vessel through which blood flows is called 'intravascular thrombus'. If the blood clot is not removed, blood flow is blocked and thrombosis occurs. When blood flow is blocked, hypoxia causes tissue necrosis, leading to fatal thrombotic diseases such as stroke, pulmonary infarction, and myocardial infarction. Therefore, if thrombosis occurs, immediate treatment is urgent.

Thrombosis is a very serious disease and has a high incidence, so various therapeutic agents have been developed. Thrombolytic agents are direct therapeutic agents for dissolving thrombi, tissue-type plasminogen activator (tPA) (US4766075, US5185259, US5587159, US5869314, US6274335), urokinase (US4259447, US4851345, US5055295, US6759042), streptokinase (US3855065, US5011686), US7105327) is an example. These thrombolytic agents, i.e., tPA (alteplase, reteplase), urokinase, and streptokinase, all cut plasminogen in the body and activate it as plasmin, and the activated plasmin breaks down the thrombus and has a thrombosis treatment effect. However, activated plasmin also 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 does not have specificity for 'intravascular thrombus'. Because of the serious bleeding side effects of plasmin, the thrombolytic agents that produce plasmin, such as tPA, urokinase, and streptokinase, are all used in very limited and 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 thrombolytic drugs for thrombotic patients. Anticoagulants and antiplatelet agents do not break up clots that have already formed, but prevent additional clots from forming in the blood vessels. In other words, anticoagulants and antiplatelet agents do not have thrombolytic properties, 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 and thus interfere with wound healing, and the side effects of bleeding are also serious.

Therefore, in order to treat thrombosis, there is an urgent need to develop a dissolving agent specific to 'intravascular thrombus', which accurately recognizes and decomposes only 'intravascular thrombus' and minimizes bleeding side effects by not interfering with normal wound healing.

After recognizing 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 that normally occurs in vascular tissue, is the most ideal thrombosis treatment. Efforts were made to develop an 'intravascular thrombus' dissolving agent. Therefore, an object of the present invention is to provide an innovative concept of 'intravascular thrombus' dissolving agent as a thrombosis treatment agent, which does not have serious bleeding side effects such as systemic bleeding by accurately dissolving only 'intravascular thrombus' causing thrombosis.

In order to achieve the above object, the present invention is composed of a thrombolytic domain comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and a thrombus recognition domain comprising the amino acid sequence shown in 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 shown in SEQ ID NO: 5 or SEQ ID NO: 6, which encodes a thrombolytic domain having an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 to provide.

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

The present invention also provides a pharmaceutical composition for treating or preventing thrombosis and related diseases, characterized in that it contains a polypeptide or a gene encoding the same as an active ingredient by recognizing 'intravascular thrombus' and dissolving the thrombus.

According to the present invention, the polypeptide for dissolving thrombus by recognizing 'intravascular thrombus' dissolves thrombus in the blood of a mammal without serious bleeding side effects and has a preventive and therapeutic effect on thrombosis, prevention of 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 of the thrombolytic enzyme SK or HtrA1 in the ex vivo thrombus according to Experimental Example 1 of the present invention (a; image in which the thrombus is dissolved (HA1) and the undissolved thrombus mass Image (SK) b; thrombus mass solubility expressed in % before treatment, c; fibrin degradation products (FDP), a degradation product of thrombus, d; amount of D-dimer, a degradation product of thrombus). (Ctrl: control, SK: streptokinase, HA1; polypeptide HtrA1 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).
Figure 2 is the result of confirming the thrombolytic ability after treatment of the thrombolytic enzyme SK or HtrA2 to the ex vivo thrombus according to Experimental Example 1 of the present invention (a; image in which the thrombus is dissolved (HA2) and the undissolved thrombus mass Image (SK), b; thrombus mass solubility expressed in % before treatment, c; fibrin degradation products (FDP), a degradation product of thrombus, d; amount of D-dimer, a degradation product of thrombus). (Ctrl: control, SK: streptokinase, HA2; polypeptide HtrA2 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).
3 is a result of confirming the plasminogen activity of the 'intravascular thrombus' lysing polypeptide HtrA1 according to Experimental Example 2 of the present invention (a; measurement of fluorescence intensity of a substrate decomposed by plasmin generated by plasminogen activation; b; SDS-PAGE image confirming the plasmin band generated by plasminogen activation). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA1; polypeptide HtrA1 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).
Figure 4 is the result of confirming the plasminogen activity of the 'intravascular thrombus' lysing polypeptide HtrA2 according to Experimental Example 2 of the present invention (a; measurement of fluorescence intensity of a substrate decomposed by plasmin generated by plasminogen activation; b; SDS-PAGE image confirming the plasmin band generated by plasminogen activation). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA2; polypeptide HtrA2 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).
5 is a result of confirming the activity of fibrinolysis components appearing in the wound healing process 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; fibrinogen An image confirming whether fibrinogen is decomposed into fibrin by SDS-PAGE after treatment with each thrombolytic enzyme, b; An image confirming the degradation of fibronectin by immuno-blot after treatment with each thrombolytic enzyme in cellular fibronectin, c ; Image confirmed by immuno-blot whether fibronectin was degraded after plasma fibronectin was treated with each thrombolytic enzyme). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA1; polypeptide HtrA1 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).
6 is a thrombosis tail image result confirming the therapeutic efficacy of the 'intravascular thrombosis' lysing 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, PL: Plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; Polypeptides HtrA1, HA2 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombi; 'Intravascular thrombus' of the present invention Polypeptide HtrA2) that recognizes and dissolves clots.
7 is an H & E staining image result of a thrombotic tail tissue confirming the therapeutic efficacy of the 'intravascular thrombosis' lysing 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, PL: plasmin, SK: streptokinase, UK; urokinase, tPA; tissue plasminogen activator, HA1; Polypeptides HtrA1, HA2 of the present invention that recognize 'intravascular thrombus' of the present invention and dissolve thrombi; Polypeptide HtrA2) that recognizes 'intravascular thrombus' and dissolves thrombus.
8 is a bleeding image result confirming the effect of the 'intravascular thrombus' lysing 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 recognizes 'intravascular thrombus' of the present invention and dissolves thrombi; solubilizing polypeptide HtrA2).
9 is a bleeding test result confirming the effect of the 'intravascular thrombus' lysing polypeptide HtrA1 of the present invention on wound healing using a wound animal model according to Example 3 of the present invention (a; bleeding time measurement, b; bleeding amount Measurements, c; measurements of the hemoglobin content of the bleeding fluid, d; measurements of time to wound healing and blood clotting). (Ctrl: control, PL: plasmin, SK: streptokinase, UK; urokinase, HA1; polypeptide HtrA1 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).
10 is a bleeding test result confirming the effect of the 'intravascular thrombus' lysing polypeptide HtrA2 of the present invention on wound healing using a wound animal model according to Example 3 of the present invention (a; bleeding time measurement, b; bleeding amount) Measurements, c; measurements of the hemoglobin content of the bleeding fluid, d; measurements of time to wound healing and blood clotting). (Ctrl: control, PL: plasmin, SK: streptokinase, tPA; tissue plasminogen activator, HA2; polypeptide HtrA2 that recognizes 'intravascular thrombus' of the present invention and dissolves thrombus).

Several drugs have been developed to remove thrombus, 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 'intravascular thrombus' is a kind of protein aggregate and that quality control proteins that decompose aggregated proteins are essential for the survival of organisms, The purpose of this study was to confirm that an endogenous proteinase having a domain that decomposes and removes exists in the body, and that this endogenous proteinase can break down blood clots in blood vessels.

Accordingly, in the present invention, a domain recognizing misfolded/aggregated protein, a domain for decomposing and removing misfolded/aggregated protein, and quality control proteins including these domains were searched, and as a result, a thrombus recognition domain and a thrombolytic domain were included. It was confirmed that a polypeptide that recognizes 'intravascular thrombus' and dissolves thrombus by recognizing 'intravascular thrombus' specifically recognizes, decomposes and removes 'intravascular thrombus' can treat thrombosis without serious bleeding side effects.

That is, in the present invention, a polypeptide that recognizes 'intravascular thrombus' containing an intravascular thrombus recognition domain and a thrombolytic domain and dissolves a thrombus (1) has excellent thrombolytic activity, and (2) is different from existing thrombolytic agents , Since it does not activate plasminogen to plasmin, there is no risk of bleeding due to plasmin production. (4) effectively treats thrombosis in an in vivo animal model, (5) unlike existing thrombolytic drugs, the survival rate after treatment for thromboembolism in an in vivo animal model is 100%, (6) Unlike existing thrombolytic agents, it was confirmed that there were no bleeding side effects as it did not interfere with blood clotting and wound healing in in vivo bleeding animal experiments.

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

In another embodiment of the present invention, a mouse model of pulmonary thromboembolism, death can be confirmed in the group treated with the existing thrombolytic agents, but the survival rate is 100% in the group treated with the polypeptide that recognizes the 'intravascular thrombus' of the present invention and dissolves the thrombus Thus, it was confirmed that the polypeptide for recognizing the 'intravascular thrombus' of the present invention and dissolving the thrombus completely treats fatal thromboembolism.

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

Accordingly, in one aspect, the present invention is characterized in that it consists of a thrombolytic domain comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and a thrombus recognition domain comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 It relates to a polypeptide that recognizes 'intravascular thrombus' and dissolves thrombus.

In the present invention, the polypeptide is composed of a thrombolytic domain comprising the amino acid sequence set forth in SEQ ID NO: 1 and a thrombolytic domain comprising the amino acid sequence set forth in SEQ ID NO: 3, or the polypeptide is It may be composed of a thrombolytic domain comprising an amino acid sequence and a thrombus recognition domain comprising an amino acid sequence set forth in SEQ ID NO: 4.

In the present invention, the polypeptide for recognizing the 'intravascular thrombus' and dissolving the thrombus is preferably a trypsin-like polypeptide, characterized in that it belongs to the High Temperature Requirement (Htr) family of serine protease, more preferably It is characterized in that it contains 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, wherein the thrombolytic domain is a GNSGGPL peptide or It is characterized in that it is a domain conferring serine protease activity, including a similar peptide.

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, wherein the thrombus recognition domain comprises: It is characterized in that it has 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 'intravascular thrombus' and regulating the activity of a lytic enzyme.

In another aspect, the present invention provides a thrombolytic domain comprising the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, which encodes a thrombolytic domain having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 It's about genes.

The thrombolytic domain gene is characterized in that it has 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 shown in SEQ ID NO: 3 or SEQ ID NO: 4, characterized in that it has a nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8. It's about genes.

The thrombus recognition domain gene is characterized in that it has 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 contains a polypeptide or a gene encoding the same as an active ingredient for recognizing 'intravascular thrombus' and dissolving the thrombus 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, acacia gum, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. can be exemplified, However, the present invention 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 agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

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

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. 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 of ordinary skill in the art to which the present invention pertains. Or it can be prepared by introducing into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in 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 stabilizer.

The pharmaceutical composition for treating or preventing thrombosis and related diseases according to the present invention has an effect of dissolving 'intravascular thrombus' 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 (cardiovascular disease) ), cerebrovascular disease, ischemic disease, and the like, preferably any one selected from the group consisting of diseases caused by 'intravascular thrombosis', but is not limited thereto. Specific examples are from the group consisting of Deep Vein Thrombosis (DVT), Pulmonary Embolism (PE), ischemic stroke, stroke, cerebral hemorrhage, cerebral infarction, myocardial infarction, heart attack, and angina (unstable angina). It is preferable that any one selected is not limited thereto.

[Example]

Hereinafter, the present invention will be described in more detail through specific examples. However, these examples are only for explaining 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 capacity

Polypeptides HtrA1 and HtrA2 that recognize 'intravascular thrombus' and dissolve thrombus were prepared as recombinant proteins. For this, the cDNA corresponding to 157-480 of the HtrA1 amino acid sequence (SEQ ID NO: 9) containing both the intravascular thrombus domain and the thrombolytic domain was used with forward primer (5'-AATTCATATGCAAGGGCAGGAAGATCCCA-3') and reverse primer (5'-TATCTCGAGCTATGGGTCAATTTCTTCGGG). -3') and amplified by PCR. For PCR conditions, after initial denaturation at 95°C for 5 minutes, amplification (30 seconds at 95°C, 1 minute at 63°C, 2 minutes at 72°C) was repeated 34 times, followed by 10 minutes at 72°C. 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 IPTG was added during culture to express HtrA1. Recombinant E. coli culture medium was sonicated to make cell lysate, passed through econo-pac chromatography column (Bio-Rad) to separate, and then purified by PD-10 column (Amersham, US) to obtain recombinant protein HtrA1. In the case of HtrA2, the cDNA corresponding to 134-458 of the HtrA2 amino acid sequence (SEQ ID NO: 10) was subjected to PCR using a forward primer (5'-GTCCTCGCCCATATGGCCGTCCCTAGCC-3') and a reverse primer (5'-GGCTCTCGAGTCATTCTGTGACCTCAGGG-3'). amplified. For PCR conditions, after initial denaturation at 95°C for 5 minutes, amplification (30 seconds at 95°C, 45 seconds at 65°C, 1 minute at 72°C) was repeated 34 times, followed by 10 minutes at 72°C. After obtaining this, the recombinant protein HtrA2 was obtained by subcloning and expressing the pET28a+ expression vector (Novagen, USA) in the same manner as for HtrA1.

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

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

Experimental Example 2: Plasmin-producing ability (thrombolytic mechanism) evaluation

The current thrombolytic mechanism of thrombolytic drugs is that plasmin-dependent fibrinolysis occurs by activating plasmininogen into plasmin. In order to confirm the mechanism of action compared with existing thrombolytic enzymes, first, each thrombolytic enzyme (0.1 mg/ml) was added and incubated. Activation of plasminogen to plasmin was confirmed by measuring the fluorescence intensity of the substrate dissolved by plasmin. Plasmin bands generated by the actual activation of plasminogen were confirmed by SDS-PAGE after each enzyme (0.15 mg/ml) was treated with plasminogen (5.14 μM).

Compared with the conventional thrombolytic enzyme, HtrA1 had no activating effect on plasminogen (FIG. 3 a), and naturally there was no plasmin production (FIG. 3 b). Similarly, HtrA2 also had no activating effect on plasminogen (FIG. 4 a), and as a result, there was no plasmin production (FIG. 4 b).

Experimental Example 3: Thrombus specificity evaluation

In order to evaluate the thrombus specificity of HtrA1, the activity of fibrinolysis components that appear in the wound healing process was checked. For this purpose, the 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. Dissolution of the fibrin clot was measured at a wavelength of 415 nm using a spectrophotometer. To see the activity of HtrA1 on 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 fibrinogen degradation was evaluated by SDS-PAGE was confirmed with In order to examine 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 to anti- Immunostaining was performed with cFN or anti-pFN antibody.

As shown in Table 1, HtrA1 and HtrA2 not only showed a statistically significant difference in absorbance when compared to the untreated control group, but also showed a statistically significant difference in absorbance for the existing thrombolytic enzyme streptokinase. For fibrin clot dissolution, HtrA1 was the best, 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 degrade fibrinogen, which is important for hemostasis at the wound site (Fig. 5a), and cellular fibronectin (Fig. 5b) and plasma fibronectin (Fig. 5c) important for wound healing also It has been shown to be preserved without decomposition.

Finally, in order to examine the activity of HtrA1 on the wound healing process, an externally incised wound (~30 mm ² ) was made from the tail skin of BALB/c mice. The excised wound pieces were incubated with control 50 mM Tris-HCl or respective thrombolytic enzymes (2 mg/ml) at 37° C. for 72 hours. Wound healing was determined by observing the liquid containing the wound pieces and measuring the absorbance at 550 nm. As such, in the wound tissue experiment using real animals, the absorbance level due to bleeding could be confirmed because the wound tissue did not heal when treated with the existing thrombolytic enzyme. 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

An animal experiment using a tail thrombosis model was performed to evaluate the thrombosis treatment efficacy 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 intraperitoneally injected into each group of mice with tail thrombosis (n = 8), and the length and proportion of the thrombosis site were measured 24 hours later, and the results are shown in Tables 3 and 4 it was

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, when compared to the existing thrombolytic enzymes, it can be seen that the length and frequency of the thrombosis tail in the HtrA1-treated group are significantly reduced, and the thrombolytic effect increases in proportion to the dose. was confirmed. That is, it was confirmed that HtrA1 had a statistically significant level of 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, in the mouse tail thrombosis model, compared to the existing thrombolytic enzymes, in the case of the HtrA2-treated group, it can be seen that the length and frequency of the thrombosis tail in the tail are also significantly reduced, and this thrombolytic effect is proportional to the dose to confirm the increase. In the case of HtrA2, it was confirmed that it had a statistically significant level of thrombosis treatment effect not only in the untreated group but also in comparison with existing thrombolytic enzymes.

In FIG. 7, as a result of observing the thrombosis tail tissue by H & E staining, as with the above thrombosis tail result, the thrombus mass and the degree of thrombus dissolution were directly confirmed. Compared with existing thrombolytic enzymes, it showed excellent thrombosis treatment.

Example 2: Therapeutic effect of pulmonary thromboembolism

Next, an animal experiment using a pulmonary thromboembolism model was performed to evaluate the prevention and treatment efficacy of thromboembolism. A model of pulmonary thromboembolism induced by adenosine 5'-diphosphate (ADP) was established in 15-week-old female C57BL/6 mice. Each group of pulmonary thromboembolism mice (n = 8) was fasted for more than 12 hours, and then saline (control) or thrombolytic enzyme was injected intravenously at a dose of 40 mg/kg. After 30 minutes, ADP (150 mg/kg) was injected to the mice 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 lung thromboembolism mouse model, the survival rates of the plasmin, streptokinase and urokinase treatment groups were 50%, 33%, and 66%, respectively, but the survival rate of the HtrA1 treatment group was 100%, from which the present invention It was found that HtrA1 of HtrA1 has perfect therapeutic and preventive effects 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 thromboembolism mouse model, the survival rates of the plasmin, streptokinase and tPA treatment groups were 60%, 20%, and 40%, respectively, but the survival rate of the HtrA2 treatment group was 100%. HtrA2 was also found to have a perfect therapeutic and preventive effect on pulmonary thromboembolism.

Example 3: Effect of removing the risk of internal bleeding

A tail bleeding test was performed using 15-week-old C57BL/6 female mice to determine whether there is a risk of internal bleeding during the wound healing process when the thrombolytic protein HtrA1 is administered. Saline (control) or each thrombolytic enzyme was intravenously injected 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 the bleeding time and amount of blood were recorded while collecting blood until the bleeding stopped, and the hemoglobin content of the collected blood was measured. In addition, the time required for blood to clot in the severed tail vein of the rat was recorded.

As shown in FIG. 8 , it was confirmed that the bleeding of HtrA1 and HtrA2 was significantly less than that of other thrombolytic enzymes in the mouse tail bleeding experiment. In the case of the HtrA1-treated group, the actual bleeding time (FIG. 9 a), bleeding amount (FIG. 9 b), lost hemoglobin amount (FIG. 9 c), and time to hemostasis (FIG. 9 d) were measured. It was confirmed that the bleeding side effects were reduced incomparably with the existing thrombolytic enzymes, so it was at a level similar to that of the control group.

In the case of the HtrA2-treated 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 incomparably with those of thrombolytic enzymes, which was similar to that of the control group. HtrA1 and HtrA2 do not interfere with the hemostasis process, which is a part of the wound healing process, and thus completely reduce the risk of bleeding.

Example 4: Evaluation of Similarity by Species

The amino acid sequences of SEQ ID NOs: 1 to 4, SEQ ID NOs: 9 and 10 of the present invention and the similarity of the mouse, rat and bovine amino acid sequences are evaluated in, and the results are shown in Tables 7 and 8 below. indicated.

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

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

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

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

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

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

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

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

The specific 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): TESHDRQAKGKAVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVISINGQSVVTANDVSDVIKKENTLNMVVRRGNE

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

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

The specific 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

SEQ ID NO: Domain Similarity (%) Human Mouse Rat bovine Seq.1 Thrombolytic domain serine protease domain -HtrA1 100 99.4 99.4 92.5 Seq.2 Thrombolytic domain serine protease domain -HtrA2 100 97.2 97.2 97.2 Seq.3 Thrombus recognition domain PDZ-HtrA1 100 95.1 95.1 97.1 Seq.4 blood clot recognition domain
PDZ-HtrA2 100 92.6 93.8 92.7

The specific 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 specific 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

SEQ ID NO: 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: Thrombolytics recognition of thrombolytics or evaluation of thrombolytic activity of thrombolytic proteins having similarity to thrombolytic domains

Mouse thrombolytic protein HtrA1 (MHT1) (SEQ ID NO: 27), which is a homologous enzyme of 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 isolated and purified, and then SATA (spectrophotometric analysis of thrombolytic activity) thrombolytic activity analysis method thrombus solubility of each thrombolytic protein was measured.

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

2. 100 μL of each thrombolytic agent to be tested was added to this thrombus solution, and then left at 37°C for 20 minutes to induce a thrombolytic reaction.

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

4. This solution was diluted 25-fold with saline solution, absorbance was measured at 405 nm, and the thrombolytic activity of HtrA1, HtrA2, and each homologous enzyme was measured compared to the blood standard curve. The results are shown in Table 9.

modified thrombolytic agent 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 thrombus in common. These results show that the function of thrombolytic protein that dissolves thrombus, which is fatal to survival, is evolutionarily preserved, so that homologous thrombolytic protein of animals having homology with human thrombolytic protein can also be used for the purpose of dissolving human thrombus. indicates that there is Therefore, in the present invention, a therapeutic agent for the purpose of dissolving human thrombolysis may include not only a human thrombolytic protein but also an animal thrombolytic protein having a similarity thereto as a therapeutic agent for the purpose of dissolving a human thrombus.

<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 Ly s 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 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 tg gggctccg caactcagac atggactaca tccagaccga cgccatcatc 360 aactatggaa actcgggagg cccgttagta aacctggacg gtgaagtgat tggaattaac 420 actttgaaag tgacagctgg DNA base lytic sequence of bodily domain 210 480 th 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 gagtgaacacac catgaaggtc 213 catgaacctt ctgaaggtc 480 acagctgtttgt> 309 c gagctgttgtt t l 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) 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 Le u 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 Ty r 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 Al a 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 A sn 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 S er 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 Se r 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 Gl u 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 Il e 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 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 P ro 85 90 95 Ala Ser Ala Thr Val Arg 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 Leu Ala Ala Pro Ala Ser Ala Gln Pro Ala 20 25 30 Arg Ala Gly Arg Ser Al a 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 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 <21 0> 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 A sp 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 H is 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 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 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 Bo vine <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 31 0 315 320 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 445Thr Leu Tyr Val Thr Pro Glu Val Thr Glu 450 455

Claims (6)

(a) a thrombolytic domain having at least 80% similarity to the amino acid sequence of SEQ ID NO: 1 and a thrombus recognition domain having at least 80% similarity to the amino acid sequence of SEQ ID NO: 3, or (b) the amino acid sequence of SEQ ID NO: 2 A polypeptide that recognizes 'intravascular thrombus' and dissolves thrombus, characterized in that it consists of a thrombolytic domain having a similarity of 80% or more and a thrombus recognition domain having 80% or more similarity to the amino acid sequence of SEQ ID NO: 4.
A polypeptide that recognizes 'intravascular thrombus' consisting of the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10 to dissolve the thrombus.
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, recognizing 'intravascular thrombus' and dissolving the thrombus.
A thrombolytic domain gene encoding a thrombolytic domain having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of claim 1, characterized in that it has 80% or more similarity to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
A thrombus recognition domain gene encoding a thrombus recognition domain having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 of claim 1, characterized in that it has 80% or more similarity to the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
A pharmaceutical composition for treating or preventing thrombosis, characterized in that it contains a polypeptide or a gene encoding the same as an active ingredient for recognizing the 'intravascular thrombus' of any one of claims 1 to 3 and dissolving the thrombus.

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