JPH0834800A - New physiologically active substance - Google Patents

Abstract

PURPOSE:To obtain a new physiologically active substance derived from Bothrops jararaca, a kind of rattlesnake native to Brazil, having properties specific in molecular weight, constituents, size and N-terminal amino acid partial sequence, capable of inhibiting blood platelet adhesion/coagulation, thus useful for antithrombotic agents, etc. CONSTITUTION:A new physiologically active substance consisting of a polypeptide derived from the snake venom of Bothrops jararaca, a kind of rattlesnake native to Brazil, capable of inhibiting blood platelet adhesion/coagulation and having antithrombotic activity, thus useful for treating and preventing a wide range of thromboembolisms etc. It is made up of subunit alpha and subunit beta. The subunit alpha has the following characteristics: molecular weight (determined by SDS-polyacrylamide gel electrophoresis): 30000+ or -5000 (17000+ or -3000 after reduction); the N-terminal amino acid partial sequence is expressed by formula I; containing an amino acid sequence which internal amino acid sequence is expressed by formula III, etc. The subunit betahas the following characteristics; molecular weight (determined by SDS- polyacrylamide gel electrophoresis): 30000+ or -5000 (13000+ or -30OO after reduction); the N-terminal amino acid partial sequence is expressed by formula II; and containing an amino acid sequence which internal amino acid sequence is expressed by formula IV, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide which inhibits adhesion and aggregation of platelets and has an antithrombotic effect.

[0002]

BACKGROUND OF THE INVENTION It is well known that arteriosclerosis, surgery, trauma, labor and infectious diseases lead to thrombosis, an increased risk of blood clots in blood vessels. Von Willebrand factor
ctor: hereinafter abbreviated as vWF) forms a complex with blood coagulation factor VIII (hemophilia factor) by non-covalent bond, stabilizes its activity as a carrier protein, and retains its activity. Contribute to the formation of. Another important function is a direct contribution to the rapid formation of platelet thrombi (primary hemostasis) at the site of vascular injury in vivo. In this primary hemostasis, adhesion of platelets to subendothelium of the blood vessel, activation of platelets and aggregation of platelets with each other via adhesion protein [attachment of platelet membrane glycoprotein (GP) IIb / IIIa and fibrinogen is regarded as important. ], VWF plays a role as a molecular glue that promotes a cross-linking reaction between the two in the first step, which is the adhesion reaction of platelets to exposed subendothelium of blood vessels. In particular, the binding reaction between vWF and platelet membrane GPIb
It is considered to be essential for the adhesion of platelets to the subendothelium. The bleeding tendency observed in von Willebrand disease (quantitative or qualitative abnormality of vWF) and Bernard-Soulier syndrome (deficiency of platelet membrane GPIb) known as hereditary hemorrhagic diseases is in vivo. This suggests the importance of this reaction.

In vitro, the binding reaction between vWF and platelet membrane GPIb is a non-physiological modulator of ristocetin (an antibiotic) or botrocetin [Bothrops jarar (Bothrops jarar from Brazil).
aca) -derived protein] and is also used for clinical diagnosis of the above-mentioned congenital diseases. However, in vivo
The details of the reaction mechanism in E. coli are not clear yet, and the following hypotheses are considered. That is, at the site of vascular rupture, vWF binds to the subendothelium of blood vessels, and as a result, structural changes occur on the vWF molecule, resulting in platelet membrane GPI.
The ability to bind to b is expressed, and it binds to platelet membrane GPIb. In this hypothesis, vWF never binds to platelet membrane GPIb in normal blood flow, and when vascular rupture occurs or under certain special fluid dynamics (high shear stress).
Then, it will be the first time to combine. High shear stress occurs in a site accompanied by strong vascular stenosis such as arteriosclerosis, and its involvement with pathological conditions has recently been particularly highlighted. High shear stress-induced platelet aggregation involves vWF and platelet membrane G
The binding of PIb and vWF to platelet membrane GPIIb / IIIa is important, while fibrinogen and platelet membrane GPIIb / II are involved in low shear stress-induced platelet aggregation.
Emphasis is placed on binding with Ia.

Under these circumstances, attempts have recently been made to create new antithrombotic agents that inhibit the binding of vWF to the platelet membrane GPIb and inhibit only platelet aggregation due to high shear stress. Peng et al. (Biochemistry, 30, 11529-115
36, 1991) reported a novel snake venom protein arboaggregin B (AL-B) that acts on platelet membrane GPIb from a certain snake venom and inhibits vWF binding.

[0005]

However, this protein AL-B causes platelet aggregation by itself,
Considering its potential as a drug, it is expected to become a major obstacle. The present invention relates to vWF and platelet membrane GPI.
It is an object of the present invention to provide a polypeptide having a property of inhibiting binding of b, inhibiting vWF-dependent platelet aggregation, and not causing platelet aggregation by itself.

[0006]

Under these circumstances, the present inventors have found that vW is added to the venom of Bottrops jaralaca.
As a result of investigating by presuming the existence of a protein that inhibits F-dependent platelet aggregation, a property of inhibiting vWF-platelet GPIb binding, inhibiting vWF-dependent platelet aggregation, and not causing platelet aggregation by itself The present invention has been completed successfully by isolating a polypeptide having

That is, the present invention is a polypeptide specified by the following physicochemical properties. (1) Molecular Weight, Constituents, Size: The molecular weight by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis is 30,000 ± 5,000 and after reduction 17,000 ± 3,000. Subunit α,
A polypeptide consisting of subunit β, which is 13,000 ± 3,000. (2) N-terminal amino acid partial sequence: (a) subunit α; sequence listing 1 (b) subunit β; sequence listing 2 (3) internal amino acid sequence: (a) subunit α; 1) sequence listing 3, 2 ) Sequence Listing 4, 3) Sequence Listing 5, 4) Sequence Listing 6, and 5) Sequence Listing 7 including the internal sequences. (B) subunit β; 1) Sequence listing 8, 2) Sequence listing 9, 3) Sequence listing 10, and 4) Sequence listing 11 including the internal sequences. (In the sequence, Xaa means an amino acid residue.)

The polypeptide of the present invention (a) inhibits aggregation of human formalin-fixed floating platelets and human platelet-rich plasma by ristocetin and / or botrocetin. (B) Inhibits the aggregation of human formalin-fixed floating platelets by bovine vWF. (C) Inhibits the binding of vWF to human formalin-fixed platelets by ristocetin and / or botrocetin. (D) Inhibits the binding of bovine vWF to human formalin-fixed platelets. (E) Inhibits high shear stress-induced platelet aggregation using human platelet-rich plasma, but not low shear stress. (F) The polypeptide alone does not cause aggregation of human fixed platelets. It is also characterized by its pharmacological properties. The polypeptide of the present invention is (g) a GPIb-binding protein whose binding site is vWF.
It is the same as or near the binding site. (H) When reduced, the vWF-dependent platelet aggregation inhibitory activity is lost. It also has the property of Therefore, in the present invention, not only the polypeptide having the structure specified by the above (1), (2) and (3), but also at one or more sites in the amino acid sequence of the polypeptide of the present invention, A polypeptide in which more amino acids have been deleted, inserted or substituted also inhibits the properties (a) to (h) of the above polypeptide, that is, the binding of vWF and platelet membrane GPIb, and vWF-dependent platelet aggregation. Since it may have the property of inhibiting the above-mentioned and not causing platelet aggregation by itself, such a polypeptide having the same effect (hereinafter referred to as the same effect substance) is also included. As a particularly preferred polypeptide in the present invention, the above-mentioned polypeptide derived from the snake venom of Botrops jaralaca or its equivalent, particularly Yoshitobin or its equivalent named by the present inventors can be mentioned.

In the present specification, the notation of a partial amino acid sequence of a peptide is customarily defined with the left end as the N-terminal,
Described on the right in the order of C-terminal direction, and IUPAC-IUB
Based on the abbreviations by the Biochemical Nomenclature Committee or the abbreviations commonly used in this field, the three-letter amino acid symbols shown below are used. Alanine Ala,
Arginine Arg, Asparagine Asn, Aspartic acid Asp, Cysteine Cys, Glutamic acid
Glu, glutamine Gln, glycine Gly, histidine His, isoleucine Ile, leucine L
eu, lysine Lys, methionine Met, phenylalanine Phe, proline Pro, serine Se
r, threonine Thr, tryptophan Trp, tyrosine Tyr, valine Val. The amino acid residue represented by Xaa may be a natural amino acid residue, and specific examples thereof include the above-mentioned amino acid residues.

Although the physiologically active polypeptide of the present invention can be produced by a synthetic method using a peptide synthesizer, it can be produced by an isolation and purification method from a natural product. The most suitable source of the natural product is the snake venom of Botrops jaralaca, which is the source of the peptide of the present invention, but is not limited to this snake venom, and the above (1), (2) and (3) A natural product containing a polypeptide having the physicochemical properties described above or a similar substance of the polypeptide having the properties (a) to (f) described above may be used. For example, when snake venom is used as a natural product, isolation / purification is carried out with or without extracting the crude venom from the snake venom, and the platelet aggregation inhibitory activity by ristocetin and botrocetin which is the characteristics of (a) to (f) above. , Ristocetin and botrocetin inhibit vWF binding to platelets, shear stress-induced platelet aggregation inhibitory activity, and platelet aggregability of the peptide itself, and if necessary, the properties (g) to (h) above are used as indicators. ,
Difference in adsorption affinity for different adsorbents, difference in solubility or solubility in different solvents, difference in partition between two immiscible liquid phases, difference in elution rate based on molecular size, from solution It is carried out by applying various means utilizing the precipitability or the difference in precipitation rate. These means
If necessary, they can be applied alone or in combination in any order, and can be applied repeatedly. When extracting the crude poison, a solvent capable of dissolving the polypeptide, particularly methanol, ethanol, an alcohol solvent such as isopropanol, benzene, toluene, xylene, acetone, methyl ethyl ketone, ether, tetrahydrofuran, dioxane, dimethylsulfoxide, It is advantageous to use an organic solvent such as ethyl acetate or acetonitrile, water, or a mixed solvent thereof with heating and stirring if necessary. Further, as the isolation / purification means, especially DEAE-
DEAE-Sepharose CL-6B, Heparin-Sepharose CL-6B,
Phenyl Superose HR 5/5, TSK G3000SW, TSK G2000S
It is advantageous to perform it by subjecting it to column chromatography using W as a column packing material or ultrafiltration.

The characteristics of the novel physiologically active substance of the present invention are determined by the following methods. (Substance) Botrops As a snake venom (lyophilized product) derived from Jalaraca, a product of Sigma was used. DEAE Sepharose, Heparin Sepharose, Phenyl Superose HR5
/ 5 for TSK G3000SW and TSK G from Pharmacia
2000SW uses Tosoh products. Aquaside II was purchased from Calbiochem.

(Preparation of Platelets) Blood was collected from healthy persons (adults and boys) with 1/10 volume sodium citrate, and DeMa
rco et al. (J. Clin. Invest., 77, 1272-1277, 198
Platelet-rich plasma according to the method of MacFarlane et al.
Fixed platelets were prepared according to mb.Diath.Haemorrh., 34, 306, 1975).

(Platelet Aggregation Assay) Ristocetin, which is an aggregation inducer, was purchased from CosmoBio and the double-chain botrocetin used was the method of Fujimura et al. (Biochemistry, 30,
1957-1964, 1991). Human vWF and bovine vWF are each described by De Marco et al. (J. Clin. Invest., 6
8, 321-328, 1981) and Mascelli et al. (Biochemistry
25, 6325-6335, 1986).
Automatic blood cell counter (ME
K-5158, Nihon Kohden Co., Ltd.) and used at 3 × 10 ⁸ / ml. The platelet aggregating ability was measured using a platelet aggregometer (NKK Hematotracer 1, SSR Engineering).
That is, in the case of platelet rich plasma, after incubating platelets (200 μl) and sample (25 μl) at 37 ° C. for 3 minutes,
Ristocetin (10 mg / ml) or Botrocetin (20
(25 μl) was added, the change in transmitted light was recorded for 5 minutes, and the aggregation inhibition rate was calculated from the maximum aggregation rate. For fixed platelets, platelets (200 μl) and purified human vWF (1
(00 μg / ml, 25 μl) and sample (25 μl) were incubated at 37 ° C for 3 minutes, and then 25 μl of ristocetin (10 mg / ml) or botrocetin (100 μg / ml) was added, and the aggregation inhibition rate was measured by the same method. Was calculated. Aggregation-inducing activity is as follows: human fixed platelets (200 μl) and purified human vWF (100 μg / m
(25 μl) was incubated at 37 ° C. for 3 minutes, 25 μl of the sample was added, and the aggregation-inducing activity was confirmed by the above aggregometer.

( ¹²⁵ I human vWF and ¹²⁵ I bovine vWF
Of ¹²⁵ I-sodium iodide (Na ¹²⁵ I; Amersham) using purified human and bovine vWF by the IodoGen method.
^{The 125} I label was applied. Radioactivity is 0.74-1.14μCi / μ
It was g. The binding assay was performed by the method of Fujimura et al. (Blood, 77, 113-120, 1991). That is, human fixed platelets (1x10 ⁸ / ml) 105 μl, sample 1
5 μl, ¹²⁵ I-human vWF (10 μg / ml) 15 μl, ristocetin (10 mg / ml) or botrocetin (20 μg / m)
l) 15 μl or human fixed platelets (1x10 ⁸ / ml) 120
μl, sample 15 μl, ¹²⁵ I-bovine vWF (10 μg / m
l) After incubating 15 μl for 30 minutes at room temperature,
% Sucrose, and the platelets were spun down by centrifugation at 10,000 g for 5 minutes, and the bound radioactivity was measured.
The non-specific binding number was determined by the radioactivity when ristocetin or botrocetin was not added, and the specific binding number was calculated by subtracting this from the total binding number. The binding to GPIb, and the binding site being the same as or near the vWF binding site, means that anti-GPIb monoclonal antibody that recognizes the vWF binding site on GPIb inhibits the binding of yocitobine to human fixed platelets. confirmed. ¹²⁵ I-sodium iodide (Na ¹²⁵ I;
Yositobin ¹²⁵ I was labeled by the IodoGen method using Amersham. The binding assay is performed by the method of Peng et al. (Biochemistry, 30,
11529-11536, 1991). The antibody used at this time is an anti-GPIb that recognizes the vWF binding site on GPIb.
Although any monoclonal antibody can be used, for example, GUR20-5 (Takara Shuzo) can be used.

(Shear Stress Dependent Platelet Aggregation Ability Evaluation System) Using a cone plate rotational viscometer, the method of Fukuyama et al.
omb.Res., 54, 253-260, 1989). That is, after addition of aforementioned platelet rich plasma 360 [mu] l and the sample 40 [mu] l, the first 15 seconds 6 dyn / cm ^2, from 6 in the next 90 seconds to 12dyn / cm ^2, from a further 12 for 2 minutes 108
The shear stress was changed to dyn / cm ² and 108 for the last 90 seconds.
Aggregation at dyn / cm ² was observed.

(Protein Concentration Measuring Method) The protein concentration of the sample was measured by using a dye-binding assay kit manufactured by Bio-Rad.
It was determined based on a standard curve using bovine serum albumin as a standard substance.

(SDS polyacrylamide gel electrophoresis) According to Laemmli's method (Nature, 227, 680-685, 1970), 15% SDS-PAGE was used to measure the protein content 1
μg, performed under non-reducing and reducing conditions. The reduced sample buffer was prepared by adding β-mercaptoethanol to a final concentration of 10%, and the sample was mixed with 1/2 volume of the sample buffer and heated at 75 ° C for 15 minutes. The gel after electrophoresis was stained with Coomassie Brilliant Blue R-250 dissolved in a 30% isopropyl alcohol solution containing 10% acetic acid, and decolorized with a 16.5% methanol solution containing 5% acetic acid.

(Amino acid sequence determination) The purified polypeptide was prepared from tri-n-butyl sulfone (reducing agent) and 4-
Reduced pyridyl ethyl with vinyl pyridine, reverse phase HP
LC [filler: Synchropak RP-18,
Synchrome Co., Ltd.] and fractionated into Yoshitobin α and β. Next, for each subunit, lysyl endopeptidase (Wako Pure Chemical Industries) and Staphylococcus
Fragmentation is performed using aureus V8 protease (Wako Pure Chemical Industries, Ltd.), and the fragmented peptides are fractionated by reverse phase HPLC in the same manner as above, and then the gas phase amino acid sequencer (Applied Biosys
tems, USA) to determine the amino acid sequence.

[0019]

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the present invention should not be limited to the examples.

Example 1 (Purification method) First step (step 1): DEAE Sepharose CL-
6B column chromatography containing 2 mM benzamidine hydrochloride, 0.1 M sodium chloride, 0.05% sodium azide (NaN ₃ ) 84
mM imidazole hydrochloride buffer (pH 7.
5. Buffer A) 3 g of crude poison (2,500 g of insoluble material removed after centrifugation for 15 minutes) dissolved in 200 ml of starting material was used as a starting material, and DEAE Sepharose FCL-6B column (2.6 x The sample was added to 35 cm 3) and washed with the same buffer at a flow rate of 60 ml / hr for 12 hours to collect a flow-through fraction. The flow-through fraction is subjected to ultrafiltration (Model 8050, Amicon) using a diaflow membrane (PM10: Amicon), and the total volume is about 20 m.
After concentrating to 1 liter, 0.02 M Mes-Tris-HCl buffer (pH 6.0 with 0.05% NaN ₃ added) (Buffer B)
It dialyzed at 4 degreeC for one day.

Second step (step 2): heparin sepharose CL-6B column chromatography The fraction obtained in step 1 was equilibrated with buffer B to a heparin sepharose CL-6B column (2.6 x).
15 cm) and washed with the same buffer at a flow rate of 60 ml / hr for 10 hours to collect a flow-through fraction. The flow-through fraction is subjected to ultrafiltration (Model 8050, Amicon) using a diaflow membrane (PM10: Amicon), concentrated to a total volume of about 10 ml, and then 50 mM phosphate buffer (pH 7.0) containing 1.7 M ammonium sulfate. , Buffer C) was dialyzed at 4 ° C. for one day. .

Third step (step 3): phenyl chromatography Phenyl S obtained by equilibrating the above active fraction with buffer C
Add to an upose HR5 / 5 column (5 mm x 5 cm), wash the column with a buffer C flow rate of 0.5 ml / min, remove the flow-through fraction, and perform linear gradient elution with 100% → 0% 1.7 M ammonium sulfate. It was performed for 60 minutes at a flow rate of 0.5 ml / min. The peak was detected at UV280 nm, and the activity was confirmed by the same assay. The active fraction was concentrated by ultrafiltration (described above) and Aquaside II, and dialyzed against 50 mM Tris buffer (pH 7.5, buffer D) containing 0.5 M NaCl at 4 ° C for one day.

Fourth stage (step 4): gel filtration HPL
C The above-mentioned active fraction was equilibrated with buffer D. TSK 3000S
W (7.5mmx30cm) and TSK2000SW (7.5mmx30cm) tandem column added and buffer D flow rate 0.2 ml / mi
Elution was performed at n. The peak was detected at UV 280 nm, and the activity was confirmed by the same assay. The active fraction was concentrated by ultrafiltration (described above) and Aquaside II, and then dialyzed against physiological saline at 4 ° C for one day. After the polypeptide was concentrated, the protein quantification described above was performed.

The results of confirming the activity of the polypeptide obtained through the process of step 4 are as follows. (1) Inhibition of Platelet Aggregation This polypeptide completely inhibited ristocetin and botrocetin aggregation of human fixed platelets and human platelet rich plasma at 3 μg / ml. Further, the aggregation of human fixed platelets by bovine vWF was completely inhibited at 3 μg / ml. Furthermore, for human ADP and collagen aggregation of platelet-rich plasma, 30 μg /
It had no effect on ml. (2) Binding Assay These polypeptides completely inhibited the binding of ¹²⁵ I-human vWF to human fixed platelets by botrocetin and ristocetin and the binding of ¹²⁵ I-bovine vWF to human fixed platelets at 3 μg / ml. . (3) Inhibition of shear stress-induced platelet aggregation These polypeptides completely inhibited platelet aggregation induced by high shear stress using human platelet-rich plasma at 3 μg / ml, but not those of low shear stress. . (4) Platelet aggregation-inducing activity These polypeptides, at 30 μg / ml, did not induce the aggregation of human fixed platelets at all.

With respect to the purified polypeptide (protein amount: 1 μg), phosphorylase B (97.4
Kd), serum albumin from bovine serum (66.2 Kd), ovalbumin from egg white (45 Kd), carbonic anhydrase from cow (31 Kd), trypsin inhibitor from soybean (21.5 Kd) and lysozyme from egg white. (14.
4 Kd) as standard 15% SDS-PAGE
When the molecular weight was accurately calculated using a calibration curve under non-reducing conditions and reducing conditions, it was found to consist of 30,000 subunits before reduction and 17,000 and 13,000 subunits after reduction.

The present inventors have recognized that the polypeptide thus obtained is a novel substance having the above-mentioned properties, and thus Yoshitobin (SD before reduction)
For the polypeptide which was found to have a molecular weight of 30,000 by S-PAGE analysis) and the polypeptide separated after reduction, Yoshitobin subunit α (after reduction SDS-PAGE
The polypeptide was identified by E analysis to have a molecular weight of 17,000), and was designated as yositobin subunit β (same 13,000).

From the above results, Yoshitobin of the present invention is
The molecular weight by SDS-PAGE analysis is 30,000.
± 5,000, 17,000 ± 3,000 after reduction
Is a heterodimer consisting of subunit α, and subunit β, which is 13,000 ± 3,000.

FIG. 1 shows the migration pattern of 15% gradient SDS-PAGE before and under the reducing conditions of yocitobine obtained through the step 4 purification step. Lanes N and R in the figure are electrophoretic patterns of yositobin (1 μg) obtained through the step 4 purification step under non-reducing (lane N) and reducing (lane R) conditions, and were calculated to be about 17.0 Kd. It is the subunit α, and similarly, the subunit β is calculated to be about 13.0 Kd.

The yositobin obtained in the step 4 purification step was dialyzed against ammonium carbonate and freeze-dried to obtain 300 μg of guanidine hydrochloride as a sample.
Add 600 mg, add 400 μl of 0.3 M Tris-HCl buffer (pH 8.3), stir, and add 200 μl of Millicure (MilliQ) water to make about 1 ml. 20 μl
Tri-n-butyl sulfone (reducing agent), then 10 μl
4-vinyl pyridine was added and left for 3 hours, then reverse phase HPL
C [filler: Synchropak RP-18] was divided into yocitovin subunits α and β, and these were separated into gas phase amino acid sequencers (Applied Biosystems, U).
The N-terminal amino acid partial sequence was confirmed by SA). Reversed phase H
The PLC elution conditions are as follows. The elution pattern obtained is shown in FIG. In addition, Yoshitobin subunit α
The N-terminal amino acid sequences of and are shown in Sequence Listings 1 and 2, respectively. Reversed phase HPLC elution conditions: Measurement wavelength: 280 nm Mobile phase: Millikyu water containing 0.1% trifluoroacetic acid (buffer E) and acetonitrile containing 0.075% trifluoroacetic acid (buffer F) at the following times: Gradient elution with the following ratios. Flow rate: 1 ml / min Next, for each subunit, lysyl endopeptidase (Wako Pure Chemical Industries) and Staphylococcus aure
fragmented using us V8 protease (Wako Pure Chemicals)
Fragment peptides were fractionated by reverse phase HPLC, and the amino acid sequence of each fragment was similarly determined. The elution conditions of the reverse phase HPLC after the lysyl endopeptidase treatment of the yositobin subunits α and β are as follows, and the obtained elution patterns are shown in FIGS. 3 and 4, respectively. Reversed phase HPLC elution conditions: Measurement wavelength: 206 nm Mobile phase: Gradient elution of the above buffer E and buffer F at the following ratio in the following time. Flow rate: 1.3 ml / min Table 1 shows the amino acid sequences of the fragments obtained from each fraction.
And shown in Table 2.

[0030]

[Table 1] Peptide fraction of lysyl endopeptidase-treated subunit α N-terminal sequence 1 Asp Thr Pro Phe Glu Cys Pro Ser Asp Trp Ser Thr His Arg Gln Tyr Cys Tyr Lys 2 Trp Ser Asp Tyr Ser Ser Val Ser Tyr Glu Asn Leu Val Arg Gly Asn Val Lys and Trp Val Asn Ile Asp Cys Val Glu Gly Asn Pro Phe Val Cys Lys 3 Glu Ser Trp Asp Asp Arg Ser Glu Tyr Asp Ala Glu Arg Phe Cys Ser Glu Gln Ala Lys

[0031]

[Table 2] Peptide fraction of lysyl endopeptidase-treated subunit β N-terminal sequence 4 Asp Cys Pro Ser Asp Trp Ser Pro Tyr Gly Gly His Cys Tyr Lys 5 Gln Arg Met Asn Trp Ala Asp Ala Glu Asn Leu Cys Ala Gln Gln Arg Lys 6 Val Asn Tyr Asn Ala Trp Ala Ser Glu Ser Glu Cys Val Ala Ser Lys 7 Thr Thr Asp Asn Gln Trp Trp Ser Phe Pro Cys Thr Arg Leu Gln Tyr Phe Val Cys Glu Phe Gln Ala

From the treatment with V8 protease, the yositobin subunit α was found to be Lys Lys GlnGly Phe Arg Lys Xaa.
Val Asn Ile Asp (where Xaa stands for amino acid residue) and Gly Asn Pro Phe Val Cys Lys Phe Ile Ar
A fragment having g Pro at the N-terminus is formed, and yocitovin subunit β is Ser His Leu Val Ser PheHis S
It was found that a fragment having er Ser Glu Glu at the N-terminal was formed. Specified yositobin subunit α
The internal sequences of β and β are shown in Sequence Listings 3 to 11.

The activity of yocitobine obtained in step 4 was confirmed. As a result, it was found that yocitotobin completely inhibits ristocetin and botrocetin aggregation of human fixed platelets and human platelet-rich plasma at 3 μg / ml, and ADP and collagen aggregation were not affected at 30 μg / ml. In addition, binding of ¹²⁵ I-human vWF to human fixed platelets and binding of ¹²⁵ I-bovine vWF to human fixed platelets by botrocetin in the binding assay were completely inhibited at 1 μg / ml. In addition, yositobin at 30 μg / ml did not cause any aggregation of human fixed platelets,
It was confirmed that it does not induce platelet aggregation by itself.

Example 2 (Reductive Alkylation of Yoshitobin) 30 μg / yosytobin obtained in step 3 of Example 1
ml to 10 mM dithiothreitol (DTT: dithiothreito
l) containing 50 mM Tris-HCl buffer (pH 8.6) 3
After incubating at 7 ° C for 30 minutes, 50 mM Tris-HCl buffer (pH 8.6) containing 80 mM iodoacetamide (IAA) was added, and the mixture was allowed to stand at room temperature for 30 minutes. The molecular weight was measured by SDS-PAGE, and it was confirmed that yocitobine was reduced to a subunit of 17,0,13,0 Kd. The vWF-dependent platelet aggregation inhibitory activity of this reduced yocitovin was confirmed by the inhibition of human fixed platelet aggregation by bovine purified vWF 5 μg / ml. As a result, the vWF-dependent platelet aggregation inhibitory activity of reduced yositobin was completely abolished.

Example 3 (Binding of ¹²⁵ I-yocitobine to human fixed platelets) ¹²⁵ I-sodium iodide (Na ¹²⁵ I; Amersham)
Was used for iodine labeling of yositobin by the iodine method. The radioactivity of the labeled protein is 0.74-1.14 μ
It was Ci / μg. This ¹²⁵ I-Yocitobin (9 μg / ml)
Bound strongly to human fixed platelets and this binding was saturable. In addition, this binding was completely inhibited by an anti-GPIb monoclonal antibody (10 μg / ml), which has already been confirmed to recognize the vWF binding site on GPIb. That is, Yoshitobin is a GPIb binding protein,
It was also found that the binding site was the same as or near the vWF binding site. Scatchard plot (Scat
As a result of analysis by chard Plot, the Kd value of the combination is 39.1.
± 2.4 nmol / l, the number of bonds per platelet is 40,
It was 629 ± 2521.

[0036]

As described above, the polypeptide of the present invention has the property of inhibiting the binding of vWF and platelet membrane GPIb, inhibiting vWF-dependent platelet aggregation, and not causing platelet aggregation by itself. As an excellent antithrombotic agent that inhibits only platelet aggregation due to high shear stress, it is useful as a preventive or therapeutic agent for a wide range of thromboembolisms caused by vascular inflammation and metabolic disorders. A pharmaceutical composition containing the polypeptide of the present invention as an active ingredient is added with a polypeptide from which bacteria and pyrogens have been completely removed and a pharmaceutically acceptable formulation component such as a diluent and a stabilizer. Agents (eg,
Physiological saline, distilled water for injection, various buffer solutions, sucrose, lactose,
Glucose, mannitol, sorbitol, xylitol and other sugars, arginine, glycine and other amino acids, monoethanolamine, diethanolamine, triethanolamine and other ethanolamines, polyoxyethylene higher fatty acid ester, polyoxyethylene sorbitan higher fatty acid ester, polyoxy Surfactants such as ethylene hydrogenated castor oil and other commonly used additives)
Usually, it is prepared as an injection (solution, lyophilized formulation that is soluble at the time of use) and parenterally administered (for example, intravenous injection or subcutaneous injection).

[0037]

[Sequence list]

 SEQ ID NO: 1 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence: 5 10 15 Asp Thr Pro Phe Glu Cys Pro Ser Asp Trp Ser Thr His Arg Gln 20 25 Tyr Cys Tyr Lys Phe Phe Gln Gln Lys Glu Ser Trp Asp Asp

SEQ ID NO: 2 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence: 5 10 15 Asp Cys Pro Ser Asp Trp Ser Pro Tyr Gly Gly His Cys Tyr Lys 20 Leu Phe Lys Gln Arg Met Asn

SEQ ID NO: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 15 Trp Ser Asp Tyr Ser Ser Val Ser Tyr Glu Asn Leu Val Arg Gly Asn Val Lys

SEQ ID NO: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 15 Trp Val Asn Ile Asp Cys Val Glu Gly Asn Pro Phe Val Cys Lys

SEQ ID NO: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 15 Glu Ser Trp Asp Asp Arg Ser Glu Tyr Asp Ala Glu Arg Phe Cys 20 Ser Glu Gln Ala Lys

SEQ ID NO: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 Lys Lys Gln Gly Phe Arg Lys Xaa Val Asn Ile Asp

SEQ ID NO: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 Gly Asn Pro Phe Val Cys Lys Phe Ile Arg Pro

SEQ ID NO: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 15 Gln Arg Met Asn Trp Ala Asp Ala Glu Asn Leu Cys Ala Gln Gln Arg Lys

SEQ ID NO: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 15 Val Asn Tyr Asn Ala Trp Ala Ser Glu Ser Glu Cys Val Ala Ser Lys

SEQ ID NO: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment Sequence: 5 10 15 Thr Thr Asp Asn Gln Trp Trp Ser Phe Pro Cys Thr Arg Leu Gln 20 Tyr Phe Val Cys Glu Phe Gln Ala

SEQ ID NO: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence: 5 10 Ser His Leu Val Ser Phe His Ser Ser Glu Glu

[0048]

[Brief description of drawings]

FIG. 1 is a drawing showing an electrophoretic pattern of yocitotobin under SDS-PAGE analysis under non-reducing conditions and reducing conditions. In the figure, lanes N and R are the migration patterns of Yoshitobin (under non-reducing conditions) and (under reducing conditions). The numerical value (Kd) on the left side shows the molecular weight of yoshitobin, and the numerical value on the right side shows the size of the yocytobin subunits α and β, respectively.

FIG. 2: Reversed phase HP of yositobin subunits α and β
It is an elution pattern by LC.

FIG. 3 is an elution pattern by reverse phase HPLC after treating yositobin subunit α with lysyl endopeptidase.

FIG. 4 is an elution pattern by reverse phase HPLC after treating yositobin subunit β with lysyl endopeptidase.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Tomihisa Kawasaki 5 Palace No. 1-18, Sengen, Tsukuba-shi, Ibaraki 203 Palace Happiness 203 (72) Yumiko Sakai 1-14, Ninomiya, Tsukuba, Ibaraki 306 Bonur Tsukuba (72) Inventor Seiji Karai 3-10-10 Namiki, Tsukuba, Ibaraki Prefecture 4 Maric Chiyokota 202

Claims (2)

[Claims] 1. A polypeptide specified by the following physicochemical properties. (1) Molecular Weight, Constituents, Size: The molecular weight by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis is 30,000 ± 5,000 and after reduction 17,000 ± 3,000. Subunit α,
A polypeptide consisting of subunit β, which is 13,000 ± 3,000. (2) N-terminal amino acid partial sequence: (a) subunit α; Asp Thr Pro Phe Glu Cys Pro Ser Asp Trp Ser Thr Hi
s Arg GlnTyr Cys Tyr Lys Phe Phe Gln Gln Lys Glu S
er Trp Asp Asp (b) subunit β; Asp Cys Pro Ser Asp Trp Ser Pro Tyr Gly Gly His Cy
s Tyr LysLeu Phe Lys Gln Arg Met Asn (3) Internal amino acid sequence: (a) Subunit α; 1) Trp Ser Asp Tyr Ser Ser Val Ser Tyr Glu Asn Le
u Val ArgGly Asn Val Lys, 2) Trp Val Asn Ile Asp Cys Val Glu Gly Asn Pro Ph
e Val CysLys, 3) Glu Ser Trp Asp Asp Arg Ser Glu Tyr Asp Ala Gl
u Arg PheCys Ser Glu Gln Ala Lys, 4) Lys Lys Gln Gly Phe Arg Lys Xaa Val Asn Ile As
p and 5) contains the internal sequence of Gly Asn Pro Phe Val Cys Lys Phe Ile Arg Pro. (B) Subunit β; 1) Gln Arg Met Asn Trp Ala Asp Ala Glu Asn Leu Cy
s Ala GlnGln Arg Lys, 2) Val Asn Tyr Asn Ala Trp Ala Ser Glu Ser Glu Cy
s Val AlaSer Lys, 3) Thr Thr Asp Asn Gln Trp Trp Ser Phe Pro Cys Th
r Arg LeuGln Tyr Phe Val Cys Glu Phe Gln Ala and 4) Contains the internal sequence of Ser His Leu Val Ser Phe His Ser Ser Glu Glu. (Xaa in the sequence means an amino acid residue) 2. Bothrops jalaraka
[Raraca] (Brazilian hibiscus snake) -derived polypeptide.