CN1289141C - Thrombogenesis inhibiting medicine - Google Patents

Abstract

The present invention discloses a medicine for inhibiting thrombus. The active component of the medicine for inhibiting thrombus of the present invention is an amino acid residue sequence of SEQ ID No. 2, or a protein which is used for substituting, deleting or adding one or a plurality of amino acid residues of the amino acid residue sequence of SEQ ID No. 2, has the same activity with the amino acid residue sequence of SEQ ID No. 2 and is derived from SEQ ID No. 2. A coded gene of a recombination platelet glycoprotein rGP302 is one of the following nucleotides: 1) a DNA sequence of SEQ ID No. 1 in a sequence list, 2) polynucleotide for coding the amino acid residue sequence of SEQ ID No. 2, and 3) a DNA sequence which has more than 95 % of homology with the DNA sequence limited by SEQ ID No. 1 in the sequence list and codes the same functional protein. The present invention provides a new approach and a new method for preventing and treating thrombus diseases, and thereby, the present invention lays the foundation for further theoretical research and novel thrombus resisting medicine preparation, and has important meanings.

Description

The thrombotic medicine of a kind of inhibition

Technical field

The present invention relates to a kind of medicine, the thrombotic medicine of particularly a kind of inhibition.

Background technology

Thrombotic disease is found in clinical each patient of section, especially cardiovascular and cerebrovascular vessel thrombotic disease, be positioned at first of the dead reason of Chinese people stomatosis, and sickness rate has increase trend, the serious harm human health.Antiplatelet drug aspirin, ticlopidine and anticoagulant heparin, Fa Hualin have brought into play very big effect in the prevention of thrombotic disease and treatment.But also there is limitation in these medicines, and particularly the curative effect effect to ischemic cardiovascular and hemorrhage complication is still waiting to improve.Along with to the pathogenetic further investigation of thrombotic disease, recently outside so-called second filial generation medicine, many new antiplatelet drugs and anticoagulant medicine (as table 1) (Gresele P, et al.Novel approachesto the treatment of thrombosis.Trends Pharmacol Sci 2002 have appearred again; 23:25-32).

Table 1. anti-thrombosis drug

	Antiplatelet drug	Anticoagulant
			First generation second filial generation new method	The activation inhibitor adp receptor antagonist of aspirin ticlopidine (resisting bolt strenuously), clopidogrel GP IIb/IIIa antagonist aspirin-clopidogrel intermixture vWF-GP Ib interaction inhibitor collagen-blood platelet interaction inhibitor thrombin induction discharges the antiplatelet substance of NO	Heparin Fa Hualin low molecular weight heparin hirudin tissue factor-VIIa factor approach restrainer selectivity factor Xa inhibitor selective thrombin inhibitors activated human protein c solubility restructuring thrombomodulin
Abbreviation: GP, glycoprotein; VWF, the von Willebrand factor

The platelet that platelet membrane surface glycoprotein Ib α (GpIb α) and combining of vWF cause and the adhesion of damaged blood vessels endothelium, hemostasis and platelet thrombus play crucial effects in forming in the early stage.

The platelet membrane surface has multiple glycoprotein, wherein GPIb α is von Willebrand factor (vonWillebrand factor, vWF) receptor of thrombin, palatelet-selectin, Mac-1, the XII factor and high molecular weight kininogen is just expressed at the disactivation platelet surface.The molecular weight of GPIb α is about 135000, each cell surface nearly 30,000 molecule, mainly be distributed in platelet, the megalokaryocyte, belong to leucine-rich glycoprotein repeated fragment (LGR) family, be made up of 610 aminoacid, GPIb α and vWF land are positioned at the aminoterminal (1-302 aminoacid) of GPIb α.

Platelet adhesion relates to three factor: GPIb, vWF and subendothelial tissue in blood vessel endothelium undertissue; In blood circulation, platelet does not adhere on the blood vessel wall, and also different other cells interact, but flow at the fluid layer of nearly tube wall; When vascular injury, subendothelial tissue exposes, and the subendothelial tissue that platelet is adhered to exposure rapidly participates in the first step that hemostasis is reacted.GPIb α is the main adhesion receptor that participates in adhesive attraction, participate in multiple physiology and pathological process, be included in sticking of blood vessel injury place, with combining of thrombin, Bernard-Soulier syndrome (BSS) or title Bernard Soulier syndrome, (Chester Q.Li, etal.Expression of the Amino-Terminal Domain of Platelet Glycoprotein Ib α: Exploitation of a Calmodulin Tag for Determination of Its FunctionalActivity.Protein Expression and Purification 2001:22:200-210) such as platelet type hemophilia and immunity thrombocytopenia.

Under the normal condition platelet can not with the blood vessel endothelium tissue bond, after the vascular injury, vWF at first is incorporated into interior subcutaneous collagen fiber, so that configuration takes place and changes in vWF, this moment, platelet GPIb promptly combined activated blood platelet with vWF, the performance adhesive attraction, discharge ADP simultaneously, improve calcium ion concentration in the kytoplasm, the GPIIb-IIIa that the activation calcium ion relies on, expose the fiber original receptor, fibre is former can be simultaneously and at least two GPIIb-IIIa combinations, cause hematoblastic (the Goto S that sticks and assemble, et al.Distinct mechanisms of platelet aggregationas a consequence of different shearing flow conditions.J Clin Invest.1998.101:479), form white thrombus (Sugimoto M, et al.Functional property of vonWillebrand factor under flowing blood.Int J Hematol 2002.75:19-24; RuggeriZM.Von Willebrand factor.Curr Opin Hematol 2003.10:142-149).This process as shown in Figure 1.

The interaction partners platelet adhesion reaction of GPIb and vWF, gathering then form thrombosis and play a part key, thereby it is the development and the exploitation of the new antithrombotic reagent of target spot that many researchs are devoted to the two.The treatment of thrombosis can realize by the interaction that following two approach suppress vWF and GPIb:the GPIb α on (1) sealing platelet; (2) the vWF-GP Ib interaction inhibitor of the present foreign study of specificity is in conjunction with vWF. has F (ab ') 2 fragments (GPIb antibody) (Cauwenberghs N.et al.Antithrombotic effect of plateletglycoprotein Ib-blocking monoclonal antibody Fab fragments in non humanprimates.Arterioscler Thrombo Vasc BIol 2000.20:1347-1353) of 6B4 monoclonal antibody, AjvW2 monoclonal antibody (GPIb antibody) (Kageyama S.et al.Antinuman von Willebrand factor monoclonal antibodyAjvW-2 prevents thrombus deposition and neointima formation after ballooninjury in guinea pigs.Arterioscler Thrombo Vasc BIol 2000.20:2303-2308), restructuring vWF fragment VCL (Gralnick HR.et al.A monomeric von Willebrand factor fragment; Leu504-Lys728, inhibits von Willebrand factor interavtion with glycoproteinIb-IX.Proc Natl Acad Sci 1992.89:7880-7884), restructuring vWF Segment A R545C (GurevitzO.et al.Recombinant von Willebrand factor fragment AR545C inhibits plateletaffregation and enhances thrombolysis with rtPA in a rabbit thrombosis model.Arterioscler Thrombo Vasc BIol 1998.18:200-207) and flavone compound (Murk JS.etal.Flavone-8-acetic acid (flavonoid) profoundly reduces platelet-dependentthrombosis and vasoconstriction after deep arterial injury in vivo.Circulation 2000.101:324-328) etc.

It is more relatively to distribute on platelet membrane based on GPIb α, and plays crucial effects in platelet and blood vessel wall adhesive attraction and thrombosis, makes GPIb α become one of important target spot of research platelet related drugs.At present many GPIb antibody of preparation can both effectively be brought into play inhibitory action under external stable or flox condition.In the report that research is successful in two examples with the Cavia porcellus are the body of animal model, 6B4 and AjvW2 confirm and can prolong the time of small artery vascular thrombosis formation under the situation in not obvious prolongation bleeding time.In another routine guinea pig model, PG-1 antibody can effectively reduce the thrombosis at damage from laser place, though this antibody can be specific in conjunction with the guinea pig blood platelet can not with human blood platelets generation cross reaction.Other has data to show, TGX-6B4 Fab section inhibitor can combine with GPIb α aminoterminal (His1-Val289) antigenic determinant, blocking-up ristomycin or the inductive platelet aggregation of the appropriate mycin of cloth.Importantly at therapeutic dose, this antibody can suppress hematoblastic adhesion and serious thrombocytopenia not take place, and does not prolong the bleeding time simultaneously.The further development of TGX-6B4 might provide a kind of antithrombotic adjuvant drug.Generally speaking, GPIb antibody is up to the present also undesirable, part may be because the difference of kind, for example, mouse-anti people monoclonal antibody prolonged application can produce the human antimouse antibody reaction, therefore having limited 6B4 and the AjvW2 application at human body, also may be because bivalent antibody and GP1b is molecule crosslinked causes serious thrombocytopenia to cause.

On the other hand, because mass data shows that the rising of plasma vWF level is relevant with the generation of thrombotic disease, so a lot of research is devoted to study the recombinant fragment of VWF molecule GPIb land and the inhibitor of VWF.Reorganization vWF fragment VCL (Leu504-Lys728) and AR545C (Ala444-Asp730) can combine with GPIb, and these two segmental thromboembolism preventing dosage have only slight prolongation for the bleeding time.This shows that other memebrane proteins such as GPIa/IIa and GPIIb-IIIa etc. have participated in the formation of thrombosis.Because this GPIb inhibitor stops platelet adhesion reaction, delay thrombosis, but do not stop thrombosis, therefore need share with aspirin or thrombolytic medicine.Because vWF is a kind of glycoprotein that is produced by endotheliocyte and megalokaryocyte, is released into blood plasma when endotheliocyte is upset, so the rising of plasma vWF level can be used as the impaired sign of endotheliocyte.Existing mass data confirms multiple vascular lesion, vascular lesion as diabetes, the plasma vWF level raises, vWF can combine with GPIb, GPIIb-IIIa, blood coagulation factor VIII, fibronectin, collagen, promote platelet to stick SE, the gathering of induced platelet promotes thrombosis.Therefore, to the raise vascular lesion of this class of plasma vWF level, the segmental effect of reorganization vWF is limited.

(Plaimauer B.et al.Cloning such as Plaimauer B, expression, and functionalcharacterization of the von Willebrand factor-cleaving protease (ADAMTS13) .Blood 2002.100:3626-3632) cloned the gene of vWF crack protein enzyme (ADAMTS13), and in the HEK293 cell, express, at external cleavable vWF polymer, and can (be contained the inhibitor of this enzyme, IgG) suppress by thrombotic thrombocytopenic purpura patient's blood plasma.But the thromboembolism preventing effect of this enzyme also is not confirmed.

The innovation and creation content

The purpose of this invention is to provide a kind of is the thrombotic medicine of inhibition of action target spot with vWF.

The thrombotic medicine of inhibition provided by the present invention, its active component is SEQ ID № in the sequence table: 2 amino acid residue sequence, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

SEQ ID №: 2 amino acid residue sequence, title is made up of 324 amino acid residues for reorganization platelet glycoprotein rGP302, and the vWF that comprises platelet glycoprotein GPIb α can combine with vWF in conjunction with the territory.

When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.

Medicine of the present invention can be made various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.

It is 10 to 30 days that the consumption of said medicine was generally for 0.1 μ g-1000mg/kg/day course of treatment.

Second purpose of the present invention provides the encoding gene of a kind of recombinant platelet glycoprotein rGP302.

The encoding gene of recombinant platelet glycoprotein rGP302 is one of following nucleotide:

1) SEQ ID № in the sequence table: 1 DNA sequence;

2) coding SEQ ID №: the polynucleotide of 2 amino acid residue sequence;

3) with sequence table in SEQ ID №: 1 DNA sequence that limits has 95% above homology, and the identical function protein DNA sequence of encoding.

The DNA sequence of sequence 1 is by 1109 base compositions in the sequence table, the reading frame of this gene be from the 17th at 5 ' end to the 1105th bit base, comprise 1089 bases.

In recombinant platelet glycoprotein rGP302 and the blood plasma and after SE vWF combines, vWF can not be combined with the GPIb on the platelet, thereby blocking platelet stick SE.VWF can be affected with combining also of GPIIb-IIIa simultaneously, and further influences hematoblastic aggregation capability, prevents thrombotic purpose thereby reach.Experiment shows that external solubility rGP302 can suppress the inductive platelet aggregation of ristocetin, prolongs clotting time, and platelet aggregation rate is reduced to 13% by 100% of contrast.

The present invention provides a kind of new thinking and method for the prevention and the treatment of thrombotic disease, thereby lays a good foundation for further theoretical research and the novel antithrombotic reagent of preparation, has great importance.

Description of drawings

Fig. 1 is the sketch map of platelet adhesion reaction and accumulation process

Fig. 2 is the electrophoretogram of the vWF factor of GPIb α in conjunction with the RT-PCR of the cDNA in territory

Fig. 3 is eukaryon expression plasmid pcDNA3.1 (+)

Fig. 4 is eukaryon expression plasmid pSecTaq2/HygroB

Fig. 5 is for identifying the electrophoretogram of eukaryon expression plasmid pGP302

Fig. 6 is the dot blot result of solubility rGP302

Fig. 7 is the Westernblot result of solubility rGP302

Fig. 8 a is the inductive curve of platelet aggregation of Ristocetin

Fig. 8 b is a CHO-empty carrier expressing protein curve of platelet aggregation

Fig. 8 c is the curve of platelet aggregation of CHO-pGP302 recombiant protein

The specific embodiment

The preparation of embodiment 1, recombinant platelet glycoprotein rGP302

The preparation of recombinant platelet glycoprotein rGP302 may further comprise the steps:

1, design primer:

Upstream: 5 '-CGCGGATCCATGCCTCTCCTCCTCTTGCTG-3 '

Downstream: 5 '-CCGGAATTCTCAGGCTTTGGTGGGGAACTTG-3 '

For ease of the directed expression vector pcDNA3.1 (+) (Invitro company product) that inserts of gene, in forward primer, introduce BamH I restriction enzyme site sequence, in downstream primer, introduce EcoR I restriction enzyme site sequence.

2, in human erythorleukemia cell line TF-I cell strain (CRL-2003Homo sapiens (human) TF-1), extract cell total rna.

3, utilizing following upstream and downstream primer, is the full-length cDNA that template utilizes RT-PCR to angle to get GPIb α with the cell total rna that extracts,

Upstream: 5 '-CGCGGATCCATGCCTCTCCTCCTCTTGCTG-3 '

Downstream: 5 '-CCGGAATTCTCAGAGGCTGTGGCCAGAGTACC-3.

4, be template with GPIb α cDNA, with the synthetic upstream and downstream of step 1 primer, the PCR method clone GPIb α vWf factor is in conjunction with the cDNA in territory, as shown in Figure 2, wherein be with the 1 demonstration GPIb α vWF factor in conjunction with the territory, be with 2 to be nucleic acid molecular weight Mark:2000,1000,750,500,250,100bp; The condition of PCR is in the 50 μ l overall reaction systems in this step, adds dNTP successively to final concentration 0.2mM, MgCl ₂To final concentration 2.5mM, adding upstream and downstream primer to final concentration is 0.4 μ M, adds 0.5 μ g template, 10 * RT-PCR buffer, 5 μ l, Taq enzyme 1 μ l (5U/ μ l).On PE-2400 type PCR instrument, increase by following condition: 94 ℃, 5Min; 94 ℃, 45s, 60 ℃, 45s, extends 1Min, totally 30 circulations by 72 ℃; At 72 ℃, extend 7Min again.

5, the GPIb α vWf factor is cloned into plasmid T-Vector (Promega company product) in conjunction with the cDNA in territory, it is correct to measure its sequence by Shanghai Bo Ya bio-engineering corporation, SEQ ID № in its sequence such as the sequence table: 1.

6, with BamH I and EcoR I double digestion the purpose fragment is downcut from T-Vector, reclaim the purpose fragment, insert and eukaryon expression plasmid pcDNA3.1 (+) (its collection of illustrative plates is as shown in Figure 3) the structure pcDNA3.1-GP302 after BamH I and the processing of EcoR I double digestion, correct through identifying.

7, the structure of eukaryon expression plasmid pGP302

With the pcDNA3.1-GP302 carrier for expression of eukaryon is template, has designed two primers of a pair of HindIII of containing or NotI:

Primer I: 5 '-GCAAGCTTCACCCCATCTGTGAGGTC

Primer I I:5 '-GAGCGGCCGCGGCTTTGGTGGGGAACTT

The GP302 gene is inserted among the carrier for expression of eukaryon pSecTaq2/HygroB (as Fig. 4, containing 6 purification tag His6 and tags detected c-myc), made up eukaryon expression plasmid pGP302.Eukaryon expression plasmid pGP302 identifies that through the HindIII/NotI enzyme action result shows that the constructed plasmid structure is correct as shown in Figure 5.

8, with pGP302 liposome transfection COS-7 and Chinese hamster ovary celI, cultivate 72 hours collecting cell culture supernatant, SDS-PAGE shows newborn protein band and occurs.Be dot blotting and Westernblot with c-myc antibody and GPIb monoclonal antibody, the result as shown in Figure 6 and Figure 7, the specificity recombinant protein that shows expression is solubility rGP302.Among Fig. 6,1 for CHO expresses supernatant, and 2 for CHO-pGP302 expresses supernatant, and 3 are CHO-p empty carrier expression supernatant; Among Fig. 7,1,3 is the GP302 recombiant protein, and 2 is the CHO culture supernatant.

9, cell line is cultured to the 80% full end of cell in the culture medium that contains 10% hyclone, washes cell with the culture medium of serum-free, and in serum-free medium, cultivated 24 hours, collect the supernatant that contains rGP302;

10, the supernatant after the collection obtains rGP302 with the nickel affinity column purification, and its aminoacid sequence is SEQ ID №: 2.

Effect anticoagulant of embodiment 2, rGP302

Gather whole blood from healthy people and mixed with 3.14% sodium citrate by 1: 10,100g collected and is rich in hematoblastic supernatant (PRP) in centrifugal 15 minutes, was 300 * 10 with serum (PPP) the adjusting PC of no platelet ⁶/ ml.With concentration be 0.5mg/ml rGP302 earlier with PRP in incubated at room after 5 minutes, with ristocetin add among the PRP to final concentration be 1.5mg/ml, on platelet aggregation instrument, measure 10 minutes coagulation rates, result such as Fig. 8 a, shown in 8b and Fig. 8 c, show that external solubility rGP302 can suppress the inductive platelet aggregation of ristocetin, prolong clotting time, platelet aggregation rate is reduced to 13% by 100% of contrast.

Sequence table

<160>2

<210>1

<211>1109

<212>DNA

＜213〉Genus Homo people (Homo sapiens)

<400>1

ctggctagcc accatggaga cagacacact cctgctatgg gtactgctgc tctgggttcc 60

aggttccact ggtgacgcgg cccagccggc caggcgcgcg cgccgtacga agcttcaccc 120

catctgtgag gtctccaaag tggccagcca cctagaagtg aactgtgaca agaggaatct 180

gacagcgctg cctccagacc tgccgaaaga cacaaccatc ctccacctga gtgagaacct 240

cctgtacacc ttctccctgg caaccctgat gccttacact cgcctcactc agctgaacct 300

agataggtgc gagctcacca agctccaggt cgatgggacg ctgccagtgc tggggaccct 360

ggatctatcc cacaatcagc tgcaaagcct gcccttgcta gggcagacac tgcctgctct 420

caccgtcctg gacgtctcct tcaaccggct gacctcgctg cctcttggtg ccctgcgtgg 480

tcttggcgaa ctccaagagc tctacctgaa aggcaatgag ctgaagaccc tgcccccagg 540

gctcctgacg cccacaccca agctggagaa gctcagtctg gctaacaaca acttgactga 600

gctccccgct gggctcctga atgggctgga gaatctcgac acccttctcc tccaagagaa 660

ctcgctgtat acaataccaa agggcttttt tgggtcccac ctcctgcctt ttgcttttct 720

ccacgggaac ccctggttat gcaactgtga gatcctctat tttcgtcgct ggctgcagga 780

caatgctgaa aatgtctacg tatggaagca aggtgtggac gtcaaggcca tgacctctaa 840

cgtggccagt gtgcagtgtg acaattcaga caagtttccc gtctacaaat acccaggaaa 900

ggggtgcccc acccttggtg atgaaggtga cacagaccta tatgattact acccagaaga 960

ggacactgag ggcgataagg tgcgtgccac aaggactgtg gtcaagttcc ccaccaaagc 1020

cgcggccgct cgaggagggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc 1080

cgtcgaccat catcatcatc atcattgag 1109

<210>2

<211>324

<212>PRT

＜213〉Genus Homo people (Homo sapiens)

<400>2

His Pro Ile Cys Glu Val Ser Lys Val Ala Ser His Leu Glu Val Asn

1 5 10 15

Cys Asp Lys Arg Asn Leu Thr Ala Leu Pro Pro Asp Leu Pro Lys Asp

20 25 30

Thr Thr Ile Leu His Leu Ser Glu Asn Leu Leu Tyr Thr Phe Ser Leu

35 40 45

Ala Thr Leu Met Pro Tyr Thr Arg Leu Thr Gln Leu Asn Leu Asp Arg

50 55 60

Cys Glu Leu Thr Lys Leu Gln Val Asp Gly Thr Leu Pro Val Leu Gly

65 70 75 80

Thr Leu Asp Leu Ser His Asn Gln Leu Gln Ser Leu Pro Leu Leu Gly

85 90 95

Gln Thr Leu Pro Ala Leu Thr Val Leu Asp Val Ser Phe Asn Arg Leu

100 105 110

Thr Ser Leu Pro Leu Gly Ala Leu Arg Gly Leu Gly Glu Leu Gln Glu

115 120 125

Leu Tyr Leu Lys Gly Asn Glu Leu Lys Thr Leu Pro Pro Gly Leu Leu

130 135 140

Thr Pro Thr Pro Lys Leu Glu Lys Leu Ser Leu Ala Asn Asn Asn Leu

145 150 155 160

Thr Glu Leu Pro Ala Gly Leu Leu Asn Gly Leu Glu Asn Leu Asp Thr

165 170 175

Leu Leu Leu Gln Glu Asn Ser Leu Tyr Thr Ile Pro Lys Gly Phe Phe

180 185 190

Gly Ser His Leu Leu Pro Phe Ala Phe Leu His Gly Asn Pro Trp Leu

195 200 205

Cys Asn Cys Glu Ile Leu Tyr Phe Arg Arg Trp Leu Gln Asp Asn Ala

210 215 220

Glu Asn Val Tyr Val Trp Lys Gln Gly Val Asp Val Lys Ala Met Thr

225 230 235 240

Ser Asn Val Ala Ser Val Gln Cys Asp Asn Ser Asp Lys Phe Pro Val

245 250 255

Tyr Lys Tyr Pro Gly Lys Gly Cys Pro Thr Leu Gly Asp Glu Gly Asp

260 265 270

Thr Asp Leu Tyr Asp Tyr Tyr Pro Glu Glu Asp Thr Glu Gly Asp Lys

275 280 285

Val Arg Ala Thr Arg Thr Val Val Lys Phe Pro Thr Lys Ala Glu Glu

290 295 300

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His

305 310 315 320

His His His His

324

Claims (4)

1, the thrombotic medicine of a kind of inhibition, its active component are SEQ ID № in the sequence table: 2 amino acid residue sequence.

2, medicine according to claim 1 is characterized in that: also contain the acceptable pharmaceutical carrier of human body in the described medicine.

3, the described SEQ ID of coding claim 1 №: the polynucleotide of 2 amino acid residue sequences.

4, polynucleotide according to claim 3 is characterized in that: its nucleotide residue sequence is the SEQ ID № in the sequence table: 1.

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