CN108339121A - Purposes of the protein kinase A inhibitor in preparing treatment platelet counts and increasing relevant disease drug - Google Patents
Purposes of the protein kinase A inhibitor in preparing treatment platelet counts and increasing relevant disease drug Download PDFInfo
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Abstract
The invention belongs to antiplatelet drug fields, disclose purposes of the protein kinase A inhibitor in preparing treatment platelet counts and increasing relevant disease drug.First passage experiment discussion of the present invention effect of protein kinase A during regulating and controlling blood platelet apoptosis.Research has shown that the inhibition of PKA Activity can lead to the generation of endogenous blood platelet apoptosis, and protein kinase A inhibitor is prompted to have the function of promoting blood platelet apoptosis, the phosphorylation blood platelet apoptosis that protein kinase A passes through regulation and control 155 site serines of BAD.The inhibitor of protein kinase A can lead to blood platelet apoptosis in vitro experiment, in vivo experiment, platelet counts in experimental animal body can be caused to reduce.Cycle platelet counts are reduced in the Mice Body of protein kinase A gene knockout.Therefore, the inhibitor of protein kinase A has exploitation at the potentiality of novel therapeutic thrombocythemia disease medicament, great scientific research and economic value.
Description
Technical field
The invention belongs to blood platelet related drugs fields, and in particular to protein kinase A inhibitor is preparing treatment blood platelet
Quantity increases the purposes in relevant disease drug.
Background technology
Blood platelet is the key factor of thrombosis and Pathology bleeding to be adjusted in the circulatory system, while also exempting from body
It plays a significant role in the pathophysiological processes such as epidemic disease reaction, infection, atherogenesis and metastases.To blood platelet
Life cycle fine-tune be the key that maintain normal human in platelet counts, platelet counts, which increase, sees a variety of diseases
Disease, such as primary thrombocytosis, polycythemia vera, and peripheral blood platelet counts increase in some pathologic processes
It is more, it increases after the risk of bleeding or thrombosis, such as chronic granulocytic leukemia, massive haemorrhage and chronic inflammation, tumour.
Therefore, the mechanism for inquiring into regulation and control platelet life span and existence reduces platelet count in peripheral blood by shortening platelet life span
Amount has important Pathological Physiology meaning to treatment thrombocythemia disease.
The Related Experimental Study of a large amount of blood platelet apoptosis emerged in large numbers in recent years discloses Austria of blood platelet life cycle
It is secret.More and more experimental studies show under pathological and physiological condition platelet destruction mainly by it apoptosis program institute
It mediates, Bak and Bax are to mediate the main molecules of eukaryocyte apoptosis, while also playing and focusing in the apoptotic process of blood platelet
It acts on.However it is verified, it is small that the Bcl-xL albumen only in Bcl-2 anti-apoptotic family can be combined the seedless blood of participation with Bak
The anti-apoptotic of plate, mutation Bcl-xL dose dependents reduce the existence of internal blood platelet, and this process can by knock out BAK and
BAX is suppressed, and P53 is by inhibiting Bcl-xL activity to be proved to participate in adjusting blood platelet apoptosis process.And have now been found that,
Only have Bad to participate in adjusting the service life of blood platelet in the pro apoptotic protein of Bcl-2 families, these researchs disclose blood platelet apoptosis egg
Play an important roll in adjusting platelet life span and blood platelet existence in vain.However, under physiology or pathological conditions, how blood is induced
Platelet apoptosis, the mechanism for reducing platelet counts it is not immediately clear.
Protein kinase A (PKA) is a kind of serine-threonine protein kinase enzyme, is regulated and controled by two catalytic subunits and two
The different tetramer of subunit composition.Cyclic adenosine monophosphate（cAMP）After being attached to regulation and control subunit, the catalytic subunit of activation is discharged, and then adjust
Control the biological behaviours such as metabolism, Growth and Differentiation, gene expression and the apoptosis of cell.It has been reported that the PKA in karyocyte
Existing apoptosis-promoting effect has Anti-G value again, and PKA expressions are higher in blood platelet and it is expressed by finely regulating,
The function point analysis of blood platelet is played an important role.But whether PKA stimulates storage or pathology the blood platelet apoptosis of induction
There is large effect, does not understand yet.
Invention content
Technical problems to be solved：For the above technical issues, present invention solves the technical problem that being further to study
Protein kinase A inhibitor discloses protein kinase A inhibitor and is preparing treatment blood to promoting the specific mechanism of blood platelet apoptosis
Platelet quantity increases relevant disease drug.
Technical solution：In view of the above-mentioned problems, the invention discloses protein kinase A inhibitors to prepare treatment platelet counts
Increase the purposes in relevant disease drug.
Preferably, the protein kinase A inhibitor is inorganic matter inhibitor, one kind in organic matter inhibitor or several
Kind.
Preferably, the inorganic matter inhibitor is one or more of hydride, oxide, acid, alkali, salt.
Preferably, the organic matter inhibitor is hydro carbons, the derivative of hydrocarbon, carbohydrate, protein, fat, nucleic acid, synthesis
One or more of high molecular material.
Preferably, the hydro carbons is one or more of alkene, alkane, alkynes, aromatic hydrocarbon；The derivative of the hydrocarbon
Object is one or more of halogenated hydrocarbons, alcohol, phenol, aldehyde, acid, ester；The carbohydrate is in monosaccharide, disaccharides, oligosaccharide, polysaccharide
It is one or more of；The protein is one or more of amino acid, polypeptide；The nucleic acid be DNA,
One or more of ribonucleic acid.
Preferably, the protein kinase A inhibitor is Fasudil, nitrogen-[2-（Phosphorylation bromine Nitro-Arginine acyl ammonia
Base）Ethyl] -5- isoquinoline sulfonaides, C94H148N32O31、C80H130N28O24、C27H21N3O5、C26H19N3O5、C20H13N3O、
C32H31N3O5、C22H22N4O、C14H17N3O2S·2HCl、C14H17N3O2S、C11H13N3O2S·HCl、C12H13ClN2O2SHCl、
C12H15N5O2S2HCl、C53H100N20O12、1-（5- quinoline sulfonyls）Piperazine, 4- cyano -3- methylisoquinoliniums, acetylaminohydroxyphenylarsonic acid 4-
Cyano -3- methylisoquinoliniums, 8- bromo -2- list acyl adenosine -3,5- rings single thiophosphate ester, adenylate 3,5- ring single thiophosphates
Ester, 2-0- only son-adenosine cyclophosphate, 8- chloros-adenosine cyclophosphate, N- [2- (cinnamoyl amino acid)] different beautiful jade quinoline ketone of -5-, reverse phase -8-
Hexylamino adenylate 3,5- single thiophosphate esters, reverse phase -8- piperidyls adenosine-adenosine cyclophosphate, reverse phase-adenylate 3,5- ring lists
It is thiophosphate, 5- iodos tuberculin, 8- hydroxyadenosine acid -3,5- single thiophosphate esters, calcium Phospoprotein C, dephnetin, anti-
Phase -8- chlorobenzenes-adenosine cyclophosphate, reverse phase-adenosine cyclophosphate, reverse phase -8-Br- adenosine cyclophosphates, 9- adenyl cyclases, 1- (5- isoquinolines
Quinoline sulphonyl) it is-pipecoline, 8- hydroxyadenosine acid -3', 5'- monophosphate, 8- hexylamino adenylate -3', 5'- monophosphate, anti-
One or more of phase-adenylate 3', 5'- ring monophosphate.
Preferably, it includes idiopathic thrombocythemia disease or secondary blood that the platelet counts, which increase relevant disease,
Platelet increases disease.
Preferably, the idiopathic thrombocythemia disease includes that primary thrombocytosis, chronic granulocyte are white
Blood disease, myelofibrosis and polycythemia vera, myelodysplastic syndrome or bone marrow proliferative tumour.
Preferably, the secondary thrombocythemia disease causes including thrombocythemia, bacterium or virus after cutting spleen
Infection, tumour or disease of immune system.
Preferably, the drug be tablet, capsule, granule, pill, sustained release preparation, controlled release preparation, oral solution or
Patch.
Preferably, the drug includes the protein kinase A inhibitor of pharmaceutical effective dose and pharmaceutically acceptable
Carrier.
Preferably, the drug is administered by oral, spraying sucking, injection or through gastrointestinal tract.
Protein kinase A inhibitor is preparing the purposes in promoting blood platelet apoptosis drug.
Advantageous effect：First passage Experimental Research of the present invention effects of PKA during regulating and controlling blood platelet apoptosis is found
In ITP, PKA activity declines in the blood platelet of infection and diabetic, and PKA is by regulating and controlling 155 site serines of BAD
Phosphorylation so that regulate and control blood platelet apoptosis.Inhibit PKA activity not only can be with blood platelet apoptosis outside inductor, but also can reduce
The quantity of body-internal-circulation blood platelet shows that PKA inhibitor can participate in the therapeutic process of thrombocythemia disease, reduces periphery
Platelet counts in circulating have exploitation at the potentiality of novel therapeutic thrombocythemia disease medicament, great scientific research and warp
Ji value.
Description of the drawings
Fig. 1 is PKA by adjusting 155 site serine phosphorylation blood platelet apoptosis related experiment result figures of Bad；
The percentage shared by blood platelet that Fig. 2 causes blood platelet apoptosis mitochondrial transmembrane potentials to depolarize for protein kinase A inhibitor
Compare test result；
Fig. 3, which is protein kinase A inhibitor, causes blood platelet apoptosis western blot to detect caspase-3, gelsolin albumen
Caspase-3 determination of activity results in expression and blood platelet；
Fig. 4 be wash blood platelet and various concentration protein kinase A inhibitor H89 be incubated after PS turn up test result；
Fig. 5 is the blood platelet scatter plot that the protein kinase A inhibitor that FSC-FL1 is collected leads to blood platelet apoptosis；
Fig. 6 is to lead to sweeping for blood platelet apoptosis after the H89 of various concentration gradient acts on washing blood platelet 160 minutes in 22 DEG C
Retouch Electronic Speculum result；
Fig. 7 is that PKA inhibition causes internal acute decrease of platelet related experiment result；
Fig. 8 is conditional gene knockout mouse building process and dependence test result；
Fig. 9 is that PKA knock-out mice blood platelets remove ratio increase correlated results；
Figure 10 is washing blood platelet and different Fasudil（Fasudil）Or blood platelet Δ ψ m and PS after negative control incubation
It turns up test result；
Figure 11 is that control group and test group inject DMSO and Fasudil respectively after mouse is taken a blood sample（Fasudil）（1.6μmol/L）
It takes a blood sample afterwards in different time and counts test result.
Specific implementation mode
1, reagent and material：
The monoclonal antibody SZ21 of anti-GpIIb/IIIa is provided by Jiangsu Province Blood Research Institute chief professor Ruan Changgeng, and dimethyl is sub-
Sulfone（DMSO）, anti-Actin primary antibodies be purchased from Sigma Co., USA, EDTA-K2 anticoagulant tubes are purchased from U.S. company BD, and isothiocyanic acid is glimmering
Light element（Fluorescein Isothiocyanate, FITC）- Annexin V are purchased from Beijing bio tech ltd Jia Mei,
FITC- sheep anti-mouse antibodies are purchased from Bioworld Technology companies of the U.S.,（Horse Radish Peroxidase, HRP）-
Sheep anti mouse, HRP- goat-antis rabbit, rabbit and mouse IgG, anti-BAX, anti-BAK, anti-Bcl-xL, anti-Bcl-2, anti-Caspase-3, anti-BAD
155 phospho-ABs are purchased from Santa Cruz biotechnologies company of the U.S., and anti-PKA C Alpha antibodies are purchased from CST companies of the U.S., N- [2-
((p-Bromocinnamyl) amino) ethyl] it is -5-isoquinolinesulfonamide (H89), Forsklin, anti-
Purchased from green skies bio tech ltd of China, E64 is purchased from U.S. Roche by GAPDH, antibodies against P 53, JC-1, ECL, PMSF
Bio tech ltd, A23187 are purchased from Calbiochem companies of the U.S..RNA oligonucleotide is synthesized by the design of Ji Ma companies.
Liposome Lipofectamine 2000 and culture medium Opti-Mem I is purchased from Invitrogen biotech firms of the U.S..
2, laboratory mice：
PKA knock out mice（B6;129X1-Prkacatm1Gsm/Mmnc）Using C57BL/6J as background, it is purchased from U.S. MMRRC
UNC.All animals experiment is ratified through First Affiliated Hospital of Soochow University,Suzhou Ethics Committee.
3, blood platelet is washed：
Adult healthy volunteers are taken a blood sample from median basilic vein, the equal informed consent of blood donor and the book that subscribes to the agreement.Experimental program is through Soviet Union
The attached First Hospital Ethics Committee approval of state university, meets Declaration of Helsinki.
When preparing washing blood platelet, healthy premenopausal volunteers venous blood and ACD are taken（2.5 % sodium citrates, 2.0 % glucose,
1.5 % citric acids）By 1：7 anti-freezings, 1300 rpm centrifuge 20 min and obtain platelet rich plasma（PRP）, by PRP with 1500 g
2 min are centrifuged, supernatant is abandoned.With CGS buffer solutions（0.123 M sodium chloride, 0.033 M glucose, 0.013 M sodium citrates,
pH 6.5）It suspends and centrifuges, wash the blood platelet precipitated, then Tyrode ' the s buffer solutions with modification（2.5 mM amphoteric ions are slow
Fliud flushing Hepes, 150 mM sodium chloride, 2.5 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesium chlorides, 12 mM sodium bicarbonates,
5.5 mM glucose, pH 7.4）Blood platelet is resuspended, obtains washing platelet suspension, blood platelet is counted with calculating instrument,
Platelet suspension is adjusted to a concentration of 3 × 108/mL, is stored at room temperature 60 min.
4, Electronic Speculum：
Blood platelet is washed to fix overnight in 4 DEG C with 2.5% glutaraldehyde.Send the sample preparation of scanning electron microscope example room.Scanning electron microscope（Japanese day
Vertical company, S-4700）Morphology analysis is carried out to blood platelet.It is observed and is taken pictures in the different visuals field of each selection 5.
5, full-body exposure and bone-marrow transplantation：
Male WT mouse（6 week old）Receive the full-body exposure of the 9.5 Gy dosage in the sources Co60.It collects from PKA genetic heterozygosis
Pregnant mouse（Pregnancy 15 days or so）Fetal liver cells, and give and inject according to the male mouse ratio of 1 tire hepatocytes correspondence, one irradiation（It connects
It is completed in 6h after exposure）, the special animal houses of IVC are put into, acidifying water, Co60 irradiated feeds and bedding and padding are given, daily observation life
Deposit situation；Survival mice measures whole blood count after 4 weeks, if restoring normally to can be used to test in next step.Transplanting whether at
Work(detects the expression of PKA albumen in Recipient mice blood platelet to determine by Western Blotting.
6, mitochondrial membrane potential（Δψm）Detection
Wash blood platelet（3 × 108 /mL）With various concentration H89 (12.5 μM, 25 μM, 37.5 μM and 50 μM) or the moon
Property control（DMSO）10 min of room temperature, later blood platelet Δ ψ m measured using lipophilic cation dyestuff JC-1.Final concentration of 2
In the JC-1 of μ g/ml is added that treated blood platelet, 37 DEG C are protected from light and are incubated 20 min, flow cytomery.Red fluorescence table
The JC-1 polymer of timberline mitochondrial membrane potential dependence, green fluorescence indicate unbonded film electricity after mitochondrial membrane potential depolarising
The JC-1 monomers of position.JC-1 monomers（529 nm of λ ex 514 nm, λ em）And polymer（590 nm of λ ex 585 nm, λ em）
By calculating streaming red fluorescence（JC-1 polymer）Or green fluorescence（JC-1 monomers）Ratio measures.
7, PS turns up
Wash the H89 (12.5 μM, 25 μM, 37.5 μM and 50 μM) or negative control of blood platelet and various concentration（DMSO）
In being incubated at room temperature 10 min.Later by Annexin V buffer solutions, H89 treated blood platelets, Annexin V-FITC according to
50: 10:1 ratio is protected from light at room temperature is incubated 15 min, flow cytomery.
8, blood platelet shrinkage is tested
Wash the H89 (12.5 μM, 25 μM, 37.5 μM and 50 μM) or negative control of blood platelet and various concentration（DMSO）
In being incubated at room temperature 10 min.Then blood platelet and SZ21 antibody at room temperature are incubated 30 min altogether.Centrifugation, with what is marked containing FITC
Blood platelet is resuspended in sheep anti-mouse antibody, and room temperature, which is protected from light, is incubated 30 min.Flow cytometer collects blood platelet, the blood that FSC-FL1 is collected
Platelet scatter plot is for analyzing blood platelet.The shrinkage of blood platelet evaluates GPIIb/IIIa positive cells by the variation of analysis FSC
FSC reduce degree and evaluate shrinkage degree.A23187 is as positive control.DMSO is as negative control.
9、Western Blotting：
Wash the H89 (25 μM, 50 μM, 100 μM) or negative control of blood platelet and various concentration（DMSO）It is incubated in room temperature
Educate 10 min.2X cell pyrolysis liquids are added（Containing 2 mM PMSF, 2 mM NaF, 2 mM Na3VO4 and protease inhibitors）Eventually
It only reacts, cracks on ice, sample preparation.Sample detects the expression of corresponding albumen with Western blot.
10, RNA interference experiments
The double-strand siRNA oligonucleotides of target PRKACA（Justice:5-GCUCCCUUCAUACCAAAGUTT-3, antisense: 5-
ACUUUGGUAUGAAGGGAGCTT-3）With negative control siRNA（Justice:5-UUCUCCGAACGUGUCACGUTT-3, instead
Justice:5-ACGUGACACGUUCGGAGAATT-3）It is designed and synthesized by Ji Ma companies.
When Hela cell transfectings, the day before transfection, by 2 × 105 hela cell inoculations in culture plate, per Kong Zhongjia
The culture medium for entering about 500 μ L antibiotic-frees, enables cell density when transfection to reach 30~50%；Take 1 holes μ L/
Lipofectamine 2000（It is gently shaken up using preceding）, dilute with 50 μ L Opti-MEM I Reduced Serum Medium
It releases.It is being incubated at room temperature 5 min after gently mixing；2 μ L FAM-siRNA are taken, with 50 μ L Opti-MEM I Reduced Serum
Medium dilutes, and is gently mixed evenly;Diluted Lipofectamine 2000 is after the incubation of 5 min, with dilution
FAM-siRNA is gently mixed, and is stored at room temperature 20 min；FAM-siRNA- transfection reagent mixed liquors are added and contain cell and culture
Liquid（Containing about 400 μ L）Hole in, jiggle orifice plate, make mixing.
When blood platelet transfection experiment, 6 × 108/mL of blood platelet is washed in sterile preparation, after standing.By 100 μ L
SiRNA oligonucleotides is added in the blood platelet that 100 μ L serum-free M199 culture mediums suspend.
In 37 DEG C of CO2It is cultivated in incubator, culture medium can be changed to the complete medium culture containing serum after 6 hours
48 h；Transfection can pass through flow cytomery transfection efficiency after 6 hours.At the end of culture, collects and crack hela cells
And blood platelet.Sample detects the expression degree of PKA C α by western blotting method, and Actin is detected for internal reference.
11, statistical analysis：
Total data derives from least 3 mutually independent experiments, and data are indicated with mean ± standard error, using Prism
Version 5.0 carries out statistical analysis to data, and non-matching T inspections, p are carried out to data<0.05 as the notable boundary of otherness
Value.
12, experimental result：
（1）PKA regulates and controls blood platelet apoptosis by adjusting 155 site phosphorylations of BAD
We explore the mechanism that PKA inhibits induced platelet apoptosis.Under room temperature, 3 × 108The washing blood platelet of/mL from it is different
H89 the or DMSO internal references of concentration gradient are incubated 160 minutes altogether, are cracked on ice 30 minutes with isometric lysate, product is through SDS-
PAGE is separated by electrophoresis to obtain different size of protein fragments, and skimmed milk power is added primary antibody after closing one hour and is incubated, final ECL hairs
Light shows destination protein band（Fig. 1 a）.Blood platelet is washed respectively with the forskin and DMSO of the H89 of 37.5 uM, 10 uM
It is pre-processed under internal reference room temperature 160 minutes, extracts the plasmosin and mitochondrial protein of blood platelet, western blot inspections respectively
Destination protein is surveyed, Image J softwares analyze the amount of destination protein, count four experiments and show result with mean ± standard deviation（Figure
1b）.After pretreated platelet lysates, 17,000 4 °C of g are centrifuged 10 minutes, and it is heavy that obtained supernatant and corresponding antibodies are incubated
It forms sediment overnight, after being incubated 2 hours with protein A/G+4 °C of sepharose 4B, protein hybridization is used for after pearl is eluted（Fig. 1 c）,
Four experiments of statistical analysis are shown with mean ± standard deviation as a result, * P< 0.05, **P < 0.01.
As a result, it has been found that PKA inhibits the phosphorylation degree of 166 site serines of dose-dependant reduction blood platelet GPIb β, into
And it proves to reduce PKA activity.However, we have discovered that, apoptosis executes egg BAK, BAX and anti-apoptotic proteins in apoptosis blood platelet
Significantly changing do not occur in the expression quantity of Bcl-2, Bcl-xL.Have been reported that display, in S49 lymphoma cells, PKA passes through
Regulation and control increase the expression of Bim, and tumour cell is made to avoid that apoptosis phenomenon occurs.And we have discovered that, participate in the blood of regulation and control in PKA
In platelet apoptotic process, Bim protein levels do not occur significant change, eliminate the regulating and controlling effect of Bim.
In karyocyte, PKA by adjust 155 site serines of BAD phosphorylation, and regulate and control 14-3-3 albumen with
The binding of anti-apoptotic proteins Bcl-xL, and then regulating cell apoptosis.Under conditions of 155 site dephosphorylations of BAD, BAD with
Bcl-xL forms dimer, release apoptosis executor BAK and BAX, and then mitochondrial membrane permeability is caused to enhance, and cell occurs and withers
It dies.We have found that there is the phenomenon that phosphorylation degree reduction under the action of PKA inhibitor H89 in 155 site serines of BAD, most
Importantly, co-immunoprecipitation result shows under H89 effects that Bcl-xL is significantly reduced with BAD, prompt H89 by regulating and controlling BAD
With the activity of the interaction regulation and control anti-apoptotic proteins Bcl-xL of Bcl-xL.Therefore, these are statistics indicate that PKA passes through regulation and control BAD
The apoptosis of 155 site serine phosphorylation blood platelets.
（2）Inhibit PKA activity inducement blood platelet endogenous apoptosis
Washing blood platelet 160 is acted in 22 DEG C with the H89 of various concentration (0,12.5,25,37.5 and 50 μM) respectively to divide
Clock, the mitochondrial transmembrane potentials depolarising and PS exposures of flow cytomery blood platelet.Experimental result is repeated four times.As a result such as
Shown in Fig. 2 to Fig. 6.The H89 for washing blood platelet various concentration gradient is pre-processed 30 minutes at 22 DEG C, while setting up DMSO
With the negative control group and positive controls blood platelet of A23187 processing, western blot detect caspase-3, gelsolin
Caspase-3 activity in protein expression and blood platelet.By the anti-CD41 antibody of FITC labels with pretreated blood platelet with 1:10
Ratio mixing, incubation 10 minutes is protected from light under room temperature, analysis Platelet Size scatter plot CD41 positive quantity, which declines, indicates that blood is small
Plate quantity is reduced.The H89 of various concentration gradient acts on washing blood platelet in 22 DEG C 160 minutes, while it is right to set up DMSO feminine genders
According to a group blood platelet.Blood platelet is after 1% glutaraldehyde fixes 30 minutes, and scanning electron microscope imaging is as a result, experimental result comes from three times
Independent experiment, scale=1 μm.As a result mean ± standard deviation is used to indicate, compared with the control group *P<0.05, as a result in triplicate with
On.
Next we have inquired into effects of the PKA in regulating and controlling blood platelet apoptosis.Blood platelet and PKA inhibitor H89 are total to
It is incubated, JC-1 dye markers variation in flow cytomery blood platelet finds that H89 dose dependent induced platelets find line
Mitochondrial membrane potential（ΔΨm）Depolarising.Moreover, H89 can also time dependent induced platelet generation △ ψ m depolarisings.ΔΨ
M depolarisings are located at caspase-3 signal paths upstream, and caspase-3 is one of caspases executioner, can be caused
The disintegration and collapse of cell.We have discovered that the dose-dependent modes of H89 induce caspase-3 activation and
The digestion of caspase-3 substrates gelsolin.Phosphatidylserine（PS）Turn up be interior approach dependent cells apoptosis another
Visible marking's molecule, we have discovered that, H89 can be turned up with dose dependent induced platelet surface PS.
In apoptotic process, mitochondria dysfunction, which causes bioenergetics, to be destroyed, and finally membrane integrity is caused to destroy
And lead to morphological change.Experimental result shows that H89 can induce the blood platelet of the GPIIb/IIIa positives, forescatering occurs
（FSC）Phenomenon is reduced, shows to shrink on platelet PLA2 after inhibiting PKA activity.In addition, the dose-dependent inductions of H89
There is typical apoptosis morphological change, including Cell membrane vesicles, pseudopodium, shrinkage, degranulation phenomenon etc. in blood platelet.In short, this
The result shows that, PKA inhibits the Apoptosis that the interior approach that can occur with induced platelet relies on a bit.
In order to further prove PKA inhibit can with the specificity of induced platelet apoptosis, exclude PKA inhibitor there may be
Non-specificity, we using siRNA methods knock out blood platelet in PKA generation.PKA catalytic subunits are made of C α or C β, but
PKA C α are considered as the active main sources of PKA.So the generation of siRNA selective depression PKA C α and design and synthesize.
Compared with control group Hela cells, PKA siRNA can effectively remove the expression of blood platelet PKA C α subunits.And as expection
As, there is PS and turns up and Δ Ψ m depolarisings in the blood platelet for knocking out PKA C α, that is, shows that blood platelet can be promoted by removing PKA
Apoptosis occurs.
（3）PKA inhibition causes internal acute decrease of platelet
Next we have further inquired into effects of the PKA in vivo in platelet life span.Male ICR mouse is through tail vein injection
The Rp-cAMPS (50 mg/kg) of single dose detects the quantity of blood platelet and netted blood platelet in different time points Mice Body.
Antiplatelet antibody R300,0.15 mg/kg of single dose is injected intraperitoneally in male ICR mouse, and removing blood platelet can cause sternly
The thrombopenia of weight, immature reticulocyte fraction quantity increase in newly synthesized intra platelet free calcium to peripheral circulation, about 3 days
Afterwards, internal platelet counts restore normal.The antiplatelet antibody R300,0.15 of single dose is injected intraperitoneally in male ICR mouse
Mg/kg, tail vein injection Rp-cAMPS (50 mg/kg) after two days and 7 days count before Rp-cAMPS is injected and inject 8 respectively
Blood platelet and immature reticulocyte fraction after hour.Every 24 hours of male ICR mouse is through tail vein injection PKA agonists 8-Br-cAMP
(2.5 mg/mL), while PBS groups are set up as negative control, the blood platelet and immature reticulocyte fraction in Mice Body are counted after 8 days,
Experimental group and control group set up 5-6 mouse * P respectively< 0.05, **P < 0.01（Fig. 7）.
By PKA inhibitor reverse phase-adenosine cyclophosphate（Rp-cAM7PS）（Non- reagent controls）It is small to ICR by tail vein injection
In mouse body.As a result, it has been found that 2 hours detect, platelet count has dropped the 30% of normal platelet count.It detects within 8 hours, blood is small
Plate counting drops to minimum.Moreover, Δ Ψ m depolarization phenomenons occurs in the blood platelet after Rp-cAMPS injections, show blood platelet
Apoptosis occurs.
Aging or storage blood platelet will appear Apoptosis, and the corresponding decline of PKA activity.It is consistent with these results,
It was found that while Rp-cAMPS injection mouse induce internal platelet count reduction, immature reticulocyte fraction quantity is promoted
Increase, prompts young blood platelet to have resistant function to the Rp-cAMPS Apoptosis induced, while also prompting, PKA presses down in vivo
Preparation is easier to induce the blood platelet of aging that apoptosis occurs.In order to verify this possibility, we mix antiplatelet monoclonal
Antibody R300 is closed, intraperitoneal injection mode is squeezed into Mice Body, and blood platelet in Mice Body is promoted to remove, artificial synchronous blood platelet production
Raw rate.As expected results, 6 hours blood platelets that can't detect cycle in Mice Body after injection, platelet counts are 7
Restore in it normal.During this period, the ratio of immature reticulocyte fraction is changed significantly.Injection Rp-cAMPS can destroy R300 antibody note
After penetrating 7 days in Mice Body 70% cycle blood platelet, however only 30% blood platelet was destroyed at second day.These results are demonstrate,proved
Real, the blood platelet of aging is easier to be stimulated by PKA inhibitor, and then induces blood platelet apoptosis and removing.Moreover, it is small to reduce blood
The PKA activity of plate can shorten cycle platelet life span.
（4）PKA knock-out mice blood platelets are removed ratio and are increased
It is conditional gene knockout mouse structure first（Fig. 8 a）.PKA, Bad in Western blot detection blood platelet,
The expression of GBIbb, phosphoBad Ser-155 and phosphorylation GBIbb Ser-166 at least set 5 mouse per experimental group（Figure
8b）.Sysmex XP-100 blood analyser platelet Countings, as a result statistical analysis 7 WT mouse, 7 PKA+/- small
Mouse and 5 PKA-/- mouse（Fig. 8 c）.Whole blood thiazole orange（0.5µg/mL）After being marked with anti-CD41 (20 μ g/mL) antibody,
It is incubated at room temperature 15 min, the quantity of flow cytomery immature reticulocyte fraction.Data come from 7 WT mouse, 7
PKA+/- mouse and 5 PKA-/- mouse（Fig. 8 d）.Antiplatelet antibody R300 (0.15 μ g/kg) through be injected intraperitoneally into
In WT and PKA+/- Mice Body, eye socket blood sampling collection whole blood, Sysmex XP-100 blood analyser platelet Counting quantity,
As a result distinguish 6 WT and 6 PKA+/- mouse of statistical analysis（Fig. 8 e）.Washing blood platelet is kept away with JC-1 (2 μ g/mL)
Light is incubated 10 minutes, flow cytomery mitochondrial transmembrane potentials depolarization level（Fig. 9 f）.The anti-CD41 of FITC labels is anti-
Body is with blood platelet with 1:10 ratio mixing, is gently incubated at room temperature 10 minutes after mixing（Fig. 9 g）.Analyze Platelet Size scatterplot
Figure, the decline of CD41 positive cell quantities represent platelet counts reduction（Fig. 9 h）.1% glutaraldehyde fixes washing blood platelet 30 minutes
Afterwards, scanning electron microscope imaging is as a result, scale=2 μm（Fig. 9 h）.*P < 0.05, **P <0.01, experimental result is from three times
Independent experiment, the figure represent at least five mouse of each genotype.
Most of PKA C α knock out mice will be dead in perinatal period, therefore we establish PKA C α gene knockouts
The liver cell transplants of mouse fetal give the marrow transplant techniques of irradiated wild mouse.The tire mouse tire liver gene type of transplanting passes through
PCR Testing and appraisals.Stable transplanting is obtained after transplanting 4 months for mouse as a result, the blood platelet with PKA defects passes through egg after transplanting
White matter trace is verified.On red blood cell, white blood cell count(WBC) and hemoglobin concentration, the PKA- of transplanting/-, PKA+/- and WT mouse
It does not differ significantly.And PKA is +/- compared with WT mouse, the quantity of blood platelet significantly reduces.The +/- netted blood with WT of PKA
For platelet relatively without difference, this shows that the reduction of platelet counts is not due to blood platelet and generates reduction, but since blood is small
The acceleration that plate is removed.And, it has been found that injection antiplatelet antibody R300 can induce blood platelet and apoptosis occur, while can be fast
The +/- blood platelets more faster than WT of speed induction PKA are removed.It is interesting that PKA -/- mouse platelets significantly reduce, and PKA-/-
Typical apoptosis variation is presented in the blood platelet of mouse.
In order to avoid there are possible interference to PKA C α knock out mice for bone-marrow transplantation, we construct PKA C α conditions
Knock-out mice, this mouse breed the mouse that can be obtained that PKA conditions knock out in blood platelet with PF4 Cre mouse altogether.Condition knocks out
C-PKA -/-, C-PKA is +/- and C-PKA+ /+mouse does not have on red blood cell, white blood cell count(WBC), hemoglobin concentration variation
There is significant difference.And PKA+/- is compared with RIP3-/- mouse, is inclined to without any spontaneous bleeding or thrombus.Heterozygote and
Dose dependent variation is presented in homozygote mouse PKA activity.On following bad platelet counts, different types of mouse is without apparent different
Often.The apparent increase however, PS of PKA knock-out mices turns up.The blood platelet of tracking biotin labeling finds that PKA is knocked out in vivo
It can dose-dependent reduction platelet life span.In order to confirm that the shortening of platelet life span comes from blood platelet internal factor, I
Will be in the platelet engraftment of PKA knock-out mices to wild-type mice body.PKA-/-, PKA+/- mouse platelets are obviously shown
The service life shortened than WT mouse platelets.
（5）Fasudil can promote the apoptosis of blood platelet
Mitochondrial membrane potential detects：
Wash blood platelet（3 × 108/mL）From different Fasudil（Fasudil）Or negative control（Physiological saline）Room temperature
10 min, later every group of addition fibrin ferment 0.1U/ml in addition to negative control, 37 DEG C of incubation 30min.Blood platelet Δ ψ m are used
Lipophilic cation dyestuff JC-1 is measured.In the JC-1 of final concentration of 2 μ g/ml is added that treated blood platelet, 37 DEG C are protected from light
It is incubated 5 min, flow cytomery.Red fluorescence indicates the JC-1 polymer of mitochondrial membrane potential dependence, green fluorescence
Indicate that mitochondrial membrane potential depolarising is not associated with the JC-1 monomers of film potential later.JC-1 monomers（λ ex 514 nm, λ em 529
nm）And polymer（590 nm of λ ex 585 nm, λ em）By calculating streaming red fluorescence（JC-1 polymer）Or green
Fluorescence（JC-1 monomers）Ratio measures（Figure 10）.
PS turns up：
Wash blood platelet（3 × 108/mL）From different Fasudil（Fasudil）Or negative control（Physiological saline）Room temperature
10 min, later every group of addition fibrin ferment 0.1U/ml in addition to negative control, 37 DEG C of incubation 30min.Annexin V are delayed later
Fliud flushing, treated blood platelet, Annexin V-FITC are according to 50: 10:1 ratio is protected from light at room temperature is incubated 15 min, stream
Formula cell instrument detects（Figure 10）.
Mouse blood sampling is given first, and as a reference value, then control group and test group inject DMSO and Fasudil respectively
（Fasudil）（1.6μmol/L）, then in 30min, 2h, 4h, 6h, time point blood sampling for 24 hours counts（Figure 11）.
In short, these are the result shows that PKA determines service life and the existence of blood platelet by modulating apoptosis, PKA inhibitor can be with
The therapeutic process of thrombocythemia disease is participated in, platelet counts in peripheral circulation blood are reduced, our research is blood platelet
The clinical treatment of increase disease provides new thinking, and PKA activity is inhibited to be likely to become the new of clinical treatment piastrenemia
Means, PKA inhibitor have exploitation at the potentiality of novel therapeutic thrombocythemia disease medicament, great scientific research and economic valence
Value.
Claims (13)
1. purposes of the egg protein kinase A inhibitor in preparing treatment platelet counts and increasing relevant disease drug.
2. protein kinase A inhibitor according to claim 1 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the protein kinase A inhibitor is inorganic matter inhibitor, one kind in organic matter inhibitor
Or it is several.
3. protein kinase A inhibitor according to claim 2 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the inorganic matter inhibitor is hydride, oxide, acid, alkali, one or more of salt.
4. protein kinase A inhibitor according to claim 2 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the organic matter inhibitor be hydro carbons, the derivative of hydrocarbon, carbohydrate, protein, fat, core
One or more of acid, synthesis high molecular material.
5. protein kinase A inhibitor according to claim 4 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the hydro carbons is alkene, alkane, alkynes, one or more of aromatic hydrocarbon；The hydrocarbon
Derivative be halogenated hydrocarbons, alcohol, phenol, aldehyde, acid, one or more of ester；The carbohydrate is monosaccharide, disaccharides, oligosaccharide, more
One or more of sugar；The protein is one or more of amino acid, polypeptide；The nucleic acid is deoxyribose
One or more of nucleic acid, ribonucleic acid.
6. protein kinase A inhibitor according to claim 1 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the protein kinase A inhibitor be Fasudil, nitrogen-[2-（Phosphorylation bromine Nitro-Arginine
Acylamino-）Ethyl] -5- isoquinoline sulfonaides, C94H148N32O31、C80H130N28O24、C27H21N3O5、C26H19N3O5、C20H13N3O、
C32H31N3O5、C22H22N4O、C14H17N3O2S·2HCl、C14H17N3O2S、C11H13N3O2S·HCl、C12H13ClN2O2SHCl、
C12H15N5O2S2HCl、C53H100N20O12、1-（5- quinoline sulfonyls）Piperazine, 4- cyano -3- methylisoquinoliniums, acetylaminohydroxyphenylarsonic acid 4-
Cyano -3- methylisoquinoliniums, 8- bromo -2- list acyl adenosine -3,5- rings single thiophosphate ester, adenylate 3,5- ring single thiophosphates
Ester, 2-0- only son-adenosine cyclophosphate, 8- chloros-adenosine cyclophosphate, N- [2- (cinnamoyl amino acid)] different beautiful jade quinoline ketone of -5-, reverse phase -8-
Hexylamino adenylate 3,5- single thiophosphate esters, reverse phase -8- piperidyls adenosine-adenosine cyclophosphate, reverse phase-adenylate 3,5- ring lists
It is thiophosphate, 5- iodos tuberculin, 8- hydroxyadenosine acid -3,5- single thiophosphate esters, calcium Phospoprotein C, dephnetin, anti-
Phase -8- chlorobenzenes-adenosine cyclophosphate, reverse phase-adenosine cyclophosphate, reverse phase -8-Br- adenosine cyclophosphates, 9- adenyl cyclases, 1- (5- isoquinolines
Quinoline sulphonyl) it is-pipecoline, 8- hydroxyadenosine acid -3', 5'- monophosphate, 8- hexylamino adenylate -3', 5'- monophosphate, anti-
One or more of phase-adenylate 3', 5'- ring monophosphate.
7. protein kinase A inhibitor according to claim 1 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the platelet counts increase relevant disease include idiopathic thrombocythemia disease or after
Hair property thrombocythemia disease.
8. protein kinase A inhibitor according to claim 7 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the idiopathic thrombocythemia disease includes that primary thrombocytosis, chronic grain are thin
Born of the same parents' leukaemia, myelofibrosis and polycythemia vera, myelodysplastic syndrome or bone marrow proliferative tumour.
9. protein kinase A inhibitor according to claim 7 increases relevant disease drug in preparation treatment platelet counts
In purposes, which is characterized in that the secondary thrombocythemia disease include cut thrombocythemia, bacterium or virus after spleen
Caused infection, tumour or disease of immune system.
10. protein kinase A inhibitor according to any one of claims 1 to 6 increases correlation in preparation treatment platelet counts
Purposes in disease medicament, which is characterized in that the drug is tablet, capsule, granule, pill, sustained release preparation, controlled release
Preparation, oral solution or patch.
11. protein kinase A inhibitor according to any one of claims 1 to 6 increases correlation in preparation treatment platelet counts
Purposes in disease medicament, which is characterized in that the drug includes the protein kinase A inhibitor and medicine of pharmaceutical effective dose
Acceptable carrier on.
12. protein kinase A inhibitor according to any one of claims 1 to 6 increases correlation in preparation treatment platelet counts
Purposes in disease medicament, which is characterized in that the drug by oral, spraying sucking, injection or through gastrointestinal tract carry out to
Medicine.
13. protein kinase A inhibitor is preparing the purposes in promoting blood platelet apoptosis drug.
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| CN201710060759.2A CN108339121A (en) | 2017-01-25 | 2017-01-25 | Purposes of the protein kinase A inhibitor in preparing treatment platelet counts and increasing relevant disease drug |
| PCT/CN2017/112898 WO2018137396A1 (en) | 2017-01-25 | 2017-11-24 | Use of protein kinase a activator and inhibitor in preparation of drugs for treating diseases associated with changes in platelet counts |
| US16/520,372 US20190343861A1 (en) | 2017-01-25 | 2019-07-24 | Method of using protein kinase a activator and inhibitor in preparation of drugs for treating diseases associated with changes in platelet counts and for inhibiting and promoting platelet apoptosis |
| US17/728,895 US20220313719A1 (en) | 2017-01-25 | 2022-04-25 | Methods of treating diseases associated with changes in platelet counts using protein kinase a activator and inhibitor |
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Citations (4)
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|---|---|---|---|---|
| CN1922148A (en) * | 2003-12-23 | 2007-02-28 | 阿斯特克斯治疗有限公司 | Pyrazole derivatives as protein kinase modulators |
| CN101969955A (en) * | 2008-01-31 | 2011-02-09 | 莫纳什大学 | Methods of treating thromboembolic disorders |
| CN102216300A (en) * | 2009-09-30 | 2011-10-12 | 浙江贝达药业有限公司 | Compounds and compositions as protein kinase inhibitors |
| CN103610684A (en) * | 2013-11-07 | 2014-03-05 | 苏州大学 | Application of saccharides in preparing medicament for treating platelet quantity related diseases |
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| CN1922148A (en) * | 2003-12-23 | 2007-02-28 | 阿斯特克斯治疗有限公司 | Pyrazole derivatives as protein kinase modulators |
| CN101969955A (en) * | 2008-01-31 | 2011-02-09 | 莫纳什大学 | Methods of treating thromboembolic disorders |
| CN102216300A (en) * | 2009-09-30 | 2011-10-12 | 浙江贝达药业有限公司 | Compounds and compositions as protein kinase inhibitors |
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