CN108339120A - Application of protein kinase A activator in preparing medicine for treating diseases related to platelet quantity reduction - Google Patents

Application of protein kinase A activator in preparing medicine for treating diseases related to platelet quantity reduction Download PDF

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CN108339120A
CN108339120A CN201710060730.4A CN201710060730A CN108339120A CN 108339120 A CN108339120 A CN 108339120A CN 201710060730 A CN201710060730 A CN 201710060730A CN 108339120 A CN108339120 A CN 108339120A
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platelet
disease
protein kinase
activator
thrombocytopenia
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CN108339120B (en
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戴克胜
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Suzhou University
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Suzhou University
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Priority to PCT/CN2017/112898 priority patent/WO2018137396A1/en
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Priority to US16/520,372 priority patent/US20190343861A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid

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Abstract

The invention discloses an application of a protein kinase A activator in preparing a medicament for treating diseases related to platelet number reduction. The invention researches the effect of protein kinase A in the process of regulating and controlling the platelet apoptosis for the first time through experiments, finds that the activity of protein kinase A in platelets in thrombocytopenic patients represented by idiopathic thrombocytopenic purpura, bacterial infection and diabetes is reduced, and researches prove that the protein kinase A regulates and controls the platelet apoptosis by regulating and controlling the phosphorylation of serine at a BAD 155 site, and the activation of the activity of the protein kinase A can inhibit the occurrence of endogenous platelet apoptosis. The protein kinase A activator can inhibit the occurrence of platelet apoptosis in vitro and improve the number of circulating platelets in vivo of experimental animals, and the PKA activator can be used for the clinical treatment process of thrombocytopenia by inhibiting platelet apoptosis, has the potential of developing novel platelet protection medicines, and has scientific research and economic values.

Description

Protein kinase A activator is preparing treatment platelet counts reduction relevant disease drug In purposes
Technical field
The invention belongs to blood platelet related drugs fields, and in particular to protein kinase A activator is preparing treatment blood platelet Quantity reduces the purposes in relevant disease drug.
Background technology
The relevant thrombus of blood platelet finely regulating blood circulation and bleeding balance.Meanwhile blood platelet is in many important diseases It plays a significant role in reason physiology course, such as：Immune, infection, artery sclerosis, tumor development and transfer etc..However, blood The service life of platelet is very short and mysterious, why blood platelet body-internal-circulation only 8-9 daysThis problem perplexs mankind's generation more than half It records.In addition, the thrombopenia of threat to life is usually happened at the disease of many high incidences, such as diabetes, and infection, ITP, And after many pharmacological treatments.The reason of platelet life span shortens in these pathologic processes not fully understands.Self limiting, service life Short, lesion when especially storing limits the term of validity of storage anti-platelet therapy thrombopenia.Therefore, regulation and control blood is found The mechanism of platelet service life and existence has important Pathophysiological Significance.
The research of blood platelet apoptosis is increasingly taken seriously in recent years, because it can reveal that the god of platelet life span and existence It is secret.More and more evidences show that the intrinsic program of Apoptosis under pathology and physiological condition leads to platelet destruction.It is similar to Eukaryocyte, BAK and BAX are intracellular two fated killers, blood platelet participate in thrombosis and stopped blooding It will not be lost in journey.However, in the anti-apoptotic proteins matter of numerous Bcl-2 families, prove only have Bcl-xL and BAK to participate in adjusting at present Control the apoptosis of anuclear platelet.Being mutated Bcl-xL dose dependents reduces internal blood platelet existence, and this process can pass through BAK and BAX is knocked out to be suppressed.P53 is by inhibiting Bcl-xL activity to be proved to participate in adjusting blood platelet apoptosis process.This Outside, BAD, i.e. anti-apoptotic proteins Bcl-2 homeodomains 3 (BH3) albumen are also found to participate in adjusting blood platelet existence.Knocking out BAD can Significantly to extend platelet life span.These researchs disclose blood platelet apoptosis albumen in the key effect for adjusting platelet life span. However, fundamental problem still has, blood platelet apoptosis how is induced or inhibited under physiology or pathological conditions, it is still unclear at present Chu.
Protein kinase A (PKA) is a kind of serine-threonine protein kinase enzyme being widely present in eukaryocyte.PKA be by The different tetramer of two catalytic subunits and two regulation and control subunit compositions.After cyclic adenosine monophosphate is attached to regulation and control subunit, release activation Catalytic subunit, and then various activities in regulating cell, including cell metabolism, growth, differentiation, gene expression and Apoptosis Deng.PKA height is expressed in blood platelet, and PKA plays an important role in the adjusting of platelet function.But whether PKA is to storage Or the blood platelet apoptosis of pathology stimulation induction has large effect, does not understand yet.
Invention content
Technical problems to be solved：Blood platelet apoptosis limits its service life, blood platelet apoptosis energy caused by many diseases Enough lead to thrombopenia, but the startup of blood platelet apoptosis and regulation mechanism do not illustrate also completely at present.We will solve The technical issues of be further research protein kinase A activator to inhibiting the specific mechanism of blood platelet apoptosis, and then open albumen Purposes of the kinases A activator in preparing treatment platelet counts and reducing relevant disease drug.
Technical solution：In view of the above-mentioned problems, the invention discloses protein kinase A activator to prepare treatment platelet counts Reduce the purposes in relevant disease drug.
Preferably, the protein kinase A activator is one or more of inorganic matter activator, organic matter activator.
Preferably, the inorganic matter activator is one or more of hydride, oxide, acid, alkali, salt.
Preferably, the organic matter activator is hydro carbons, the derivative of hydrocarbon, carbohydrate, protein, fat, nucleic acid, synthesis high score One or more of sub- material.
Preferably, the hydro carbons is one or more of alkene, alkane, alkynes, aromatic hydrocarbon；The derivative of the hydrocarbon is One or more of halogenated hydrocarbons, alcohol, phenol, aldehyde, acid, ester；The carbohydrate is one kind in monosaccharide, disaccharides, oligosaccharide, polysaccharide Or it is several；The protein is one or more of amino acid, polypeptide；The nucleic acid is DNA, ribose One or more of nucleic acid.
Preferably, the protein kinase A activator is Pimobendane, adenyl cyclase agonist, ring phosphorus gland One or more of glycosides.
Preferably, the protein kinase A activator be drug Amrinone, milrinone, Enoximone, aminophylline, promise forefront One or more of ketone, iloprost, Cilostazol, cilostamide, Dipyridamole.Preferably, the protein kinase A Activator be ginkgo biloba p.e, Quercetin, meglumine adenosine cycle phosphate, adenosine cyclophosphate, Forskolin, 8- bromines adenosine -3 ', 5 '-rings Monophosphate, 8- bromos-adenosine cyclophosphate, 8- piperidyls adenosine-adenosine cyclophosphate, 8- chloros-adenosine cyclophosphate, adenylate 3,5- ring list phosphorus Hydrochlorate, N6- benzoyls-adenosine cyclophosphate, (S)-adenylate, ring 3', 5'- (hydrogen thiophosphate) triethyl group, 3- isobutyl groups -1- Methyl xanthine, 8- chlorobenzenes-adenosine cyclophosphate, adenylate 3,5- rings monophosphate, adenylate 3,5- rings single thiophosphate ester, 8- It is bromo-adenosine cyclophosphate, specificity 5,6-4,5- dicyano imidazoles-ring phosphorus bismuth glycosides, specificity 8- chlorobenzenes-cyclic guanylic acid sodium, special Sexual gland thuja acid 3', 5'- ring single thiophosphate ester triethyl group salt, specific adenosine cyclophosphate, dibutyryl-adenosine cyclophosphate, the mono- acyls of N6- Adenosine 3', 5'- ring monophosphate, 8- bromo adenylate 3', 5'- ring monophosphates thioesters, 8- bromo adenylate 3', 5'- ring list phosphorus Hydrochlorate, N6- benzoyls-adenosine cyclophosphate, red -9- amino-betas-hexyl-α-methyl -9H- purine -9- acidic alcohol -9- glands are fast One or more of purine hydrochloric acid.
Preferably, platelet counts reduction relevant disease includes that the blood caused by immune thrombocytopenia, infection is small Plate reduce disease, secondary thrombocytopenia, drug-induced thrombocytopenia, thrombocytopoiesis shortcoming disease or Non-immunity thrombocytopenia.
Preferably, the immune thrombocytopenia includes mutually Idiopathic Thrombocytopenic Purpura.
Preferably, the thrombocytopenia caused by the infection includes bacterium infection thrombocytopenia or virus infection Thrombocytopenia.
Preferably, the secondary decrease of platelet relevant disease include thrombocytopenia in diabetic body, Thrombocytopenia, cardiovascular and cerebrovascular disease in tumour patient body thrombocytopenia in patient body, drug therapy mistake Caused thrombocytopenia in journey, hypersplenia disease, during pregnancy thrombocytopenia, hinder secondary to regeneration The thrombocytopenia of impenetrability anaemia, the thrombocytopenia secondary to hypersplenia, the blood secondary to leukaemia are small Plate reduces disease, the thrombocytopenia secondary to systemic loupus erythematosus, the decrease of platelet secondary to dry syndrome Disease or thrombocytopenia secondary to ionising radiation.
Preferably, in the drug-induced thrombocytopenia, which is antitumor drug, quinine, quinindium, liver One or more of element, antibiotic, anticonvulsant drug.
Preferably, thrombocytopoiesis shortcoming disease includes that congenital platelet generation is bad, small without megakaryocytic blood Bernard's-Su Liye synthesis caused by plate reduction, fanconi syndrome, platelet membrane glycoprotein Ib-IX shortages or dysfunction (Bernard-Soulier syndromes), gray platelet syndrome, eczema decrease of platelet are levied with immunologic deficiency syndrome Decrease of platelet caused by (Wiskott-Aldrich syndromes), alpastic anemia and myelodysplastic syndrome Disease, acquired thrombocytopoiesis be bad, caused by the thrombocytopoiesis shortcoming disease caused by chemotherapeutics or radiation insult Thrombocytopoiesis is short of disease.
Preferably, it includes that thrombocytopoiesis reduces caused disease, blood platelet is broken that the platelet counts, which reduce relevant disease, It is bad to increase caused disease or thrombotic thrombocytopenic purpura.
Preferably, disease includes chronic aplastic anemia, myeloproliferative disorder caused by the thrombocytopoiesis is reduced Thrombocytopoiesis caused by syndrome, radiotherapy reduces thrombocytopoiesis caused by disease or chemotherapy and reduces disease；The blood is small Disease includes that platelet destruction caused by autoimmune disease increases disease, antiphospholipid syndrome draws caused by plate destruction increases The platelet destruction risen increases platelet destruction caused by disease, human immunodeficiency virus and increases disease or Drug blood platelet Platelet destruction increases disease caused by reducing disease.
Preferably, the drug is tablet, capsule, granule, pill, sustained release preparation, controlled release preparation, oral solution or patch Agent.
Preferably, the drug includes the protein kinase A activator and pharmaceutically acceptable carrier of pharmaceutical effective dose.
Preferably, the drug is administered by oral, injection, spraying sucking or through gastrointestinal tract.
Advantageous effect：The present invention is the study found that PKA is located at the early stage for starting or inhibiting pathophysiological condition induced platelet apoptosis The regulation and control stage.PKA is enhanced by the serine residue in 155 sites of phosphoBad pro apoptotic protein and the combination of 14-3-3, from And promote anti-apoptotic proteins Bcl-xL releases to inhibit blood platelet apoptosis.Therefore, our results of study confirm, inside and outside it is various Pathophysiological factors all can be with induced platelet apoptosis, and PKA is in the upstream of apoptosis regulation, can by improving PKA activity Significantly to protect storage or pathology to stimulate the blood platelet apoptosis of induction, it is special that technical scheme of the present invention is particularly applicable to treatment The platelet counts such as hair property thrombocytopenic purpura, diabetes and bacterium infection reduce relevant disease, and potential applicability in clinical practice is non- It is often wide, and the protein kinase A activator of the present invention can be widely using in the storage of blood platelet.
Description of the drawings
Fig. 1 be ITP patient, diabetic and septic patient blood platelet in phosphorylation GPIb β, GPIb β total proteins and PKA active testing results；
Fig. 2 is platelets analysis GPIb β phosphorylated proteins, GPIb β total proteins and PKA active testing results after bacterium infection；
Fig. 3 inhibits the percentage shared by the blood platelet for causing blood platelet apoptosis mitochondrial transmembrane potentials to depolarize for protein kinase A Test result；
Fig. 4, which is protein kinase A inhibition, causes blood platelet apoptosis western blot to detect caspase-3, gelsolin albumen table Up to caspase-3 determinations of activity result in blood platelet；
Fig. 5 be wash blood platelet and various concentration H89 be incubated after, protein kinase A inhibition causes blood platelet apoptosis PS to turn up survey Test result；
Fig. 6 is that protein kinase A inhibits the blood platelet scatter plot for causing blood platelet apoptosis FSC-FL1 to collect；
Fig. 7 is that the protein kinase A inhibitor H89 of various concentration gradient acts on sweeping after washing blood platelet 160 minutes in 22 DEG C Retouch Electronic Speculum result；
Fig. 8 is PKA by adjusting 155 site serine phosphorylation blood platelet apoptosis related experiment result figures of Bad；
Fig. 9 is that the 0-8 days blood counted in Mice Body is small afterwards by male ICR mouse injection PKA agonists 8-Br-cAMP (2.5mg/mL) Plate and immature reticulocyte fraction measurement result；
Figure 10 is conditional gene knockout mouse building process and dependence test result；
Figure 11 is that PKA knock-out mice blood platelets remove ratio increase correlated results；
Figure 12 depolarizes the percentage correlated results shared by blood platelet and PS positive blood platelets for mitochondrial transmembrane potentials；
Figure 13 is blood after washing blood platelet is incubated with protein kinase A activator drug milrinone (8 μM), negative control, fibrin ferment Platelet Δ ψ m results；
Figure 14 is after washing blood platelet is incubated with protein kinase A activator drug aminophylline (0.48mM), negative control, fibrin ferment Blood platelet Δ ψ m results；
Figure 15 is that washing blood platelet and protein kinase A activator medicated disinfecting prostaglandin E2 solution (10ng/ml), feminine gender are right Blood platelet Δ ψ m results after being incubated according to, fibrin ferment；
Figure 16 is washing blood platelet and protein kinase A activator drug cyclic AMP injecta (24 μ g/mL), negative control, coagulates Blood platelet Δ ψ m results after hemase is incubated；
Figure 17 is that different time blood is small afterwards for mouse tail vein injection protein kinase A activator drug milrinone (1mg/kg) (or NS) Plate count results；
Figure 18 is that different time blood is small afterwards by mouse tail vein injection protein kinase A activator drug PGE2 (20ng/ml) (or NS) Plate count results；
Figure 19 is that different time blood is small afterwards by mouse tail vein injection protein kinase A activator drug cAMP (12 μ g/ml) (or NS) Plate count results；
When Figure 20 is that mouse tail vein injection protein kinase A activator drug aminophylline (0.24mmol/L) (or NS) is different afterwards Between platelet count 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 are purchased from Sigma Co., USA, and 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] -5-isoquinolinesulfonamide (H89), Forskolin (Forsklin), anti-GAPDH, antibodies against P 53, JC-1, ECL, PMSF are purchased from green skies bio tech ltd of China, E64 Purchased from the bio tech ltd Roche of the U.S., A23187 is purchased from Calbiochem companies of the U.S..RNA oligonucleotide is by lucky agate Company's design synthesis.Liposome LipofectamineTM2000 and culture medium Opti-Mem I give birth to purchased from U.S. Invitrogen Object company.
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, take healthy premenopausal volunteers venous blood and ACD (2.5% sodium citrate, 2.0% glucose, 1.5% Citric acid) press 1：7 anti-freezings, 1300rpm centrifugations 20min obtain platelet rich plasma (PRP), and PRP is centrifuged 2min with 1500g, Abandon supernatant.With CGS buffer solutions (0.123M sodium chloride, 0.033 M glucose, 0.013M sodium citrates, pH 6.5) suspend and from The blood platelet of the heart, washing precipitation, then Tyrode ' s buffer solutions (2.5mM zwitterionic buffers Hepes, the 150mM chlorine with modification Change sodium, 2.5 mM potassium chloride, 1mM calcium chloride, 1mM magnesium chlorides, 12mM sodium bicarbonates, 5.5mM glucose, pH 7.4) blood is resuspended Platelet obtains washing platelet suspension, is counted to blood platelet with calculating instrument, and adjustment platelet suspension to a concentration of 3 × 108/mL is stored at room temperature 60min.
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 9.5Gy dosage in the sources Co60.It collects from PKA genetic heterozygosis Pregnant mouse (pregnancy 15 days or so) fetal liver cells, and give to inject according to the male mouse ratio of 1 tire hepatocytes correspondence, one irradiation and (connect 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 (Δ ψ m) detects
Wash blood platelet (3 × 108/mL) and various concentration H89 (12.5 μM, 25 μM, 37.5 μM and 50 μM) or negative control (DMSO) room temperature 10min, later blood platelet Δ ψ m measured using lipophilic cation dyestuff JC-1.Final concentration of 2 μ g/ml's JC-1 is added in treated blood platelet, and 37 DEG C are protected from light and are incubated 20 min, flow cytomery.Red fluorescence indicates mitochondria The JC-1 polymer of film potential dependence, green fluorescence indicate the JC-1 of unbonded film potential after mitochondrial membrane potential depolarising Monomer.JC-1 monomers (λ ex 514 nm, λ em 529nm) and polymer (λ ex 585nm, λ em 590nm) are flowed by calculating Formula red fluorescence (JC-1 polymer) or green fluorescence (JC-1 monomers) ratio measure.
7, PS turns up
The H89 (12.5 μM, 25 μM, 37.5 μM and 50 μM) or negative control (DMSO) of blood platelet and various concentration are washed in room Temperature is incubated 10min.Later by Annexin V buffer solutions, H89 treated blood platelet, Annexin V-FITC according to 50:10:1 Ratio be protected from light at room temperature be incubated 15min, flow cytomery.
8, blood platelet shrinkage is tested
The H89 (12.5 μM, 25 μM, 37.5 μM and 50 μM) or negative control (DMSO) of blood platelet and various concentration are washed in room Temperature is incubated 10min.Then blood platelet and SZ21 antibody at room temperature are incubated 30min altogether.Centrifugation, with the sheep anti mouse marked containing FITC Blood platelet is resuspended in antibody, and room temperature, which is protected from light, is incubated 30min.Flow cytometer collects blood platelet, the blood platelet scatterplot that FSC-FL1 is collected Figure is for analyzing blood platelet.The shrinkage of blood platelet passes through the variation of analysis FSC, the FSC drops of evaluation GPIIb/IIIa positive cells Low degree evaluates shrinkage degree.A23187 is as positive control.DMSO is as negative control.
9、Western Blotting：
The H89 (25 μM, 50 μM, 100 μM) or negative control (DMSO) of blood platelet and various concentration are washed in incubation at room temperature 10min.2X cell pyrolysis liquids (PMSF containing 2mM, 2mM NaF, 2mM Na3VO4 and protease inhibitors) are added to terminate instead It answers, cracks on ice, sample preparation.Sample detects the expression of corresponding albumen with Western blot.
10, RNA interference experiments
The double-strand siRNA oligonucleotides (justice of target PRKACA:5-GCUCCCUUCAUACCAAAGUTT-3, antisense: 5- ACUUUGGUAUGAAGGGAGCTT-3) and negative control siRNA is (just:5-UUCUCCGAACGUGUCACGUTT-3, antisense: 5-ACGUGACACGUUCGGAGAATT-3) designed and synthesized by Ji Ma companies.
When Hela cell transfectings, the day before transfection is added about by 2 × 105 hela cell inoculations in culture plate in every hole The culture medium of 500 μ L antibiotic-frees, enables cell density when transfection to reach 30~50%；Take 1 holes μ L/ Lipofectamine 2000 (is gently shaken up) using preceding, is diluted with the low blood serum mediums of 50 μ L Opti-MEM I.Gently mix Afterwards in incubation at room temperature 5min；2 μ L FAM-siRNA are taken, are diluted with the low blood serum mediums of 50 μ L Opti-MEM I, are gently mixed equal It is even；Diluted Lipofectamine 2000 is gently mixed with dilution FAM-siRNA, is stored at room temperature after the incubation of 5min 20min；FAM-siRNA- transfection reagent mixed liquors are added in the hole containing cell and culture solution (containing about 400 μ L), are gently shaken Orifice plate is shaken, mixing is made.
When blood platelet transfection experiment, 6 × 108/mL of blood platelet is washed in sterile preparation, after standing.By 100 μ L siRNA few nucleosides Acid is added in the blood platelet that 100 μ L serum-free M199 culture mediums suspend.
It is cultivated in 37 DEG C of CO2 incubators, culture medium can be changed to the complete medium culture 48h containing serum after 6 hours；Turn Dye can pass through flow cytomery transfection efficiency after 6 hours.At the end of culture, collects and crack hela cells and blood is small Plate.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) septicemia, diabetes or patient's ITP blood plasma were incubated or the blood platelet of bacterium infection will appear the decline of PKA activity Phenomenon
Thrombopenia frequently occurs in some high incidence diseases, such as diabetes, ITP, septicemia or bacterium infection etc. Deng.We have studied PKA in these effects in there is decrease of platelet disease.ITP patient, patient of diabetes is collected by centrifugation The blood platelet of person and septic patient, and compared with the healthy population blood platelet of corresponding age, gender, by blood platelet MTB After resuspension, a concentration of 3 × 108/mL is adjusted, the total protein after platelet lysates is for detecting the total egg of phosphorylation GPIb β, GPIb β White and PKA is active, compared with the control group * P<0.05,**P<0.01.By MTB 1 × 107/mL of diluted washing blood platelet and phase The bacterium answered (uses MTB buffer solutions 1:20 dilutions) 37 DEG C co-culture 90 minutes, while setting no Bacteria Culture group and doing negative control, GPIb β phosphorylated proteins, GPIb β total proteins and PKA activity are detected, as a result comes from different platelet donors four times independently in fact It tests, the result is shown in Figure 1 and Fig. 2, * P<0.05,**P<0.01.
We can be found that, it has been found that after the blood platelet of Healthy People is incubated with diabetes or ITP patients blood plasmas, can induce normal Blood platelet apoptosis, meanwhile, PKA activity significantly reduces.Newest evidence shows the Escherichia coli and gold detached in sepsis patient Staphylococcus aureus can be with external evoked blood platelet apoptosis (Kraemer etc., 2012).We have discovered that Escherichia coli and Portugal Grape coccus not only can be with induced platelet apoptosis, but also can significantly reduce PKA activity.In short, these results indicate that ITP, losing Mass formed by blood stasis and diabetic or bacterium infection can induce blood platelet apoptosis, while PKA activity declines.
(2) inhibit PKA activity inducement blood platelets that the Apoptosis that interior approach relies on occurs
Act on washing blood platelet in 22 DEG C with the H89 of various concentration (0,12.5,25,37.5 and 50 μM) respectively 160 minutes, stream Formula cell instrument detects mitochondrial transmembrane potentials depolarising and the PS exposures of blood platelet.Experimental result is repeated four times.As a result extremely such as Fig. 3 Shown in Fig. 7.By wash blood platelet various concentration gradient H89 pre-process 30 minutes at 22 DEG C, while set up DMSO with The negative control group and positive controls blood platelet of A23187 processing, western blot detect caspase-3, gelsolin egg Caspase-3 activity in white expression and blood platelet.By the anti-CD41 antibody of FITC labels with pretreated blood platelet with 1:10 Ratio mixing is protected from light incubation 10 minutes under room temperature, and analysis Platelet Size scatter plot CD41 positive quantity, which declines, indicates blood platelet Quantity is reduced.The H89 of various concentration gradient acts on washing blood platelet in 22 DEG C 160 minutes, while setting up DMSO negative controls 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 incubated altogether It educates, JC-1 dye markers change in flow cytomery blood platelet, it is found that H89 dose dependent induced platelets find line grain Body film potential (Δ Ψ m) depolarizes.Moreover, H89 can also time dependent induced platelet generation △ ψ m depolarisings.ΔΨm Depolarising is 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 is finally caused membrane integrity to destroy and is led Cause morphological change.Experimental result shows that H89 can induce the blood platelet of the GPIIb/IIIa positives, forescatering (FSC) occurs and subtracts Few phenomenon shows to inhibit to shrink on platelet PLA2 after PKA activity.In addition, the blood platelet of the dose-dependent inductions of H89 There is typical apoptosis morphological change, including Cell membrane vesicles, pseudopodium, shrinkage, degranulation phenomenon etc..In short, these results Show that PKA inhibits the Apoptosis that can be relied on the interior approach that induced platelet occurs.
(3) PKA regulates and controls blood platelet apoptosis by adjusting 155 site phosphorylations of BAD
Next we further explore the mechanism that PKA inhibits induced platelet apoptosis.Under room temperature, the washing of 3 × 108/mL Blood platelet and H89 the or DMSO internal references of various concentration gradient are incubated 160 minutes altogether, and 30 points are cracked on ice with isometric lysate Clock, product are separated by electrophoresis to obtain different size of protein fragments through SDS-PAGE, and primary antibody is added after closing one hour in skimmed milk power It is incubated, final ECL luminescence displays destination protein band (Fig. 8 a).Blood platelet is washed respectively with H89,10uM of 37.5uM It is pre-processed under forskin and DMSO internal reference room temperature 160 minutes, extracts the plasmosin and mitochondrial protein of blood platelet respectively, Western blot testing goal albumen, Image J softwares analyze the amount of destination protein, count four experiments with mean ± standard Difference shows result (Fig. 8 b).After pretreated platelet lysates, 17,000g 4 DEG C centrifuge 10 minutes, by obtained supernatant and phase Antibody incubation precipitates overnight is answered, after the incubation 2 hours of 4 DEG C of protein A/G+ sepharose 4Bs, albumen is used for after pearl is eluted Hybridize (Fig. 8 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 dose-dependant to reduce the phosphorylation degree of 166 site serines of blood platelet GPIb β, and then prove Reduce PKA activity.However, we have discovered that, apoptosis executes egg BAK, BAX and anti-apoptotic proteins Bcl- in apoptosis blood platelet 2, significantly changing does not occur in the expression quantity of Bcl-xL.Have been reported that display, in S49 lymphoma cells, PKA passes through regulation and control The expression for increasing Bim makes tumour cell avoid that apoptosis phenomenon occurs.And we have discovered that, participate in the blood platelet of regulation and control in PKA In apoptotic process, Bim protein levels do not occur significant change, eliminate the regulating and controlling effect of Bim.
Have been reported that proof, PKA inhibits can to promote the expression of P53, and the P53 of phosphorylation can be in Concentration In Platelets of Diabetic Patients The directly inactivation of induction anti-apoptotic proteins Bcl-xL, and then promote blood platelet that apoptosis occurs.However, the blood platelet participated in PKA In apoptosis, the P53 of P53 or phosphorylation does not detect to change.
In karyocyte, PKA regulates and controls 14-3-3 albumen and withers with anti-by adjusting the phosphorylation of 155 site serines of BAD Die the binding of protein B cl-xL, and then regulating cell apoptosis.Under conditions of 155 site dephosphorylations of BAD, BAD and Bcl-xL Dimer, release apoptosis executor BAK and BAX are formed, and then mitochondrial membrane permeability is caused to enhance, Apoptosis occurs.I Find 155 site serines of BAD PKA inhibit the phenomenon that induction blood platelet in there is phosphorylation degree reduction.Moreover, H89 is reduced, Forskolin enhances the phosphorylation of 155 site serines of BAD, and then influences Bcl-xL albumen on mitochondrial membrane It reduces and increases.Most of all, co-immunoprecipitation result shows under H89 or Forskolin stimulation that Bcl-xL is apparent with BAD It reduces or enhancing, prompt H89 or Forskolin regulates and controls anti-apoptotic proteins Bcl-xL by regulating and controlling the interaction of BAD and Bcl-xL Activity.Therefore, these are statistics indicate that the apoptosis that PKA passes through regulation and control 155 site serine phosphorylation blood platelets of BAD.
(4) PKA activator improves cycle platelet counts
On the other hand, PKA activation can promote 155 serine sites phosphorylation levels of BAD, and then prevent the hair of Apoptosis It is raw.Therefore, PKA activators may prevent the blood platelet apoptosis of aging, and extend the service life of blood platelet.In order to verify this It is assumed that we by every 24 hours of male ICR mouse through tail vein injection PKA agonists 8-Br-cAMP (2.5mg/mL), simultaneously PBS groups are set up as negative control, the blood platelet and immature reticulocyte fraction in Mice Body, experimental group and control component are counted after 8 days 5-6 mouse * P is not set up<0.05, **P<0.01 (Fig. 9).
As a result, it has been found that platelet count increases by day, reached peak value at the 8th day.Corresponding immature reticulocyte fraction is reduced by day, is shown The increase of blood platelet is not that blood platelet generates the result (Fig. 9) increased.Moreover, JC-1 label blood platelets do not change significantly. These statistics indicate that, the increase of PKA activity protects blood platelet that apoptosis phenomenon does not occur, and can be with the longevity of blood platelet in extension body Life.
(6) PKA knock-out mices blood platelet is removed ratio and is increased
It is conditional gene knockout mouse structure (Figure 10 a) first.PKA, Bad, GBIb in Western blot detection blood platelets The expression of β, phosphoBad Ser-155 and phosphorylation GBIb β Ser-166 at least set 5 mouse (Figure 10 b) per experimental group. Sysmex XP-100 blood analyser platelet Countings, 7 WT mouse of as a result statistical analysis, 7 PKA+/- mouse and 5PKA-/- mouse (Figure 10 c).After whole blood thiazole orange (0.5 μ g/mL) and anti-CD41 (20 μ g/mL) antibody label, in room temperature Lower incubation 15min, the quantity of flow cytomery immature reticulocyte fraction.Data come from 7 WT mouse, 7 PKA+/- mouse With 5PKA-/- mouse (Figure 10 d).Antiplatelet antibody R300 (0.15 μ g/kg) is through being injected intraperitoneally into WT and PKA+/- Mice Body Interior, whole blood, Sysmex XP-100 blood analyser platelet Counting quantity, as a result statistical analysis 6 respectively are collected in eye socket blood sampling WT and 6 PKA+/- mouse (Figure 10 e).Washing blood platelet is protected from light incubation 10 minutes, flow cytometer with JC-1 (2 μ g/mL) Detect mitochondrial transmembrane potentials depolarization level (Figure 11 a).The anti-CD41 antibody of FITC labels is with blood platelet with 1:10 ratio Mixing is gently incubated at room temperature 10 minutes (Figure 11 b) after mixing.Platelet Size scatter plot is analyzed, under CD41 positive cell quantities Drop represents platelet counts reduction.1% glutaraldehyde fixed washing blood platelet after 30 minutes, and scanning electron microscope imaging is as a result, mark Ruler=2 μm (Figure 11 c).*P<0.05, * * P<0.01, experimental result is from independent experiment, the figure represent each genotype three times At least five mouse.
Most of PKA C α knock out mice will be dead in perinatal period, therefore we establish PKA C α knock out mice The liver cell transplants of fetus give the marrow transplant techniques of irradiated wild mouse.The tire mouse tire liver gene type of transplanting passes through PCR Testing and appraisal.Stable transplanting is obtained after transplanting 4 months for mouse as a result, the blood platelet with PKA defects passes through albumen after transplanting Matter trace is verified.On red blood cell, white blood cell count(WBC) and hemoglobin concentration, the PKA- of transplanting/-, PKA+/- and WT mouse do not have There is significant difference.And PKA+/- compared with WT mouse, the quantity of blood platelet significantly reduces.PKA+/- small with the netted blood of WT For plate relatively without difference, this shows that the reduction of platelet counts is not due to blood platelet and generates reduction, but due to blood platelet The acceleration of removing.And, it has been found that injection antiplatelet antibody R300 can induce blood platelet and apoptosis occur, while can be rapid PKA+/- more faster than WT blood platelet is induced to remove.It is interesting that PKA-/- mouse platelets significantly reduce, and PKA-/- mouse Blood platelet the variation of typical apoptosis is presented.
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 knockout Mouse, this mouse breed the mouse that can be obtained that PKA conditions knock out in blood platelet with PF4Cre mouse altogether.The C- that condition knocks out PKA-/-, C-PKA+/- and C-PKA+ /+mouse in red blood cell, without notable in white blood cell count(WBC), hemoglobin concentration variation Difference.And PKA+/- is compared with RIP3-/- mouse, is inclined to without any spontaneous bleeding or thrombus.Heterozygote and homozygote Dose dependent variation is presented in mouse PKA activity.On following bad platelet counts, different types of mouse Non Apparent Abnormality.So The apparent increase and PS of PKA knock-out mices turns up.The blood platelet of tracking biotin labeling is found in vivo, and PKA knockouts can agent Measure the reduction platelet life span of dependence.In order to confirm that the shortening of platelet life span comes from blood platelet internal factor, we are by PKA In the platelet engraftment of knock-out mice to wild-type mice body.PKA-/-, PKA+/- mouse platelets obviously show smaller than WT The service life that mouse blood platelet shortens.
(7) occur BAD phosphorylations in PKA knock-out mices body and reduce phenomenon
Then we have inquired into the mechanism of PKA knock-out mice blood platelet apoptosis.With us before in human platelet's experiment in vitro Result it is consistent, in heterozygote and homozygote mouse P53 expression dose gradient increase, and PKA catalytic subunits expression and PKA activity significantly reduces.Moreover, 155 site serine phosphorylations of PKA knock-out mice blood platelets BAD reduce.However, PKA C α Blood platelet is knocked out compared with wild type platelets, BAK, BAD, BAX, Bcl-xL is without significant change.In short, these mouse results Demonstrate our discoveries in human platelet, it was demonstrated that the inhibition of PKA leads to the blood of 155 site serine mediated phosphorylations of BAD Platelet apoptosis occurs.
(8) pathological conditions induction does not occur for PKA active protections blood platelet Apoptosis and removing are improved
It washs blood platelet and uses 22 DEG C of 5uM forskin and DMSO pretreatment 5 minutes respectively, then at room temperature with ITP patients serums It co-cultures 12 hours, while setting up healthy adult human serum and comparing, the depolarising of flow cytomery mitochondrial transmembrane potentials Percentage shared by blood platelet (Figure 12 a) and PS positive blood platelets (Figure 12 b).ICR mouse mainline single doses 8-Br- CAMP (0.0625,1.25,2.5mg/kg) and internal reference, then to remove the blood platelets that Fc section mediate clear through Fc inhibitor is injected intraperitoneally It removes, after ten minutes, antiplatelet antibody R300 (0.2 μ g/kg) enters through intraperitoneal injection in Mice Body, in different time eye droppings Socket of the eye venous collection Mouse whole blood, Sysmex XP-100 blood analyser platelet Counting quantity, every group sets 6 mouse, as a result uses Mean ± standard deviation indicates (Figure 12 c).It washs blood platelet and uses 22 DEG C of 5uM forskin and DMSO pretreatment 5 minutes respectively, so 90min is co-cultured at room temperature with staphylococcus aureus suspension afterwards, while setting feminine gender without staphylococcus aureus liquid handling Negative control group blood platelet, flow cytomery mitochondrial transmembrane potentials depolarize blood platelet (Figure 12 d) and PS positive bloods are small Percentage shared by plate (Figure 12 e).It more than experiment in triplicate, is as a result indicated with mean ± standard deviation.Wash blood platelet difference With 5uM forskin and DMSO 22 DEG C of pretreatments, 5 minutes clocks, it is small that 12 are then co-cultured at room temperature with the serum of diabetic When, while setting up healthy adult human serum and comparing, flow cytomery mitochondrial transmembrane potentials depolarize blood platelet (figure 12a) and PS positive blood platelets (Figure 12 b) shared by percentage.
Blood platelet apoptosis seemingly stores the main reason for blood platelet occurs dysfunction and quickly removed.In order to verify PKA In the effect of Storage lesion of platelet, PKA activator or inhibitor are added in platelet reserve.As expected, PKA inhibitor Cause blood platelet apoptosis at first.It is interesting that PKA activator Forskolin obviously postpones the generation of blood platelet apoptosis.Many institutes Known, the intrinsic program apoptosis of mitochondrial membrane potential depolarising regulation and control is an irreversible process.These data are not only Further verification PKA adjusts the effect of blood platelet apoptosis, and also indicates that PKA is located at mitochondrial depolarization and adjusts Apoptosis Upstream.
In addition to Apoptosis, also other storage damage variations cause to store the removing of blood platelet in vivo.Therefore, Wo Menyan Studied carefully whether PKA activation protection blood platelet apoptosis while, can prevent storage blood platelet be eliminated.As a result, it has been found that Forsklin Storage blood platelet can be obviously protected, it is inhibited to be removed in vivo, on the other hand, H89 accelerates the speed of removing.These data Show that the Apoptosis of PKA regulation and control plays key effect in Storage lesion of platelet, and has prompted a kind of effective extension blood bank Store the new method of blood platelet.
ITP, particularly the autoantibody of intractable ITP patient's bodies meeting induced platelet apoptosis, cause platelet destruction.With elder generation Preceding report is consistent, we have discovered that apoptosis occurs for the apparent induced platelet of ITP patient's antiplatelet sera.However, Blood platelet after Forskolin preincubates significantly reduces the generation of the blood platelet apoptosis of Serum-induced.In order to which clear PKA is activated Agent removed in blood platelet body in effect, we establish an ITP mouse with antiplatelet monoclonal mixed antibody R300 Model.R300 is incubated with blood platelet altogether in vitro can induced platelet apoptosis.Fc receptor blockade agent is injected into Mice Body and can seal Close the platelet destruction of Fc- dependences.The blood platelet that PKA activator can be induced with dose-dependent inhibition antiplatelet antibody is clear Except phenomenon.These are as a result, not only confirm effects of the PKA in protection antibody induction blood platelet apoptosis and in removing, but also prompt Treat the new strategy of ITP.
Then it was found that PKA activation effectively prevent staphylococcus aureus separation strains and diabetes in sepsis patient body The Apoptosis induced after patient's blood plasma and blood platelet hatching.In a word these statistics indicate that, PKA be blood platelet apoptosis early stage prison Pipe modulin, and most of all, this result of study to the thrombopenia of different pathologic, physiologic Induced by Stimulation The service life of blood platelet has great importance in treatment and control volume.
(9) experiment of same type Drug inhibition blood platelet apoptosis and result
Mitochondrial membrane potential detects：
Washing blood platelet (3 × 108/mL), (aminophylline 0.48mM, sterilize prostaglandin E2 solution from different PKA agonists 10ng/ml, 8 μM of milrinone, 24 μ g/mL of cyclic AMP injecta) or negative control (physiological saline) room temperature 10min, later Every group of addition the fibrin ferment 0.1U/ml, 37 DEG C of incubation 30min in addition to negative control.Blood platelet Δ ψ m are contaminated using lipophilic cation Expect that 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 and are incubated 5min, fluidic cell Instrument detects.Red fluorescence indicates that the JC-1 polymer of mitochondrial membrane potential dependence, green fluorescence indicate that mitochondrial membrane potential is gone The JC-1 monomers of unbonded film potential after polarization.JC-1 monomers (λ ex 514nm, λ em 529nm) and polymer (λ ex 585 nm, λ em 590nm) by calculating streaming red fluorescence (JC-1 polymer) or green fluorescence (JC-1 monomers) ratio To measure (Figure 13 to Figure 16).
PS turns up：
Washing blood platelet (3 × 108/mL), (aminophylline 0.48mM, sterilize prostaglandin E2 solution from different PKA agonists 10ng/ml, 8 μM of milrinone, 24 μ g/ml of cyclic AMP injecta) or negative control (physiological saline) room temperature 10min, later Every group of addition the fibrin ferment 0.1U/ml, 37 DEG C of incubation 30min in addition to negative control.It later will be after Annexin V buffer solutions, processing Blood platelet, Annexin V-FITC are according to 50:10:1 ratio is protected from light at room temperature is incubated 15min, flow cytomery (figure 13 to Figure 16).
Milrinone can inhibit to remove in blood platelet body
ICR mouse 12, every group each 6.Experimental group milrinone 1mg/kg 6,6, control group physiological saline (NS).Mouse After tail vein injection milrinone 1mg/kg 10min (or NS), mouse peritoneal injects 0.1mg/kg R300 antibody.Then exist respectively Each time point blood sampling counts.From result, it will be seen that 1mg/kg milrinones are obviously improved mouse peripheral blood blood platelet It counts (Figure 17).
PGE2 can inhibit to remove in blood platelet body
Mouse blood sampling is given first, and as a reference value, then control group and test group inject 0.9%NS and PGE2 respectively (20ng/ml), injection R300 (0.1 μ g/g) after 10min, then in 30min, 2h, 4h, 6h, time point blood sampling for 24 hours counts. When 30min, the platelet count P of NS groups and PGE2 groups<0.05, there is significant difference (Figure 18).
CAMP can inhibit to remove in blood platelet body
Mouse blood sampling is given first, and as a reference value, then control group and test group inject 0.9%NS and cAMP (12 μ respectively ), g/ml R300 (0.1 μ g/g) is injected after 10min, then in 30min, 2h, 4h, 6h, time point blood sampling for 24 hours counts.In 30min When, the platelet count P of NS groups and cAMP groups<0.05, there is significant difference (Figure 19).
Aminophylline can inhibit to remove in blood platelet body
Give mouse blood sampling first, as a reference value, then control group and test group inject respectively 0.9%NS and R300 (0.1 μ g/g) is injected after Aminophylline (aminophylline) (0.24mmol/L), 10min, then in 30min, 2h, 4h, 6h, for 24 hours time point blood sampling count (Figure 20).

Claims (19)

1. purposes of the protein kinase A activator in preparing treatment platelet counts and reducing relevant disease drug.
2. protein kinase A activator according to claim 1 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the protein kinase A activator is inorganic matter activator, one kind in organic matter activator Or it is several.
3. protein kinase A activator according to claim 2 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the inorganic matter activator is hydride, oxide, acid, alkali, one or more of salt.
4. protein kinase A activator according to claim 2 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the organic matter activator be hydro carbons, the derivative of hydrocarbon, carbohydrate, protein, fat, core One or more of acid, synthesis high molecular material.
5. protein kinase A activator according to claim 4 is preparing treatment platelet counts reduction relevant disease drug 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 activator according to claim 1 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the protein kinase A activator be Pimobendane, adenyl cyclase excitement One or more of agent, adenosine cyclophosphate.
7. protein kinase A activator according to claim 1 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the protein kinase A activator be drug Amrinone, milrinone, Enoximone, aminophylline, One or more of dinoprostone, iloprost, Cilostazol, cilostamide, Dipyridamole.
8. protein kinase A activator according to claim 1 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the protein kinase A activator be ginkgo biloba p.e, Quercetin, meglumine adenosine cycle phosphate, Adenosine cyclophosphate, Forskolin, 8- bromines adenosine -3 ', 5 '-ring monophosphates, 8- bromos-adenosine cyclophosphate, 8- piperidyls adenosine-ring phosphorus gland Glycosides, 8- chloros-adenosine cyclophosphate, adenylate 3,5- rings monophosphate, N6- benzoyls-adenosine cyclophosphate, (S)-adenylate, ring 3', 5'- (hydrogen thiophosphate) triethyl group, 3-isobutyl-1-methylxanthine, 8- chlorobenzenes-adenosine cyclophosphate, adenylate 3, 5- rings monophosphate, adenylate 3,5- rings single thiophosphate ester, 8- bromos-adenosine cyclophosphate, specificity 5,6- 4,5- dicyanos Imidazoles-ring phosphorus bismuth glycosides, specificity 8- chlorobenzenes-cyclic guanylic acid sodium, specific adenylate 3', 5'- ring single thiophosphate ester triethyl group Salt, specific adenosine cyclophosphate, dibutyryl-adenosine cyclophosphate, mono- acyl adenosine 3', the 5'- ring monophosphates of N6-, 8- bromo adenylates 3', 5'- ring monophosphate thioesters, 8- bromo adenylate 3', 5'- rings monophosphate, N6- benzoyls-adenosine cyclophosphate, red -9- ammonia One or more of base-β-hexyls-Alpha-Methyl -9H- purine -9- acidic alcohol -9- adenine hydrochloric acid.
9. protein kinase A activator according to any one of claims 1 to 8 is preparing the related disease for the treatment of platelet counts reduction Purposes in medicine, which is characterized in that the platelet counts reduce relevant disease include immune thrombocytopenia, Thrombocytopenia, secondary thrombocytopenia, drug-induced thrombocytopenia, blood platelet caused by infection Generate shortcoming disease or non-immunity thrombocytopenia.
10. protein kinase A activator according to claim 9 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the immune thrombocytopenia includes Idiopathic Thrombocytopenic Purpura.
11. protein kinase A activator according to claim 9 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the thrombocytopenia caused by the infection includes bacterium infection thrombocytopenia Or virus infection thrombocytopenia.
12. protein kinase A activator according to claim 9 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that the secondary decrease of platelet relevant disease includes the blood platelet in diabetic body Reduce disease, the thrombocytopenia in tumour patient body, thrombocytopenia, the medicine of cardiovascular and cerebrovascular disease in patient body Caused thrombocytopenia in object therapeutic process, hypersplenia disease, during pregnancy thrombocytopenia, secondary Thrombocytopenia in alpastic anemia, the thrombocytopenia secondary to hypersplenia, secondary to white blood Thrombocytopenia, the thrombocytopenia secondary to systemic loupus erythematosus, the blood secondary to dry syndrome of disease Platelet reduces disease or the thrombocytopenia secondary to ionising radiation.
13. protein kinase A activator according to claim 9 is preparing treatment platelet counts reduction relevant disease drug In purposes, which is characterized in that in the drug-induced thrombocytopenia, the drug be antitumor drug, quinine, One or more of quinindium, heparin, antibiotic, anticonvulsant drug.
14. protein kinase A activator according to claim 9 is preparing treatment platelet counts reduction relevant disease medicine Purposes in object, which is characterized in that the described thrombocytopoiesis shortcoming disease include congenital platelet generate it is bad, without macronucleus Bernard-caused by cellularity decrease of platelet, fanconi syndrome, platelet membrane glycoprotein Ib-IX shortages or dysfunction Su Liye syndromes, gray platelet syndrome, eczema decrease of platelet with immunologic deficiency syndrome, alpastic anemia with Thrombocytopenia, acquired thrombocytopoiesis caused by myelodysplastic syndrome be bad, caused by chemotherapeutics Thrombocytopoiesis shortcoming disease or radiation insult caused by thrombocytopoiesis be short of disease.
15. protein kinase A activator according to any one of claims 1 to 8 is preparing treatment platelet counts reduction correlation Purposes in disease medicament, which is characterized in that it includes that thrombocytopoiesis reduction is led that the platelet counts, which reduce relevant disease, Disease or thrombotic thrombocytopenic purpura caused by the disease of cause, platelet destruction increase.
16. protein kinase A activator according to claim 15 is preparing treatment platelet counts reduction relevant disease medicine Purposes in object, which is characterized in that disease includes chronic aplastic anemia, bone caused by the thrombocytopoiesis is reduced Thrombocytopoiesis caused by marrow hyperplasia exception syndrome, radiotherapy reduces thrombocytopoiesis caused by disease or chemotherapy and reduces disease； Disease includes that platelet destruction caused by autoimmune disease increases disease, anti-phosphorus caused by the platelet destruction increases Platelet destruction caused by fat syndrome increases platelet destruction caused by disease, human immunodeficiency virus and increases disease or medicine Platelet destruction increases disease caused by physical property thrombopenia.
17. protein kinase A activator according to any one of claims 1 to 8 is preparing treatment platelet counts reduction correlation 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.
18. protein kinase A activator according to any one of claims 1 to 8 is preparing treatment platelet counts reduction correlation Purposes in disease medicament, which is characterized in that the drug includes the protein kinase A activator and medicine of pharmaceutical effective dose Acceptable carrier on.
19. protein kinase A activator according to any one of claims 1 to 8 is preparing treatment platelet counts reduction correlation Purposes in disease medicament, which is characterized in that the drug by oral, injection, spraying sucking or through gastrointestinal tract carry out to Medicine.
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