CN108339120B

CN108339120B - Application of protein kinase A activator in preparing medicine for treating diseases related to platelet quantity reduction

Info

Publication number: CN108339120B
Application number: CN201710060730.4A
Authority: CN
Inventors: 戴克胜
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2021-08-06
Anticipated expiration: 2037-01-25
Also published as: CN108339120A

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

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

Technical Field

The invention belongs to the field of platelet-related medicines, and particularly relates to application of a protein kinase A activator in preparation of a medicine for treating platelet quantity reduction-related diseases.

Background

Platelets fine-regulate the blood circulation-related thrombotic and hemorrhagic balance. At the same time, platelets play an important role in many important pathophysiological processes, such as: immunity, infection, arteriosclerosis, tumorigenesis, metastasis, and the like. However, the life span of platelets is short and mysterious, why is the platelet circulating in vivo only 8-9 days? This problem afflicts humans for more than half a century. In addition, life-threatening thrombocytopenia commonly occurs in a number of high-incidence diseases, such as diabetes, infections, ITP, and after numerous pharmacological treatments. The reasons for the shortened platelet life in these pathological processes are not fully understood. Self-limiting, short life, especially storage-time lesions, limit the effective period of storing platelets for treating thrombocytopenia. Therefore, finding a mechanism for regulating platelet life and survival has important pathophysiological significance.

The study of platelet apoptosis has become more and more important in recent years because it can reveal the mystery of platelet life and survival. There is increasing evidence that the intrinsic process of apoptosis in pathological and physiological conditions leads to platelet destruction. Like eukaryotic cells, BAK and BAX are two of the hits of intracellular killers that are not lost during thrombosis and hemostasis processes in which platelets participate. However, of the many anti-apoptotic proteins of the Bcl-2 family, only Bcl-xL and BAK are currently demonstrated to be involved in regulating apoptosis in anucleated platelets. Dose-dependent reduction of platelet survival in vivo by mutant Bcl-xL can be inhibited by knock-out of BAK and BAX. P53 was shown to be likely involved in the regulation of platelet apoptosis by inhibiting Bcl-xL activity. In addition, BAD, an anti-apoptotic protein Bcl-2 homeodomain 3(BH3) protein, was also found to be involved in regulating platelet survival. Knockout of BAD can significantly prolong platelet life. These studies reveal a key role for apoptotic proteins in the regulation of platelet life. However, the fundamental problem still remains, and how to induce or inhibit platelet apoptosis in physiological or pathological conditions is still unclear at present.

Protein kinase a (pka) is a serine-threonine protein kinase that is widely found in eukaryotic cells. PKA is a heterotetramer consisting of two catalytic subunits and two regulatory subunits. After cyclic adenosine monophosphate is combined with the regulation subunit, the activated catalytic subunit is released, and various activities in the cell, including cell metabolism, growth, differentiation, gene expression, apoptosis and the like, are further regulated and controlled. PKA is highly expressed in platelets and plays an important role in the regulation of platelet function. However, it is unknown whether PKA has a major effect on platelet apoptosis induced by storage or pathological stimulation.

Disclosure of Invention

The technical problem to be solved is as follows: platelet apoptosis limits its lifespan, and many diseases cause platelet apoptosis that can lead to thrombocytopenia, but the mechanisms that initiate and regulate platelet apoptosis are not fully elucidated at present. The technical problem to be solved is to further research the specific mechanism of the protein kinase A activator for inhibiting platelet apoptosis, and further disclose the application of the protein kinase A activator in preparing the medicines for treating the diseases related to platelet number reduction.

The technical scheme is as follows: in view of the above problems, the present invention discloses the use of a protein kinase a activator for the manufacture of a medicament for the treatment of a disease associated with a decreased platelet count.

Preferably, the protein kinase A activator is one or more of an inorganic activator and an organic activator.

Preferably, the inorganic activator is one or more of hydride, oxide, acid, alkali and salt.

Preferably, the organic activator is one or more of hydrocarbons, hydrocarbon derivatives, saccharides, proteins, fats, nucleic acids, and synthetic polymer materials.

Preferably, the hydrocarbon is one or more of olefin, alkane, alkyne and aromatic hydrocarbon; the hydrocarbon derivative is one or more of halogenated hydrocarbon, alcohol, phenol, aldehyde, acid and ester; the saccharide is one or more of monosaccharide, disaccharide, oligosaccharide and polysaccharide; the protein is one or more of amino acid and polypeptide; the nucleic acid is one or more of deoxyribonucleic acid and ribonucleic acid.

Preferably, the protein kinase A activator is one or more of phosphodiesterase inhibitor, adenylate cyclase agonist and cyclic adenosine monophosphate.

Preferably, the protein kinase A activator is one or more of the drugs amrinone, milrinone, enoximone, aminophylline, dinoprostone, iloprost, cilostazol, cilostamide and dipyridamole. Preferably, the protein kinase A activator is ginkgo biloba extract, quercetin, meglumine adenosine cyclophosphate, forskolin, 8-bromoadenosine-3 ',5' -cyclic monophosphate, 8-bromo-adenosine cyclophosphate, 8-piperidyl adenosine-adenosine cyclophosphate, 8-chloro-adenosine cyclophosphate, adenosine 3, 5-cyclic monophosphate, N6-benzoyl-adenosine cyclophosphate, (S) -adenosine, cyclic 3',5' - (hydrogen sulfur phosphate) triethyl, 3-isobutyl-1-methylxanthine, 8-chlorobenzene-adenosine cyclophosphate, adenosine 3, 5-cyclic monophosphate, adenosine 3, 5-cyclic monothiophosphate, 8-bromo-adenosine cyclophosphate, specificity 5,6-4, 5-dicyanoimidazole-cyclic bismuth phosphate glycoside, specific 8-chlorobenzene-cyclic guanylate sodium, specific adenosine 3',5' -cyclic monosulfuric phosphate triethyl salt, specific adenosine cyclophosphate, butyryl-adenosine cyclophosphate, N6-monoacyladenosine 3',5' -cyclic monophosphate, 8-bromoadenosine 3',5' -cyclic monophosphate thioester, 8-bromoadenosine 3',5' -cyclic monophosphate, N6-benzoyl-adenosine cyclophosphate, erythro-9-amino-beta-hexyl-alpha-methyl-9H-purine-9-ethanol hydrochloride-9-adenine hydrochloride.

Preferably, the disease associated with the decrease of platelet number comprises immune thrombocytopenia, thrombocytopenia caused by infection, secondary thrombocytopenia, drug-induced thrombocytopenia, thrombocytopenia deficiency or non-immune thrombocytopenia.

Preferably, the phase of immune thrombocytopenia includes idiopathic thrombocytopenic purpura.

Preferably, the thrombocytopenia caused by infection includes bacterial or viral infection.

Preferably, the secondary thrombocytopenia-related disease includes thrombocytopenia in diabetic patients, thrombocytopenia in oncological patients, thrombocytopenia in cardio-cerebrovascular disease patients, thrombocytopenia resulting from drug therapy, splenic hyperactivity disease, thrombocytopenia during pregnancy, thrombocytopenia secondary to aplastic anemia, thrombocytopenia secondary to splenic hyperactivity, thrombocytopenia secondary to leukemia, thrombocytopenia secondary to systemic lupus erythematosus, thrombocytopenia secondary to sjogren's syndrome, or thrombocytopenia secondary to ionizing radiation.

Preferably, in the thrombocytopenia caused by the medicine, the medicine is one or more of antitumor drugs, quinine, quinidine, heparin, antibiotics and anticonvulsant drugs.

Preferably, the thrombocytopenia disease includes congenital thrombocytopenia, megakaryocytic thrombocytopenia, fanconi syndrome, Bernard-Soulier syndrome caused by deficiency or dysfunction of platelet membrane glycoprotein Ib-IX, Gray platelet syndrome, eczema thrombocytopenia with immunodeficiency syndrome (Wiskott-Aldrich syndrome), thrombocytopenia disease caused by aplastic anemia and myelodysplastic syndrome, acquired thrombocytopenia, thrombocytopenia disease caused by chemotherapy drugs or thrombocytopenia disease caused by radiation injury.

Preferably, the disease associated with a decrease in platelet count includes a disease caused by a decrease in platelet production, a disease caused by an increase in platelet destruction, or thrombotic thrombocytopenic purpura.

Preferably, the disease caused by thrombocytopenia includes chronic aplastic anemia, myelodysplastic syndrome, thrombocytopenia caused by radiotherapy or thrombocytopenia caused by chemotherapy; the diseases caused by the platelet destruction increase comprise the platelet destruction increase diseases caused by autoimmune diseases, the platelet destruction increase diseases caused by antiphospholipid syndrome, the platelet destruction increase diseases caused by human immunodeficiency virus or the platelet destruction increase diseases caused by drug thrombocytopenia.

Preferably, the medicament is tablets, capsules, granules, pills, sustained release preparations, controlled release preparations, oral liquid or patches.

Preferably, the medicament comprises a pharmaceutically effective dose of a protein kinase A activator and a pharmaceutically acceptable carrier.

Preferably, the medicament is administered orally, by injection, by inhalation spray or through the gastrointestinal tract.

Has the advantages that: the research of the invention finds that PKA is positioned in the early regulation stage of starting or inhibiting the platelet apoptosis induced by pathophysiological conditions. PKA inhibits platelet apoptosis by phosphorylating serine residue at position 155 of Bad pro-apoptotic protein to enhance binding to 14-3-3, thereby promoting release of anti-apoptotic protein Bcl-xL. Therefore, the research results prove that various in vivo and in vitro pathophysiology factors can induce platelet apoptosis, PKA is positioned at the upstream of apoptosis regulation, and the platelet apoptosis induced by storage or pathological stimulation can be remarkably protected by improving the activity of PKA.

Drawings

FIG. 1 shows the results of assays for phosphorylated GPIb β, GPIb β total protein and PKA activity in platelets from ITP patients, diabetic patients and septic patients;

FIG. 2 shows the results of platelet detection GPIb β phosphorylated protein, GPIb β total protein and PKA activity assays after bacterial infection;

FIG. 3 is a graph of the results of a percentage test of platelets in which protein kinase A inhibition causes depolarization of the mitochondrial transmembrane potential of platelet apoptosis;

FIG. 4 shows the results of western blot assay for detecting caspase-3, gelsolin protein expression and caspase-3 activity in platelets, which results from inhibition of protein kinase A to cause platelet apoptosis;

FIG. 5 shows the result of PS valgus assay for inhibition of protein kinase A resulting in apoptosis of platelets after incubation of washed platelets with varying concentrations of H89;

FIG. 6 is a platelet scatter plot of FSC-FL1 collection with protein kinase A inhibition leading to platelet apoptosis;

FIG. 7 shows SEM results of different concentration gradients of protein kinase A inhibitor H89 after washing platelets for 160 minutes at 22 ℃;

FIG. 8 is a graph showing the results of experiments relating to PKA regulation of platelet apoptosis through the regulation of serine phosphorylation at the Bad 155 site;

FIG. 9 is a graph of the results of platelet and reticulocyte assays in male ICR mice counted 0-8 days after injection of PKA agonist 8-Br-cAMP (2.5 mg/mL);

FIG. 10 shows the construction process and the related test results of conditional knockout mice;

FIG. 11 is a graph showing the results associated with an increased rate of platelet clearance in PKA knockout mice;

FIG. 12 is a graph correlating the percentage of mitochondrial transmembrane potential depolarized platelets and PS positive platelets;

FIG. 13 is a result of platelet Δ ψ M after incubation of washed platelets with the protein kinase A activator drug milrinone (8 μ M), a negative control, thrombin;

FIG. 14 is a result of platelet Δ ψ m after incubation of washed platelets with the protein kinase A activator drug aminophylline (0.48mM), negative control, thrombin;

FIG. 15 shows the results of platelets Δ ψ m after incubation of washed platelets with prostaglandin E2 solution (10ng/ml) sterilized with protein kinase A activator drug, negative control, and thrombin;

FIG. 16 shows the results of platelets Δ ψ m after incubation of washed platelets with an injection of protein kinase A activator drug adenosine cyclophosphate (24 μ g/mL), a negative control, and thrombin;

FIG. 17 shows the results of platelet counts at various times following injection of the protein kinase A activator drug Milrinone (1mg/kg) (or NS) into the tail vein of mice;

FIG. 18 shows the results of platelet counts at various times after intravenous injection of protein kinase A activator drug PGE2(20ng/ml) (or NS) into the tail of mice;

FIG. 19 shows the results of platelet counts at various times after intravenous injection of protein kinase A activator drug cAMP (12. mu.g/ml) (or NS) into the tail of mice;

FIG. 20 shows the results of platelet counts at various times after intravenous injection of the protein kinase A activator drug aminophylline (0.24mmol/L) (or NS) into the tail of mice.

Detailed Description

1. Reagents and materials:

monoclonal antibody SZ21 against GpIIb/IIIa was provided by professor Rankine, Chang Gunn, national institute of hematology, Jiangsu province, dimethyl sulfoxide (DMSO), anti-Actin primary antibody was purchased from American Sigma, EDTA-K2 anticoagulant tube was purchased from American BD, Fluorescein Isothiocyanate (FITC) -Annexin V was purchased from Beijing Gm Mei science Co., Ltd, FITC-sheep anti-mouse antibody was purchased from American Bioworld Technology, Horse radiation Peroxidase, HRP) -sheep anti-mouse, HRP-sheep anti-rabbit, rabbit and mouse IgG, anti-BAX, anti-BAK, anti-Bcl-xL, anti-Bcl-2, anti-BAD-155 antibody was purchased from American SanCruz science Co., anti-PKA C alpha antibody was purchased from American CST, N- [2- ((amino-2) pasyl) phosphorylation]5-isoquinonesulfonamide (H89), forskolin (Forsklin), anti-GAPDH, anti-P53 antibody, JC-1, ECL, PMSF were purchased from Biyuntian Biotech, Inc., China, E64 from Roche Biotech, Inc., USA, and A23187 from Calbiochem, USA. RNA oligonucleotides were designed and synthesized by Gima corporation. Liposome Lipofectamine^TM2000 and Medium Opti-Mem I were purchased from Invitrogen, USA.

2. Experimental mice:

PKA knockout mice (B6; 129X1-Prkacatm1Gsm/Mmnc) were purchased from U.S. MMRRC UNC in the background of C57 BL/6J. All animal experiments were approved by the ethical committee of the first hospital affiliated with suzhou university.

3. Washing the blood platelets:

healthy adult volunteers collected blood from the median elbow vein and blood donors gave informed consent and signed a protocol. The protocol was approved by the ethical committee of the first hospital affiliated suzhou university, in compliance with the declaration of helsinki.

To prepare washed platelets, healthy volunteer venous blood was taken and mixed with ACD (2.5% sodium citrate, 2.0% glucose, 1.5% citric acid) at a ratio of 1: 7 anticoagulated, centrifuged at 1300rpm for 20min to obtain Platelet Rich Plasma (PRP), PRP was centrifuged at 1500g for 2min, and the supernatant was discarded. The precipitated platelets were suspended and centrifuged with CGS buffer (0.123M sodium chloride, 0.033M glucose, 0.013M sodium citrate, pH 6.5), and the precipitated platelets were washed, then resuspended with modified Tyrode's buffer (2.5mM zwitterionic buffer Hepes, 150mM sodium chloride, 2.5mM potassium chloride, 1mM calcium chloride, 1mM magnesium chloride, 12mM sodium bicarbonate, 5.5mM glucose, pH 7.4) to give a washed platelet suspension, the platelets were counted with a counter, the platelet suspension was adjusted to a concentration of 3X 108/mL, and left to stand at room temperature for 60 min.

4. Electron microscope:

washed platelets were fixed with 2.5% glutaraldehyde at 4 ℃ overnight. And (5) sending the sample to a sample room of a scanning electron microscope for sample preparation. The morphology of platelets was analyzed by scanning electron microscopy (Hitachi, Japan, S-4700). Each of 5 different fields of view was selected for observation and photographed.

5. Whole body irradiation and bone marrow transplantation:

male WT mice (6 weeks old) received total body irradiation at a dose of 9.5Gy from Co 60. Collecting fetal liver cells from a PKA gene heterozygote pregnant mouse (about 15 days of pregnancy), injecting according to the proportion that 1 fetal liver cell corresponds to one irradiated male mouse (completed within 6 hours after irradiation), putting into an IVC special animal room, adding acidified water, Co60 irradiated feed and padding, and observing the survival condition every day; the surviving mice were assayed for whole blood cell count after 4 weeks and used for the next experiment if normal. Whether the transplantation was successful was determined by Western Blotting testing the expression of PKA protein in the recipient mouse platelets.

6. Mitochondrial membrane potential (Δ ψ m) detection

Platelets (3X 108/mL) were washed with different concentrations of H89 (12.5. mu.M, 25. mu.M, 37.5. mu.M and 50. mu.M) or negative control (DMSO) for 10min at room temperature, after which the platelets. delta. psi.m were determined using the lipophilic cationic dye JC-1. JC-1 with final concentration of 2 mug/ml is added into the treated blood platelets, incubation is carried out for 20min at 37 ℃ in the dark, and detection is carried out by a flow cytometer. Red fluorescence indicates a mitochondrial membrane potential-dependent JC-1 polymer, and green fluorescence indicates a JC-1 monomer unbound to membrane potential after depolarization of mitochondrial membrane potential. JC-1 monomer (. lamda. ex 514nm,. lamda. em 529nm) and polymer (. lamda. ex 585nm,. lamda. em 590nm) were determined by calculating the ratio of flow red fluorescence (JC-1 polymer) or green fluorescence (JC-1 monomer).

7. PS eversion

Washed platelets were incubated with different concentrations of H89 (12.5. mu.M, 25. mu.M, 37.5. mu.M and 50. mu.M) or negative control (DMSO) at room temperature for 10 min. Then, the Annexin V buffer solution, the platelet treated by H89 and the Annexin V-FITC are incubated for 15min at room temperature in a dark place according to the proportion of 50:10:1, and the detection is carried out by a flow cytometer.

8. Platelet shrinkage test

Washed platelets were incubated with different concentrations of H89 (12.5. mu.M, 25. mu.M, 37.5. mu.M and 50. mu.M) or negative control (DMSO) at room temperature for 10 min. The platelets were then co-incubated with SZ21 antibody for 30min at room temperature. Centrifugation, resuspension of platelets with goat anti-mouse antibody containing FITC label, and incubation in the dark at room temperature for 30 min. Platelets were collected by flow cytometry and a platelet scattergram collected by FSC-FL1 was used to analyze platelets. Shrinkage of platelets the degree of shrinkage was evaluated by analyzing changes in FSC and evaluating the degree of FSC reduction in GPIIb/IIIa positive cells. A23187 served as a positive control. DMSO served as a negative control.

9、Western Blotting：

Washed platelets were incubated with different concentrations of H89 (25. mu.M, 50. mu.M, 100. mu.M) or negative control (DMSO) for 10min at room temperature. The reaction was stopped by adding 2X cell lysate (containing 2mM PMSF, 2mM NaF, 2mM Na3VO4, and protease inhibitor), and the sample was prepared by cleavage on ice. The samples were tested for expression of the corresponding protein by immunoblotting.

10. RNA interference assay

Double-stranded siRNA oligonucleotides (sense: 5-GCUCCCUUCAUACCAAAGUTT-3, antisense: 5-ACUUUGGUAUGAAGGGAGCTT-3) and negative control siRNA (sense: 5-UUCUCCGAACGUGUCACGUTT-3, antisense: 5-ACGUGACACGUUCGGAGAATT-3) of the target PRKACA were designed and synthesized by Gima corporation.

When the Hela cells are transfected, 2 multiplied by 105 Hela cells are inoculated in a culture plate one day before transfection, and about 500 mu L of antibiotic-free culture medium is added into each hole, so that the cell density during transfection can reach 30-50%; mu.L/well of Lipofectamine 2000 (gently shaken before use) was taken and diluted with 50. mu.L of Opti-MEM I low serum medium. Gently mixing and incubating at room temperature for 5 min; diluting 2. mu.L FAM-siRNA with 50. mu.L Opti-MEM I low serum medium, and mixing gently; incubating the diluted Lipofectamine 2000 for 5min, gently mixing the incubated Lipofectamine 2000 with diluted FAM-siRNA, and standing the mixture at room temperature for 20 min; the FAM-siRNA-transfection reagent mixture was added to wells containing cells and culture medium (approximately 400. mu.L), and the wells were gently shaken to mix.

For platelet transfection experiments, platelets were washed 6X 108/mL aseptically and allowed to stand. 100 μ L of siRNA oligonucleotide was added to 100 μ L of platelets suspended in serum-free M199 medium.

Culturing in a CO2 incubator at 37 deg.C, and after 6 hr, changing the culture medium to complete culture medium containing serum for 48 hr; the transfection efficiency was determined by flow cytometry 6 hours after transfection. At the end of the culture, hela cells and platelets were collected and lysed. The expression degree of PKA C alpha of the sample is detected by a western blotting method, and Actin is used for internal reference detection.

11. Statistical analysis:

all data are derived from at least 3 mutually independent experiments, the data are expressed by mean +/-standard error, the data are subjected to statistical analysis by Prism Version 5.0, the data are subjected to non-pairing T test, and p is less than 0.05 and is used as a difference significance threshold value.

12. The experimental results are as follows:

(1) the PKA activity is reduced when blood plasma incubated or bacterially infected platelets are used for septicemia, diabetes or ITP patients

Thrombocytopenia is often found in some high-incidence diseases, such as diabetes, ITP, sepsis or bacterial infections, among others. We investigated the role of PKA in these diseases in which thrombocytopenia occurs. Platelets from patients with ITP, diabetes and sepsis were collected by centrifugation and controlled to healthy population platelets of the corresponding age and sex, were resuspended in MTB and adjusted to a concentration of 3 × 108/mL, and total protein from platelet lysis was used to detect phosphorylated GPIb β, total protein of GPIb β and PKA activity, P <0.05 and P <0.01 compared to the control. Washed platelets diluted with MTB 1X 107/mL were co-incubated with the corresponding bacteria (diluted 1:20 in MTB buffer) at 37 ℃ for 90 minutes, while the bacteria-free culture group was used as a negative control to detect GPIb β phosphorylated protein, GPIb β total protein and PKA activity, and the results were from four independent experiments with different platelet donors, as shown in FIGS. 1 and 2,. P <0.05,. P < 0.01.

We have found that platelets from healthy humans induce apoptosis in normal platelets following incubation with plasma from diabetic or ITP patients, and that PKA activity is significantly reduced. Recent evidence suggests that isolated escherichia coli and staphylococcus aureus in sepsis patients can induce platelet apoptosis in vitro (Kraemer et al, 2012). We found that E.coli and Staphylococcus could not only induce platelet apoptosis but also significantly reduce PKA activity. Taken together, these results indicate that ITP, sepsis and diabetes patients or bacterial infections can induce platelet apoptosis with reduced PKA activity.

(2) Inhibition of PKA activity induces pathway-dependent apoptosis in thrombopoiesis

Flow cytometry examined mitochondrial transmembrane potential depolarization and PS exposure of platelets by washing the platelets with varying concentrations of H89(0, 12.5, 25, 37.5, and 50 μ M) at 22 ℃ for 160 minutes. The results of the experiment were repeated four times. The results are shown in FIGS. 3 to 7. The washed platelets were pretreated with different concentration gradients of H89 at 22 ℃ for 30 minutes, and DMSO and A23187 treated negative and positive control platelets were simultaneously established, and western blots were used to detect caspase-3, gelsolin protein expression and caspase-3 activity in platelets. The FITC labeled anti-CD 41 antibody and the pretreated platelets are mixed uniformly in a ratio of 1:10, incubated for 10 minutes at normal temperature in the dark, and analyzed by the decrease of the number of positive CD41 in the platelet size scatter diagram, the decrease of the number of platelets is shown. Different concentration gradients of H89 were used to wash platelets for 160 minutes at 22 ℃ while DMSO negative control platelets were established. After the platelets were fixed for 30 minutes with 1% glutaraldehyde, the results were observed by scanning electron microscopy imaging from three independent experiments with a scale of 1 μm. Results are expressed as mean ± sd, P <0.05 compared to control, and results were repeated three more times.

Next we explored the role of PKA in the regulation of platelet apoptosis. Platelets were incubated with PKA inhibitor H89 and flow cytometry detected JC-1 dye marker changes within platelets, and H89 was found to dose-dependently induce depolarization of the mitochondrial membrane potential (Δ Ψ m) found by platelets. Furthermore, H89 can also induce Δ ψ m depolarization of platelets time-dependently. Δ Ψ m depolarization lies upstream of the caspase-3 signaling pathway, whereas caspase-3 is one of the executives of the caspase family, leading to cell disassembly and collapse. We found that the H89 dose-dependent manner induced caspase-3 activation and cleavage of the caspase-3 substrate, gelsolin. Phosphatidylserine (PS) eversion is another obvious marker molecule for intrinsic pathway-dependent apoptosis, and we found that H89 can induce PS eversion on the surface of platelets in a dose-dependent manner.

During apoptosis, mitochondrial dysfunction triggers bioenergetic destruction, ultimately leading to disruption of plasma membrane integrity and resulting morphological changes. The experimental result shows that H89 can induce GPIIb/IIIa positive platelets to reduce Forward Scattering (FSC), which indicates that platelets shrink morphologically after PKA activity is inhibited. In addition, H89 dose-dependent induced platelets exhibited typical apoptotic morphological changes including cell membrane vesicles, pseudopodia, shrinkage, degranulation, and the like. Taken together, these results indicate that PKA inhibition can induce intrinsic pathway-dependent apoptosis of thrombopoiesis.

(3) PKA regulates platelet apoptosis by regulating BAD 155 site phosphorylation

Next we explored further the mechanism by which PKA inhibition induces platelet apoptosis. At normal temperature, 3X 108/mL of washed platelets are incubated with H89 or DMSO internal reference with different concentration gradients for 160 minutes, the platelets are lysed on ice by using an equal volume of lysate for 30 minutes, protein fragments with different sizes are obtained by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic separation of products, primary antibody is added for incubation after skim milk powder is sealed for one hour, and finally ECL (electron cyclotron resonance) luminescence shows a target protein band (figure 8 a). The washed platelets were pretreated with 37.5uM H89, 10uM forskin and DMSO for 160 minutes at room temperature, cytoplasmic protein and mitochondrial protein of the platelets were extracted, western blot was used to detect the target protein, Image J software was used to analyze the amount of the target protein, and the results were shown as mean. + -. standard deviation in four experiments (FIG. 8 b). After lysis of the pretreated platelets, centrifugation was carried out at 17,000G for 10min at 4 ℃, the resulting supernatants were incubated with the corresponding antibodies overnight, incubated with protein a/G + agarose beads for 2h at 4 ℃, the beads were eluted and used for protein hybridization (fig. 8c), and statistical analysis of the four experiments showed results as mean ± standard deviation, # P <0.05, # P < 0.01.

As a result, PKA inhibition dose-dependently reduced the phosphorylation of serine at the GPIb beta 166 site of platelet, thereby demonstrating the reduction of PKA activity. However, we found that there was no significant change in the expression levels of the apoptosis-inducing proteins BAK, BAX and the anti-apoptotic proteins Bcl-2, Bcl-xL in the apoptotic platelets. It has been reported that in S49 lymphoma cells, PKA increases the expression of Bim by regulation, which protects tumor cells from apoptosis. However, the research finds that the protein level of Bim does not change obviously in the process of platelet apoptosis regulated by PKA, and the regulation effect of Bim is eliminated.

The report proves that the PKA inhibition can promote the expression of P53, and the phosphorylated P53 in the blood platelet of the diabetic patient can directly induce the inactivation of the anti-apoptotic protein Bcl-xL, thereby promoting the blood platelet to generate apoptosis. However, no changes were detected in either P53 or phosphorylated P53 in platelet apoptosis in which PKA was involved.

In nucleated cells, PKA regulates apoptosis by regulating phosphorylation of serine at BAD 155 site and regulating binding of 14-3-3 protein and anti-apoptotic protein Bcl-xL. Under the condition of dephosphorylation of BAD 155 locus, BAD and Bcl-xL form a dimer to release BAK and BAX of apoptosis performers, thereby leading to enhancement of mitochondrial membrane permeability and apoptosis. We found that the BAD 155 site serine exhibits a reduced degree of phosphorylation in platelets induced by PKA inhibition. Furthermore, H89 decreases and forskolin enhances phosphorylation of serine at BAD 155 site, thereby affecting decrease and increase of Bcl-xL protein on mitochondrial membrane. Most importantly, the co-immunoprecipitation result shows that Bcl-xL and BAD are obviously reduced or enhanced under the stimulation of H89 or forskolin, which indicates that H89 or forskolin regulates the activity of the anti-apoptotic protein Bcl-xL by regulating the interaction of BAD and Bcl-xL. Thus, these data indicate that PKA regulates apoptosis of platelets by regulating phosphorylation of serine at the BAD 155 site.

(4) PKA activators increase circulating platelet counts

On the other hand, PKA activation can promote the phosphorylation level of BAD 155 serine site, thereby preventing the occurrence of apoptosis. Thus, PKA activators may prevent apoptosis of aging platelets and prolong platelet life. To test this hypothesis, male ICR mice were injected with PKA agonist 8-Br-cAMP (2.5mg/mL) every 24 hours via tail vein, PBS group was set as negative control, platelets and reticulocytes in mice were counted after 8 days, and 5-6 mice P <0.05, P <0.01 were set in experimental and control groups, respectively (fig. 9).

As a result, the platelet count was found to increase day by day, reaching a peak on day 8. The corresponding decrease in reticulocytes from day to day indicates that the increase in platelets is not a result of increased platelet production (fig. 9). Furthermore, there was no significant change in JC-1 marker platelets. These data indicate that increased PKA activity protects platelets from apoptosis and can extend platelet life in vivo.

(6) Increased platelet clearance rates in PKA knockout mice

First was the construction of conditional knockout mice (FIG. 10 a). Western blot was used to detect the expression of PKA, Bad, GBIb β, phosphorylated Bad Ser-155 and phosphorylated GBIb β Ser-166 in platelets, with at least 5 mice per experimental group (FIG. 10 b). The Sysmex XP-100 hematology analyzer counted platelets and resulted in statistical analysis of 7 WT mice, 7 PKA +/-mice and 5 PKA-/-mice (FIG. 10 c). Whole blood was labeled with thiazole orange (0.5. mu.g/mL) and anti-CD 41 (20. mu.g/mL) antibody, incubated at room temperature for 15min, and the number of reticulocytes was detected by flow cytometry. Data were from 7 WT mice, 7 PKA +/-mice and 5 PKA-/-mice (FIG. 10 d). Anti-platelet antibody R300 (0.15. mu.g/kg) was injected intraperitoneally into WT and PKA +/-mice, whole blood was collected by orbital bleeding, and platelet counts were counted by Sysmex XP-100 hematology analyzer, resulting in statistical analysis of 6 WT and 6 PKA +/-mice, respectively (FIG. 10 e). Washed platelets were incubated with JC-1 (2. mu.g/mL) protected from light for 10 minutes and the level of mitochondrial transmembrane potential depolarization was examined by flow cytometry (FIG. 11 a). FITC-labeled anti-CD 41 antibody was mixed with platelets at a ratio of 1:10, gently mixed and incubated at room temperature for 10 minutes (FIG. 11 b). Analysis of the platelet size scatter plot, a decrease in the number of CD41 positive cells represents a decrease in platelet number. After washing the platelets for 30 minutes with 1% glutaraldehyde fixation, the results were observed by scanning electron microscopy, with a scale of 2 μm (fig. 11 c). P <0.05, P <0.01, the results from three independent experiments, the graph represents at least five mice per genotype.

Most of the PKA C alpha gene knockout mice die in perinatal period, so we established a bone marrow transplantation method for transplanting liver cells of fetuses of the PKA C alpha gene knockout mice into irradiated wild mice. The genotype of the transplanted fetal liver of the fetal mouse is identified by PCR detection. Stable transplantation results were obtained 4 months after transplantation of mice, and platelets with PKA deficiency after transplantation were confirmed by western blotting. There were no significant differences in red blood cell, white blood cell counts and hemoglobin concentrations, in the transplanted PKA-/-, PKA +/-, and WT mice. Whereas PKA +/-the number of platelets was significantly reduced compared to WT mice. PKA +/-is not different from the WT reticulocytes, indicating that the decrease in platelet number is not due to decreased platelet production, but to accelerated platelet clearance. Furthermore, we have found that injection of the anti-platelet antibody R300 induces apoptosis of platelets while rapidly inducing platelet clearance faster than PKA +/-WT. Interestingly, there was a significant reduction in platelets in PKA-/-mice, and platelets in PKA-/-mice exhibited typical apoptotic changes.

To avoid possible interference of bone marrow transplantation with PKA C α knockout mice, we constructed PKA C α conditional knockout mice, which were co-bred with PF4Cre mice to give in-platelet PKA conditional knockout mice. The C-PKA-/-, C-PKA +/-, and C-PKA +/+ mice that were conditional knockouts did not show significant differences in red blood cell, white blood cell count, hemoglobin concentration changes. Furthermore, PKA +/-and RIP 3-/-mice did not have any tendency to bleed spontaneously or to thrombus. Heterozygous and homozygous mouse PKA activity exhibits dose-dependent variation. There were no obvious abnormalities in different types of rats in the number of circulating platelets. However, PS eversion was significantly increased in PKA knockout mice. Following biotin-labeled platelets in vivo, PKA knockdown can dose-dependently reduce platelet life. To confirm that the shortened platelet life comes from platelet intrinsic factors, we transplanted platelets from PKA knockout mice into wild-type mice. PKA-/-, PKA +/-mouse platelets clearly show a shortened lifespan compared to WT mouse platelets.

(7) The phenomenon of reduced BAD phosphorylation in PKA knockout mice

We then explored the mechanism of platelet apoptosis in PKA knockout mice. Consistent with our previous results in vitro in human platelets, the expression dose gradient of P53 was increased in both heterozygous and homozygous mice, while PKA catalytic subunit expression and PKA activity were significantly reduced. Furthermore, phosphorylation of serine at the BAD 155 site of the PKA knockout mouse platelets is reduced. However, there was no significant change in BAK, BAD, BAX, Bcl-xL for PKA C α knockout platelets compared to wild type platelets. Taken together, these mouse results confirm our findings in human platelets, demonstrating that inhibition of PKA leads to the development of BAD 155 site serine phosphorylation-mediated platelet apoptosis.

(8) Increasing PKA activity protects platelets from pathological condition-induced apoptosis and clearance

The washed platelets were pretreated with 5uM forskin and DMSO, respectively, at 22 ℃ for 5 minutes, then co-incubated with serum from ITP patients for 12 hours at room temperature, while a healthy adult serum was set as a control and the flow cytometer was used to measure the percentage of mitochondrial transmembrane potential depolarized platelets (FIG. 12a) and PS positive platelets (FIG. 12 b). ICR mice intravenous injection single dose of 8-Br-cAMP (0.0625, 1.25, 2.5mg/kg) and internal reference, and intraperitoneal injection of Fc inhibitor to remove Fc segment mediated platelet clearance, 10 minutes later, antiplatelet antibody R300(0.2 ug/kg) through intraperitoneal injection into mice, in the different time points of orbital vein collection of mice whole blood, Sysmex XP-100 blood analyzer counting platelet amount, each group of 6 mice, the results with mean + -standard deviation (figure 12 c). The washed platelets were pretreated with 5uM forskin and DMSO at 22 ℃ for 5 minutes, co-cultured with staphylococcus aureus suspension at room temperature for 90 minutes, and meanwhile, the flow cytometer was used to detect the percentage of mitochondrial transmembrane potential depolarized platelets (fig. 12d) and PS positive platelets (fig. 12e) with negative control group platelets treated with no staphylococcus aureus liquid. The experiment was repeated three more times and the results are expressed as mean ± standard deviation. The washed platelets were pretreated with 5uM forskin and DMSO (DMSO) at 22 ℃ for 5 minutes, and then co-incubated with diabetic serum for 12 hours at room temperature, while a healthy adult serum was set as a control, and the flow cytometer was used to detect the percentage of mitochondrial transmembrane potential depolarized platelets (FIG. 12a) and PS positive platelets (FIG. 12 b).

Platelet apoptosis appears to be the major cause of dysfunction and rapid clearance of stored platelets. To ascertain the effect of PKA on the impairment of platelet storage, PKA activators or inhibitors were added during platelet storage. As expected, PKA inhibitors first trigger platelet apoptosis. Interestingly, the PKA activator Forskolin significantly delayed the onset of platelet apoptosis. It is well known that intrinsic apoptosis regulated by mitochondrial membrane potential depolarization is an irreversible process. These data not only further demonstrate the role of PKA in modulating platelet apoptosis, but also indicate that PKA is located upstream of mitochondrial depolarization in modulating apoptosis.

In addition to apoptosis, there are other storage impairment changes that result in the clearance of stored platelets in vivo. Therefore, we investigated whether PKA activation protects platelets from being cleared from storage while at the same time protecting them from apoptosis. It was found that Forsklin clearly protected stored platelets from in vivo clearance, while H89 accelerated the rate of clearance. These data indicate that PKA-regulated apoptosis plays a key role in platelet storage injury and suggest a new approach to effectively prolong platelet storage in blood banks.

Autoantibodies in ITP, particularly in refractory ITP patients, induce platelet apoptosis, leading to platelet destruction. Consistent with previous reports, we found that anti-platelet serum from ITP patients significantly induced apoptosis of platelets. However, platelets after Forskolin preincubation significantly reduced the incidence of serum-induced platelet apoptosis. To clarify the role of PKA activators in vivo platelet clearance, we established an ITP mouse model with the anti-platelet monoclonal mixed antibody R300. In vitro co-incubation of R300 with platelets induces platelet apoptosis. Injection of Fc receptor blocking agents into mice blocks Fc-dependent platelet destruction. PKA activators can dose-dependently inhibit antiplatelet antibody-induced platelet clearance. These results, not only confirm the role of PKA in protecting antibodies from inducing platelet apoptosis and clearance, but also suggest a new strategy for treating ITP.

We then found that PKA activation was effective in preventing apoptosis induced following incubation of staphylococcus aureus isolates in sepsis patients and plasma and platelets in diabetic patients. Taken together, these data indicate that PKA is an early regulatory protein of platelet apoptosis and, most importantly, that the results of this study are of great significance for the treatment of thrombocytopenia induced by different pathophysiological stimuli and for controlling the life of platelets in vivo.

(9) Experiment and result for inhibiting platelet apoptosis by same type of drugs

Mitochondrial membrane potential detection:

platelets were washed (3X 108/mL) with different PKA agonists (aminophylline 0.48mM, sterile prostaglandin E2 solution 10ng/mL, milrinone 8. mu.M, adenosine cyclophosphate injection 24. mu.g/mL) or negative controls (saline) for 10min at room temperature, after which thrombin was added to each group at 0.1U/mL except negative controls and incubated for 30min at 37 ℃. The platelets Δ ψ m were determined using the lipophilic cationic dye JC-1. JC-1 with final concentration of 2 mug/ml is added into the treated blood platelets, incubation is carried out for 5min at 37 ℃ in the dark, and detection is carried out by a flow cytometer. Red fluorescence indicates a mitochondrial membrane potential-dependent JC-1 polymer, and green fluorescence indicates a JC-1 monomer unbound to membrane potential after depolarization of mitochondrial membrane potential. JC-1 monomer (. lamda. ex 514nm,. lamda. em 529nm) and polymer (. lamda. ex 585nm,. lamda. em 590nm) were determined by calculating the ratio of flow red fluorescence (JC-1 polymer) or green fluorescence (JC-1 monomer) (FIGS. 13 to 16).

PS eversion:

platelets were washed (3X 108/mL) with different PKA agonists (aminophylline 0.48mM, sterile prostaglandin E2 solution 10ng/mL, milrinone 8. mu.M, adenosine cyclophosphate injection 24. mu.g/mL) or negative controls (saline) for 10min at room temperature, after which thrombin was added to each group at 0.1U/mL except negative controls and incubated for 30min at 37 ℃. Then, Annexin V buffer solution, the treated platelets and Annexin V-FITC are incubated for 15min at room temperature in a ratio of 50:10:1 in the absence of light, and detected by a flow cytometer (FIGS. 13 to 16).

Milrinone can inhibit platelet clearance in vivo

ICR mice were 12, 6 each per group. The test group has 6 milrinone at 1mg/kg, and the control group has 6 Normal Saline (NS). After the mouse tail is injected with milrinone 1mg/kg 10min (or NS), the mouse is injected with R300 antibody 0.1mg/kg in the abdominal cavity. Blood was then counted at each time point. From the results, we can see that 1mg/kg milrinone significantly increased the peripheral platelet count of mice (fig. 17).

PGE2 can inhibit platelet clearance in vivo

The mice were first given blood sampling as a baseline, then the control and test groups were injected with 0.9% NS and PGE2(20ng/ml), respectively, R300(0.1 μ g/g) after 10min, and then blood sampling counts were taken at 30min,2h,4h,6h, and 24h time points. At 30min, the platelet count P of the NS group and the PGE2 group was <0.05, with statistical differences (fig. 18).

cAMP can inhibit platelet clearance in vivo

The rats were first given blood sampling as a reference value, and then the control group and the test group were injected with 0.9% NS and cAMP (12. mu.g/ml), respectively, and R300 (0.1. mu.g/g) was injected after 10min, and then blood sampling counting was performed at 30min,2h,4h,6h, and 24h time points. At 30min, the platelet counts P <0.05 were statistically different between NS and cAMP groups (fig. 19).

Aminophylline can inhibit platelet clearance in vivo

The mice were first given blood sampling as baseline values, then the control and test groups were injected with 0.9% NS and Aminophylline (0.24mmol/L), respectively, and R300(0.1 μ g/g) was injected after 10min, and then blood sampling counts were taken at 30min,2h,4h,6h,24h time points (fig. 20).

Claims

1. Use of a protein kinase a activator for the preparation of a medicament for the treatment of a disease associated with a reduced platelet count, which is idiopathic thrombocytopenic purpura; the protein kinase A activator comprises one or more of aminophylline, sterile prostaglandin E2, milrinone or adenosine cyclophosphate; the medicine comprises a pharmaceutically effective dose of a protein kinase A activator and a pharmaceutically acceptable carrier.

2. The use of a protein kinase a activator in the preparation of a medicament for the treatment of a condition associated with a decreased platelet count according to claim 1, wherein the medicament is in the form of a tablet, capsule, granule, pill, sustained release formulation, controlled release formulation, oral liquid, or patch.

3. Use of a protein kinase a activator in the manufacture of a medicament for the treatment of a condition associated with a reduced platelet count according to claim 1, wherein said medicament is for oral, injectable, inhaled by nebulization or for administration via the gastrointestinal tract.