CN107047538B

CN107047538B - Application of protein kinase A activator in platelet preservation and platelet preservation method

Info

Publication number: CN107047538B
Application number: CN201710158221.5A
Authority: CN
Inventors: 戴克胜
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2021-04-13
Anticipated expiration: 2037-03-16
Also published as: CN107047538A

Abstract

The invention discloses an application of a protein kinase A activator in platelet preservation and a platelet preservation method. The embodiment of the invention can see that the invention discloses the application of the protein kinase A activator in inhibiting platelet apoptosis or maintaining platelet activity for the first time under the condition that the induction and regulation mechanism of platelet apoptosis is unknown, the protein kinase A activator can inhibit the function loss caused by platelet apoptosis and can protect platelets from apoptosis under the conditions of storage and pathological stress, thereby prolonging the survival time of the platelets. The method is a new strategy for improving the survival period of the stored platelets, and the result shows that the method determines the life and survival of the platelets by regulating apoptosis, has profound significance for prolonging the storage time of the platelets, improving the clinical application efficiency of the platelets and avoiding the rejection of the platelets in stock due to the inactivation of the platelets caused by the loss of the function of the platelets.

Description

Application of protein kinase A activator in platelet preservation and platelet preservation method

Technical Field

The invention belongs to the technical field of platelet preservation, and particularly relates to an application of a protein kinase A activator in platelet preservation and a platelet preservation method.

Background

Platelets are key factors in the regulation of thrombosis and pathological bleeding in the circulatory system, and play an important role in the pathophysiological processes of the body, such as immune response, infection, atherosclerosis formation, tumor metastasis and the like. However, platelets have a short life cycle and survive only 8-9 days in peripheral blood. The mechanism of platelet life shortening caused by thrombocytopenia is not completely clear, and the fatal bleeding caused by thrombocytopenia is often infected, immune thrombocytopenia, diabetes mellitus, some drug therapies and other common diseases. In addition, the short-lived platelets, especially storage lesions, limit the shelf life of clinical applications of platelets. Therefore, the research on the mechanism for regulating and controlling the platelet life and survival has important pathophysiological significance.

The damage caused by stored platelets severely limits the clinical use of platelets in thrombocytopenia, a well-recognized worldwide problem, apoptosis appears to be the leading cause of storage damage, and attempts have been made to mitigate platelet storage disorders by inhibiting caspases and related enzymes, but no substantial progress has been made to date. The research shows that apoptosis protein plays an important role in regulating platelet life and platelet survival, and the starting and inhibiting mechanisms of platelet apoptosis under physiological or pathological conditions are still not clear at present.

The blood platelet is one of the main components of blood, participates in the blood coagulation process of the organism, plays the normal hemostatic function, prevents the blood from losing after injury, so the blood platelet infusion is a common method in clinical treatment. Platelets must be stored as soon as possible after collection if they cannot be used in the clinic sooner. Therefore, the platelets must have a proper storage technology to save blood resources and store the platelets. However, since platelets have a short life and are susceptible to various factors in structure and function, they need to be stored reasonably and effectively before clinical application. The washing of the platelet can not only solve the problem of ineffective treatment of platelet transfusion of patients with plasma allergy, IgA deficiency, alloimmunity and the like, but also play a certain role in preventing non-hemolytic fever reaction and acute intravascular hemolytic reaction in the platelet transfusion, simultaneously concentrate the remaining leucocytes in the platelet product to compete with the platelet for nutrition in plasma, can accelerate the storage and damage of the platelet, and can directly generate harmful effects to cause alloimmunity and EB virus infection. At present, the temperature for storing the blood platelets at room temperature is 22 +/-2 ℃, but the storage time is about 3 days.

Disclosure of Invention

The invention aims to provide an application of a protein kinase A activator in platelet preservation and a platelet preservation method, so as to prolong the platelet preservation life.

The invention adopts the following technical scheme that the protein kinase A activator is applied to inhibiting platelet apoptosis or maintaining platelet activity.

The invention also discloses application of the protein kinase A activator in platelet preservation.

The invention also discloses application of the protein kinase A activator in prolonging the survival time of platelets.

The invention also discloses application of the protein kinase A activator in preparing an additive reagent for platelet preservation.

In the technical scheme, the blood platelets are stored at 4-15 ℃, preferably 10 ℃. The synergistic effect of the environment at 10 ℃ and the protein kinase A activator can further inhibit the activation of the apoptosis protein caspase-3 and up-regulate the expression of the anti-apoptosis protein Bcl-xl, and prolong the storage time of platelets.

In the technical scheme, the protein kinase A activator comprises a phosphodiesterase inhibitor, an adenylate cyclase activator, adenosine cyclophosphate and adenosine cyclophosphate derivatives; the phosphodiesterase inhibitor comprises a phosphodiesterase inorganic inhibitor and a phosphodiesterase organic inhibitor; the adenylate cyclase activator comprises an adenylate cyclase inorganic substance activator and an adenylate cyclase organic substance activator; the adenosine cyclophosphate derivative comprises a modifier taking adenosine cyclophosphate as a core component. The embodiment of the invention can show that the protein kinase A activator can reduce PS eversion and up-regulate the expression of anti-apoptotic protein Bcl-xl, which shows that the protein kinase A activator plays a role in inhibiting the apoptosis of platelets.

For example, the protein kinase A activator of the present invention is amrinone, milrinone, enoximone, aminophylline, dinoprostone, iloprost, cilostazol, cilostamide, dipyridamole, ginkgo biloba extract, quercetin, adenosine cyclophosphate, forskolin, 8-bromoadenosine-3 ',5' -cyclic monophosphate, 8-bromo-adenosine cyclophosphate, 8-piperidinyl adenosine cyclophosphate, 8-chloro-adenosine cyclophosphate, adenosine 3, 5-cyclic monophosphate, N6-benzoyl adenosine cyclophosphate, (S) -adenosine, cyclic 3',5' - (hydrogen thio phosphate) triethyl, 3-isobutyl-1-methylxanthine, 8-chlorobenzene adenosine cyclophosphate, adenosine adenylate 3, 5-cyclic monophosphate, adenosine 3, 5-cyclic monothiophosphate, 8-bromo-cyclic adenosine monophosphate, specific 5, 6-4, 5-dicyanoimidazole-cyclic bismuth phosphate glycoside, specific 8-chlorobenzene-cyclic guanosine sodium, specific adenosine 3',5' -cyclic monothiophosphate triethyl salt, specific cyclic adenosine monophosphate, dibutyryl-cyclic adenosine monophosphate, N6-monoacyladenosine 3',5' -cyclic monophosphate, 8-bromoadenosine 3',5' -cyclic thioester monophosphate, 8-bromoadenosine 3',5' -cyclic monophosphate, N6-benzoyl-cyclic adenosine monophosphate, erythro-9-amino-beta-hexyl-alpha-methyl-9H-purine-9-ethanol hydrochloride-9-adenine hydrochloride Seed growing; wherein forskolin (Forsklin), forskolin, is a natural diterpene product isolated from Indian plant Coleus forskohlii, and is an activator of eukaryotic Adenylate Cyclase (AC).

The invention also discloses a platelet preservation method, which comprises the following steps of adding the protein kinase A activator into the washed platelets and then preserving the platelets.

In the technical scheme, the blood platelet is stored at 4-15 ℃, preferably 10 ℃.

The invention further discloses a preparation method of the reagent for prolonging the storage time of the blood platelets, which is prepared from a protein kinase A activator. The invention also discloses an additive reagent for platelet preservation, which comprises a protein kinase A activator and can also comprise a dispersion medium, such as a buffer medium.

The invention discloses the application of a protein kinase A activator in inhibiting platelet apoptosis or maintaining platelet activity for the first time under the condition that the induction and regulation mechanism of platelet apoptosis is unknown, can inhibit phosphatidylserine eversion, up-regulate the expression of anti-apoptosis protein Bcl-xl, and can protect platelets from apoptosis under the conditions of storage and pathological stress, thereby prolonging the survival time of the platelets.

Drawings

FIG. 1 is a graph of the results of flow cytometry in detecting PS evagination of washed platelets after Forskolin and DMSO treatment;

FIG. 2 is a Western Blot method for detecting the expression of the platelet anti-apoptotic protein Bcl-xl after treatment by different methods;

FIG. 3 is a Western blot method for detecting the expression pattern of the anti-apoptotic proteins Bcl-xl of the washed platelets after treatment by the two methods;

FIG. 4 is a graph of the results of washing platelet Δ Ψ m depolarization;

FIG. 5 is a graph of PS eversion results for washed platelets;

FIG. 6 is a graph showing the results of flow cytometry analysis;

FIG. 7 is a photograph of electrophoresis of platelets after H89, forskin, and DMSO treatments;

FIG. 8 is an electropherogram of pretreated platelets after lysis;

FIG. 9 is a diagram showing the coexistence of BAD and Bcl-xL in mitochondrial binding;

FIG. 10 is a diagram showing the expression of BAD on mitochondria;

FIG. 11 is a graph of ristocetin-induced platelet aggregation;

FIG. 12 is a graph of collagen-induced platelet aggregation;

FIG. 13 is a diagram showing thrombus formation and fluorescence labeling of platelets in a thrombus.

Detailed Description

Reagents and materials for this example:

JC-1, Forskolin, calcium ionophore A23187 and anti-caspase-3 monoclonal antibody are products of Biyunnan company, and solvent dimethyl sulfoxide (DMSO) is a product of Sigma company in America. The Annexin V-FITC apoptosis detection kit is a product of Jiamei biology company. The phenylmethylsulfonyl fluoride (PMSF) is a product of America Amresco, the polyvinylidene fluoride membrane (PVDF membrane) is a product of America Bio-Rad, the rabbit anti-human Bcl-xl and beta-actin are products of America CST, the horseradish peroxidase-labeled goat anti-rabbit antibody is a product of America Santa Cruz, and the ECL is a product of America Advansta. Suffruter centrifuge types 1-16 (Sigma, USA); flow cytometer FC 500 (Beckman coulter, USA), vertical electrophoresis apparatus (Bio-Rad, USA).

Washing blood platelets, extracting venous blood of healthy blood donators without hemorrhagic diseases, and adding anticoagulant glucose sodium citrate (ACD 2.5% trisodium citrate, 2.0% glucose, 1.5% citric acid) according to a volume ratio of 7: 1. The anticoagulated whole blood was centrifuged at 100g (1100 r.p.m) for 11 minutes, and after centrifugation, the lower layer was red blood cells and the upper layer was platelet-rich plasma. Transfer the supernatant to a fresh centrifuge tube. Platelet rich plasma was centrifuged at 825 g (3000 r.p.m) for 2 minutes to pellet platelets and the upper layer was platelet poor plasma. After discarding the supernatant, the platelets were resuspended in an equal volume of CGS buffer (0.123 mol/L sodium chloride, 0.033 mol/L glucose, 0.013 mol/L sodium citrate, pH 6.5) to platelet-rich plasma and centrifuged at 270 g (2000 r.p.m.) for 2 minutes to wash out plasma proteins. The precipitated platelets were finally resuspended in a volume of modified Tyrode buffer (2.5 mmol/L Hepes, 150 mmol/L sodium chloride, 2.5 mmol/L potassium chloride, 12 mmol/L sodium bicarbonate, 5.5 mmol/L glucose, 1 mmol/L calcium chloride, 1 mmol/L magnesium chloride, pH 7.4) at a concentration of 3X 108/ml. The resuspended washed platelets were allowed to stand at room temperature for 1 hour to return to physiological state before use in subsequent experiments.

Flow cytometry was used to detect the depolarization of Δ Ψ m by aseptically preserving untreated washed platelets at 5 temperatures, 0 ℃, 4 ℃, 10 ℃, 22 ℃ and 37 ℃ for 5 days. Washed platelets (50. mu.L) stored at different temperatures and washed platelets (50. mu.L) treated with A23187 were added at room temperature at 1d, 2d, 3d, 4d and 5d, respectively, and then incubated in a mixture of JC-1 (2. mu.g/ml), DMSO and MTB at room temperature for 10min, and finally subjected to flow cytometry analysis.

Flow cytometry measurement of PS eversion, washing of platelets under sterile conditions was divided into 3 groups: adding Forskolin into an untreated group, (II) adding Forskolin into the untreated group, adding DMSO (0.5%) into the untreated group, storing three kinds of platelets at 5 temperatures of 0 ℃, 4 ℃, 10 ℃, 22 ℃, 37 ℃ and the like for 5 days, taking three kinds of platelets (5 mu L) respectively at 1d, 2d, 3d, 4d and 5d and adding washed platelets (5 mu L) treated by A23187 into the platelets at normal temperature, respectively adding Annexin V-FITC 5 mu L and 150 mu L of 1 Xloading Buffer into the platelets, uniformly mixing, incubating for 15 minutes in a dark place at room temperature, and adding 350 mu L of 1 Xloading Buffer before Loading. And finally, carrying out flow cytometry analysis.

Western Blot to detect Bcl-xl protein expression, washing platelets under aseptic conditions into 3 groups: adding Forskolin (I) into an untreated group, (II) adding Forskolin into the untreated group, adding DMSO (0.5%) into the untreated group, storing three kinds of platelets at 5 temperatures of 0 ℃, 4 ℃, 10 ℃, 22 ℃, 37 ℃ and the like for 5 days, taking three kinds of platelets (50 mu L) from 1d, 2d, 3d, 4d and 5d respectively and adding fresh washed platelets (50 mu L) treated by A23187 at normal temperature, adding isovolumetric lysate (containing PMSF) into ice for cracking for 30 min, centrifuging for 2 min at 3500 r/min, taking supernatant, adding 4x sample loading buffer solution and beta-mercaptoethanol into the supernatant, and boiling for 10 min. The sample was subjected to SDS-PAGE and transferred to a PVDF membrane. After 5% milk blocking, TBST membrane washing, and incubating at 4 ℃ overnight with anti-Bcl-xl monoclonal antibody (1: 1000 dilution) and anti-beta-actin monoclonal antibody (1: 1000 dilution). After washing the membrane with TBST, incubating the goat anti-rabbit antibody (diluted 1: 8000) marked by horseradish peroxidase for 1h at room temperature. And washing the membrane again by TBST and detecting Bcl-xl and beta-actin bands by adopting an ECL method.

Statistical analysis of the data, all experimental results were repeated at least 3 times. Statistical analysis is carried out on experimental data by using SPSS 16.0 software, the experimental data are expressed by mean +/-standard deviation, statistical analysis is carried out on comparison among different groups by using one-way ANOVA, and P <0.05 represents that statistical difference exists.

Examples

Under aseptic conditions, the washed platelets were divided into three groups, untreated, 5 μ M Forsklin added, 0.5% DMSO added; the three groups of platelets were divided equally, stored at 0 deg.C, 4 deg.C, 10 deg.C, 22 deg.C, 37 deg.C for 5 days, 5 μ L each day, and 5 μ L of washed platelets treated with A23187 at room temperature was added as a control.

FIG. 1 shows the results of flow cytometry to detect PS eversion after Forskolin and DMSO treatment of washed platelets stored for five days. The DMSO group is used as a control, the percent PS eversion is expressed as mean plus or minus standard deviation, the experiment is repeated for 5 times, and P is less than 0.05; compared with the platelets in a DMSO group, the PS eversion percentage of the washed platelets in the Forskolin group in the same time and storage environment is reduced, the PS eversion percentage is more obvious along with the time extension, and the Forskolin on the 5 th day has statistical significance on the PS eversion action difference of the washed platelets stored in various temperature environments; the results show that Forskolin has an inhibitory effect on apoptosis of washed platelets.

FIG. 2 shows the expression of the anti-apoptotic protein Bcl-xl in the Western Blot method after the storage of platelets for five days after treatment with different methods, wherein (-) in each figure represents fresh untreated washed platelets, A represents fresh washed platelets treated with A23187, and 0, 4, 10, 22 and 37 represent the corresponding storage temperatures (repeated 3 times), respectively; the different degrees of apoptosis exist in platelet washing at different temperatures, and Forskolin has an inhibiting effect on platelet PS eversion. The Western Blot result shows that the expression level of washed platelets Bcl-xl in the Forskolin group at the same time and temperature is increased compared with that in the control group, and the Forskolin has the function of up-regulating the expression of washed platelets BCL-xl.

FIG. 3 shows Western blot analysis of the expression of the anti-apoptotic proteins Bcl-xl after washing platelets treated by the two methods, (A) showing the change in expression of the anti-apoptotic proteins Bcl-xl for 5 consecutive days, and (B) showing the change in expression of the anti-apoptotic proteins Bcl-xl for washing platelets on day 1 and day 5 (repeated 3 times); it can be seen that the expression of the anti-apoptotic protein Bcl-xl is obviously reduced along with the time extension when the DMSO group washes the platelets for 5 days at 22 ℃, while the Forskolin group washes the platelets for 1 day without obvious reduction compared with the 5 day (FIG. 3B), which indicates that the Forskolin plays a role in promoting the anti-apoptosis of the washed platelets.

Platelet apoptosis is the main cause of dysfunction and rapid clearance of stored platelets, and intrinsic apoptosis regulated by mitochondrial membrane potential depolarization is an irreversible process. FIG. 4 is a graph of the results of washing platelet Δ Ψ m depolarization; FIG. 5 is a graph of PS eversion results for washed platelets; FIG. 6 is a graph showing the results of flow cytometry analysis; wherein 3 is multiplied by 10 in figures 4 and 5⁸The washed platelets/mL are pretreated with 25uM H89, 5uM forskin and DMSO at 22 ℃ for different time points, the JC-1 added in the graph 4 enables the final concentration to be 2 mug/mL, and the platelets are incubated for 10min in a dark place; FIG. 5 was incubated with 5 μ g/mL Lactadherin in dark for 30 min, and the differently treated platelets were examined by flow cytometry for the percentage of mitochondrial transmembrane potential depolarization (FIG. 4) and PS positive platelets (FIG. 5), the results being expressed as mean. + -. standard deviation, 3X 10 in FIG. 6⁸The method comprises the following steps of (1) incubating the washed platelets per mL for 72 hours at 22 ℃ by using 25uM H89, 5uM forskin and DMSO internal references respectively, marking the platelets, infusing the labeled platelets into a mouse body through a tail vein, collecting blood from an orbital vein, analyzing and detecting the labeled platelets by using a flow cytometer, and repeating the experiment for more than three times; the results show that the addition of Protein Kinase A (PKA) activator during platelet storage, according to the invention, the PKA activator significantly inhibited the occurrence of platelet apoptosis; 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.

FIG. 7 is a photograph of electrophoresis of platelets after H89, forskin, and DMSO treatments; washing platelets, pretreating the platelets with 37.5 uM H89, 10 uM forskin and DMSO (dimethylsulfoxide) at normal temperature for 160 minutes, respectively extracting cytoplasmic protein and mitochondrial protein of the platelets, detecting target protein by western blot, analyzing the amount of the target protein by Image J software, and counting four experiments to display results in terms of mean +/-standard deviation; FIG. 8 is an electrophoretogram of pretreated platelets after lysis, centrifuged at 17000G 4 ℃ for 10 minutes, the resulting supernatant was incubated with the corresponding antibody overnight, incubated with protein A/G + agarose beads at 4 ℃ for 2 hours, the beads were eluted and used for protein hybridization, and the results were shown as mean. + -. standard deviation for four statistical analyses with P <0.05 and P < 0.01; FIG. 9 is a diagram showing the coexistence of BAD and Bcl-xL in mitochondrial binding; FIG. 10 is a diagram showing the expression of BAD on mitochondria. Research shows that the PKA agonist forskolin can enhance the serine phosphorylation level of a Bad 155 site, regulate the binding of a 14-3-3 protein and an anti-apoptosis protein Bcl-xL and further regulate apoptosis. The co-immunoprecipitation result shows that the effect between Bcl-xL and BAD is obviously reduced or enhanced under the stimulation of H89 or Forsklin, and the combination between 14-3-3 and BAD is opposite to the effect; furthermore, BAD coexists with Bcl-xL in mitochondrial binding, H89 was able to increase and forskolin could decrease BAD expression on mitochondria, data indicating that PKA slowed apoptosis in platelets by modulating serine phosphorylation.

Human washed platelets (3X 10)⁸/mL) were pretreated with 5uM of forskin and DMSO of internal control at 22 ℃ for 96 hours, respectively, and platelet aggregation induced by ristocetin (fig. 11) and collagen (fig. 12) was detected using a platelet aggregometer. The results show that forskin is able to maintain platelet aggregation function. Mouse washed platelets (3X 10)⁸/mL) was incubated with 25uM H89, 5uM forskin and DMSO, respectively, at 22 ℃ for 96 hours, platelets were labeled and then infused back into the mice via the tail vein, the mesenteric vessels of the mice, and after FeCl3 injury, the presence of thrombus formation and fluorescently labeled platelets in the thrombus was recorded (FIG. 13), indicating that forskin can maintain platelet adhesion and thrombus formation.

The addition amount of Forsklin group is changed, such as 2uM and 8 uM; incubating for 72 hours at 22 ℃, marking the platelets, infusing the marked platelets into a mouse body through a tail vein, collecting whole blood by sampling blood from an orbital vein, analyzing and detecting the marked platelets by a flow cytometer, and repeating the experiment for more than three times; the result shows that the survival rate of the blood platelet is more than 70%; after incubation for 72 hours at 10 ℃, the survival rate reaches more than 82 percent, and the survival rate reaches more than 71 percent after 120 hours; the survival rate reaches more than 77 percent and 78 percent after incubation for 72 hours at the temperature of 4 ℃ and 15 ℃. Pretreating for 96 hours at 22 ℃, detecting the ristocetin and collagen-induced platelet aggregation by using a platelet aggregation instrument, wherein the results show that forskin can maintain the platelet aggregation function, the aggregation rate reaches 70%, and the incubation at 4 ℃ and 15 ℃ reaches 75%; pretreating at 10 ℃ for 96 hours and 140 hours, and detecting the platelet aggregation induced by ristomycin and collagen by using a platelet aggregation instrument, wherein the results show that forskin can maintain the platelet aggregation function, and the aggregation rates reach 81% and 68%.

The method comprises the steps of replacing forskin with amrinone and meglumine adenosine cyclophosphate for storing platelets, adding 5uM, incubating for 72 hours at 22 ℃, marking the platelets, infusing the marked platelets into a mouse body through a tail vein, collecting blood through orbital vein blood collection, analyzing and detecting the marked platelets by a flow cytometer, repeating the experiment for more than three times, and displaying that the survival rate of the platelets is more than 70%, incubating for 72 hours at 10 ℃, the survival rate reaches more than 82%, incubating for 72 hours at 15 ℃, and the survival rate reaches more than 75%.

The invention divides washed platelets into 3 groups: (I) untreated group, (II) adding protein kinase A activator such as Forskolin, (III) adding DMSO, and storing three kinds of platelets at 5 temperatures of 0 deg.C, 4 deg.C, 10 deg.C, 22 deg.C and 37 deg.C for 5 days. And detecting the relevant indexes of each sample for five consecutive days. Changes in mitochondrial membrane potential (Δ Ψ m) depolarization and Phosphatidylserine (PS) eversion were detected using flow cytometry. Western Blot was used to detect the expression of caspase-3 and Bcl-xl, indicators of apoptosis in washed platelets. The results show that: under the same time and temperature, the untreated group washed platelets are continuously stored at five temperatures for 5 days to generate mitochondrial membrane potential depolarization and phosphatidylserine eversion, and Forskolin can inhibit phosphatidylserine eversion; when only the storage temperature is different, the expression of the anti-apoptotic protein Bcl-x of the washed platelet stored at 10 ℃ is not obviously reduced, but the Forskolin up-regulates the expression of the anti-apoptotic protein Bcl-xl; the results show that Forskolin has the effects of inhibiting apoptosis on platelets, prolonging the time for storing the platelets, and can further inhibit the activation of the apoptotic protein caspase-3 and up-regulate the expression of the anti-apoptotic protein Bcl-xl under the synergistic effect with the environment at 10 ℃.

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. According to the technical scheme of the invention, the PKA activity of the stored platelet is improved, the endogenous programmed death of the platelet is slowed down and started, and the thrombocytopenia caused in vivo is avoided. More importantly, the invention not only can protect the stored platelets, but also can improve the platelet damage caused by various pathological stimuli in vivo and prolong the service life of the platelets; the results demonstrate that the present invention has a great effect on the stabilization of platelets under pathophysiological conditions. According to the technical scheme, the PKA is enhanced to be combined with 14-3-3 through phosphorylating the serine residue at the 155 site of the Bad apoptosis-promoting protein, so that the release of the anti-apoptosis protein Bcl-xL is promoted to inhibit the apoptosis of platelets, the degradation of the Bcl-xL in the platelets is inhibited, the apoptosis of stored platelets is prevented, the apoptosis of platelets in vitro and the acute thrombocytopenia in vivo are avoided, the platelets can be protected from apoptosis induced by various stimuli, and the number of platelets in peripheral blood can be increased by moving objects. The damage caused by the storage of platelets severely limits the clinical use of platelets in thrombocytopenia, a recognized worldwide problem. Apoptosis is the leading cause of storage damage, and attempts have been made to mitigate platelet storage pathology by inhibiting apoptotic proteins and related enzymes, but no substantial progress has been made to date. The technical scheme disclosed by the invention is a new strategy for improving the survival period of the stored platelets, and the result shows that the life and survival of the platelets are determined by regulating apoptosis, so that the method has profound significance for improving platelet storage and improving clinical application of the platelets.

Claims

1. The use of a protein kinase a activator in platelet preservation; the preservation temperature of the platelets is 10 ℃; the protein kinase A activator is adenylate cyclase activator Forskolin.

2. The application of a protein kinase A activator in preparing an additive reagent for platelet preservation; the preservation temperature of the platelets is 10 ℃; the protein kinase A activator is adenylate cyclase activator Forskolin.