阅读说明：本技术 一种血小板的保存方法 (Method for preserving blood platelets ) 是由 戴克胜 于 2021-04-02 设计创作，主要内容包括：本发明公开了一种血小板的保存方法。本发明从保存所致血小板的凋亡、活化角度,血小板生理功能丧失,回输至体内止血血栓形成能力及清除效率角度探讨不同温度对保存血小板的影响,最终选择血小板保存的最佳温度,高于4℃不高于8℃,优选为6℃,该温度下保存的血小板生理功能最佳,血栓形成能力最佳,体内清除效率最慢。相比现有认可的保存温度4℃及22℃,延长了血小板的保存期限,最大限度保持了血小板的生理功能,同时也满足了血小板储存方法简便、长期大量保存、运输方便等方面的要求,适于临床上普遍推广与应用。同时,本发明还研究了血小板在不同保存温度发生凋亡的机制,得出了低温(4℃及6℃)以及22℃保存血小板所致凋亡的原因。(The invention discloses a method for preserving platelets. The invention discusses the influence of different temperatures on the stored platelets from the aspects of apoptosis and activation of the platelets caused by storage, loss of physiological functions of the platelets, capability of stopping bleeding and forming thrombus in vivo and clearing efficiency, and finally selects the optimal temperature for storing the platelets, wherein the optimal temperature is higher than 4 ℃ and not higher than 8 ℃, preferably 6 ℃, the physiological function of the platelets stored at the temperature is optimal, the capability of forming thrombus is optimal, and the clearing efficiency in vivo is slowest. Compared with the existing approved storage temperature of 4 ℃ and 22 ℃, the storage life of the platelets is prolonged, the physiological functions of the platelets are maintained to the maximum extent, and meanwhile, the requirements of the platelets on the aspects of simple and convenient storage method, long-term mass storage, convenient transportation and the like are met, so that the method is suitable for clinical popularization and application. Meanwhile, the invention also researches the apoptosis mechanism of the platelets at different storage temperatures, and obtains the causes of apoptosis caused by storing the platelets at low temperature (4 ℃ and 6 ℃) and 22 ℃.)

1. A method for preserving platelets, characterized in that the platelet-rich plasma obtained is preserved at a temperature higher than 4 ℃ and not higher than 8 ℃.

2. The preservation method according to claim 1, wherein the platelet-rich plasma obtained is preserved at a temperature of 6 ℃.

3. The preservation method according to claim 1, characterized in that the platelet rich plasma is prepared as follows: aseptically pumping healthy human venous blood, mixing whole blood with an anticoagulant, slowly inverting and uniformly mixing; the anticoagulated whole blood is centrifuged at room temperature, and the upper layer after centrifugation is the plasma rich in platelets.

Technical Field

The invention relates to a preservation method, in particular to a platelet preservation method.

Background

Platelets are anucleated cellular components that come off the cytoplasm of mature megakaryocytes, play a crucial role in hemostasis and thrombosis, and are widely used in the treatment of hemorrhagic diseases due to thrombocytopenia and platelet dysfunction, and in patients undergoing myelosuppressive chemotherapy. With the development of medical technology, the clinical application of the platelet product is increasingly widespread, however, the platelet product is still in a condition of severe short supply and short demand in clinic for a long time, and the contradiction between supply and demand in the future is more prominent, so that the guarantee of platelet transfusion is still a leading problem in the world. Because platelets have a short life span and are susceptible to a variety of factors in structure and function, preservation of platelets is urgently needed to achieve the following goals: simple storage method, long-term mass storage, convenient transportation and obvious immediate hemostatic effect. The long-term and effective storage problem of the platelets is always a hotspot and a difficulty of the research of international blood transfusion workers, and is a world problem.

In recent years, the fully-established storage condition at home and abroad is that the platelet is stored at 22 ℃ under continuous oscillation, the activity and the stability of the platelet are well maintained, and the 22 ℃ is used as the conventional temperature in many countries. However, platelets stored at ambient temperature at 22 ℃ are susceptible to bacterial contamination, with 1 being contaminated with bacteria per 1000 to 2000 platelet products, which may cause sepsis after transfusion; in addition, the progressive activation of platelets leads to the accumulation of metabolic byproducts of platelets, resulting in a progressive decline in platelet function and viability. The united states Food and Drug Administration (FDA) stipulates that platelets are stored for 3-5 days and will be discarded after expiration, which results in a large amount of platelet waste. Due to the short time for storing the platelets by asphyxiation, the optimization and improvement of the existing platelet storage conditions are continuously researched for a long time.

Because low temperature can inhibit bacterial growth, some researchers have proposed storing platelets at low temperature, however, since platelets are living cells, although preservation period of platelets at deep low temperature (-80 ℃) can be prolonged, clinical application proves that the platelets have obvious immediate hemostatic effect, but the defects are that: 1) the refrigerator needs to be preserved at extra-high and expensive deep low temperature (-80 ℃) or consumes a large amount of liquid nitrogen; 2) is inconvenient to carry; 3) difficulty in long-distance transportation; especially 4) the biological activity of the frozen platelets can be fatally impaired. In addition, some attempts have been made to maintain platelets at 4 ℃ which, when stored at 22 ℃, reduces the bacterial contamination rate of platelets and extends the shelf life of platelets, but platelets refrigerated at 4 ℃ are easily activated and aggregated and no longer functionally active, and are rapidly phagocytosed and cleared by macrophages in the body after being infused into the body, resulting in a significantly shortened survival time in the body. Therefore, due to the lack of necessary theoretical guidance, technical means and equipment conditions, no conclusive and instructive research results for systematically researching the influence of temperature on platelets exist in the world.

Disclosure of Invention

In order to solve the above problems of the prior art, it is an object of the present invention to provide a method for preserving platelets, which is capable of screening conditions optimal for platelet preservation by examining the influence of different temperatures on the preserved platelets from the viewpoints of apoptosis and activation of platelets, loss of physiological functions, and thrombogenicity and removal efficiency of platelets after being returned to the body during preservation. Selecting 10 kinds of temperature of 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 15 deg.C and 22 deg.C to store platelets; separating human platelet-rich plasma (hPRP) by centrifugation; continuously detecting the apoptosis and activation degree of the platelets from 1 st to 7 th days of storage by using a flow cytometer; detecting the platelet aggregation function stored at different temperatures by an aggregation instrument for 7 days continuously; in vivo FeCl ₃Detecting the thrombus formation capability of platelets stored at different temperatures by using an injured mesenteric thrombus model; the in vivo reinfusion model in immunodeficient (SCID) mice measures survival time and clearance efficiency in maintenance platelets.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for preserving blood platelet comprises preserving the blood plasma rich in blood platelet at a temperature higher than 4 deg.C and not higher than 8 deg.C.

Preferably, the platelet preservation method of the present invention is a method for preserving the platelet-rich plasma obtained by subjecting the platelet-rich plasma to a temperature of 6 ℃.

Preparation of human platelet rich plasma (hPRP):

sterile drawing 25-50mL of healthy human venous blood, mixing the whole blood with sterile 1/9 vol% human 3.8% sodium citrate anticoagulant, slowly inverting and mixing. Centrifuging the anticoagulated whole blood at the room temperature of 1100rpm for 11 minutes, wherein the lower layer is erythrocytes, the middle layer is leukocytes and the upper layer is leukocytesThe layer is Platelet Rich Plasma (PRP). Carefully remove the upper PRP to a sterile 15mL centrifuge tube. And (4) continuously centrifuging the middle-lower layer at room temperature of 3500rpm for 10 minutes, taking the supernatant as platelet-free plasma (PPP), and taking the supernatant into a sterile centrifuge tube for later use (keeping to dilute PRP). Counting by a full-automatic human blood cell counter, and adjusting the concentration to be (1-3)' 10 after counting ⁸/mL。

Different conditional preservation of hPRP:

the prepared hPRP/hPLT was dispensed into sterile 1.5mL EP tubes at 200. mu.L/tube. Then the packed platelets are respectively placed in different temperature environments, including 0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃, 15 ℃ and 22 ℃, and then the apoptosis activation indexes of hPRP/hPLT are detected after being respectively stored for 1, 2, 3, 4, 5, 6 and 7 days. The prepared hPRP/hPLT was dispensed into a sterile 1.5mL EP tube at 1.2 mL/tube. Then the packed platelets are respectively placed in different temperature environments including 4 ℃, 6 ℃ and 22 ℃, and then the aggregation of hPRP/hPLT under different inducer induction conditions is detected after the platelets are respectively stored for 1, 2, 3, 4, 5, 6 and 7 days, and the optimal storage temperature is selected to be 6 ℃.

Has the advantages that: the invention discloses a method for preserving platelets, which discusses the influence of different temperatures on the preservation of platelets from the aspects of apoptosis activation, aggregation function loss, thrombus formation capability and clearance efficiency of returning to the body of the platelets caused by preservation, and finally selects 6 ℃ as the optimal temperature for preserving the platelets, and simultaneously can still achieve better preservation effect at the temperature higher than 4 ℃ and not higher than 8 ℃, and the performance of the platelets after being preserved for 7 days is obviously superior to other preservation temperatures in the temperature range. Meanwhile, the mechanism of platelet preservation is researched, apoptosis caused by platelet preservation at low temperature (4 ℃ and 6 ℃) is the decrease of PKA activity caused by the increase of cAMP hydrolysis by activating PDE3A through PKC pathway activation, but platelet preservation at 6 ℃ is better because of lower activation degree of PKC pathway. Apoptosis due to the storage of platelets at 22 ℃ is due on the one hand to changes in enzyme activity and on the other hand to degradation of the proteins due to prolonged storage times.

The optimal temperature for storing the selected platelets is higher than 4 ℃ and not higher than 8 ℃, and is preferably 6 ℃, compared with the standard temperature of 22 ℃ for storing the platelets in China at the present stage and 4 ℃ which is most widely researched by scientists, the platelets have the best aggregation function, the best thrombosis forming capability and the slowest in vivo clearing efficiency after being stored for 1 to 7 days. Therefore, the method for storing the platelets at the temperature interval, particularly 6 ℃, prolongs the storage life of the platelets compared with the existing approved storage temperature, meets the requirements of simple and convenient platelet storage method, long-term large-scale storage, convenient transportation and the like, and is suitable for clinical popularization and application.

Drawings

FIG. 1 is a graph showing the change in JC1 index after hPRP storage for one to seven days at 0, 2, 4, 6, 8, 10, 12, 14, 15 and 22 ℃. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 2 is a graph showing the change in PS index after hPRP storage at 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 15 deg.C and 22 deg.C for one to seven days. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 3 is a graph showing the change in CD62P after hPRP storage for one to seven days at 0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃, 15 ℃ and 22 ℃. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 4 is a graph showing changes in the PAC1 index after hPRP storage for one to seven days at 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 15 deg.C, and 22 deg.C. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 5 is a diagram showing the change in aggregation function of hPRP after 7 days of continuous storage. (A) Ristocetin-induced platelet aggregation; (B) statistics of Ristocetin induced platelet aggregation; (C) platelet aggregation induced by Collagen; (D) statistics of platelet aggregation induced by Collagen; (E) ADP-induced platelet aggregation; (F) statistical results of ADP-induced platelet aggregation; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 6 is a graph showing the thrombogenicity of hPRP stored at 4 ℃, 6 ℃ and 22 ℃ for 4 days in the reinfused mice.

FIG. 7 is a graph showing the measurement of the clearance efficiency of hPRP reinfused into SCID mice stored at 4 ℃, 6 ℃ and 22 ℃ for 4 days; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 8 is a graph showing the dynamic expression of caspase 3 protein in hPLT cells stored continuously for 7 days at 4 ℃, 6 ℃ and 22 ℃. (A) The change of caspase 3 protein from the first day to the seventh day of platelets preserved at 4 ℃, 6 ℃ and 22 ℃ respectively; (B) changes in the platelet caspase 3 protein stored at 4 ℃, 6 ℃ and 22 ℃ from day one to day seven, respectively.

FIG. 9 is a graph showing the dynamic expression changes of BCL-XL, BAK, BAX proteins in hPLT stored continuously at 4 ℃, 6 ℃ and 22 ℃ for 7 days. (A) The change of BCL-XL, BAK and BAX proteins of platelets preserved at 4 ℃, 6 ℃ and 22 ℃ respectively from the first day to the seventh day; (B) changes in the platelets BCL-XL, BAK, BAX proteins stored at 4 ℃, 6 ℃ and 22 ℃ from day one to day seven, respectively.

FIG. 10 is a graph showing the change in PKA activity of hPLT stored for 7 days at 4 ℃, 6 ℃ and 22 ℃. (A) Change of pVASP (Ser 157), VASP, p-GPIb beta (Ser 166) and GPIb beta protein from the first day to the seventh day of platelets preserved at 4 ℃, 6 ℃ and 22 ℃ respectively; (B) changes in the VASP proteins of platelets pVASP (Ser 157) stored at 4 ℃, 6 ℃ and 22 ℃ from day one to day seven, respectively.

FIG. 11 is a graph showing the results of ELISA detection of PKA activity after continuous storage of hPLT for seven days at 4 ℃, 6 ℃ and 22 ℃; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 12 is a graph showing the results of ELISA detection of PKA activity after continuous storage of PKAC α recombinant protein at 4 ℃, 6 ℃ and 22 ℃ for seven days; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 13 is a graph showing the results of ELISA detection of cAMP content after platelets were continuously stored at 4 deg.C, 6 deg.C and 22 deg.C for seven days; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 14 is a graph showing the results of ELISA detection of PDE3A after continuous seven-day platelet storage at 4 ℃, 6 ℃ and 22 ℃; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 15 is a graph showing changes in the dynamic expression of PDE3A protein in hPLT stored continuously for 7 days at 4 ℃, 6 ℃ and 22 ℃. (A) Change of PDE3A protein from day one to day seven in platelets stored at 4 ℃, 6 ℃ and 22 ℃, respectively; (B) changes in platelet PDE3A protein from day one to day seven, stored at 4 ℃, 6 ℃ and 22 ℃ respectively.

FIG. 16 is a graph showing the effect of 8-Br-cAMP pretreatment on platelet preservation. (A) WesternBlot detects changes in PKA activity; (B) a change in JC 1; (C) a change in PS; (D) changes in CD 62P; (E) a change in PAC 1; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 17 is a graph showing the effect of Forskolin pretreatment on the status of stored platelets. (A) WesternBlot detects changes in PKA activity; (B) a change in JC 1; (C) a change in PS; (D) changes in CD 62P; (E) a change in PAC 1; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 18 is a graph showing the effect of Milliprone pretreatment on the status of stored platelets. (A) WesternBlot detects changes in PKA activity; (B) a change in JC 1; (C) a change in PS; (D) changes in CD 62P; (E) a change in PAC 1; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 19 is a graph of the effect of Enoxidone pretreatment of platelets on the preservation of platelet status. (A) WesternBlot detects changes in PKA activity; (B) a change in JC 1; (C) a change in PS; (D) changes in CD 62P; (E) a change in PAC 1; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 20 is a graph showing the results of PKC activity assay in hPLT storage at 4 deg.C, 6 deg.C and 22 deg.C for 7 days. (A) Changes in p-PKC substrate protein from day one to day seven in platelets stored at 4 ℃, 6 ℃ and 22 ℃, respectively; (B) changes in platelet p-PKC substrate protein from day one to day seven, stored at 4 ℃, 6 ℃ and 22 ℃, respectively.

Fig. 21 is a graph showing the results of downstream pathway detection after pretreatment of platelets with PKC inhibitors.

Fig. 22 is a graph of the effect of GO6983 pretreatment of platelets on the preservation of platelet status. (A) A change in JC 1; (B) a change in PS; (C) changes in CD 62P; (D) a change in PAC 1; differences were significant when p < 0.05. The levels of significance were p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

FIG. 23 is a graph showing the results of detection of platelet apoptosis activation indexes in WT and Bad knockout mice stored at 4 ℃, 6 ℃ and 22 ℃. (A) Membrane potential depolarization test (JC 1) of WT and Bad knockout mice platelets stored continuously at 4 ℃, 6 ℃ and 22 ℃ for seven days; (B) PS eversion of WT and Bad knockout mice platelets is continuously preserved at 4 ℃, 6 ℃ and 22 ℃ for seven days; (C) continuously storing WT and Bad knockout mouse blood platelets at 4 ℃, 6 ℃ and 22 ℃ for seven days for detecting CD 62P; (D) PAC1 assay of platelets from WT and Bad knockout mice stored continuously at 4 deg.C, 6 deg.C and 22 deg.C for seven days.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

First, selection of optimal platelet preservation temperature

The invention provides a method for prolonging the preservation of platelets, which starts from the aspects of platelet apoptosis activation, aggregation function, thrombus formation capability and platelet removal rate in vivo and selects the optimal temperature for preserving the platelets. Four indices of JC1, PS, CD62P, PAC1 were selected to detect the apoptotic activation status of platelets. JC1 can detect the depolarization of membrane potential, which is the specific index of platelet apoptosis. PS eversion, i.e., phosphatidylserine eversion, can occur after activation and apoptosis of platelets and is a non-specific indicator for the detection of platelet apoptosis. CD62P, p-selectin, is platelet alpha-granule protein that reflects the level of platelet release. PAC1, which detects activated IIBIIA, reflects the degree of platelet activation. The optimal preservation temperature of the platelets is selected by the change of these indices.

To further confirm the preliminary results, different inducers such as Ristocetin, Collagen and ADP were used to induce platelet aggregation at different temperatures for different periods of time, and hPRP treated under different conditions was returned to SCID mice for testing of thrombogenicity and clearance of hPRP stored at different temperatures to SCID mice.

1.1 Experimental methods

1.1.1 preparation of human platelet-rich plasma (hPRP)

Sterile drawing 25-50mL of healthy human venous blood, mixing the whole blood with sterile 1/9 vol% human 3.8% sodium citrate anticoagulant, slowly inverting and mixing. The anticoagulated whole blood is centrifuged at 1100rpm for 11 minutes at room temperature, and after centrifugation, the lower layer is red blood cells, the middle layer is white blood cells, and the upper layer is platelet-rich plasma (PRP). Carefully remove the upper PRP to a sterile 15mL centrifuge tube. And (4) continuously centrifuging the middle-lower layer at room temperature of 3500rpm for 10 minutes, taking the supernatant as platelet-free plasma (PPP), and taking the supernatant into a sterile centrifuge tube for later use (keeping to dilute PRP). The full-automatic human blood cell counter counts and adjusts after countingConcentration to be (1-3)' 10⁸/mL。

1.1.2 storage of hPRP under different conditions

The prepared hPRP/hPLT was dispensed into sterile 1.5mL EP tubes at 200. mu.L/tube. Then the packed platelets are respectively placed in different temperature environments, including 0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃, 15 ℃ and 22 ℃, and then the apoptosis activation indexes of hPRP/hPLT are detected after being respectively stored for 1, 2, 3, 4, 5, 6 and 7 days. The prepared hPRP/hPLT was dispensed into a sterile 1.5mL EP tube at 1.2 mL/tube. The packed platelets are then placed in different temperature environments, including 4 ℃, 6 ℃ and 22 ℃, and then tested for aggregation of hPRP/hPLT under different inducer-induced conditions after storage for 1, 2, 3, 4, 5, 6 and 7 days.

1.1.3 flow-type detection of platelet Membrane potential depolarization

And (3) measuring the mitochondrial membrane potential depolarization of the platelets by using a lipophilic cationic dye JC-1, adding JC-1 into the treated platelets to enable the final concentration to be 2 mu g/mL and the total volume to be 350 mL per tube, incubating at room temperature in a dark place for 5 min, and detecting by using a flow cytometer. Platelets show a decrease in JC-1 aggregate content following apoptosis, which is reflected in a decrease in the amount of FL2 channel fluorescence, while FL1 channel fluorescence increases.

1.1.4 flow assay for platelet Phosphatidylserine (PS) eversion

Detecting PS (polystyrene) eversion on the surface of the platelet by using FITC-labeled lactadherin, adding 10 mu g/mL of FITC-lactadherin into the platelet to be detected, fully mixing uniformly, incubating at room temperature for 30 minutes, adding MTB to complement the total volume to 350 mu L before flow detection, and turning out phosphatidylserine after the platelet is apoptotic, wherein the increase of FL1 channel fluorescence quantity is shown on a flow cytometer.

1.1.5 flow assay platelet CD62P Release

P-selectin release is detected by using FITC labeled CD62P antibody, 20 mu g/mL FITC-CD62P is added into the blood platelets to be detected, after fully mixing, the mixture is incubated for 15 minutes at room temperature, and MTB is added before flow detection to make up the total volume of 350 mu L. Activation of platelets can be manifested as increased release, with P-selectin being one of the releases. The flow cytometer showed an increase in the amount of FL1 channel fluorescence.

1.1.6 flow assay for platelet activation

And adding FITC-labeled PAC-1 labeled activated alpha IIb beta 3, 20 mu g/mL FITC-PAC-1 into the platelets to be detected, fully mixing uniformly, incubating for 15 minutes at room temperature, adding MTB to complement the total volume to 350 mu L before flow detection, wherein the platelet activation is characterized in that the FL1 channel fluorescence quantity is increased.

1.1.7 platelet aggregation assay

After platelets were stored at different temperatures, i.e., 4 ℃, 6 ℃ and 22 ℃ for 1, 2, 3, 4, 5, 6 and 7 respectively, a platelet aggregometer was used to measure the aggregation ability of hPRP induced by different inducers (Ristocetin 1.25mg/mL; ADP 20. mu.M; Collagen 5. mu.g/mL). Agonist-induced platelet aggregation was monitored in a two-channel Chrono-Log aggregation instrument. Platelet Poor Plasma (PPP) obtained from centrifugation of whole blood was used as a control well. Samples of hPRP stored at different temperatures were pipetted 250 μ L into cuvettes. Two cuvettes were placed in PRP wells and reference wells, respectively, and a stir bar and 250. mu.L of sample were added to the sample wells, and the other was referenced with 250. mu.L of PPP, followed by incubation at 37 ℃ for 3 minutes. The channels were calibrated for 0 to 100% light transmission by pressing the SET BASELINES button with 1200 rpm agitation. Aggregation was initiated by addition of the agonist described above and monitored by optical density, which was converted to percent activity within 5 minutes after addition of the agonist.

1.1.8 platelet reinfusion model

The hPRP stored at different temperatures, i.e. 4 deg.C, 6 deg.C and 22 deg.C for 4 days is concentrated by centrifugation to a concentration of 4X 10⁹The suspension was allowed to stand at room temperature for 1 hour. To anesthetized recipient SCID mice, 100. mu.L of 4X 10 was reinfused through the retrobulbar venous plexus⁹hPRP per mL; the whole blood was collected from the mouse orbital vein at 30s (basal), 20 min, 40 min, 2h, 4h and 24h after injection, the labeled platelets were analyzed by flow cytometry, and after anticoagulation by mouse ACD, the whole blood was labeled with PE-anti-mouse CD41, APC-anti-human CD41 antibody for 15 min at room temperature, diluted with 350. mu.L MTB and flow-detected. Preservation of hPRP-proportion = anti-human CD41 positive platelets/(anti-mouse CD 41)Positive platelets + anti-human CD41 positive platelets). The final ratio was calculated and counted by dividing the saved hPRP ratio at each time point by the saved hPRP ratio at 30s based on 30 s.

1.1.9 calcein labeling of human platelets

(1) The hPRP stored for 4 days at different temperatures, namely 4 ℃, 6 ℃ and 22 ℃, is centrifuged at 3500rpm for 2 minutes, then the supernatant is removed, and the CGS buffer is used for resuspension;

(2) at room temperature 2500 rpm, 2 minutes, removing supernatant, and carrying out CGS buffer heavy suspension;

(3) at room temperature 2500 rpm, 2 minutes, removing supernatant, and resuspending MTB buffer;

(4) The washed platelets were adjusted to 1X 10⁹(iii) mL, incubated with calcium yellow-green-AM (calcein-AM) 5 μ g/mL for 15 min at room temperature;

(5) adding an equal volume of PGI containing 20. mu.g/mL₂CGS buffer dilution;

(6) centrifugation at 2500 rpm for 2 min at room temperature, removal of supernatant, CGS (containing 10. mu.g/mL PGI)₂) Resuspending the buffer;

(7) centrifuging at room temperature 2500 rpm for 2 minutes, removing supernatant, and resuspending through PPP;

(8) hPRP adjusted to 1X 10⁹and/mL, and standing for 1 hour for later use.

1.1.10 thrombosis in vivo

The hPRP stored at different temperatures, i.e. 4 deg.C, 6 deg.C and 22 deg.C for 4 days was concentrated by centrifugation to a concentration of 1X 10⁹The suspension was allowed to stand at room temperature for 1 hour. To anesthetized recipient SCID mice, 100. mu.L 10 was reinfused through the retrobulbar venous plexus⁹hPRP in/mL, using a model of ferric chloride-induced (FeCl 3-induced) mesenteric arteriole thrombosis. Briefly, the washed hPRP after 4 days of storage at 4, 6 and 22 ℃ was labeled with calcein-AM (5. mu.g/mL), recipient male mice were anesthetized, and the mesenteric vascular bed was externalized. Followed by pretreated hPRP (100. mu.L.times.10)⁹/mL) orbital intravenous injection of SCID mice. By topical application of 3 mm impregnated with 5% FeCl3²The filter paper induced thrombosis. An arteriole was selected and visualized with an inverted fluorescence microscope (Leica Microsystems) and recorded on videotape 。

1.1.11 statistical analysis

All data are expressed as mean ± standard deviation and the data are statistically analyzed using one-way analysis of variance or multi-way analysis of variance. Two groups were compared using a two-tailed t-test. The significance of the data was assessed using GraphPad Prism 5 software. When in usepThe difference was significant < 0.05. The different significance levels arep＜0.05，** p＜0.01，***p＜0.001，****pIs less than 0.0001. All experiments requiring the use of animals were randomly grouped by litter size, and the sample size was predetermined based on the variability observed in previous experiments and preliminary data.

1.2 results of the experiment

1.2.1 alteration of apoptosis activation index after one to seven days of preservation of hPRP

Platelet apoptosis is an intrinsic form of mitochondrially mediated programmed cell death, and BCL-2 family proteins interact with the outer mitochondrial membrane, leading to depolarization of the transmembrane potential, loss of the transmembrane potential difference, mitochondrial swelling and rupture, and release of cytochrome C in the mitochondria into the cytosol. PS eversion is another apparent marker molecule for intrinsic pathway-dependent apoptosis. The platelet is wrapped by lipid bilayer adventitia, when resting, more than 90% of phosphatidylserine distributes in the intima, when the platelet is apoptotic, the surface tension of the cell membrane is reduced, PS can overturn to the adventitia surface through different ways, and the cell exposed PS can be phagocytized by phagocyte as 'eat me' signal. Then selecting the indexes of mitochondrial membrane potential depolarization and PS valgus to detect the level of platelet apoptosis caused by storage. CD62P release and IIBIIIA (PAC-1) switching from resting to active for testing blood resulting from storage

Level of platelet activation.

As shown in FIGS. 1 to 4, they are graphs showing the change in the platelet apoptosis activation index after preservation of hPRP at different temperatures (0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃, 15 ℃, 22 ℃) for 1 to 7.

As shown in fig. 1-4, after the platelets were stored at different temperatures for 1 day, the degree of membrane potential depolarization of the platelets stored at different temperatures was low (fig. 1); the PS eversion degree is higher when the PS is stored at 0 ℃ than other temperatures; the positive rates of CD62P and PAC1 of the platelets preserved at 0 ℃ are both more than 10 percent and are obviously higher than the positive rates of the platelets preserved at other temperatures, so that the activation level of the platelets preserved at 0 ℃ is higher than that of the platelets preserved at other temperatures after the platelets are preserved at different temperatures for 1 day, and the state of the platelets preserved at other temperatures for 1 day is not obviously damaged by the platelets.

After the platelets are stored for 2 days at different temperatures, the degree of depolarization of membrane potential of the platelets stored at 0 ℃ is the highest, and the degree of PS eversion is the highest when the platelets are stored at 0 ℃, which preliminarily shows that the state of the platelets stored at 0 ℃ is inferior to that of other platelets; the positive rates of CD62P and PAC1 of the platelets preserved at 0 ℃ and 2 ℃ are obviously higher than those of other temperatures, so that the activation level of the platelets preserved at 0 ℃ and 2 ℃ is higher, and the primary indication shows that the platelet preservation damage caused by the platelets preserved at 0 ℃ and 2 ℃ is higher, and the platelet preservation damage is mainly reflected in the aspect of platelet activation.

After the platelets are stored at different temperatures for 3 days, the degree of membrane potential depolarization of the platelets stored at 0 ℃ is the highest, and the mitochondrial membrane potential depolarization levels of the platelets stored at 2 ℃ and 22 ℃ are lower than 0 ℃ but still higher than other temperatures, which preliminarily shows that the degree of platelet storage damage caused by the platelets stored at 0 ℃, 2 ℃ and 22 ℃ is higher than other temperatures. The trend of PS eversion is substantially consistent with the trend of mitochondrial membrane potential depolarization, further indicating that the degree of apoptosis is higher than other temperatures when platelets are stored at 0 ℃, 2 ℃ and 22 ℃ for 3 days. From the positive rate levels of CD62P and PAC1, the activation levels of platelets were higher when stored at 0 ℃, 2 ℃ and 22 ℃. In combination with the results of the four indicators of the apoptosis activation assay, the degree of platelet storage damage is high when platelets are stored at 0 ℃, 2 ℃ and 22 ℃ for 3 days, which may explain why platelets are required to be stored at 22 ℃ for no more than 3 days in japan.

After the platelets are stored for 4 days at different temperatures, the degree of depolarization of membrane potential of the platelets stored at 0 ℃ and 22 ℃ is the highest, and the degree of depolarization of membrane potential of the platelets stored at 2 ℃, 4 ℃, 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃ and 15 ℃ is lower than that of the platelets stored at 22 ℃; compared with other temperatures, the PS valgus degree of the platelets stored at 8 ℃ is lower, and the PS valgus shows double peaks when the platelets are stored for 4 days at 0 ℃ and 2 ℃, which indicates that the platelet state is poor and the difficulty is higher when the platelets are stored for a longer time. According to the detection results of CD62P and PAC1, the storage at 6 ℃ and 8 ℃ is better than other temperatures, and the storage time can be longer.

After the platelets are stored for 5 days at different temperatures, compared with the platelets stored for 5 days at 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃ and 15 ℃, the degree of depolarization of the membrane potential is lower, and the PS eversion trend is basically consistent with the depolarization trend of the mitochondrial membrane potential. The degree of platelet activation was still low when stored at 6 ℃ and 8 ℃ compared to the other temperatures, as measured by CD62P and PAC 1.

As a result of storing the platelets for 6 days and 7 days at different temperatures, the degree of the storage injury of the platelets is gradually increased along with the prolonging of the storage time, and compared with other temperatures, the degree of depolarization of the platelet membrane potential at 6 ℃ is lowest, and the degree of PS eversion is lowest. The platelet activation was minimal when stored at 6 ℃ compared to other temperatures as measured by CD62P and PAC 1. Therefore, after 6 days and 7 days of storage, the level of apoptosis activation of platelets stored at 6 ℃ is still lower than other temperatures, which is the optimal temperature for platelet storage. Meanwhile, as can be seen from the experimental results of fig. 1 to 4, when platelets are stored at a temperature not higher than 8 ℃ higher than 4 ℃, although the level of apoptosis activation of platelets is lower than that of platelets stored at 6 ℃, the level of apoptosis activation thereof is still lower than that of platelets stored at other temperatures, and is also suitable as the storage temperature of platelets.

1.2.2 changes in the function of hPRP to preserve aggregation at different times

The above flow cytometry results of apoptosis activation of stored platelets have preliminarily shown that 6 ℃ is the optimal temperature for storing platelets. To further demonstrate that 6 ℃ is the optimal temperature for platelet preservation, the aggregation function after platelet preservation was next examined. Because 4 ℃ is the most common temperature for cryopreserving the platelets currently under discussion, 22 ℃ is the standard temperature for domestic platelet preservation at present, and three temperatures of 4 ℃, 6 ℃ and 22 ℃ are finally selected to discuss the functional conditions of the platelets.

As shown in FIG. 5, the aggregation function of platelets stored at 4 ℃ and 22 ℃ induced by Ristocetin (FIGS. 5A-B) was decreased from the first day to the seventh day, and the aggregation function of platelets stored at 6 ℃ was less changed from the first day to the seventh day. After being stored for 4, 5, 6 and 7 days, the platelet stored at 6 ℃ has the best aggregation function and has statistical significance. The aggregation function of platelets stored at 4 ℃, 6 ℃ and 22 ℃ induced by Collagen (FIGS. 5C-D) was reduced from day one to day seven, but the decrease in aggregation function of platelets stored at 6 ℃ was minimal. After 1, 2, 3, 4, 5, 6 and 7 are stored, the platelet aggregation function is optimal after 6 ℃ storage, and the statistical significance is achieved. The aggregation function of the platelets stored at 4 ℃, 6 ℃ and 22 ℃ induced by ADP (FIGS. 5E-F) was decreased from day one to day seven, and the aggregation function of the platelets stored at 6 ℃ was less changed from day one to day seven. After being stored for 4, 5, 6 and 7 days, the platelet stored at 6 ℃ has the best aggregation function and has statistical significance. These results all further indicate that 6 ℃ is the optimal temperature for platelet preservation.

1.2.3 testing of thrombogenicity in the reinfusion of hPRP treated under different conditions into SCID mice

The apoptosis activation index and aggregation result obtained by the flow cytometry indicate that the temperature of 6 ℃ is the optimal temperature for storing the platelets. To further demonstrate that 6 ℃ is the optimal temperature for hPRP preservation, the thrombogenicity of hPRP stored at different temperatures for reinfusion into the body was subsequently examined. The hPRP stored at different temperatures, i.e. 4 deg.C, 6 deg.C and 22 deg.C for 4 days is concentrated by centrifugation to a concentration of 1 × 10⁹suspension/mL, and washing platelets with calcein-AM (5. mu.g/mL) marker, and resuspension to 1X 10 using PPP⁹and/mL, standing for 1h at room temperature. Anaesthesia recipient SCID mice were ventilated and mice mesenteric vascular beds were externalized, followed by pre-treated hPRP (100. mu.L × 10)⁹/mL) orbital intravenous injection of SCID mice. Impregnated with 5% FeCl by topical application₃3 mm of²The filter paper induced thrombosis. An arteriole was selected and observed and recorded with an inverted fluorescence microscope. As shown in FIG. 6, the thrombogenicity of hPRP stored at 6 ℃ was higher than that of 4 ℃ and 22 ℃ and that of hPRP stored at 22 ℃ was higher than 4 ℃.

1.2.4 measurement of the Effect of hPRP Return to SCID mice after storage at different temperatures

The apoptosis activation index, aggregation result and in vivo thrombosis result obtained by the flow cytometry indicate that the temperature of 6 ℃ is the optimal temperature for storing the platelets. Finally, the hPRP stored at different temperatures is detected and returned to the body to observe the platelet removal efficiency. After storage of hPRP at 4 deg.C, 6 deg.C and 22 deg.C for 4 days, hPRP was concentrated to 4X 10⁹The suspension was allowed to stand at room temperature for 1 hour. To anesthetized recipient SCID mice, 100. mu.L of 4X 10 was reinfused through the retrobulbar venous plexus⁹hPRP/mL, whole blood was collected from the orbital vein of mice 30s (basal), 20min, 40min, 2h, 4h and 24h after injection, and the clearance efficiency of stored platelets by reinfusion into the body was analyzed by flow cytometry. As shown in FIG. 7, the hPRP was gradually cleared after storage by reinfusion into SCID mice over time, and was completely cleared within 24 h. However, platelets stored at different temperatures were cleared at different rates by reinfusion into SCID mice, with hPRP clearance rates at 6 ℃ being less than 4 ℃ and 22 ℃ for reinfusion into SCID mice for 20min, 40min, 2h, 4h, and 24h, and hPRP clearance rates at 22 ℃ being less than 4 ℃ for reinfusion into SCID mice for 20min, 40min, 2h, and 4 h. And has statistical significance at 20 min.

In summary, the above results indicate that the degree of apoptosis activation of platelets varies when stored at different temperatures for the same time. From the membrane potential depolarization level index: hPRP stored at 0 deg.C has membrane potential depolarization degree higher than other temperatures in the third day; the hPRP preserved at 22 ℃ has the membrane potential depolarization degree obviously higher than other temperatures from the fourth day of preservation; the blood platelets preserved at 2 ℃ and 4 ℃ have obviously increased membrane potential depolarization level from the fifth day of preservation; the level of depolarization of membrane potential of the platelets preserved at 8 ℃ is obviously increased from the sixth day of preservation; platelets stored at other temperatures changed little from day one to day seven in JC1, with the least change being platelets stored at 6 ℃. From PS valgus index: hPRP stored at 0 ℃ with significant PS eversion from the first day of storage; hPRP stored at 22 ℃ exhibited significant PS eversion from the fourth day of storage; platelets stored at 2 ℃ and 4 ℃ undergo significant PS eversion from the fifth day of storage; while other temperatures have relatively low PS eversion levels from day one to day seven, with the least change in PS eversion being platelets stored at 6 ℃. From the CD62P index: platelets stored at 0 ℃ and 2 ℃ had undergone significant release of CD62P from the day of storage; platelets stored at 4 ℃ had a significant release of CD62P since the fourth day of storage; platelets stored at 10 ℃, 12 ℃, 14 ℃, 15 ℃ and 22 ℃ had significant CD62P release from the third day of storage; platelets stored at 8 ℃ had a significant release of CD62P from the fifth day of storage. From the PAC1 index: hPRP stored at 0 deg.C, had significant IIBIIIA activation from the first day of storage; platelets stored at 2 ℃, 4 ℃, 8 ℃ and 10 ℃ had significant IIBIIIA activation from the next day; platelets stored at 22 ℃ had significant libiia activation from day four, suggesting that low temperature (4 ℃) at 22 ℃ and most studied was not the optimal choice for storing platelets, 6 ℃ being the optimal temperature for platelets. This also explains why the cryopreserved platelets previously studied did not work well and had many drawbacks, such as rapid clearance of cold platelets from circulation by kupffer cells, resulting in a half-life of about 1.3 days, and a 4 day RT-PLT half-life.

The aggregation ability of platelets stored at different temperatures for different times is induced by using different inducers such as Ristocetin, Collagen and ADP, and as expected, platelets stored at 6 ℃ are the strongest in aggregation ability from the first day to the seventh day. Using FeCl₃In the thrombus model of the damaged mesenteric vein, it was also confirmed that 6 ℃ is the temperature at which the platelet function is preserved most completely. By constructing a model for detecting the clearance efficiency after human hPRP is returned to SCID mice, the clearance efficiency of platelets stored at 6 ℃ is the lowest, the platelet circulation in vivo is the longest, the platelet circulation is 22 ℃ times, and the platelet clearance at 4 ℃ is the worst.

Second, study of mechanism of influence of temperature on platelet preservation

Platelet apoptosis is a hot point of research in recent years and has important significance for disclosing the regulation mechanism of platelet life and survival. There is increasing evidence that intrinsic apoptotic programs lead to platelet destruction under both pathological and physiological conditions. Depolarization of platelet mitochondrial membrane potential, increased caspase activity, and phosphatidylserine exposure result in platelets undergoing apoptotic cell death. Platelets have been shown to contain a variety of apoptosis-related proteins, such as Bcl-2 family proteins and a variety of caspase family proteins. A study by Takahiro Kodama et al showed that spontaneous apoptosis of human platelets occurred after storage at room temperature (22 ℃) and that Bcl-xl expression gradually decreased. Platelets deficient in Bak/Bax or overexpressed in Bcl-xl were less apoptotic during storage, whereas platelets deficient in Bid/Bim did not undergo apoptosis, indicating that platelet life is regulated by a fine balance of anti-apoptotic and pro-apoptotic multi-domain Bcl-2 family proteins. Inhibition of PKA activity can lead to dephosphorylation of the pro-apoptotic protein BAD at the Ser155 site, resulting in the retention of the apoptotic protein bcl-xl in the mitochondria and thus in apoptosis. In this study, the mechanism of apoptosis in platelets stored at different temperatures is described in detail below.

2.1 Experimental methods

2.1.1 human washed platelet (hPLT) preparation

Aseptically extracting 25-50mL of healthy human venous blood, mixing the whole blood with sterile 1/7 volume percent of ACD anticoagulant, slowly inverting and uniformly mixing. The anticoagulated whole blood is centrifuged at 1100rpm for 11 minutes at room temperature, and after centrifugation, the lower layer is red blood cells, the middle layer is white blood cells, and the upper layer is platelet-rich plasma (PRP). Carefully remove the upper PRP by aspiration into an autoclaved 1.5mL Ep tube. After centrifugation at 3500rpm for 2 minutes, the supernatant was discarded, the platelet pellet was left at the bottom of the tube, and the platelets were resuspended in CGS buffer pre-warmed at 37 ℃ and gently pipetted until well mixed. Centrifuging at 2500rpm for 2 min, washing off plasma proteins, finally resuspending the precipitated platelets in a certain volume of 37 ℃ pre-warmed MTB buffer solution, counting, and adjusting the concentration to 3' 10⁸/mL, adding CaCl₂、MgCl₂The final concentration was 1 mM. The resuspended washed platelets were allowed to stand at 22-26 ℃ for 1 hour to restore their physiological state for subsequent experiments.

2.1.2 preparation of mouse washed platelets

0.2g/mL 10% chloral hydrate was intraperitoneally injected into a mouse at room temperature, the mouse was anesthetized for about 3 minutes, the limbs of the mouse were fixed on a test bed with the abdomen facing up, after alcohol sterilization, the lower abdominal cavity was opened with ophthalmic scissors to expose the inferior vena cava, a 1mL syringe needle containing a mouse ACD anticoagulant (whole blood to ACD volume ratio 7: 1) was inserted into the inferior vena cava of the mouse with the needle tip inclined downward, after thorough mixing in an empty needle, the needle tip was removed and the whole blood was injected into a 1.5mL EP tube, the whole blood of 3 mice was mixed into a 15mL centrifuge tube, twice the volume of physiological saline was added to dilute the whole blood, after thorough mixing, centrifugation at 1100rpm for 11 minutes, the supernatant was platelet-rich plasma, and the upper PRP was carefully removed and transferred to a autoclaved 1.5mL Ep tube. After subsequent centrifugation at 3500rpm for 2 minutes, the supernatant was discarded and the bottom platelets were resuspended in 37 ℃ prewarmed CGS buffer and gently pipetted until well mixed. Centrifuging at 2500rpm for 2 min, washing to remove plasma protein, washing once again by the same method, finally suspending in a certain volume of MTB buffer solution pre-warmed at 37 ℃, counting, and adjusting the concentration to be 3' 10 ⁸/mL, adding CaCl₂、MgCl₂The final concentration was 1 mM. The resuspended washed platelets were allowed to stand at room temperature, i.e., 22-24 ℃, for 2 hours to restore their physiological state for subsequent experimental studies.

2.1.3 storage of hPLT under different conditions

The prepared hPLT was dispensed into sterile 1.5mL EP tubes at 200. mu.L/tube. The packed platelets are then placed in different temperature environments, including 4 ℃, 6 ℃ and 22 ℃, and stored for 1, 2, 3, 4, 5, 6 and 7 days before being used in subsequent experimental studies.

2.1.4 different Condition preservation of mouse PLT

Prepared mouse PLTs, including Wild Type (WT) and Bad knock-out (KO) mouse platelets were aliquoted into sterile 1.5mL EP tubes at 200. mu.L/tube. Then, the packed platelets were placed in different temperature environments including 4 ℃, 6 ℃ and 22 ℃, and then the apoptosis activation indexes of WT and KO mice were examined after being stored for 1, 2, 3, 4, 5, 6 and 7 days, respectively.

2.1.5 flow-type detection of platelet Membrane potential depolarization

And (3) measuring the mitochondrial membrane potential depolarization of the platelets by using a lipophilic cationic dye JC-1, adding JC-1 into the treated platelets to enable the final concentration to be 2 mu g/mL and the total volume to be 350 mL per tube, incubating at room temperature in a dark place for 5 min, and detecting by using a flow cytometer. Platelets show a decrease in JC-1 aggregate content following apoptosis, which is reflected in a decrease in the amount of FL2 channel fluorescence, while FL1 channel fluorescence increases.

2.1.6 flow-type detection of platelet Phosphatidylserine (PS) eversion

Detecting PS (polystyrene) eversion on the surface of the platelet by using FITC-labeled lactadherin, adding 10 mu g/mL of FITC-lactadherin into the platelet to be detected, fully mixing uniformly, incubating at room temperature for 30 minutes, adding MTB to complement the total volume to 350 mu L before flow detection, and turning out phosphatidylserine after the platelet is apoptotic, wherein the increase of FL1 channel fluorescence quantity is shown on a flow cytometer.

2.1.7 flow assay of human platelet CD62P Release

P-selectin release is detected by using an FITC-labeled anti-human CD62P antibody, 20 mu g/mL FITC-CD62P is added into the blood platelets to be detected, the mixture is fully mixed, the mixture is incubated for 15 minutes at room temperature, and MTB is added before flow detection to make up the total volume of 350 mu L. Activation of platelets can be manifested as increased release, with P-selectin being one of the releases. The flow cytometer showed an increase in the amount of FL1 channel fluorescence.

2.1.8 flow assay for mouse platelet CD62P release

P-selectin release is detected by using FITC-labeled anti-mouse CD62P antibody, 2 mu L of FITC-CD62P is added into the blood platelets to be detected, after fully mixing, the mixture is incubated for 15 minutes at room temperature, and MTB is added before flow detection to make up the total volume of 350 mu L. Activation of platelets can be manifested as increased release, with P-selectin being one of the releases. The flow cytometer showed an increase in the amount of FL1 channel fluorescence.

2.1.9 flow-type detection of human platelet activation

And adding FITC-labeled PAC-1 labeled activated alpha IIb beta 3, 20 mu g/mL FITC-PAC-1 into the platelets to be detected, fully mixing uniformly, incubating for 15 minutes at room temperature, adding MTB to complement the total volume to 350 mu L before flow detection, wherein the platelet activation is characterized in that the FL1 channel fluorescence quantity is increased.

2.1.10 flow assay for mouse platelet activation

Adding 2 muL PE-JON/A marked and activated alpha IIb beta 3 by using PE mark JON/A into the blood platelet to be detected, fully mixing uniformly, incubating for 15 minutes at room temperature, adding MTB to complement the total volume to 350 muL before flow detection, wherein the characteristic of blood platelet activation is that the FL2 channel fluorescence quantity is increased.

2.1.11 detection of activation of platelet Caspase-3

Storing at different temperatures, namely 4 deg.C, 6 deg.C and 22 deg.C for 1, 2, 3, 4, 5, 6, and 7 days, respectively, to obtain 200 μ L of 3' 10⁸Perml platelets, 1/9 volumes of 10 × cell lysate (containing 2mM PMSF, 2mM NaF, 2mM Na) were added₃VO₄And protease inhibitor) is subjected to ice-bath lysis for 30 minutes, then 4' -protein loading buffer solution with the total volume of 1/3 is added, and after metal bath at 99 ℃ for 5 minutes, the mixture is cooled to room temperature and stored at-80 ℃; after proteins are separated by SDS-PAGE gel, transferring the bands to a PVDF membrane, sealing with 5% skimmed milk powder for 1 hour, co-incubating the PVDF membrane with anti-caspase-3 primary antibody diluted by 1:500, incubating overnight at 4 ℃, washing for 5 minutes by TBST, repeating for three times, co-incubating with a secondary antibody coupled with HRP, detecting the protein level after ECL luminescence, and simultaneously detecting the GAPDH protein expression condition of each group of platelets, and analyzing whether the sample loading amount of each group of samples is consistent or not by taking the protein as an internal reference.

2.1.12 Western Blot protein detection

Storing at different temperatures, namely 4 deg.C, 6 deg.C and 22 deg.C for 1, 2, 3, 4, 5, 6, and 7 days, respectively, to obtain 200 μ L of 3' 10⁸Perml platelets, 10 Xlysate (containing 2mM PMSF, 2mM NaF, 2mM Na)₃VO₄And protease inhibitor) is cracked on ice for 30 minutes, then protein loading buffer solution is added and mixed evenly, after metal bath is carried out for 5 minutes at 99 ℃, protein is separated by SDS-PAGE gel, after the protein is transferred to a PVDF membrane, 5% skimmed milk powder is sealed for 1 hour, the PVDF membrane is incubated with primary antibody (GAPDH, beta-actin, BAK, BAX, BCL-XL, p-VASP (Ser157), VASP, p-PKC substrate, p-GPIb beta (Ser 166), GPIb beta, PDE 3A) and secondary antibody with corresponding dilution concentration, and bands are displayed by ECL luminescence.

2.1.13 detection of the Activity of PKA recombinant proteins

PKA activity was detected by examining PKA activity by ELISA using the PKA activity kit (catalog ADI-EKS-390A; Enzo Life Science Inc).

(1) Reagents contained in the cassette: PKA Substrate microtiter plates, phosphorylated Substrate antibody, Anti-Rabbit IgG: HRP Conj. mu.gate, antibody dilution Buffer, kinase assay dilution Buffer, ATP, Active PKA, 20X Wash Buffer, TMB Substrate, stop Buffer 2

(2) Sample treatment: the activity of the active PKA recombinant protein was detected at 0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃, 10 ℃, 12 ℃, 14 ℃, 15 ℃ and 22 ℃ on the first, second, third, fourth, fifth, sixth and seventh days

(3) The method comprises the following steps:

a. and (3) returning to room temperature: PKA substrate microtiter plates, antibody dilution buffer, kinase assay dilution buffer, TMB substrate and stop buffer 2.

b. Wells of a PKA substrate microtiter plate were soaked with 50 μ Ι _ of kinase assay dilution for 10 min at room temperature. Carefully aspirate the liquid from each well.

c. The treated sample, i.e. 30 μ L of PKA recombinant protein, was added to the appropriate wells of the PKA substrate microtiter plate.

d. The reaction was initiated by adding 10 μ L of diluted ATP (excluding blank samples) to each well.

e. Incubate at 30 ℃ for 90 minutes.

f. The reaction was stopped by emptying the contents of each well.

g. In addition to the blank, 40 μ L of phosphorylated substrate antibody was added to each well.

h. Incubate at room temperature for 60 minutes.

i. Wells were washed 4 times with 100 μ L1X wash buffer.

j. Adding 40 μ L of diluted anti-rabbit IgG to the blank wells: HRP conjugate except blank wells.

k. Incubate at room temperature for 30 minutes.

Wells were washed 4 times with 100 μ L1X wash buffer.

Add 60 μ L TMB substrate to each well.

n. incubate at room temperature for 30 min.

Add 20 μ L of stop solution 2 to each well.

Measure absorbance at 450 nm.

(4) And (3) calculating:

The relative PKA kinase activity was calculated by the following formula: (sample average absorbance-blank average absorbance)/amount of crude protein used per measurement.

2.1.14 PKA Activity assay for stored platelets

Sample treatment: mu.L of 3' 10 was added⁸the/mL hPLT was stored at 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 15 deg.C and 22 deg.C for 1, 2, 3, 4, 5, 6 and 7 days. Preparing 2' lysine buffer, and mixing according to the volume 1: 1 was added to the hPLT sample along with NaF, Na3VO3, PMSF, Cocktail. The mixture is cracked on ice for 30min, centrifuged at 1000g for 10min to obtain supernatant, and the sample is frozen in a refrigerator at minus 80 ℃. The rest steps are the same as above.

2.1.15 cAMP detection

cAMP content was checked by ELISA using cAMP detection kit (catalog RPN 225; GE Healthcare Life Sciences).

(1) Reagents contained in the cassette: a microporous plate coated with the donkey anti-rabbit antibody; assay buffer (0.05M sodium acetate buffer); a cAMP standard; antiserum; rabbit anti-cAMP antibody; cAMP-horseradish peroxidase coupling; a Wash buffer; TMB substrate; lysine reagent 1; lysine reagent 2.

(2) Sample treatment: take 180. mu.L of platelets stored at different temperatures for different periods of time, add 20. mu.L of Lysis reagent 1A, and shake for 10min at room temperature to facilitate platelet Lysis. The completely lysed samples were frozen in a-80 ℃ freezer for use.

(3) Diluting the standard substance:

a. 8 1.5mL EP tubes were labeled with 25, 50, 100, 200, 400, 800, 1600, and 3200 fmol.

b. 500 μ L of Lysis reagent Lysis reagent 1B was transferred into these tubes.

c. Add 500. mu.L CAMP standard (64 pmol/mL) to a 3200 fmol pipette and mix well.

d. 500 μ L was transferred from 3200 fmol tube to 1600 fmol tube and vortexed thoroughly.

e. This double dilution was repeated sequentially with the remaining tubes and vortexed after each dilution.

f. Aliquots of 100. mu.L in each series of dilutions will produce 8 standard levels of cAMP from 25-3200 fmol.

(4) The method comprises the following steps:

a. standards were prepared for Assay buffer, lysine reagent and concentrations ranging from 25-6400 fmol.

b. All reagents were brought to room temperature and mixed thoroughly before use. Especially the enzyme substrate TMB.

c. Microplates with sufficient wells were set up to run all blanks, standards and samples as needed. Recommended substrate blank (B), non-specific binding (NSB), standard (0-6400 fmol) and sample (S) wells.

d. Remove 100. mu.L of lysis reagent 1B and 100. mu.L of lysis reagent 2B into NSB wells.

e. 100 μ L of lysis reagent 1B was pipetted into a zero standard (0) well.

f. Pipette 100. mu.L of each standard into the appropriate well using a clean pipette.

g. Remove 100 μ L of unknown sample into the appropriate well.

h. Remove 100 μ L of Antiserum to all wells except blank and NSB wells.

i. The petri dish was covered with the provided lid, mixed gently, and incubated at 3-5 ℃ for 2 hours.

j. Carefully move 50 μ L of cAMP-peroxidase conjugate into all wells except the blank.

k. Cover the plate, mix gently, incubate for 60 minutes at 3-5 ℃.

Discard supernatant, aspirate and Wash all wells four times with 400 μ L Wash Buffer. The plates were blotted dry on a tissue paper to ensure that all residual volume was removed during blotting. Implement for curing diabetes

Immediately dispense 150 μ Ι _ of enzyme substrate into all wells, cover the plate and mix on a microplate shaker at room temperature (15-30 ℃) for 60 minutes. The mixture will turn blue and read at 630 nm.

(5) And (3) calculating:

a standard curve is calculated and drawn by the following formula: % B/B0 = (standard or sample OD-NSB OD)/(0 standard OD-NSB OD) x 100.

2.1.16 PDE3A Activity assay

PDE activity was examined by ELISA using the PDE activity detection kit (catalog ab 139460; Abcam).

(1) Reagents contained in the cassette: PDE Enzyme; 5' -Nucleotidase (5 kU/. mu.L); 3 ', 5' -cAMP Substrate (1 mM); PDE Assay Buffer; green Assay Reagent; 5' -AMP Standard (100. mu.M); 96-well Clear Microplate.

(2) Sample treatment: taking 100mL of 3' 10⁸Platelets stored at different temperatures for different times per mL were lysed on ice for 30 minutes by adding 15. mu.L of 10 × lysate (containing 2mM PMSF, 2mM NaF, 2mM Na3VO4, and protease inhibitor, IBMX). 1000g, centrifuging for 10min, taking the supernatant, and freezing and storing at-80 ℃ for later use.

(3) Sample desalting (Zeba Spin desalting):

a. the bottom seal of the column or the bottom seal of the plate is removed. The cap is released (and not removed).

b. The column was placed in a 1.5mL centrifuge tube and centrifuged at 1500g for 1min to remove the stock solution.

c. The flow-through was discarded and the column was then replaced in the collection tube.

d. Wash/equilibration buffer was added on top of the resin. Centrifuge the tube and discard the flow through. This step was repeated two more times.

Note that: after each revolution the resin should appear white and free of liquid. If liquid is present, please ensure that the correct centrifugation speed and time are used. Incomplete centrifugation may result in poor sample recovery or dilution of the sample.

e. The bottom of the column or plate is blotted to remove excess liquid. Transfer the column to a new 1.5mL centrifuge tube.

f. The sample was placed on the resin. Centrifuging at 1500g for 2min, and collecting the lower layer liquid.

g. The spin column was discarded.

(4) Preparing a standard curve: preparing 5' -AMP standard substance with the concentration of 75, 50, 37.5, 25, 18.75, 12.5 and 6.25 μ M; 75. concentrations of 5'-AMP standards of 50, 37.5, 25, 18.75, 12.5, and 6.25. mu.M correspond to 3, 2, 1.5, 1.0, 0.75, 0.50, and 0.25 nmol of 5' -AMP, respectively.

a) 80 μ L of 75 μ M5' -AMP standard solution was added to well A of the assay plate, and 80 μ L of 50 μ M standard solution was added to well B.

b) Add 40. mu.L of 1 × PDE assay buffer to the C-to-H wells.

c) Remove 40. mu.L from well A and then add it to well C. They were mixed well by pipetting up and down several times.

d) Remove 40 μ L from the C well and add it to the E well. Mix well E well then remove 40. mu.L and discard.

e) Remove 40 μ L from well B and then add it to well D. It was thoroughly mixed by blowing up and down several times. This procedure was repeated, moving 40 μ L from well D to F, and then from F to G.

Remove 40. mu.L from the G well and discard. Do not enter the blank H well.

f) To each well was added 10. mu.L of 5' -nucleotidase (5 kU/. mu.L), and mixed well.

g) Incubate at 30 ℃ for 30 minutes.

(5) The method comprises the following steps:

a. samples containing the PDE enzyme, substrate and test compound, dissolved in PDE assay buffer, are prepared. Namely cAMP Substrate 20. mu.L + 5' -Nucleotidase (5 kU/. mu.L) 10. mu.L + sample 20. mu.L.

b. The samples were incubated at 30 ℃ for 30 min.

c. The reaction was stopped by adding 100. mu.L of Green Assay Reagent.

Stir plate or gently stir the well for mixing.

Note that: avoiding the generation of bubbles in the pores.

d. Development was carried out for 30 minutes.

e. Microplate reader OD620nm readings.

(6) And (3) calculating: 5' -AMP Release = (OD 620 nm-y intercept)/slope

2.1.17 PKA pathway activation assay

After washing hPLT, it was pretreated with 8-Br-cAMP (12.5. mu.M, 25. mu.M, 50. mu.M) and Forskolin (1.25. mu.M, 1.875. mu.M, 2.5. mu.M) and DMSO (control), and after storing at 6 ℃ for 4 days, it was examined for the apoptosis and activation index of platelets by flow cytometry, and at the same time, protein samples were prepared to examine the effect on PKA by Wsetron blot.

2.1.18 PDE3A Activity inhibition assay

After washing hPLT, it was pretreated with Millinone (6.25. mu.M, 12.5. mu.M, 25. mu.M) and Enoximinone (0.5. mu.M, 1. mu.M, 2. mu.M) and DMSO (control), and stored at 6 ℃ for 4 days, and then the indicators of apoptosis and activation of platelets were examined by flow cytometry, and protein samples were prepared so as to examine the effect on PKA by Wsetren blot.

2.1.19 PKC Activity inhibition assay

Washing hPLT, pre-treating with GO6983 (50 nM, 100nM, 200 nM) and DMSO (control), storing at 6 deg.C for 4 days, detecting the apoptosis and activation of platelets by flow cytometry, and preparing protein samples to detect the effect on PKC by Wsetern blot.

2.1.20 statistical analysis

All data are expressed as mean ± standard deviation and the data are statistically analyzed using one-way analysis of variance or multi-way analysis of variance. Two groups were compared using a two-tailed t-test. The significance of the data was assessed using GraphPad Prism 5 software. When in usepThe difference was significant < 0.05. The different significance levels are p＜0.05，** p＜0.01，***p＜0.001，****pIs less than 0.0001. All experiments requiring the use of animals were randomly grouped by litter size, and the sample size was predetermined based on the variability observed in previous experiments and preliminary data.

2.2 results of the experiment

2.2.1 increase in caspase3 cleavage fragments and increase in apoptosis after hPLT storage for different periods of time at different temperatures

To further demonstrate that platelets undergo apoptosis during storage, expression of the apoptosis executive protein caspase3 was also examined in addition to membrane potential depolarization and PS eversion. During apoptosis, mitochondrial dysfunction triggers bioenergetic destruction, ultimately leading to disruption of plasma membrane integrity and resulting morphological changes. Mitochondrial Δ Ψ m depolarization is located upstream of the caspase-3 signaling pathway, and caspase-3 is one of the executives of the caspase family, leading to cell disassembly and collapse, and is the executive protein of apoptosis. As shown in FIG. 8A, it was found that no cleavage of caspase3 was detected after 1, 2, 3, 4, 5, and 6 days of storage of platelets stored at 4 ℃ and that cleavage of caspase3 was detected on the seventh day of storage; after the platelets preserved at the temperature of 6 ℃ are preserved for 1, 2, 3, 4, 5 and 6 days, no enzyme digestion fragment of caspase3 is found; after the platelets preserved at the temperature of 22 ℃ are preserved for 1, 2 and 3 days, no enzyme-cut fragment of caspase3 is found, but after the platelets are preserved for 4, 5, 6 and 7 days, an enzyme-cut fragment of obvious caspase3 is detected, and the enzyme-cut fragments increase along with the prolonging of the preservation time.

Also, the change in the apoptosis executive protein caspase3 when stored for the same time at different temperatures, i.e., 4 ℃, 6 ℃ and 22 ℃, was examined. As shown in FIG. 8B, no cleavage bands were detected in any of the three temperature-stored platelet caspase3 on days 1, 2, and 3 of storage. Caspase3 bands were detected on day 4, 5, 6, and 7 of storage by storing platelets at 22 ℃. These results further indicate that platelets undergo apoptosis during long-term storage, but platelets stored at different temperatures undergo apoptosis to a different extent, with platelets stored at 22 ℃ undergoing apoptosis earliest and increasing in time. Platelets stored at 4 ℃ began to undergo apoptosis on day seven. The platelets stored at 6 ℃ did not detect the caspase3 band within seven days of storage. These results also indirectly indicate that 6 ℃ is the optimal temperature for preserving platelets.

2.2.2 detection of apoptosis-related proteins after seven days of continuous hPLT storage

Next, the molecular mechanism of apoptosis during platelet storage was investigated. It has been shown that when platelets are apoptotic when stored at room temperature, the expression of the BCL-2 family of proteins, including BCL-XL and BAX proteins, decreases over time, but the decrease in BAK expression occurs at a later time point, indicating that BCL-XL, BAX, BAK are degraded. Therefore, the expression of BCL-XL, BAX and BAK proteins in the platelets preserved at different temperatures is detected.

Consistent with previous studies, platelet BCL-XL, BAK, BAX protein expression at 22 ℃ storage decreased with longer storage time. Protein expression of BCL-XL decreased from day 6 and more at day 7. BAX protein expression decreased from day 4, and the decrease was more and more pronounced with longer storage time. Protein expression of BAK decreased from day 6. However, protein expression of BCL-XL, BAK and BAX was not changed from the first day to the seventh day of storage in platelets stored at 4 ℃ and 6 ℃ (FIG. 9A). Meanwhile, the protein expression conditions of the platelets BCL-XL, BAK and BAX stored at different temperatures after the same time of storage are also detected. As shown in FIG. 9B, the expression levels of BAX, BAK, and BCL-XL were consistent in platelets stored at 4 ℃ and 6 ℃ on days 6 and 7, while the expression levels of BAX, BAK, and BCL-XL were lower in platelets stored at 22 ℃ than in platelets at 4 ℃ and 6 ℃. It was preliminary shown that platelets stored at different temperatures have different mechanisms of apoptosis.

2.2.3 PKA Activity after seven consecutive days of hPLT storage gradually decreased with time

According to earlier researches, PKA activity is found to be a main regulation link for regulating platelet apoptosis, and thus, it is suspected that apoptosis caused by cryopreserving platelets can be caused by the fact that PKA activity is reduced along with the prolonging of preservation time. PKA activity can be expressed in terms of phosphorylation of the PKA substrate GPIb β at Ser166 (p-GPIb β (Ser 166)) and phosphorylation of VASP at Ser157 (pVASP (Ser 157)). When PKA activity is reduced, GPIb β (Ser 166) and pVASP (Ser 157) are dephosphorylated.

As shown in fig. 10A, the PKA activity decreased to different degrees with the time of storage of the platelets stored at 4 ℃, 6 ℃ and 22 ℃, the platelets pVASP (Ser 157) stored at 4 ℃ underwent significant dephosphorylation from day 4, the platelets pVASP (Ser 157) stored at 4 ℃ underwent dephosphorylation from day six, and the total protein expression of the VASP was unchanged, indicating that the PKA activity decreased. Although the phosphorylation of platelet pVASP (Ser 157) stored at 22 ℃ decreased, the total VASP protein expression also decreased at the same time, indicating that the decrease in PKA activity may be due to protein degradation. Similarly, although dephosphorylation occurred at different times, GPIb β (Ser 166) also dephosphorylated with longer storage time, indicating a decrease in PKA activity.

Meanwhile, the protein expression conditions of the platelets BCL-XL, BAK and BAX stored at different temperatures after the same time of storage are also detected. As shown in FIG. 10B, the level of pVASP (Ser 157) was significantly reduced in the platelets stored at 4 ℃, 6 ℃ and 22 ℃ on days 4, 5, 6 and 7 of storage, but the reduction at 6 ℃ was most gradual, and the expression of VASP was consistent in the platelets stored at 4 ℃ and 6 ℃, and the expression level of VASP was lower in the platelets stored at 22 ℃ than in the platelets at 4 ℃ and 6 ℃. It was further shown that apoptosis of platelets stored at 4 ℃ and 6 ℃ was due to a decrease in PKA activity, whereas 22 ℃ was due to degradation of proteins.

2.2.4 hPLT seven days after continuous storage PKA activity gradually decreased with time

To further tamp the protein results of the Western Blot assay described above, the change in PKA activity was next detected using an ELISA enzyme-linked assay kit. As shown in fig. 11, based on the platelet PKA activity after one day of storage, the platelet PKA activities at 4 ℃ and 6 ℃ decreased gradually with the increase of the storage time, but the decrease rate of the platelet PKA activity at 6 ℃ was relatively slow, and the PKA activity decreased gradually and at a rate higher than 4 ℃ with the platelets stored at 22 ℃ for 1, 2, 3, 4, and 5 days, but the platelet PKA activity did not decrease at 6 and 7 days, which was inversely proportional to the increase at 5 days, and this may be related to the storage state of the platelets. After 6 and 7 days of storage at 22 ℃ the platelets are somewhat activated and even aggregate formation is associated with an increased frequency of PKA activity.

2.2.5 PKAC alpha recombinant protein after seven days of continuous storage, the PKA activity gradually decreased with time

The above results have revealed that a decrease in PKA activity is a regulatory link in platelet apoptosis. To investigate whether PKA is a direct influence factor for different degrees of apoptosis of platelets stored at different temperatures. Constructing active PKAC alpha recombinant protein, continuously storing PKAC alpha at different temperatures including 4 deg.c, 6 deg.c and 22 deg.c for 7 days, and detecting the PKA activity with ELISA enzyme linked detection kit for seven days.

As shown in fig. 12, the activity of the PKAC α recombinant proteins stored at different temperatures decreased with the increase in the storage time, but the decrease rates of the activity of the PKAC α recombinant proteins stored at the temperatures of 4 ℃, 6 ℃ and 22 ℃ were very small, indicating that the decrease in the PKA activity was not a direct factor affecting the apoptosis of the stored platelets.

2.2.6 hPLT continuous storage seven days later, cAMP content gradually decreases with storage time

The PKA activity change result of the PKAC alpha recombinant protein stored for seven days at different temperatures shows that the PKA activity reduction is a downstream link of the temperature influence on the platelet apoptosis. The upstream links of the PKA pathway are discussed further below. Increased or decreased cAMP binds in a synergistic manner to PKA (cAMP dependent kinase) resulting in its activation or inactivation. Then, platelet samples stored at 4 ℃, 6 ℃ and 22 ℃ for 7 days were continuously collected, and the cAMP content of platelets stored at different temperatures for different times was measured. As shown in FIG. 13, the cAMP content was gradually decreased in platelets stored at 4 ℃, 6 ℃ and 22 ℃ as the storage time was prolonged. In addition, the decrease rate of cAMP content of platelets stored at 22 ℃ is the fastest, 4 ℃ times the decrease rate of cAMP content of platelets stored at 6 ℃ is the slowest, and the decrease rate of cAMP content of platelets stored at different temperatures is consistent with the decrease rate of PKA activity of platelets stored at different temperatures along with the time.

2.2.7 hPLT seven days after continuous storage PDE Activity increases with extended storage time

The above results indicate that the decrease in cAMP levels is responsible for different degrees of apoptosis in platelets stored at different temperatures, followed by the detection of the upstream mechanism of cAMP. Intracellular levels of cAMP are dependent on the hydrolysis of Phosphodiesterases (PDEs) and the synthesis of Adenylate Cyclase (AC). Since PDE3A is the most abundant PDE in platelets, changes in PDE3A activity were subsequently detected in platelets stored at 4 deg.C, 6 deg.C and 22 deg.C for 7 days.

As shown in fig. 14, the PDE activity in platelets increased in fold with increasing storage time at different temperatures. And platelets stored at 4 ℃ increased with days, with a rate of PDE activity increase higher than 6 ℃, consistent with a trend of decreasing cAMP levels being detected. However, platelets stored at 22 ℃ showed an increase in PDE activity over time, but the increase did not match the decrease in cAMP.

2.2.8 detection of Total protein level in PDE3A after seven consecutive days of hPLT storage

As a result of the above PDE activity assay, we found that platelets stored at 22 ℃ had increased PDE activity with prolonged storage time, but this was not as expected. It is hypothesized that this phenomenon may be associated with degradation of proteins over time in platelets stored at 22 ℃.

As shown in FIG. 15A, the protein expression of PDE3A began to gradually decrease in platelets stored at 22 ℃ from day 2, whereas the expression of PDE3A was unchanged in platelets stored at 4 ℃ and 6 ℃ from day 1 to day 7. Meanwhile, the protein expression of platelet PDE3A stored at different temperatures after the same time is tested. As shown in FIG. 15B, the expression of PDE3A was consistent between platelets stored at 4 ℃ and 6 ℃ at 7 days of storage, whereas PDE3A expression levels were lower in platelets stored at 22 ℃ than in platelets at 4 ℃ and 6 ℃, indicating that the PDE3A protein was degraded in platelets stored at 22 ℃.

2.2.9 PKA activator 8-Br-cAMP pretreatment of hPLT can partially restore platelet-induced apoptosis activation

The above results have revealed that preservation of platelets at different temperatures can further enhance the activity of PDE3A by activating the PKC pathway, thereby accelerating the hydrolysis of cAMP, resulting in a decrease in PKA activity, and thus leading to apoptosis of platelets to varying degrees. To further tamp this result, the stored platelets were then pretreated with PKA activators, PDE3A inhibitors and PKC inhibitors, respectively, and Western blot assays for effects on downstream PKA or PKC activity; and simultaneously detecting the change of the platelet apoptosis activation index pretreated by the activator or the inhibitor by using a flow cytometer. This section mainly discusses the change in the index of apoptosis activation after platelets were pretreated with different concentrations of cAMP stimulator, i.e., PKA activator 8-Br-cAMP, and stored at 6 ℃ for 4 days. As shown in FIG. 16, 8-Br-cAMP can dose-dependently activate PKA pathway and inhibit the degree of apoptosis activation of platelets stored at 6 ℃.

2.2.10 partial Retention of platelet-induced activation of apoptosis after pretreatment of hPLT with the PKA activator forkolin

Forskolin is an activator of Adenosine Cyclase (AC) that stimulates cAMP production to further activate the PKA pathway. The extent of apoptotic activation of platelets was dose-dependently reversed after storage of platelets for 4 days at 6 ℃ with three concentrations of forskolin pre-treated platelets selected. The activity of PKA was also reflected in p-GPIb β (Ser 166) and pVASP (Ser 157), and PKA was found to be dose-dependent activated, suggesting that reversal of platelet apoptotic activation is the result of enhanced PKA activity (figure 17).

2.2.11 partial reversion of platelet-induced apoptosis activation following Milirone pretreatment of hPLT as PDE3A inhibitor

Milirone is an inhibitor of PPDE3A, which inhibits the activity of PDE3A by a decrease in cAMP hydrolysis, thereby indirectly increasing PKA activity. In this section, 6.25. mu.M, 12.5. mu.M and 25. mu.M Milirone-pretreated platelets were selected, DMSO group was used as a control, the platelets were stored at 6 ℃ for 4 days, and the apoptosis activation results of the platelets were examined by flow cytometry, and it was confirmed from the protein level that the PKA pathway was activated by the selected concentration of Milirone. As shown in fig. 18, Milirone can dose-dependently activate the PKA pathway and inhibit the extent of apoptotic activation of platelets stored at 6 ℃.

2.2.12 partial reversion to platelet-induced apoptosis activation following Enoxidone pretreatment of hPLT with PDE3A inhibitors

Enoximinone is also an inhibitor of PDE3A, in the research, the platelets are pretreated by selecting three concentrations of Enoximinon, namely 0.5 mu M, 1 mu M and 2 mu M, and the influence of Western blot detection on the activity of downstream PKA is detected; and simultaneously detecting the platelet apoptosis activation index by a flow cytometer. As shown in fig. 19, Enoximone can dose-dependently activate the PKA pathway and inhibit the extent of platelet apoptosis stored at 6 ℃, but had no effect on the level of platelet activation.

2.2.13 hPLT seven days after continuous preservation, PKC pathway is gradually activated with time

The above results have demonstrated that increased PDE3A activity is a major component in the development of different degrees of apoptosis in platelets after storage at different temperatures. Next, it is further examined why the activity of PDE3A increases after treatment at different temperatures. The literature has reported. PDE3A activation requires activation of PKC, and PDE3A is a target for PKC in platelets. Thus, the change in PKC activity was measured at different temperatures and for different time periods for platelets. As shown in FIG. 20A, the PKC activity of platelets stored at 4 ℃ and 6 ℃ was gradually enhanced with prolonged storage time. And platelet PKC activity was greater than 6 ℃ from day one to day seven of storage at 4 ℃ (fig. 20B).

2.2.14 PKC inhibitor GO6983 post-hPLT pretreatment activation of PKA pathway

The above results have demonstrated that platelet preservation time is prolonged, PKA activity is decreased, PKC activity is enhanced, and activation of PDE3A requires enhanced PKC activity. To further verify that PKC is upstream of PKA. After platelets were pretreated with different concentrations of PKC inhibitor GO6983 and then stored for 4 days, PKA and PKC activities were simultaneously detected, and the results showed that PKA activity was increased after PKC activity was inhibited, indicating that PKC is an upstream signaling pathway for platelet apoptosis resulting from storage (fig. 21).

Finally, the PKC inhibitor GO6983 is used for pretreating platelets, and Western blot is used for detecting the influence of GO6983 on the activity of downstream PKC, so that the inhibition of the PKC activity is determined. The flow cytometer detects the apoptosis activation index of platelets and further verifies the enzyme-cleaved fragment of the apoptosis executive protein caspase3 from the protein level. As shown in fig. 22, GO6983 can dose-dependently inhibit PKC activity and inhibit levels of apoptotic activation of platelets.

2.2.16 Bad knockout mice show less post-platelet-storage apoptosis activation than WT mice

Previous studies have found that PKA activity leads to dephosphorylation of downstream Bad ser155, serine at sites 112, 136 and 155 of pro-apoptotic protein Bad can be phosphorylated under the action of Protein Kinase A (PKA) and Akt, so that a Bcl-xL/Bad heterodimer complex is depolymerized, phosphorylated Bad can be combined with 14-3-3 to stay in cytoplasm, and released anti-apoptotic protein Bcl-xL is combined with Bak on the outer mitochondrial membrane, so that the occurrence of mitochondrial pathway apoptosis is inhibited. Therefore, the phosphorylation level of the pro-apoptotic protein Bad plays a key regulatory role in the occurrence of platelet apoptosis. In addition, anti-GPIb α antibody-induced apoptotic events were significantly reduced in platelets from Bad knockout mice that lack PKA substrates.

Therefore, the detection of apoptosis activation indexes after the platelets of WT mice and Bad knockout mice are stored at different temperatures is discussed. As shown in FIG. 23, it can be seen that platelets from WT mice stored at 22 ℃ showed higher and higher IIBIIIA levels with increased membrane potential depolarization, PS eversion, CD62P release and activation as the storage time increased. The Bad mouse blood platelet stored at the same temperature has a slow increase of the apoptosis activation index along with the prolonging of the storage time, has a lower apoptosis activation degree than the WT mouse blood platelet, and has statistical significance. The WT mouse platelets stored at 4 ℃ and 6 ℃ have little change in membrane potential depolarization and CD62P release levels with the storage time being prolonged, the PS valgus level begins to increase obviously until day 7, and the activated IIBIIIA level gradually rises with the storage time being prolonged. The IIBIIIA levels of membrane potential depolarization, PS eversion, CD62P release and activation of Bad mouse platelets stored at the same temperature were essentially identical to WT.

In conclusion, the following conclusions can be drawn in this section by studying the mechanism of apoptosis in platelet preservation: apoptosis caused by platelet preservation at low temperature (4 ℃ and 6 ℃) is caused by PKC pathway activation, PDE3A is further activated, PKA activity is reduced due to cAMP hydrolysis increase, but platelet preservation at 6 ℃ has better platelet state due to lower activation degree of PKC pathway. Apoptosis due to the storage of platelets at 22 ℃ is due on the one hand to changes in enzyme activity and on the other hand to degradation of the proteins due to prolonged storage times.