Application of pentose phosphate pathway regulator in resisting platelet storage injury

Abstract

The invention relates to the field of biological medicine, and provides application of a pentose phosphate pathway regulator in resisting platelet storage damage. According to the invention, a non-targeted metabonomics technology is utilized to systematically analyze the metabolic change of the singly-collected platelets during the in-vitro storage period. The relationship between the blood platelet storage damage and the blood platelet storage damage is discussed by intervening the significantly enriched metabolic pathway in the blood platelet storage process. Research finds that the pentose phosphate pathway (PPP) has a potential effect in regulating platelet storage damage, and a new research idea is provided for improving the platelet storage quality by intervening the platelet metabolism process.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and relates to the application of pentose phosphate pathway regulators in anti-platelet storage injury. Background Art

[0002] Platelet transfusion, as a treatment method, is widely used in the treatment of patients with thrombocytopenia, blood diseases, cancers, etc. [1,2] . Platelets are formed in vivo by the cytoplasmic shedding of bone marrow megakaryocytes and lung megakaryocytes [3,4] , circulate in the blood for 7 to 10 days, and are then recognized and cleared by hepatocytes and hepatic macrophages due to activation or senescence [5-8] . During in vitro storage, due to the active metabolism of platelets and the fact that cold storage can lead to the rapid clearance of platelets after transfusion, its transfusion effect is often affected [8,9] . Clinically, platelets are usually placed in a special breathable storage bag and stored with oscillation at a temperature of 22±2°C, and the storage time generally does not exceed 5 days. Platelet storage lesion (PSL) refers to the structural and functional changes of platelets caused by factors such as storage environment or metabolic changes during in vitro storage, and its manifestations include impaired mitochondrial function, increased generation of mtROS (mitochondrial reactive oxygen species), apoptosis, shedding of surface membrane proteins, and release of microvesicles, etc. [10-12] . Platelet storage lesion significantly affects its lifespan and transfusion effect. Therefore, exploring intervention means that can regulate platelet storage lesion has important clinical significance for optimizing the clinical application of platelets.

[0003] Many studies have shown that during the in vitro storage of platelets, significant changes occur in their intracellular metabolic levels, including the rapid consumption of metabolites such as glucose, glutamine, acetate, etc., and the accumulation of lactate, etc. [13,14] Mitochondrial oxidative phosphorylation and aerobic glycolysis are the main energy sources of platelets

[15] . Fatty acids, as important substrates for mitochondrial energy metabolism, have been found in some studies that their metabolism is inhibited during storage, and further exacerbates mitochondrial damage [16,17] . Reactive oxygen species (ROS) are a class of oxygen-containing chemical substances, mainly generated during the electron transfer process on the inner mitochondrial membrane, and participate in processes such as intracellular signal transduction and oxidative stress

[18] . However, some studies have found that the level of ROS increases significantly during platelet storage, and has an adverse effect on platelet quality [19,20] . These research results indicate that there is a potential association between changes in cell metabolism and platelet storage lesion. However, how to relieve platelet storage lesion by regulating cell metabolism is still unclear at present. Summary of the Invention

[0004] The present invention is based on the above research. In view of the technical problem of platelet storage damage, it provides the application of pentose phosphate pathway regulators in anti-platelet storage damage. The non-targeted metabolomics technology was used to systematically analyze the metabolic changes of apheresis platelets during in vitro storage. By intervening in the metabolic pathways significantly enriched during platelet preservation, the relationship between them and platelet storage damage was explored. It was found that the pentose phosphate pathway (PPP) has a potential role in regulating platelet storage damage, providing a new research idea for improving the quality of platelet preservation by intervening in the platelet metabolic process.

[0005] The research process of the present invention is as follows: First, it was found that the metabolic characteristics of platelets changed significantly during preservation. Further, through KEGG pathway enrichment analysis, it was found that the pentose phosphate pathway (PPP), arachidonic acid metabolism pathway, and linoleic acid metabolism pathway were significantly enriched during preservation. Among them, the activity of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose phosphate pathway, increased significantly at the initial stage of platelet preservation. Subsequently, after inhibiting the function of G6PD with a G6PD-specific inhibitor, the mitochondrial function of platelets was damaged, and at the same time, the degree of phosphatidylserine externalization increased significantly; on the contrary, after further using the G6PD activator AG1 to enhance the G6PD activity, the generation of mitochondrial reactive oxygen species (ROS) could be significantly inhibited, and the externalization of phosphatidylserine could be effectively alleviated. In addition, by constructing a platelet-specific G6PD knockout mouse model and conducting in vitro preservation experiments on its platelets, obvious platelet storage damage was also shown. In summary, the research of the present invention reveals the key role of PPP in regulating platelet storage damage, providing a potential target for improving the quality of platelet storage.

[0006] Based on the above research, the technical solutions to be protected by the present invention are as follows:

[0007] The main purpose of the present invention is to provide the application of pentose phosphate pathway regulators in the preparation of anti-platelet storage damage preparations.

[0008] In the first aspect disclosed by the present invention, it provides the uses of G6PD, the nucleic acid molecule encoding this protein, and its promoter in the preparation of anti-platelet storage damage drugs.

[0009] In the first aspect disclosed by the present invention, it also provides a product with G6PD, the nucleic acid molecule encoding this protein, and its promoter as active components.

[0010] In the third aspect disclosed by the present invention, it also provides a platelet storage method, which includes applying an effective amount of G6PD, the nucleic acid molecule encoding this protein, its promoter, or its recombinant expression vector into the container for storing platelets in vitro.

[0011] Preferably, the G6PD promoter is selected from any one or more of the following: exogenous G6PD protein or nucleic acid encoding the same, substances that promote the expression or active function of G6PD protein, substances that promote the overexpression of nucleic acid molecules encoding G6PD protein, or liposomes or nanomaterials encapsulating nucleic acids encoding G6PD protein.

[0012] More preferably, the G6PD protein is selected from any of the following:

[0013] (a) a polypeptide having an amino acid sequence shown in any one of SEQ ID NO.1 - 3, and the sequences of the RNAs corresponding to the polypeptide are shown in SEQ ID NO.4 - 6 respectively;

[0014] (b) a substance having homology or sequence identity with the amino acid sequence shown in any one of SEQ ID NO.1 - 3 and having the function of promoting G6PD;

[0015] (c) a substance in which one or several amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b) and having the function of promoting G6PD, or a protein or polypeptide derived from (a) or (b),

[0016] The nucleic acid molecule encoding G6PD protein is selected from any of the following:

[0017] (i) a nucleic acid molecule having the nucleotide sequence shown by Genbank accession number NG_009015.2;

[0018] (ii) a molecule that hybridizes with the nucleotide sequence defined in (i) under stringent conditions;

[0019] (iii) a nucleic acid molecule having homology or sequence identity with the nucleotide sequence shown by NG_009015.2;

[0020] (iv) a nucleic acid molecule in which one or several nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii).

[0021] The substance that promotes the expression or active function of G6PD protein is selected from G6PD activator AG1.

[0022] In some embodiments, G6PD is: a naturally purified protein, a chemically synthesized product, or a product produced using recombinant techniques from prokaryotic or eukaryotic hosts. The host is selected from: bacteria, yeast, higher animals and mammalian cells. Preferably, it is human G6PD.

[0023] Furthermore, the present invention also provides a G6PD recombinant expression vector, comprising an expression vector and a nucleic acid molecule encoding G6PD protein inserted into the expression vector, and the nucleic acid molecule encoding G6PD protein is as described above.

[0024] Among them, the expression vector is a conventional vector such as a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the specific type is selected from the prior art according to the actual situation. The "viral vector" includes adeno-associated virus and lentivirus. Suitable viral vectors are well known to those of ordinary skill in the art. The remaining "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers and nanoparticle carriers, etc. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0025] In the second aspect disclosed by the present invention, a product for resisting platelet storage damage is provided, which comprises an active component and a pharmaceutically or immunologically acceptable carrier or excipient, and the active component includes the above-mentioned G6PD promoter or G6PD recombinant expression vector.

[0026] Preferably, the pharmaceutical composition or kit of the present invention comprises:

[0027] (A) A therapeutically or prophylactically effective amount of G6PD, a nucleic acid molecule encoding this protein, its promoter and / or its inhibitor herein;

[0028] (B) A carrier or excipient that helps maintain the activity of the active component in vitro and is harmless to cells.

[0029] When used in the process of in vitro preservation of platelets, the product is directly applied into the platelet storage container, and the application amount can be obtained through experimental exploration.

[0030] In terms of the application time, it is found that in the initial stage of platelet preservation (days 1-3), significant changes occurred in PPP, manifested as a significant increase in the levels of the intermediate metabolite 6-phosphogluconic acid and the end product glyceraldehyde 3-phosphate. As a key enzyme of PPP, G6PD shows dynamic activity changes during platelet preservation. The experimental results show that the G6PD activity significantly increases on the second day of preservation, then gradually decreases with the prolongation of the preservation time, and is lower than the initial level at the end of the preservation. Therefore, it is appropriate to apply at the end of the second day of preservation.

[0031] Beneficial guarantees and effects of the present invention:

[0032] The present invention verifies the crucial role of PPP in platelet storage. By constructing megakaryocyte / platelet-specific G6PD knockout mice (G6PDPF4-cre), it is shown that G6PD knockout platelets exhibit obvious characteristics of storage damage during in vitro preservation, further demonstrating the important position of PPP in regulating platelet storage damage. Further, the present invention reveals the mechanism by which PPP regulates storage damage by maintaining redox balance during platelet storage, and this finding provides a new research direction and potential intervention strategies for improving the quality of platelet preservation. Brief Description of the Drawings

[0033] The following further illustrates the present disclosure in conjunction with the drawings, where these displays are only for illustrating the embodiments of the present disclosure and not for limiting the scope of the present disclosure.

[0034] Figure 1 It shows that during the in vitro preservation of platelets, the metabolite levels in platelets under positive ion mode show a stable and significant change trend: (A, D) Principal component analysis of the overall metabolites in platelets and plasma identified under positive ion mode. The abscissa represents the explanatory degree of the first principal component, and the ordinate represents the explanatory degree of the second principal component. The points represent experimental samples, and the colors represent different groups. The more the samples within a group are aggregated and the more dispersed the samples between groups are, the more reliable the results are; (B, E) Partial least squares discriminant analysis of the overall metabolites in platelets and plasma identified under positive ion mode; (C, F) After performing differential analysis on the metabolites identified by mass spectrometry in platelets and plasma at different time points, an intersection is made. Yellow, green, and blue are the numbers of metabolites obtained by performing differential analysis on the metabolites identified on the first day and the third day, the third day and the fifth day, and during the entire preservation period, respectively.

[0035] Figure 2 It shows that during the in vitro preservation of platelets, the metabolite levels in platelets under negative ion mode show a stable and significant change trend: (A, C) Principal component analysis of the overall metabolites in platelets and plasma identified under negative ion mode. The abscissa represents the explanatory degree of the first principal component, and the ordinate represents the explanatory degree of the second principal component. The points represent experimental samples, and the colors represent different groups. The more the samples within a group are aggregated and the more dispersed the samples between groups are, the more reliable the results are; (B, D) Partial least squares discriminant analysis of the overall metabolites in platelets and plasma identified under negative ion mode.

[0036] Figure 3Shows metabolites with significant changes during platelet storage in vitro: (A) Heat map of overall differential metabolites in platelets during apheresis platelet storage in vitro. Note: The relative content in the figure is shown by different colors. The redder the color, the higher the expression level, and the bluer the color, the lower the expression level. Among them, the columns represent samples, the rows represent metabolite names, and the clustering tree on the left side of the figure is the clustering tree of differential metabolites. The same applies hereinafter. (B) Heat map of overall differential metabolites in plasma during apheresis platelet storage in vitro.

[0037] Figure 4 Shows that during the initial stage of platelet storage (days 1 to 3), 6 metabolic pathways such as the pentose phosphate pathway and arachidonic acid metabolism were significantly enriched: (A) Results of KEGG pathway enrichment analysis of metabolites obtained from differential analysis of platelets stored for 1 day and 3 days. The color depth represents the P value, and the numbers represent the number of differential metabolites contained in the pathway; (B) Results of KEGG pathway enrichment of metabolites obtained from differential analysis of plasma of apheresis platelets stored for 1 day and 3 days.

[0038] Figure 5 Shows metabolic pathways with significant changes during platelet storage in vitro: (A) Results of KEGG pathway enrichment analysis of metabolites obtained from differential analysis of platelets stored for 3 days and 5 days. The color depth represents the P value, and the numbers represent the number of differential metabolites contained in the pathway. (B) Results of KEGG pathway enrichment of metabolites obtained from differential analysis of plasma of apheresis platelets stored for 1 day and 5 days.

[0039] Figure 6 Shows that the activities of G6PD (glucose-6-phosphate dehydrogenase) and COX1 (cyclooxygenase 1) were significantly increased during the initial stage of platelet storage: (A - B) Commercial kits were used to detect the activities of G6PD and COX1 during the in vitro storage of apheresis platelets; (C - E) Changes in the relative levels of 6-phospho-D-gluconic acid and cycloendoperoxides PGG2 and PGB2 in platelets during in vitro storage.

[0040] Figure 7 Shows that G6PDi significantly promoted the externalization of platelet phosphatidylserine, increased the generation of mitochondrial ROS, and exacerbated the depolarization of mitochondrial membrane potential: (A - C) The externalization rate of platelet phosphatidic acid serine, mitochondrial ROS, and mitochondrial membrane potential were detected 1 hour, 3 days, and 5 days after adding 1 μm and 10 μm G6PD; (D - F) The externalization rate of platelet phosphatidic acid serine, mitochondrial ROS, and mitochondrial membrane potential were detected 1 hour, 3 days, and 5 days after adding 100 μm Aspirin.

[0041] Figure 8It shows that increasing G6PD activity can effectively alleviate platelet storage damage: (A-C) The phosphatidylserine externalization rate, mitochondrial ROS, and mitochondrial membrane potential of platelets were detected 1 hour, 3 days, and 5 days after adding 5 mM Acetylcysteine (NAC) and 10 μM G6PD activator AG1 respectively; (D-F) After obtaining washed platelets, they were stored in a 37°C constant temperature water bath, and the phosphatidylserine externalization rate, mitochondrial ROS, and mitochondrial membrane potential of platelets were detected during storage. Detailed implementation manners

[0042] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications.

[0043] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the detailed implementation manners are for illustrative purposes only.

[0044] I. Materials and methods

[0045] 1. Collection and omics submission of platelet and plasma samples

[0046] Apheresis platelets were from the Shanghai Blood Center, and the blood donors had signed the "Blood Donor Informed Consent and Health Status Inquiry Form", agreeing that the blood center could conduct scientific research-related tests and use of blood specimens according to regulations. This scientific research was approved by the Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. (2022) Lin Lun Shen No. (168)). Apheresis platelets were stored in standard disposable gas-permeable platelet storage bags, stored with oscillation at 22 ± 2°C, and quantitative platelets and an equal volume of supernatant (plasma) were centrifuged on the 1st day, 3rd day, and 5th day, and the metabolism was stopped by rapid freezing in liquid nitrogen. Before detection, 100 mg of glass beads and 1 ml of acetonitrile: methanol: H 2O mixed solution (2:2:1, v / v / v), vortex for 30 s; then freeze in liquid nitrogen for 5 minutes, take out and thaw at room temperature, and grind with a tissue grinder at 55 Hz for 2 min. Repeat the liquid nitrogen freezing and grinding steps 3 times, and then centrifuge at 12,000 rpm and 4 °C for 10 min. After concentrating and drying the supernatant, add 300 μL of a solution of 2-chloro-L-phenylalanine (4 ppm) containing acetonitrile: 0.1% formic acid (1:9, v / v) pre-cooled at 4 °C to re-dissolve the sample. Filter the supernatant through a 0.22 μm membrane, and add the filtrate to a detection bottle and wait for on-machine detection.

[0047] 2. In vitro storage of platelets

[0048] Mouse whole blood was centrifuged at 250 g for 10 min at room temperature to obtain platelet-rich plasma, and further centrifuged at 800 g for 10 min to obtain washed platelets, which were transferred to a 50 ml centrifuge tube. Cover the mouth of the centrifuge tube with a pre-sterilized breathable membrane and tightly tie the breathable membrane with a rubber band. Place the centrifuge tube at a 30° angle on a horizontal shaker for shaking and storage.

[0049] 3. Flow cytometry

[0050] Apoptosis, mitochondrial reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) of platelets were detected by flow cytometry (Becton, Dickinson and Company, BD). Apoptosis on the platelet surface was detected by the exposure of annexin V. Mitochondrial ROS was detected by mitoSOX, which selectively targets mitochondria. MMP was measured by TMRM. Platelets (5×10 7 platelets per milliliter) were stained with annexin V-FITC, mitoSOX, and TMRM in the dark at 37 °C for 30 minutes, and the staining system was 50 μL. The reaction was stopped by adding 450 μL of Tyrode buffer, and then analyzed by flow cytometry.

[0051] II. Detection results

[0052] 1. Overall metabolic characteristics during in vitro storage of platelets

[0053] To visually display the changing characteristics of the overall metabolite levels of platelets and plasma during in vitro storage of apheresis platelets, we used non-targeted metabolomics technology to systematically analyze the metabolite information of apheresis platelets from 6 healthy blood donors at different in vitro storage times. Based on the metabolome data, we used unsupervised pattern recognition methods (principal component analysis, PCA) and supervised pattern recognition methods (partial least squares discriminant analysis, PLS-DA) to perform multivariate statistical analysis on the metabolic changes. The results showed that during the in vitro storage of platelets, the metabolite levels in platelets showed a stable and significant changing trend ( Figure 1 A, B, Figure 2 A, B). Similarly, obvious differences in metabolite levels were also observed in plasma ( Figure 1 D, E, Figure 2 C, D). The above results indicate that during the in vitro storage of apheresis platelets, the metabolic level of platelets does not remain constant, but changes dynamically with the extension of storage time.

[0054] 2. Metabolite changes and KEGG pathway enrichment analysis during in vitro storage of platelets

[0055] Through liquid chromatography separation and mass spectrometry analysis, a total of 4832 metabolites were identified in platelets, and 44 metabolites showed significant differential changes during in vitro storage ( Figure 1 C). Among them, the levels of 18 metabolites such as Prostaglandin B2 (PGB2) and 6-Phosphogluconic acid (6PG) continued to increase with the extension of storage time; while the levels of 5 metabolites such as Sphingosine-1-phosphate (S1P) and N-Acetyl-L-glutamate continued to decrease ( Figure 3 A).

[0056] In plasma, a total of 6468 metabolites were identified, and 108 metabolites showed significant changes during in vitro storage ( Figure 1 F). Among them, the levels of 36 metabolites such as D-Ribose and the arachidonic acid metabolites 11,12-DiHETrE and 8,9-DiHETrE increased significantly; while the levels of 38 metabolites such as Prostaglandin E2 (PGE2), the linoleic acid metabolite Gamma-Linolenic acid, conjugated linoleic acid (10E,12Z-Octadecadienoic acid), and 9-OxoODE gradually decreased ( Figure 3 B).

[0057] To deeply explore the potential biological significance of the above differential metabolites in platelet preservation and metabolism, we adopted KEGG pathway enrichment analysis. The analysis results showed that in the initial stage of platelet preservation (days 1 to 3), 6 metabolic pathways such as the pentose phosphate pathway and arachidonic acid metabolism changed significantly ( Figure 4 A), suggesting that there was a certain degree of spontaneous activation of platelets in the initial stage of preservation. Compared with day 3, the metabolic changes of platelets were more severe after 5 days of in vitro preservation. 14 metabolic pathways, including platelet activation, linoleic acid metabolism, purine metabolism, and the pentose phosphate pathway, showed significant changes ( Figure 5 A), indicating that in the later stage of preservation, platelets underwent metabolic stress and were further activated. Compared with platelets, the metabolic changes in plasma were more significant. A total of 15 metabolic pathways changed significantly during preservation, including metabolic pathways such as linoleic acid metabolism and arachidonic acid metabolism ( Figure 4 B, Figure 5 B). It is worth noting that the linoleic acid metabolism pathway showed continuous differential changes throughout the in vitro preservation process, suggesting that it may have important clinical research value in platelet in vitro preservation. Overall, these data revealed the dynamic changes of metabolites and related metabolic pathways in platelets and plasma during platelet preservation, providing a basis and direction for further in-depth study of metabolic pathways.

[0058] 3. Changes in G6PD and COX1 activities during platelet preservation

[0059] The main metabolites of the PPP are NADPH and pentose sugars, which are used to maintain the reduced state of glutathione (GSH) and nucleic acid biosynthesis respectively

[21] . As anucleate cells, platelets rely on the PPP to maintain the redox balance in their physiological state

[22] . In addition, the arachidonic acid metabolism pathway is closely related to the generation of thromboxane A 2 (Thromboxane A 2 , TXA 2 ), which plays an important role in promoting platelet activation and recruiting platelets to the damaged site, and is also an important target for inhibiting platelet function [23,24] . G6PD (glucose-6-phosphate dehydrogenase) and COX1 (cyclooxygenase 1) are the key enzymes of the PPP and the cyclooxygenase pathway of arachidonic acid metabolism respectively. Their metabolites 6-phospho-D-gluconic acid and cyclo-peroxidation compounds PGG2 and PGB2 accumulated significantly during platelet in vitro preservation ( Figure 6 C-E). To verify the changes in these two metabolic pathways during platelet preservation, we detected the activities of G6PD and COX1. Surprisingly, the results showed that the activities of the two enzymes increased significantly in the initial stage of platelet preservationFigure 6 A, B). These results further support the potential key role of the PPP and arachidonic acid metabolic pathways in the occurrence of platelet storage lesion.

[0060] 4. Inhibition of PPP by G6PD enzyme inhibitor leads to mitochondrial damage and PS exposure of stored platelets

[0061] To investigate the roles of the PPP and arachidonic acid metabolic pathways during platelet storage, we separately used the specific inhibitor G6PDi of G6PD

[25] and the specific inhibitor aspirin of COX1 for intervention. Platelet storage lesion is closely related to impaired mitochondrial function, generation of mitochondrial reactive oxygen species (mtROS), and intrinsic apoptosis of platelets, among which the exposure of phosphatidylserine on the platelet surface is importantly associated with rapid clearance after in vivo infusion [8] . In this study, we detected the changes in platelet membrane potential, mtROS level, and phosphatidylserine exposure after adding G6PDi and aspirin. The results showed that G6PDi significantly promoted the exposure of phosphatidylserine of platelets, increased the generation of mitochondrial ROS, and exacerbated the depolarization of mitochondrial membrane potential ( Figure 7 A - C). While aspirin showed no significant effect on the metabolic changes during platelet storage ( Figure 7 D - F). Taken together, these results indicate that inhibition of G6PD function exacerbates platelet storage lesion, revealing the important regulatory role of the PPP in platelet storage.

[0062] 5. G6PD activator alleviates the generation of mitochondrial reactive oxygen species and PS exposure in stored platelets

[0063] Platelet storage lesion is closely related to the excessive generation of mitochondrial reactive oxygen species (mtROS), and this kind of lesion can be alleviated by ROS scavengers

[26] The main function of the PPP in platelets is to regulate the intracellular redox balance by generating NADPH. In addition, studies have shown that NADPH not only participates in the generation of ROS, but also promotes platelet activation as a signaling molecule

[27] To investigate the potential role of the PPP in platelet storage lesion, we used the G6PD activator AG1

[28] to enhance the G6PD activity during platelet storage. Meanwhile, the known ROS inhibitor N - acetylcysteine (NAC) was selected as a positive control

[29] . The results showed that after adding AG1, the level of platelet mitochondrial ROS was significantly reduced, and at the same time, the exposure of phosphatidylserine was also significantly inhibited ( Figure 8A-C). This phenomenon indicates that increasing G6PD activity can effectively alleviate platelet storage damage. In summary, these results further support that the PPP plays an important role in regulating platelet storage damage by maintaining intracellular redox balance.

[0064] 6. Platelets with G6PD knockout exhibit poorer platelet storage quality

[0065] To further investigate the role of the PPP in platelet ex vivo preservation, we constructed megakaryocyte / platelet-specific knockout mice (G6PDPF4-cre) and obtained platelets from them for ex vivo preservation experiments. The results showed that, similar to the experimental results of adding G6PD inhibitor in vitro, the platelets of G6PDPF4-cre mice showed a higher degree of phosphatidylserine externalization after 6 hours of preservation ( Figure 8 D-F). In summary, the PPP plays a key role in platelet ex vivo preservation and is closely related to the generation of mitochondrial reactive oxygen species (ROS). Modulating the PPP to alleviate platelet storage damage may be a potential and promising intervention strategy.

[0066] III. Discussion

[0067] Platelet transfusion is a commonly used clinical treatment method. However, currently, platelet transfusion faces problems such as increasing demand, storage damage, and short storage time. Existing studies have shown that the metabolic state of platelets changes significantly during storage, generating a large amount of lactic acid through aerobic glycolysis, leading to a decrease in the pH value in the storage system, thus affecting the storage quality of platelets

[30] . Although researchers have been concerned about the metabolic changes during platelet storage for many years, the metabolic pathways or key metabolites directly related to platelet storage damage have not been fully clarified. In this study, we used non-targeted metabolomics technology to systematically analyze the overall metabolite changes during platelet storage and explored the potential relationship between the significantly enriched metabolic pathways during storage and platelet storage damage. The results showed that the PPP and arachidonic acid metabolic pathways were significantly enriched in the early stage of platelet preservation. Further verification of the potential roles of these two pathways in platelet storage using inhibitors and activators of key enzymes in the pathways provided a new research direction for improving platelet storage quality.

[0068] As a bypass pathway of glycolysis, the PPP does not directly produce energy but generates NADPH and phosphoribose through oxidation and group transfer. As a hydrogen donor, NADPH not only participates in the synthesis of lipids and amino acids but also protects cells from oxidants by maintaining the reduced state of glutathione

[21] Meanwhile, NADPH generates reactive oxygen species (ROS) under the action of NADPH oxidase (NOX2), and the latter promotes platelet activation as a signaling molecule.

[27] Phosphoribose can not only serve as a raw material for nucleic acid biosynthesis, but also generate glyceraldehyde 3-phosphate through group transfer and re-enter the glycolytic pathway to produce energy. Existing studies have shown that inhibiting PPP with small molecule inhibitors will lead to a decrease in platelet aggregation, granule release, and thrombin generation ability.

[31] .

[0069] Research findings showed that significant changes occurred in PPP during the initial stage of platelet preservation (days 1 - 3), manifested as a significant increase in the levels of intermediate metabolite 6 - phosphogluconic acid and end - product glyceraldehyde 3 - phosphate. As a key enzyme of PPP, G6PD exhibited dynamic changes in activity during platelet preservation. The experimental results showed that the activity of G6PD increased significantly on the second day of preservation, then gradually decreased with the prolongation of preservation time, and was lower than the initial level at the end of preservation. This dynamic change suggested that the fluctuation of G6PD activity might be closely related to platelet storage damage. To further investigate the role of G6PD in platelet storage, we treated apheresis platelets with a specific inhibitor of G6PD, G6PDi, and evaluated storage damage indicators such as mitochondrial function and apoptosis of platelets at different preservation time points (days 1, 3, 5). The results showed that after inhibiting the function of G6PD, compared with the control group, the level of mitochondrial ROS in platelets increased significantly, and the degree of cell apoptosis increased significantly, aggravating platelet storage damage. An important manifestation of platelet storage damage is the massive generation of mitochondrial reactive oxygen species, which significantly affects the function and lifespan of platelets. Given that the main function of PPP is to generate NADPH to maintain intracellular redox balance, we speculated that the generation of ROS during platelet storage might activate PPP by inducing cellular oxidative stress. To verify this hypothesis, we used the G6PD activator AG1 to study whether PPP could regulate platelet storage damage, and at the same time used the known drug NAC (N - acetylcysteine) that inhibits mitochondrial ROS generation as a positive control. The experimental results showed that AG1 significantly reduced the generation of mitochondrial ROS, maintained the mitochondrial membrane potential, and reduced the externalization of phosphatidylserine, thus effectively alleviating platelet storage damage. In addition, to further verify the key role of PPP in platelet storage, we constructed megakaryocyte / platelet - specific G6PD - knockout mice (G6PDPF4 - cre). Experiments showed that G6PD - knockout platelets exhibited obvious storage damage characteristics during in vitro preservation, further demonstrating the important position of PPP in regulating platelet storage damage. In summary, through metabolomics analysis and functional verification, this study revealed the mechanism by which PPP regulates storage damage by maintaining redox balance during platelet storage. This finding provides a new research direction and potential intervention strategies for improving the quality of platelet preservation.

[0070] The present invention systematically analyzed the overall metabolic pattern during platelet preservation using non - targeted metabolomics technology, revealing the important role of PPP in regulating platelet storage damage. This finding provides a new research direction and ideas for improving the quality of platelet preservation by intervening in the platelet metabolic process.

[0071] We discovered the role of PPP in regulating platelet storage damage. By using G6PD-specific inhibitors and activators, we observed the direct effects of G6PD on platelet mitochondrial function and phosphatidylserine externalization within the platelet membrane, further demonstrating the criticality of G6PD activity in maintaining platelet quality and function. Collectively, these findings not only enhanced our understanding of platelet storage mechanisms but also provided new strategies and targets for optimizing platelet preservation methods and improving the quality of clinical transfusions.

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[0107] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of pentose phosphate pathway regulators in the preparation of anti-platelet storage injury preparations.

2. The use according to claim 1, characterized in that The pentose phosphate pathway regulator is selected from the group consisting of G6PD promoters.

3. The use according to claim 1, characterized in that: The G6PD promoter is selected from any one or more of the following: exogenous G6PD protein or nucleic acid encoding it, a substance that promotes the expression or activity function of the G6PD protein, a substance that promotes the overexpression of nucleic acid molecules encoding the G6PD protein, or a liposome or nanomaterial that encapsulates the nucleic acid encoding the G6PD protein.

4. The use according to claim 3, Features: in, The G6PD protein is selected from any of the following situations: (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 3; (b) a substance having homology or sequence identity with the amino acid sequence shown in any one of SEQ ID NOs. 1 to 3 and having the function of promoting G6PD; (c), a substance having a G6PD-promoting function in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b), or a protein or polypeptide derived from (a) or (b), The nucleic acid molecule encoding the G6PD protein is selected from any of the following situations: (i) a nucleic acid molecule having a nucleotide sequence shown in Genbank accession number NG_009015.2; (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions; (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in NG_009015.2; (iv) A nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii).

5. The use according to claim 3, characterized in that: in, The substance that promotes the expression or activity function of G6PD protein is selected from the G6PD activator AG1.

6. A G6PD recombinant expression vector, characterized in that: The recombinant vector comprises an expression vector and a nucleic acid molecule encoding a G6PD protein inserted into the expression vector. The nucleic acid molecule encoding a G6PD protein is as described in claim 4.

7. The G6PD recombinant vector according to claim 6, characterized in that: in, The expression vector is a plasmid vector, a cosmid vector, a phage vector or a virus vector.

8. Use of the G6PD recombinant expression vector according to claim 6 in the preparation of an anti-platelet storage injury preparation.

9. A preparation for preventing platelet storage damage, characterized in that: The active component comprises the G6PD promoter according to any one of claims 1 to 5, or the G6PD recombinant expression vector according to claim 6 or 7.

10. A method for storing platelets in vitro, characterized in that: An effective amount of an anti-platelet storage injury preparation is administered into a platelet storage container, wherein the anti-platelet storage injury preparation is as described in claim 9.

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