PubMed · 3162053
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K Watanabe. 1985. [Membrane surface proteins].. https://pubmed.ncbi.nlm.nih.gov/3162053/
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OBJECTIVES: Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. METHODS: We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. RESULTS: We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. CONCLUSION: By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.
Platelets labeled with 2',7'-dihydrodichlorofluorescein diacetate (DCF-DA) and stimulated with 50-400nM peroxynitrite (ONOO(-)) produced a rapid increase of the fluorescence signal at 523nm with good linearity and reproducibility. Platelet fluorescence was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suggesting that HCO(3)(-)/Cl(-) transporter mediated ONOO(-) transport into the platelets. Exposure of platelets to potassium superoxide, hydrogen peroxide, and sodium nitroprusside at concentrations of up to 100 microM did not generate a fluorescence signal. We also studied other nitrating compounds to establish the specificity of the DCF-DA-labeled platelet ONOO(-) assay. A rapid increase of fluorescence was observed when sodium hypochlorite (0.15 to 0.75mM) was added to platelets suspended in a buffered nitrite solution. Exposure of platelets to NO(2), nitroglycerin, and tetranitromethane produced a slow sustained increase of fluorescence. Endogenous glutathione appeared to be an essential factor in the generation of fluorescence by ONOO(-) and other nitrating compounds. We further studied other conditions that increased platelet fluorescence. Stimulation of platelets with thrombin (1U/mL) produced a rapid increase in fluorescence that corresponded to the formation of 20.5nmol ONOO(-) per 10(7) cells, whereas stimulation with collagen and arachidonic acid was without effect. Hypoxia of platelets for 20 and 40min followed by 5min of reoxygenation doubled the fluorescence from these platelets compared with control platelets. Thus, thrombin produced an effect that was likely due to the formation of ONOO(-) in platelets, whereas hypoxia-reoxygenation was likely to cause the formation of an active nitroglutathione-like molecule.
Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, and amputation remains the single best treatment. Previous studies have demonstrated that tissue destruction follows C. perfringens phospholipase C (PLC)-induced, platelet gpIIbIIIa-mediated formation of occlusive intravascular platelet/leukocyte aggregates. In this study, the intracellular signaling events leading to activation of gpIIbIIIa by PLC were investigated. PLC activated surface expressed gpIIbIIIa and mobilized gpIIbIIIa from internal stores. Chelation of intracellular calcium or inhibition of store-operated calcium entry each blocked PLC-induced activation of gpIIbIIIa, whereas inhibition of protein kinase C was without effect. Thus, PLC initiates an "inside-out" signaling cascade that begins with depletion of internal calcium stores, is sustained by an influx of calcium through store-sensitive channels, and culminates in the functional activation of gpIIbIIIa. These findings suggest that calcium-channel blockade and strategies targeting gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with gas gangrene.