PubMed · 7221812
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R Castillo. 1980. [Platelet structure. Platelet function].. https://pubmed.ncbi.nlm.nih.gov/7221812/
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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.
Interactions between human platelets and human umbilical vein endothelial cells (HUVEC) were studied by monitoring changes in cytosolic [Ca2+]i in both cell types. Confluent monolayers of Fura-2-loaded HUVEC, grown on gelatin-coated coverslips, responded to repeated addition of a suspension of unstimulated platelets by transient increases in cytosolic [Ca2+]i. These platelet-evoked Ca2+ responses were not caused by products secreted from the platelets and were insensitive to inhibitors of platelet activation and/or platelet aggregation. The platelet-evoked rises in [Ca2+]i in endothelial cells, similarly as the thrombin-evoked rises, were blocked by preincubation of HUVEC with the phospholipase C inhibitor U73122 or the Ca2+ influx blocker Ni2+. In contrast, treatment with the protein tyrosine kinase inhibitor genistein was without effect. Video imaging experiments, in which the fluorescence signal was analysed from the individual cells of an endothelial monolayer, showed that only 2-20% of the cells, scattered over the monolayer, responded to the addition of platelets by a transient increase in [Ca2+]i, whereas most of the cells responded to thrombin. This leads to the conclusion that unstimulated platelets can activate HUVEC putatively by mechanical interaction with individual endothelial cells in the monolayer.
By employing an ELISA for detection of glutathione S-transferase-pi (GST-pi) established in our laboratory, gel filtration profiles of GST-pi in the plasma of normal subjects and patients with malignant tumors were investigated. The results showed that the plasma GST-pi for both of these groups was approximately half the molecular size of placental GST-pi used as a standard control. Similar analyses were performed on GST-pi of platelets and cultured cancer cells, which are considered to be the main sources of the GST-pi in the plasma of normal subjects and cancer patients, respectively. The results indicated that the GST-pi in both the centrifuged supernatants of aggregated platelets and in the culture medium of cancer cells was about half of the molecular size on intact GST-pi. Moreover, the GST-pi in the culture medium was shown to have an N-terminus and a C-terminus, by analysis with specific ELISA. Western blot analysis of the GST-pi in the culture medium detected a single band migrating at 23 kDa, confirming that the extracellular GST-pi was the monomer, not a cleaved form of intact GST-pi. The release of GST-pi from cancer cells was suppressed at 4 degrees C, or by sodium azide, but not suppressed by colchicine or cytochalasin B. These findings suggest that GST-pi may be released by an energy-dependent, active process, and not by a secretion mechanism.