PubMed · 7947298
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J G White, G Escolar. 1994. Restoring platelet adhesion function.. https://doi.org/10.1111/j.1365-2141.1994.tb04943.x
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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.
G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.
BACKGROUND: Long-term exposure of platelets to endogenous or exogenous prostacyclin or its analogues might result in desensitization of the platelet prostacyclin receptor in vitro and in vivo accompanied by a loss in receptor density on the platelet surface and a reduced sensitivity toward the inhibitory effects of prostacyclins. However, the reversibility of this process in platelets has not yet been investigated. METHODS AND RESULTS: Human platelets desensitized by the chemically stable prostacyclin analogue iloprost showed a significant reduction in [3H]-iloprost binding sites that was reversed by saponin permeabilization. This indicates functionally active internalized prostacyclin receptors. To assess whether the internalized prostacyclin receptors recycle to the cell surface after withdrawal of the agonist, iloprost sensitivity and prostacyclin receptor binding properties of iloprost (30 nmol/L, 2 hours) desensitized platelets incubated in iloprost-free autologous plasma were investigated. While desensitized platelets showed a significant increase in IC50 for iloprost inhibition of thrombin-induced platelet aggregation, serotonin release, and p-selectin expression and a reduced iloprost-stimulated cAMP formation, platelet iloprost sensitivity was restored 3 hours after iloprost withdrawal. In addition, the significant reduction in Bmax and the increase in K(D) of prostacyclin receptors in desensitized platelets as revealed by [3H]-iloprost binding studies also returned to the initial values. CONCLUSIONS: These results indicate that prostacyclin receptors internalized during short-term desensitization are not degraded but can be recycled rapidly to the platelet surface in a functionally active form after withdrawal of the agonist.