PubMed · 6459720
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M J Gawel, F C Rose. 1982. Platelet function in migraineurs.. https://pubmed.ncbi.nlm.nih.gov/6459720/
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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.
Potential sphingosine (Sph) metabolites include phosphorylated, N-acylated, and N-methylated derivatives. Phosphorylated Sph, i.e., sphingosine 1-phosphate (Sph-1-P), may act as an autocrine stimulator of blood platelets, as it is abundantly stored in platelets and released extracellularly and its exogenous addition induces platelet activation. In this study, we evaluated Sph-1-P formation and its effects in human platelets in the presence of other Sph metabolites. On addition of [3H]Sph to intact platelets, the label was rapidly converted to Sph-1-P. This conversion into [3H]Sph-1-P was inhibited by N,N-dimethylsphingosine (DMS) in a dose-dependent manner, but not by other structurally related Sph derivatives, including ceramide. The inhibition of Sph-1-P formation by DMS was reproduced using a cell-free system (Sph kinase obtained from platelet cytosolic fractions) and much stronger than that by DL-threo-dihydrosphingosine, which had been considered to be the strongest inhibitor of Sph kinase. Administration of DMS to intact platelets resulted in a decrease in Sph-1-P mass and an increase in Sph mass. Furthermore, DMS inhibited the release of Sph-1-P from platelets stimulated with 12-O-tetradecanoylphorbol 13-acetate and inhibited platelet aggregation induced by exogenous addition of Sph-1-P. Collectively, our results indicate that DMS is useful as a Sph kinase inhibitor and that Sph-1-P actions as an autocrine stimulator of platelets are inhibited by DMS.
Human platelets possess a specific membrane-bound leukotriene (LT) C4 synthase, which catalyzes the conversion of LTA4 to LTC4. Stimulation of the receptors for thrombin, collagen or thromboxane A2 provoked inhibition of this enzyme, as judged by suppressed transformation of exogenous LTA4 to LTC4. Similarly, direct activation of protein kinase (PK) C with nanomolar concentrations of 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited the production of LTC4. Kinetic studies demonstrated that the inhibition induced by thrombin and PMA was non-competitive. Elevation of intracellular cAMP levels with carbacyclin did not affect basal LTC4 formation, but abolished the attenuation of platelet LTC4 synthase activity induced by the thromboxane receptor agonist U-46619. The unselective protein kinase inhibitor staurosporine prevented both receptor-mediated and PMA-induced suppression of LTC4 formation. In contrast, two selective PKC inhibitors, Ro 31-8220 and GF 109203X, reversed the inhibitory effect provoked by PMA, but failed to prevent thrombin-induced inhibition. Furthermore, the protein tyrosine phosphatase inhibitor, sodium orthovanadate, induced dose-dependent inhibition of LTC4 production in platelet sonicates. In conclusion, receptor-mediated activation of human platelets leads to decreased LTC4 synthase activity via phosphoregulation. Although the present results demonstrate that platelet LTC4 synthase can be regulated via PKC-dependent events, alternative mechanisms appears to be involved in the physiological regulation of this enzyme. The findings suggest the possible importance of protein tyrosine phosphorylations in this process.