PubMed · 15308812
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Robert Flaumenhaft. 2004. Platelet permeabilization.. https://doi.org/10.1385/1-59259-783-1%3A365
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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.
The synthesis and structure-activity relationship study of a series of 1-indol-1-yl-3-phenoxypropan-2-one inhibitors of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) are described. The compounds were evaluated in a vesicle assay with isolated cPLA(2)alpha and in cellular assays with intact human platelets. Systematic variation led to 3-methylhydrogen 1-[3-(4-decyloxyphenoxy)-2-oxopropyl]indole-3,5-dicarboxylate (57), which revealed the highest activity against the isolated enzyme. With an IC(50) value of 4.3 nM in this assay, it is one of the most potent in vitro cPLA(2)alpha inhibitors known today.
Platelets play an important role in carcinogenesis, but the underlying molecular mechanisms remain poorly understood. To investigate the effects of platelets on in vitro invasion of MCF7 human breast cancer cells, human MCF7 cells were used to study their interactions with platelets using aggregometry and cell invasion chambers. Zymography and quantitative polymerase chain reaction (PCR) were used to study matrix metalloproteinases (MMPs), whereas Western blot was used to study protein kinase C (PKC) delta in MCF7 cells. We observed that platelets promoted invasion of MCF7 cells (3-fold increase, p<0.05, n=3) and that this process correlated with a dramatic increase in MMP-9 (8 fold-increase, p<0.001, n=3), which is known to facilitate cancer cell invasion. Because both platelets and MCF7 cells have been shown to release MMP-9, we investigated the cellular source that accounted for this increase. The time course and the use of specific protein synthesis inhibitors demonstrated that most of the increase in MMP-9 levels derived from de novo synthesis of this protease by cancer cells. Furthermore, platelets activated PKCdelta in MCF7 cells after 1 h of incubation (18.45+/-4.75% increase, p<0.05, n=4-7), which, in turn, led to an up-regulation of MMP-9 mRNA (from 60+/-20 to 1040+/-100 pg, p<0.001, n=3) and protein levels (18-fold increase, p<0.001, n=3), with the subsequent cell invasion-promoting effects. PKCdelta plays a crucial role in transducing the invasion-promoting effects of platelets in breast cancer cells, and the specific inhibition of PKCdelta may be a strategy to decrease platelet-mediated cancer cell invasion.