PubMed · 1633739
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H Ishii, F Umeda, H Nawata. 1992. Platelet function in diabetes mellitus.. https://doi.org/10.1002/dmr.5610080106
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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.
S-nitrosothiols (RSNOs) regulate several aspects of platelet physiology including inhibition of activation, adhesion and aggregation. PDI (protein disulphide-isomerase) has recently been found to be localized to the cell surface, where it exhibits both disulphide-exchange and denitrosation activities. The disulphide-exchange activity of PDI has been linked to aspects of platelet aggregation. The present study suggests that the metabolism of RSNOs by platelets is a function of PDI denitrosation activity. Exposure of washed human platelets to increasing concentrations of GSNO (S-nitrosoglutathione) resulted in saturable denitrosation kinetics. The presence of known PDI inhibitors phenylarsine oxide and anti-PDI antibodies prevented GSNO denitrosation. The fact that, in the presence of GSNO plus the cell-permeable guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one, the initial rates of ADP-induced platelet aggregation and the maximum DeltaOD were diminished by approximately 40% shows that RSNOs have dual inhibitory effects on platelets, which are mediated through PDI. First, PDI denitrosates RSNOs, releasing NO that, via the guanylate cyclase/G-kinase route, attenuates platelet activation. Secondly, RSNOs are denitrosated at the same PDI-active site that catalyses the disulphide bond formation between integrins and their ligands, thereby attenuating irreversible aggregation.
Intrinsic tenase consists of activated Factors IX (IXa) and VIII (VIIIa) assembled on a negatively charged phospholipid surface. In vivo, this surface is mainly provided by activated platelets. In vitro, phosphatidylcholine/phosphatidylserine vesicles are often used to mimic natural pro-coagulant membranes. In the present study, we developed a quantitative mathematical model of Factor X activation by intrinsic tenase. We considered two situations, when complex assembly occurs on either the membrane of phospholipid vesicles or the surface of activated platelets. On the basis of existing experimental evidence, the following mechanism for the complex assembly on activated platelets was suggested: (i) Factors IXa, VIIIa and X bind to their specific platelet receptors; (ii) bound factors form complexes on the membrane: platelet-bound Factor VIIIa provides a high-affinity site for Factor X and platelet-bound Factor IXa provides a high-affinity site for Factor VIIIa; (iii) the enzyme-cofactor-substrate complex is assembled. This mechanism allowed the explanation of co-operative effects in the binding of Factors IXa, VIIIa and X to platelets. The model was reduced to obtain a single equation for the Factor X activation rate as a function of concentrations of Factors IXa, VIIIa, X and phospholipids (or platelets). The equation had a Michaelis-Menten form, where apparent V(max) and K(m) were functions of the factors' concentrations and the internal kinetic constants of the system. The equation obtained can be used in both experimental studies of intrinsic tenase and mathematical modelling of the coagulation cascade. The approach of the present study can be applied to research of other membrane-dependent enzymic reactions.