PubMed · 579684
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E P Kirby. 1977-12-15. Factor VIII-associated platelet aggregation.. https://pubmed.ncbi.nlm.nih.gov/579684/
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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.
Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for mu-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.
We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.