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Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management. [ABSTRACT FROM AUTHOR]

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1–4 fractions, and non-rafts were distributed on 5–12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned. [ABSTRACT FROM PUBLISHER]

Lipid-rafts are defined as membrane microdomains enriched in cholesterol and glycosphingolipids within platelet plasma membrane. Lipid raft-mediated clot retraction requires factor XIII and other interacting proteins. The aim of this study was to investigate the proteins that interact with factor XIII in raft and non-raft domains of activated and non-activated platelet plasma membrane. By lipidomics analysis, we identified cholesterol- and sphingomyelin-enriched areas as lipid rafts. Platelets were activated by thrombin. Proteomics analysis provided an overview of the pathways in which proteins of rafts and non-rafts participated in the interaction network of FXIII-A1, a catalytic subunit of FXIII. “Platelet activation” was the principal pathway among KEGG pathways for proteins of rafts, both before and after activation. Network analysis showed four types of interactions (activation, binding, reaction, and catalysis) in raft and non-raft domains in interactive network of FXIII-A1. FXIII-A1 interactions with other proteins in raft domains and their role in homeostasis highlight the specialization of the raft domain in clot retraction via the Factor XIII protein network. [ABSTRACT FROM PUBLISHER]