Identification of key lipids critical for platelet activation by comprehensive analysis of the platelet lipidome.
ABSTRACT: Platelet integrity and function critically depend on lipid composition. However, the lipid inventory in platelets was hitherto not quantified. Here, we examined the lipidome of murine platelets using lipid-category tailored protocols on a quantitative lipidomics platform. We could show that the platelet lipidome comprises almost 400 lipid species and covers a concentration range of 7 orders of magnitude. A systematic comparison of the lipidomics network in resting and activated murine platelets, validated in human platelets, revealed that <20% of the platelet lipidome is changed upon activation, involving mainly lipids containing arachidonic acid. Sphingomyelin phosphodiesterase-1 (Smpd1) deficiency resulted in a very specific modulation of the platelet lipidome with an order of magnitude upregulation of lysosphingomyelin (SPC), and subsequent modification of platelet activation and thrombus formation. In conclusion, this first comprehensive quantitative lipidomic analysis of platelets sheds light on novel mechanisms important for platelet function, and has therefore the potential to open novel diagnostic and therapeutic opportunities.
Project description:Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.
Project description:Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe<sup>-/-</sup> and Ldlr<sup>-/-</sup> mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe<sup>-/-</sup> mice). Bone marrow from wild-type or Ldlr<sup>-/-</sup> mice was transplanted into irradiated Ldlr<sup>-/-</sup> recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe<sup>-/-</sup> and Ldlr<sup>-/-</sup> mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype.
Project description:Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via ?-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ?5,600 unique species, with only ?50% being putatively identified. Thrombin elevated ?900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ?50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented.
Project description:Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism.
Project description:Ovarian cancer patients have a high risk of developing venous thrombosis. The membrane lipid bilayer of platelets and platelet-derived microparticles (PMP) provides a platform for assembly of coagulation proteins and generation of blood clots.We compared the lipid composition of platelets and PMPs in patients with ovarian cancer to those in healthy subjects. We used shotgun lipidomics to quantify 12 classes and 177 species of lipids.We found a significant change in 2 classes of lipids in platelets and PMPs isolated from ovarian cancer patients: higher phosphatidylinositol and lower lyso-phosphatidylcholine. The level of 28 species of lipids was also significantly altered in the direction of an increase in the pro-coagulant and a reduction in the anticoagulant lipids. We found that cancer platelets expressed less lipid phosphate phosphatase 1 (LPP1), a key enzyme in phospholipid biosynthesis pathways, than normal platelets. The reduction in LPP1 might contribute to the changes in the lipid profile of cancer platelets.Our results support a procoagulant lipid profile of platelets in ovarian cancer patients that can play a role in the increased risk of venous thrombosis in these patients.As far as we are aware, our study is the first study on platelet lipidomics in ovarian cancer. The importance of our findings for the future studies are: 1) a similar change in lipid profile of platelets and PMP may be responsible for hypercoagulability in other cancers, and 2) plasma level of high-risk lipids for venous thrombosis may be useful biomarkers.
Project description:Lipidomics of human blood plasma is an emerging biomarker discovery approach that compares lipid profiles under pathological and physiologically normal conditions, but how a healthy lipidome varies within the population is poorly understood. By quantifying 281 molecular species from 27 major lipid classes in the plasma of 71 healthy young Caucasians whose 35 clinical blood test and anthropometric indices matched the medical norm, we provided a comprehensive, expandable and clinically relevant resource of reference molar concentrations of individual lipids. We established that gender is a major lipidomic factor, whose impact is strongly enhanced by hormonal contraceptives and mediated by sex hormone-binding globulin. In lipidomics epidemiological studies should avoid mixed-gender cohorts and females taking hormonal contraceptives should be considered as a separate sub-cohort. Within a gender-restricted cohort lipidomics revealed a compositional signature that indicates the predisposition towards an early development of metabolic syndrome in ca. 25% of healthy male individuals suggesting a healthy plasma lipidome as resource for early biomarker discovery.
Project description:Arterial foam cells are central players of atherogenesis. Cholesterol acceptors, apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL), take up cholesterol and phospholipids effluxed from foam cells into the circulation. Due to the high abundance of cholesterol in foam cells, most previous studies focused on apoA-I/HDL-mediated free cholesterol (FC) transport. However, recent lipidomics of human atherosclerotic plaques also identified that oxidized sterols (oxysterols) and non-sterol lipid species accumulate as atherogenesis progresses. While it is known that these lipids regulate expression of pro-inflammatory genes linked to plaque instability, how cholesterol acceptors impact the foam cell lipidome, particularly oxysterols and non-sterol lipids, remains unexplored. Using lipidomics analyses, we found cholesterol acceptors remodel foam cell lipidomes. Lipid subclass analyses revealed various oxysterols, sphingomyelins, and ceramides, species uniquely enriched in human plaques were significantly reduced by cholesterol acceptors, especially by apoA-I. These results indicate that the function of lipid-poor apoA-I is not limited to the efflux of cholesterol and phospholipids but suggest that apoA-I serves as a major regulator of the foam cell lipidome and might play an important role in reducing multiple lipid species involved in the pathogenesis of atherosclerosis.
Project description:Untargeted omics analyses aim to comprehensively characterize biomolecules within a biological system. Changes in the presence or quantity of these biomolecules can indicate important biological perturbations, such as those caused by disease. With current technological advancements, the entire genome can now be sequenced; however, in the burgeoning fields of lipidomics, only a subset of lipids can be identified. The recent emergence of high resolution tandem mass spectrometry (HR-MS/MS), in combination with ultra-high performance liquid chromatography, has resulted in an increased coverage of the lipidome. Nevertheless, identifications from MS/MS are generally limited by the number of precursors that can be selected for fragmentation during chromatographic elution. Therefore, we developed the software IE-Omics to automate iterative exclusion (IE), where selected precursors using data-dependent topN analyses are excluded in sequential injections. In each sequential injection, unique precursors are fragmented until HR-MS/MS spectra of all ions above a user-defined intensity threshold are acquired. IE-Omics was applied to lipidomic analyses in Red Cross plasma and substantia nigra tissue. Coverage of the lipidome was drastically improved using IE. When applying IE-Omics to Red Cross plasma and substantia nigra lipid extracts in positive ion mode, 69% and 40% more molecular identifications were obtained, respectively. In addition, applying IE-Omics to a lipidomics workflow increased the coverage of trace species, including odd-chained and short-chained diacylglycerides and oxidized lipid species. By increasing the coverage of the lipidome, applying IE to a lipidomics workflow increases the probability of finding biomarkers and provides additional information for determining etiology of disease. Graphical Abstract ?.
Project description:Major alterations in metabolism occur during pregnancy enabling the mother to provide adequate nutrients to support infant development, affecting birth weight (BW) and potentially long-term risk of obesity and cardiometabolic disease. We classified dynamic changes in the maternal lipidome during pregnancy and identified lipids associated with Fenton BW z-score and the umbilical cord blood (CB) lipidome. Lipidomics was performed on first trimester maternal plasma (M1), delivery maternal plasma (M3), and CB plasma in 106 mother-infant dyads. Shifts in the maternal and CB lipidome were consistent with the selective transport of long-chain polyunsaturated fatty acids (PUFA) as well as lysophosphatidylcholine (LysoPC) and lysophosphatidylethanolamine (LysoPE) species into CB. Partial correlation networks demonstrated fluctuations in correlations between lipid groups at M1, M3, and CB, signifying differences in lipid metabolism. Using linear models, LysoPC and LysoPE groups in CB were positively associated with BW. M1 PUFA containing triglycerides (TG) and phospholipids were correlated with CB LysoPC and LysoPE species and total CB polyunsaturated TGs. These results indicate that early gestational maternal lipid levels influence the CB lipidome and its relationship with BW, suggesting an opportunity to modulate maternal diet and improve long-term offspring cardiometabolic health.
Project description:Cell type-resolved proteome analyses of the brain, heart and liver have been reported, however a similar effort on the lipidome is currently lacking. Here we applied liquid chromatography-tandem mass spectrometry to characterize the lipidome of major lung cell types isolated from human donors, representing the first lipidome map of any organ. We coupled this with cell type-resolved proteomics of the same samples (available at Lungmap.net). Complementary proteomics analyses substantiated the functional identity of the isolated cells. Lipidomics analyses showed significant variations in the lipidome across major human lung cell types, with differences most evident at the subclass and intra-subclass (i.e. total carbon length of the fatty acid chains) level. Further, lipidomic signatures revealed an overarching posture of high cellular cooperation within the human lung to support critical functions. Our complementary cell type-resolved lipid and protein datasets serve as a rich resource for analyses of human lung function.