Quantification of bulk lipid species in human platelets and their thrombin-induced release

Abstract

Lipids play a central role in platelet physiology. Changes in the lipidome have already been described for basal and activated platelets. However, quantitative lipidomic data of platelet activation, including the released complex lipids, are unavailable. Here we describe an easy-to-use protocol based on flow-injection mass spectrometry for the quantitative analysis of bulk lipid species in basal and activated human platelets and their lipid release after thrombin activation. We provide lipid species concentrations of 12 healthy human donors, including cholesteryl ester (CE), ceramide (Cer), free cholesterol (FC), hexosylceramide (HexCer), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM) and triglycerides (TG). The assay exhibited good technical repeatability (CVs < 5% for major lipid species in platelets). Except for CE and TG, the inter-donor variability of the majority of lipid species concentrations in platelets was < 30% CV. Balancing of concentrations revealed the generation of LPC and loss of TG. Changes in lipid species concentrations indicate phospholipase-mediated release of arachidonic acid mainly from PC, PI, and PE but not from PS. Thrombin induced lipid release was mainly composed of FC, PS, PC, LPC, CE, and TG. The similarity of the released lipidome with that of plasma implicates that lipid release may originate from the open-canalicular system (OCS). The repository of lipid species concentrations determined with this standardized platelet release assay contribute to elucidating the physiological role of platelet lipids and provide a basis for investigating the platelet lipidome in patients with hemorrhagic or thrombotic disorders.

Plasma Instead of Serum Avoids Critical Confounding of Clinical Metabolomics Studies by Platelets

“…The following platelet aggregation was found to be accompanied by the significant deregulation of 786 proteins, as well as 28 fatty acids and lipid mediators in the platelet supernatant (Figure , Supplementary Table S7). Most of these findings are compatible with or reproduce the results of previous reports. ,− As expected, all proteins except antibodies with increased abundance values in serum, when compared to those in plasma, were also found to be released from platelets upon aggregation. Similarly, most eicosanoids with high abundance values in serum were found to be released from platelets upon aggregation (Supplementary Table S8).…”

Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation

“…Nevertheless, critical precursors for platelet signaling like PI 18:0_20:4 are already abundantly available in MKs. Comparing fully mature MKs against the lipidome of human 31 and mouse 12 platelets reveals an equal basis for various lipid classes such as PE, PS, ST, cholesterol ester (SE), PC-ether lipid (PCO), triacylglycerol (TG) and lyso-PE (LPE) (Fig. 2e).…”

Serum lipid metabolism characteristics and potential biomarkers in patients with unilateral sudden sensorineural hearing loss

“…GPLs are the predominant phospholipids found in mammalian cell membranes and fulfill critical functions across diverse physiological processes, such as cell signaling, platelet activation, endoplasmic reticulum regulation, and mitochondrial stress management [ 17 , 18 ]. Animal studies have suggested that the imbalance in PC and PE and their ratios might contribute to multiple metabolic disorders, such as endoplasmic reticulum stress, obesity, and insulin resistance [ 19 – 21 ].…”