Clinical Platelet Lipidomics in Targeted and Untargeted Approach by Liquid Chromatography Coupled to Mass Spectrometry

Abstract

Platelets are small cellular components of blood with a primary role in hemostasis which in contrary are also responsible for a pathological condition called thrombosis that might results in cardiovascular disease (CAD) such as heart attack and stroke. During the hemostasis, lipids play important roles, especially the fatty acids and their derivatives such as oxylipins which are involved in platelets activation. Therefore, the analysis of platelets lipidomics is particularly interesting and the platelet lipidomic landscape might be considered as a powerful tool for diagnostic and prognostic biomarkers for CAD. The thesis is mainly divided into two parts. Part I involved in liquid chromatography and mass spectrometry (LC-MS) based analytical method developments including 1) the optimization of sample preparation procedure for large-scale clinical lipidomics, 2) the method development for targeted analysis of 3-OH-FAs in biological samples human plasma and platelets, 3) the method development for profiling of branched chain and straight chain saturated fatty acids in different types of biological samples including human plasma, platelets and Staphylococcus aureus, 4) the method development for targeted analysis of oxylipins with microLC coupled with MS and 5) the method development for chiral separation of oxylipins. Part II involved in the application of the developed methods to clinical lipidomics study of CAD patients including 1) investigation of the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome by targeted and untargeted lipidomics analysis, 2) investigation of the platelet lipidome by untargeted approach to highlight the significant changes between acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) patients, 3) investigation of the platelet lipidome of CAD patients by untargeted lipidomics and highlighting significant changes between statin-treated and naïve patients. As a result, an advanced monophasic extraction protocol with methanol/methyl tert-butylether/ isopropanol, MeOH/MTBE/IPA (1.3:1:1, v/v/v) as extraction solvents, bead homogenizer for cell disruption and MeOH/MTBE (1:1, v/v) as reconstitution solvent which provides optimal cellular and subcellular extraction efficiencies for both polar (e.g. acylcarnitines) and apolar lipids (e.g. triglycerides TGs) and several targeted LC-MS methods with selected reaction monitoring (SRM) mode for fatty acids and their derivatives (3-OH-FAs and oxylipins) were reported. Further, the developed methods were successfully applied for the clinical platelets lipidomics studies and some conclusions were made: 1) platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation which may offer a novel therapeutic strategy in CAD, 2) Lipids alteration was observed between acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) patients and between CAD patients treated with statin and statin naïve patients.