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Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post–myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A–mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia–sera/immunoglobulin G–triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and