Common anti-platelet therapies modulate procoagulant phospholipids in arterial disease

Abstract

Enzymatically oxygenated phospholipids (eoxPL) formed by lipoxygenases (LOX) and cyclooxygenase (COX) in platelets and leukocytes are pro-coagulant in multiple model systems. However, their generation in arterial thrombotic disease, and how their levels are modulated by common therapies is unknown. Here, eoxPL were first characterized in isolated platelets and leukocytes from an arterial vascular disease cohort, a healthy cohort administered low dose aspirin, and from retrieved human arterial thrombi. In both cohorts, aspirin reduced platelet COX-1-derived eoxPL, while elevating diacyl 12-LOX-derived eoxPL in males, through enhanced Lands cycle esterification. Conversely, P2Y12 inhibition reduced 12-LOX-derived eoxPL in leukocytes. Complex aspirin-dependent gender and seasonal effects on platelet eoxPL were seen in healthy subjects. Limb or coronary (STEMI) thrombi showed a platelet eoxPL signature while carotid thrombi had a white cell profile. Mice genetically lacking leukocyte 12/15-LOX, which are deficient in eoxPL, generated smaller carotid thrombi in vivo. In summary, pro-coagulant eoxPL generation is altered in human arterial vascular disease by commonly used cardiovascular therapies. These changes to the phospholipid composition of blood cells in humans at risk of thrombotic events may be clinically significant where the pro-coagulant membrane plays a central understood role in driving elevated thrombotic risk.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease

“…EV numbers are higher in patients with cardiovascular disease, including in this cohort ( 44 ). To investigate whether this accounted for elevated PT levels, lipid levels were normalized to EV counts, previously published in a related study using this cohort ( 29 ). After normalization, PT species were no longer significantly different in the disease groups ( Fig.…”

“…Separately, blood was collected from a clinical cohort as described in Supplementary Methods . Participants were recruited from Cardiff University and Cardiff and Vale University Health Boards, as described ( 29 ). Ethical approval was from Health and Care Research Wales (HCRW, IRAS 243701; REC reference 18/YH/0502).…”

“…Lipid extracts of platelets, EV, and leukocytes from the clinical cohort were generated as outlined in Supplementary Methods and stored at −80°C ( 29 ). Following LC-MS/MS analysis for a previous study ( 29 ), the stored extracts were retrieved and added to an equal volume of methanol containing SPLASH mix (PS 15:0/18:1-D7: 0.05 ng/μl, final conc), then analyzed using LC-MS/MS as described in Supplementary Methods for PT ( supplemental Table S2 ). As IS was added post-extraction, amounts of PT are not quantified, and data is presented as relative levels, normalized across lipid species, or patient groups.…”

“…Details on the cohort were recently reported by Protty et al. ( 29 ) and full information is in Supplementary Methods .…”

“…To address whether PT could also be associated with CAD, we measured PT in platelets and EVs from a cohort of coronary artery disease patients ( 29 ), to investigate whether the lipid is altered in line with increased thrombotic risk in vascular disease.…”