Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease

BACKGROUND: Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. METHODS: Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. RESULTS: External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. CONCLUSIONS: The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.