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

Supplemental Material isa available online at: https://doi.org/10.1161/ATVBAHA.124.320902 .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.This work was supported by the Wellcome Trust (GW4-CAT fellowship to M.B. Protty, 216278/Z/19/Z) and the British Heart Foundation (program grant to P.W. Collins and V.B. O’Donnell, RG/F/20/110020). V.J. Tyrrell was supported, in part, by the Welsh Government/EU Ser Cymru Programme. A.A. Hajeyah is supported by a grant from the Kuwait University. P.V. Jenkins and A. Sharman were funded by the Government of Kingdom of Saudi Arabia

Relationship between lipoproteins, thrombosis and atrial fibrillation.

The prothrombotic state in atrial fibrillation (AF) occurs as a result of multifaceted interactions, known as Virchow's triad of hypercoagulability, structural abnormalities and blood stasis. More recently, there is emerging evidence that lipoproteins are implicated in this process, beyond their traditional role in atherosclerosis. In this review, we provide an overview of the various lipoproteins and explore the association between lipoproteins and AF, the effects of lipoproteins on haemostasis, and the potential contribution of lipoproteins to thrombogenesis in AF. There are several types of lipoproteins based on size, lipid composition and apolipoprotein category, namely: chylomicrons, very low density lipoprotein, low density lipoprotein (LDL), intermediate density lipoprotein and high density lipoprotein. Each of these lipoproteins may contain numerous lipid species and proteins with a variety of different functions. Furthermore, the lipoprotein particles may be oxidised causing an alteration in their structure and content. Of note, there is a paradoxical inverse relationship between total cholesterol and LDL-C levels, and incident AF. The mechanism by which this occurs may be related to the stabilising effect of cholesterol on myocardial membranes, along with its role in inflammation. Overall, specific lipoproteins may interact with haemostatic pathways to promote excess platelet activation and thrombin generation, as well as inhibiting fibrinolysis. In this regard, LDL-C has been shown to be an independent risk factor for thromboembolic events in AF. The complex relationship between lipoproteins, thrombosis and AF warrants further research with an aim to improve our knowledge base and contribute to our overall understanding of lipoprotein-mediated thrombosis

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

Data availability: All data produced in the present study are available upon reasonable request to the authors (contact Ali A. Hajeyah, [email protected]).Supplemental Data are available online at: https://www.sciencedirect.com/science/article/pii/S0022227523001578?via%3Dihub#sec5 and https://www.sciencedirect.com/science/article/pii/S0022227523001578?via%3Dihub#appsec1 .Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography–tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.A. A. H. acknowledges funding from Kuwait University and support from Kuwait Cultural Office (KCO) in London. M. B. P. was funded by the Wellcome Trust (GW4-CAT fellowship 216278/Z/19/Z) and Academy of Medical Sciences Starter Grant (SGL026∖1037). D. C., P. W. C., and V. B. O. D. acknowledge funding from British Heart Foundation (Programme Grant RG/F/20/110020). Funding from EU Marie Sklodowska-Curie ITN ArthritisHeal is acknowledged (V. B. O. D., D. C.). D. P. and J. H. M. acknowledge support from the NIH Common Fund (Grant R01 GM123455) and the National Heart, Lung, and Blood Institute of NIH (Grant R35 HL135823)