12-HETE, a Platelet Specific Oxylipin, As an Early Diagnostic Indicator for Thrombotic Risk

Thrombosis is one of the primary causes of morbidity and mortality in cardiovascular diseases. While thrombosis places a major burden on the healthcare system, there are no currently accepted biomarkers that can define the risk of arterial thrombosis or thromboembolism amongst patients with symptomatic vascular disease. Clinical D-dimer tests measure the breakdown products of a clot and are only used to diagnose if venous thrombosis has already occurred. Thromboelastography (TEG) and total-thrombus formation analysis system (T-TAS) are additional clinical tests that can be used to assess coagulation parameters and thrombogenicity, respectively, but are not traditionally utilized as a thrombosis diagnostic predictor. Given the high rate of major adverse cardiovascular events amongst patients with symptomatic vascular disease, they are commonly prescribed antiplatelet agents or dual pathway inhibition. However, outcomes are still imperfect, largely due to attendant bleeding rate associated with antiplatelets and anticoagulants that preclude more aggressive therapy. Therefore, there is a critical need for an early detection biomarker that could reliably identify patients at highest risk of thrombosis and major cardiovascular events is needed to identify patients in whom more aggressive therapy is warranted. Our lab identified a significant increase in 12-HETE, an oxidized lipid specifically produced in activated platelets, in thrombotic COVID-19 patients, and previous studies have shown 12-HETE plays a critical role in thrombus formation. 12-HETE is stable in the blood, has a dynamic range, is not altered by aspirin treatment, and increased with disease progression, suggesting it could be used as an early diagnostic predictor for thrombosis.

To evaluate 12-HETE levels in patients with symptomatic vascular disease, we measured plasma 12-HETE levels in healthy donors and patients with peripheral arterial disease (PAD). Additional indicators of platelet activation, integrin α_IIbβ₃ activation and α-granule secretion, were analyzed via flow cytometry, and current clinically available D-dimer, TEG, and T-TAS tests were conducted. Our findings show PAD patients have elevated 12-HETE levels compared to healthy donors, but no differences in basal levels of integrin α_IIbβ₃ activation and α-granule secretion. D-dimer levels are increased in PAD patients but are within the typical range based on age. TEG analysis shows PAD patients and healthy donors have no difference in initial reaction time and clot strength, but PAD patients have elevated rates of clot formation. Further, PAD patients have reduced thrombus formation analyzed via T-TAS compared to healthy donors, which is likely due to treatment with antiplatelet therapies such as aspirin. To account for the presence of aspirin in PAD patients, whole blood from healthy donors was spiked ex vivo prior to analysis which resulted in attenuated thrombus formation comparable to PAD patients.

Here, we show for the first time the platelet-specific oxidized lipid, 12-HETE, is stable in the blood, is elevated in patients at risk for thrombosis, and demonstrates greater sensitivity in detecting platelet activation. Importantly, 12-HETE can be detected prior to a thrombotic event, which is a limitation of current clinically available tests as they can only determine if venous thrombosis has already occurred and are sensitive to antiplatelet therapies. Overall, our findings suggest 12-HETE could be used as an early diagnostic biomarker for thrombosis. This discovery will improve thrombotic interventions by identifying patients at highest risk for thrombosis that require more aggressive treatments prior to the occurrence of an occlusive thrombotic event.

Holinstat:Veralox Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties, Research Funding; Lexicon Pharmaceuticals: Research Funding; Cereno Scientific: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.