Extracellular Vesicles in Cardiovascular Disease – Modulation of Circulating Extracellular Vesicle Signatures by Rivaroxaban Therapy

Thrombosis remains a major cause of mortality and morbidity worldwide. Common risk factors include pro-inflammatory conditions, endothelial dysfunction, and aberrant platelet activation, all augmenting the patient’s risk of developing a thrombus. Intriguingly, these risk factors also manifest in increased levels of circulating extracellular vesicles (EVs). EVs are a heterogenous group of membrane vesicles released from all cells. Due to the expression of tissue factor and phospholipids on their surface, EVs can accelerate thrombus formation. To circumvent adverse events in thrombotic disease, pharmacological thromboprophylaxis is frequently indicated. Rivaroxaban, a direct FXa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties, however, these remain poorly characterized. Given the prevalent role of EVs in thrombotic diseases, we hypothesized that Rivaroxaban’s anti-inflammatory properties are reflected upon differential molecular profiles of circulating EVs. Here, we used single vesicle analysis and comparative proteomics to, for the first time, characterise small (<200nm) and large (200-1000nm) plasma EVs from patients with non-valvular atrial fibrillation (AF), venous thromboembolism (VTE) and stable cardiovascular disease (CVD) treated with Rivaroxaban compared with cohort-specific controls. We identified profound changes in circulating EV profiles and proteomic signatures that may contribute to Rivaroxaban’s pleiotropic effects. Circulating EV profiles were fundamentally altered across the cohorts analysed. While AF patients displayed profoundly reduced levels of total small EVs, we identified a decrease in the proportion of large EVs between 400-700 nm in VTE patients. Patients with stable CVD, on the other hand, indicated increased small EV mode size with a concomitant increase in the proportion of large EVs between 600-800 nm. Comparative proteomic characterisation of enriched EV fractions revealed differential expression of proteins involved in the regulation of complement activation, vascular integrity, and inflammation, collectively mirroring the reported anti-inflammatory and cardioprotective characteristics associated with Rivaroxaban therapy. In summary, we have determined that circulating EV profiles are powerful surrogate markers of the Rivaroxaban-mediated pleiotropic effects. Regardless of the underlying thrombotic disease, Rivaroxaban appears to confer vascular protective effects in both venous and arterial thrombotic conditions. While several mechanisms, including inhibition of protease activated receptor or NF-¿B signalling, have been proposed to contribute to these vascular protective characteristics mediated by Rivaroxaban, we here provide original evidence that the modulation of circulating EV signatures may pose a distinct mechanism through which Rivaroxaban therapy may elicit such pleiotropic effects, an exciting finding that may be leveraged when planning future studies exploring personalized management strategies, such as identification of those patients most likely to benefit from this therapy, particularly in jurisdictions where access to therapy may be restricted. Alleviating the underlying pro-inflammatory state of patients with thrombosis poses a promising future therapeutic target to avert major adverse thrombotic events. Further investigations into Rivaroxaban-mediated changes in circulating EV signatures may provide a future avenue to open Rivaroxaban’s use for extended patient cohorts, such as rheumatoid arthritis.