The Role of Plasma Extracellular Vesicles and Procoagulant Phospholipid Activity in Venous Thromboembolism

Abstract

Venous thromboembolism (VTE) is the formation of a blood clot in, most commonly, the deep veins of the lower extremities and the pulmonary circulation. VTE is a prevalent disease associated with severe short- and long-term complications. Negatively charged procoagulant phospholipids (PPL), and phosphatidylserine (PS) in particular, are vital to efficient coagulation activation, and found expressed on the surface of extracellular vesicles (EVs) and activated platelets. The overall aim of the present thesis was to develop an easily available and reproducible FXa-dependent clotting assay to measure PPL activity in plasma, and further use the assay to investigate the association between plasma PPL activity and the risk of VTE. In paper I, we investigate the impact of several pre-analytical conditions on EV concentration and size measured by Nanoparticle Tracking Analysis (NTA) and scanning electron microscopy (SEM). In paper II, we developed a modified FXa-dependent clotting assay by substituting the chemically phospholipid depleted plasma with PPL-depleted plasma obtained by ultracentrifugation. In paper III, we used our modified PPL assay to investigate the association between PPL clotting time (PPLCT) and the risk of incident VTE in a nested case-control study derived from a population based cohort (the Tromsø study). Previous studies have suggest that statin treatment reduced the risk of recurrent VTE. In paper IV, we investigated the impact of statin treatment (rosuvastatin) on PPL activity, using the modified PPL assay and plasma samples from the STAtins Reduce Thrombophilia trial. The impact of pre-analytical conditions (i.e. anticoagulants, centrifugation protocols, and fasting status) on EV measurements was demonstrated, and the obstacle of post-prandial lipoproteins interfering with NTA analysis was particularly highlighted. We found that the modified PPL assay displayed similar sensitivity and reproducibility compared to commercial assays based on chemically phospholipid-depleted plasma. We observed an inverse association between plasma PPLCT, assessed by the modified assay, and the risk of future VTE in a population-based nested case-control study. Additionally, rosuvastatin treatment caused a substantial decrease in plasma PPL activity in subjects with a history of VTE. The development of the modified PPL assay enabled us to perform high-quality measurements in large-scale studies. The inverse association between PPLCT and VTE risk supports an important role of plasma PPL in the pathogenesis of VTE and may partly explain the reduced risk of VTE recurrence observed by statin treatment.