Date Published: January 14, 2019
Publisher: Public Library of Science
Author(s): Thøger Nielsen, Søren Risom Kristensen, Henrik Gregersen, Elena Manuela Teodorescu, Gunna Christiansen, Shona Pedersen, Giovanni Camussi.
Multiple myeloma (MM) patients have increased risk of developing venous thromboembolism, but the underlying mechanisms and the effect on the coagulation system of the disease and the current cancer therapies are not known. It is possible that cancer-associated extracellular vesicles (EV), carrying tissue factor (TF) and procoagulant phospholipids (PPL) may play a role in thrombogenesis. The aim of this study was to perform an in-depth analysis of procoagulant activity of small and large EVs isolated from 20 MM patients at diagnosis and after receiving first-line treatment compared with 20 healthy control subjects. Differential ultracentrifugation at 20,000 × g and 100,000 × g were used to isolate EVs for quantitative and phenotypical analysis through nanoparticle tracking analysis, Western blotting and transmission electron microscopy. The isolated EVs were analyzed for procoagulant activity using the calibrated automated thrombogram technique, a factor Xa-based activity assay, and the STA Procoag-PPL assay. In general, MM patients contained more EVs, and immunoelectron microscopy confirmed the presence of CD9- and CD38-positive EVs. EVs in the 20,000 × g pellets from MM patients exerted procoagulant activity visualized by increased thrombin generation and both TF and PPL activity. This effect diminished during treatment, with the most prominent effect observed in the high-dose chemotherapy eligible patients after induction therapy with bortezomib, cyclophosphamide, and dexamethasone. In conclusion, the EVs in patients with MM carrying TF and PPL are thus capable of exerting procoagulant activity.
Cancer patients have a 4–7-fold higher risk of venous thromboembolism (VTE) than does the general population, but the risk in different cancer types varies, and the frequency of VTE in cancer patients is between 1–8% [1–3]. Patients with multiple myeloma (MM) have a considerably increased risk of VTE, partly because the associated treatment may be thrombogenic [4–6]. Although several factors, such as age, acquired protein C resistance, coagulation factor VIII, von Willebrand factor, and interleukin-6, have been proposed as contributors to this hypercoagulable state, the mechanisms causing VTE in patients with MM are not clearly understood [7–9]. A possible contributing factor is an increased level of tissue factor (TF), a central coagulation factor in initiating haemostasis that triggers thrombin generation . It has been reported that aberrant TF expression is linked to cancer pathophysiology, e.g., angiogenesis . Anionic procoagulant phospholipids (PPL), such as phosphatidylserine, act as important cofactors necessary for the formation of coagulation complexes but have also been proposed to be involved in cancer pathogenesis . TF and PPL can be present in plasma in circulating extracellular vesicles (EV) with procoagulant properties. In malignancy, EVs from the cancer cells are involved in several pleiotropic processes, such as metastasis, angiogenesis, and immunomodulation [13,14]. Because they may also carry TF and PPL, likely on the large EVs, so-called microvesicles (MV), these EVs may play a significant role in haemostasis and VTE-risk in various diseases, including MM [15–18]. Auwerda et al  reported a microparticle-associated TF-activity in MM patients receiving high-dose chemotherapy (HDCT). The aim of this study was to investigate the procoagulant effect of EVs from patients with newly diagnosed MM compared with controls, hypothesizing that EVs in patients with MM are procoagulant.
This setup to investigate procoagulant activity demonstrated a substantially higher thrombin generation and both TF and PPL activity in EVs in patients with MM than in healthy control subjects. This increase in procoagulant activity, however, diminished markedly in the patients receiving VCD induction therapy and to a lesser extent in those that received the conventional treatment. These results indicate that the procoagulant activity in MM can be ascribed to the larger EVs, which likely exert their procoagulant activity through PPL and TF. Furthermore, we demonstrated that some of the EVs possibly originate from the cancerous B cells.