Extracellular vesicle-associated procoagulant phospholipid and tissue factor activity in multiple myeloma

Abstract

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.

CD38, a Receptor with Multifunctional Activities: From Modulatory Functions on Regulatory Cell Subsets and Extracellular Vesicles, to a Target for Therapeutic Strategies

“…Such conclusion was supported by the direct correlation measured between the percentage of CD38 hi EV and malignant plasma cells in the BM [70]. Notably, the presence of CD38 and CD39 on EV isolated from BM of MM patients has been recently confirmed by electron microscopy analysis [72].…”

Novel Blood-Derived Extracellular Vesicle-Based Biomarkers in Alzheimer’s Disease Identified by Proximity Extension Assay

“…Transmission Electron Microscopy (TEM) was performed to detect and confirm vesicular CD9 + structures in the EV pellets. TEM was performed as previously described [ 35 ]. Briefly, five microliters of pooled EV pellets were mounted for 30 seconds on carbon-coated, glow discharged 400 mesh Ni grids (SPI Supplies, Chester, PA, USA), stained with one drop 1% ( w / v ) phosphotungstic acid (PTA) pH 7.0 (Ted Pella, Caspilor AB, Lindingö, Sweden), and then dry-blotted using filter papers.…”

Targeted Approaches to Inhibit Sialylation of Multiple Myeloma in the Bone Marrow Microenvironment

“…It has been proposed that EVs have an important role in different steps of the metastatic process (Colombo et al, 2019). Due to their pro-coagulant activity, EVs could lead to platelet activation and polymerization of fibrinogen to fibrin, which in turn would enhance MM dissemination by protecting the malignant plasma cells in the circulation, favoring their seeding to distant sites and pre-conditioning the metastatic niche with platelet-derived cytokines (Labelle et al, 2014; Remiker and Palumbo, 2018; Nielsen et al, 2019). It has also been shown that MM-EVs contribute to neo-angiogenesis by inducing endothelial cell proliferation and formation of new blood vessels (Liu et al, 2014; Wang et al, 2016; Li et al, 2019; Zarfati et al, 2019).…”