Towards standardization of assays measuring extracellular vesicle‐associated tissue factor activity

The clinical and scientific interest in extracellular vesicles (EVs) is growing exponentially. The term EVs is an umbrella term for various types of vesicles that are present in body fluids and other (bio)fluids. This umbrella term is used because clear hallmarks to distinguish different types of EVs from each other are lacking. Thus, the term “EVs” encompasses earlier “microparticles” or “microvesicles” and exosomes, which are vesicles released directly from the plasma membrane or by secretion of intraluminal vesicles stored in multivesicular endosomes, respectively.1, 2 There is evidence that EVs play a role in intercellular communication and contribute to coagulation and likely inflammation.3, 4, 5, 6 The oldest‐known function of “platelet dust,” now known as platelet‐derived EVs, is their ability to support coagulation by exposing negatively charged phospholipids, such as phosphatidylserine (PS). Such PS exposing EVs facilitates formation of tenase and prothrombinase complexes. Furthermore, different subtypes of EVs, such as leukocyte, endothelial, or tumor‐derived EVs, can also trigger coagulation by exposing tissue factor (TF).7 Tissue factor‐exposing EVs (TF‐EVs) are present in body fluids, such as saliva and urine, under physiological conditions. The presence of TF‐EVs in saliva may explain the reflex to lick a wound, thereby exposing blood to extravascular TF and accelerating hemostasis and reducing the risk of infection.8 Although Tissue factor was initially thought to be exclusively present outside the vasculature (“envelope model”); there is increasing evidence that during medical intervention and in various clinical conditions, such as surgery, or in patients suffering from sepsis or cancer, the presence of coagulant TF‐EVs is associated with disseminated intravascular coagulation and venous thrombosis.9, 10 There are two reasons why a proposed standardization is timely and relevant. First, there is a growing interest to improve the reproducibility of results in science in general, and this also holds true for the new field of EV research. During the last few years, “minimal requirements” have been published by the International Society of Extracellular Vesicles (ISEV) regarding the reporting on studies involving EVs,11, 12, 13 as well as a structure to record and score reporting of preanalytical variables 14, 15, 16, 17 In addition, guidelines and position papers have been published18, 19 and an increasing number of standardization studies have been and are being performed involving various aspects of EV detection and characterization.2, 14, 20, 21 At present, various in‐house and commercially available assays have been developed to measure the EV‐associated TF (EV‐TF) activity, but hitherto the results of these methods have not been easily compared and required standardization. Second, to identify cancer patients at risk of developing venous thromboembolism, an EV‐TF‐based factor Xa generation assay and an EV‐TF‐based plasma clotting test have been developed and applied in clinical trials and have shown promising results for the prediction of VTE in pancreatic cancer patients. This underscores the relevance of studying TF‐EVs as a potential clinically relevant biomarker.22, 23 Taken together, we provide a summary of the outcomes of the questionnaire and discussion with the goal to improve future standardization of studies measuring the TF activity of EVs.