ABSTRACT
Figure 7.1 MVs generated in blood circulation can originate from various blood cells, although the majority are produced by platelets; they can be markers of pathology, but they also contribute to the progression of disease. Capture-based assays, as first reported by Aupeix et al.,19 also use surface-coated A5 for capturing these MVs in the presence of calcium and then simply measuring their procoagulant potential through thrombin generation, in the presence of factors Xa, Va, and prothrombin. Capture-based assays unlike flow cytometry measure the total procoagulant activity associated with all sizes of EVs that express PS. Theoretically EVs with the lowest size will also support a much higher procoagulant potential than larger vesicles because of the increase in surface area. Capture-based assays can also be designed for specifically measuring EVs derived from defined blood cells by using specific monoclonal antibodies to appropriate cell surface antigens (CDs). Of specific interest are EVs exposing tissue factor (TF), as they also associate the PS-dependent procoagulant activity with the blood coagulation-triggering effect of TF.20-23
A third class of assays has also been described for measuring the plasma procoagulant potential of cell-released phospholipids, mainly microparticles (MPs), using homogeneous clotting assays.24 Although these assays are nonspecific unlike capture methods they can still measure intrinsic phospholipid-based procoagulant potential in plasma. In this chapter, the generation of EVs in health and disease and the technical characteristics of capture-based assays are described, as well as the preanalytical variables required. 7.2 Occurrence of EVs in Health and DiseaseMicro-and nanoparticles can be generated in many pathological conditions, originating from many cell types, including platelets, monocytes, leukocytes, erythrocytes, or endothelial cells. More par-ticularly, EVs exposing TF have been reported in cancer patients.25They are also associated with the high rate of thrombotic complica-tions observed in malignancy26 and also in infectious diseases27 or in sepsis.28 Many papers have been published that show the genera-tion of various EV types in pathology and their possible contribu-tion to disease progression and to thrombo-embolic complications. In physiological conditions, procoagulant activity increases with age (although the total number looks to remain stable). For example, in women, a significant change is observed at menopause, especially in metabolic syndrome.29,30 However, development of pathologi-cal conditions can have important repercussions on the generation of various types of EVs and on their thrombogenicity.31,9 Smoking habits can promote their release from monocytes/macrophages.32Abnormal levels of procoagulant activity and cell interactions have been described in obesity and metabolic syndrome.33,34 As expect-ed, these procoagulant vesicles and their thrombogenic effect are elevated in diabetic patients.35-37 The presence of EVs from differ-ent cell origins can also stimulate some interactions and synergy between these cells.38,39 Other clinical conditions, where EVs con-tribute to symptoms, have been reported, including Crohn disease40and injection of activated coagulation factors, such as factor VIIa.41In this latter case, the EVs can not only contribute to the hemostatic efficacy of factor VIIa but also may cause thrombotic complica-tions. Similarly, their contribution to the development of thrombosis in patients with heparin-induced thrombocytopenia has also been suggested.42 Conversely, in Scott syndrome, there is a defect in the
procoagulant activity of platelets with failure of PS exposure and EV generation, resulting in a lifelong bleeding diathesis.43 In cardiovascular diseases (myocardial infarction, atrial fibrillation) they can remain a predictor of possible recurrence and of poorer prog-nosis,44-46 and those exposing TF are associated with poorer recov-ery.47,48 In patients with cancer, blood activation and inflammation are important components, which contribute to pathological com-plications, and EVs are intimately involved in these processes. They can be derived from various cells and can expose TF in many cir-cumstances.26,49,21,25 This process can be potentiated by treatments implemented for cancer, such as chemotherapy. Therapeutic drugs used can also release EVs through the destruction of malignant cells, or they can directly activate blood cells.50 However, the contribution of the membrane environment seems to be more important than TF expression for promoting thrombin formation.51 EVs can also par-ticipate in a hypercoagulable state by allowing increased survival of the generated factor Va, as factor Va bound to EVs is protected from inactivation by activated protein C.52 The participation of MPs in disease evolution is also supported by a reduction of their con-centration and/or activity following initiation of targeted therapies, which potentially improve patient outcome.53-57 This effect is also supported by laboratory studies that show a reduction in EVs or the use of PS-targeted proteins (such as A5) to reduce their hemostatic potential.58-60 7.3 Capture-Based Assays for Procoagulant EVs
Capture-based assays exhibit many advantages for the specific measurement of the procoagulant potential of EVs. The basic capture-based assay used for EV procoagulant activity is derived from the method described by Aupeix et al.19 and is reported here next. 7.3.1 Assay Principle
Capture-based assays are designed using a specific PS capture protein (e.g., A5) or a monoclonal antibody (MoAb) coated on the solid phase for binding EVs within the tested specimens. Initially an assay of general use was developed for measuring procoagulant EVs. Basically, A5 is coated on a solid surface, usually a micro-
enzyme-linked immunosorbent assay (ELISA) plate (Maxisorb, Nunc, Roskilde, Denmark), and then stabilized. A diluted, tested plasma or specimen, a calibration standard of EVs, and controls are introduced into the A5-coated wells within a buffer diluent containing calcium. Any EVs within the samples therefore specifically bind with high affinity onto the A5 in the presence of calcium. Any unbound material is washed away with a washing solution also containing calcium in order to preserve the interaction of A5 with the EVs. Bound EVs are subsequently measured through thrombin generation. For this step, a solution containing factor Xa, factor Va, and calcium is added into the washed wells, and then a constant concentration of prothrombin is introduced. Prothrombinase forms on the EV surface, and its activity is dependent not only on the EV concentration but also on the exposure of PS. The amount of thrombin generated is proportional to the EV procoagulant activity, which is directly related to the PS concentration on the EV surface. The thrombin generated is measured through its activity on a specific chromogenic substrate. Results are expressed in nanomoles (nM) of PS equivalent. Calibration is performed using liposomes with a defined homogeneous size (e.g., 0.1 µm) and with a well-defined PS content (expressed in nanomoles). A direct linear relationship is obtained between EV concentration (expressed in nanometer PS) and the amount of procoagulant EVs. This assay principle and a typical calibration curve obtained are represented in Fig. 7.2.
