**5. Microvesiculation of erythrocytes and fragmentation of platelets**

It was found that in *ex vivo* conditions washed erythrocytes undergo transformation into echinocytes (**Figure 6A**) and eventually budding takes place on the top of echinocyte spicules (**Figure 6 B** and **C**) [25, 26, 34]. Vesiculation was accelerated by the addition of amphiphilic molecules into the suspension of washed erythrocytes [25]. The shape of the buds as well as of the vesicles found in the isolates depended on the type of amphiphilic molecules added (**Figure 6B**–**E**). Vesicles matching in size and shape could be found in the isolate (**Figure 6D** and **E**) [34]. It was assumed that the amphiphilic molecules intercalate into the erythrocyte membrane and change the identity of the membrane constituents which in turn causes shape transformation. While dodecylzwittergent induced budding of globular structures and globular shape of isolated CVs (**Figure 6B** and **D**), dodecylmaltoside induced tubular shape of the buds and the CVs (**Figure 6C** and **E**). Dodecylmaltoside is composed of a carbohydrate tail and a bulky multipolar headgroup. The orientational ordering of the constituents involving dodecylmaltoside can explain the stable shape of tubular buds and vesicles that were observed in experiments. Budding erythrocytes were found also in CV isolates from blood [30] indicating that a part of CVs harvested from blood could be erythrocyte microvesicles.

#### **Figure 6.**

*A: Echinocytes, B: Spheroechinocyte with glubular buds induced by dodecyl-zwittergent, C: Spheroechinocyte with tubular buds induced by dodecylmaltoside, as observed with SEM; D and E: Respective isolated spherical and tubular CVs as observed by TEM. From [34].*

The relevance of the model lies in agreement with experiments. The model of isotropic bending describes well the discocyte-stomatocyte transformation of erythrocytes, but cannot distinguish between tubular and spherical budding of the vesicle and therefore the formation of tubular/spherical vesicles. To our best knowledge, the presented model is by now the only model that explained the stability of different nanostructures with strongly anisotropically curved membranes (tubes, thin necks, hexagonal and cubic stacks).

Observations of isolates from blood indicate the presence of a major pool of CVs which shape corresponds to the membrane free energy and can be described as colloidal CVs. It was found [30] that the size of the colloidal CVs in blood isolates was different for different isolation protocols indicating a transient identity of colloidal CVs. In contrast, erythrocyte microvesicles shed from washed erythrocytes were uniform in size and were sensitive to intrinsic curvatures of the membrane constituents. The identity of CVs depends on the processing of samples. As stated above, the most commonly used method for CV isolation involves (differential) centrifugation/ ultracentrifugation, which can be followed by ultracentrifugation on a sucrose or iodixanol gradient [4, 5, 49]. Ultrafiltration, dialysis, and size exclusion chromatography are used to harvest fractions of EVs, and immunoaffinity isolation and precipitation methods are used to harvest CVs with particular compositions [50, 51]. But transformation of the material may occur during any of these harvesting procedures, due to chemical/mechanical/thermal stress, as the nanostructures are fragile and prone to interact. Furthermore, the same principle applies to assessment methods. Commonly used methods are flow cytometry, SEM, TEM, atomic force microscopy, light scattering, fluorescence microscopy with analysis of Brownian motion (nanoparticle tracking analysis), and immunoblotting. Also, CV contents are analyzed for high-resolution molecular profiling of protein, microRNA, and lipid content (reviewed in [51]).

Besides colloidal CVs, other types of particles can be expected in biological samples, such as lipoproteins [52] and viruses [53]. Recently, besides the colloidal CVs, rod-like viruses were identified in CV isolates from tomato homogenate. The CVs were isolated by differential centrifugation and by size exclusion chromatography and observed by SEM [53] and by cryo-TEM (**Figure 7**). Three different fractions of the isolate obtained by using iodixanol gradient are shown (denoted by A, B, and C, respectively). The fraction observed in Panel A contained mostly colloidal CVs (black arrow); the fraction observed in Panel B contained colloidal CVs (black arrow) and rod-like virions (white arrow); the fraction observed in Panel C contained mostly

#### **Figure 7.**

*Cryo-TEM images of three fractions of CV isolate from homogenate of tomato infected by the viruses. Panels A - C show three different fractions of the EV isolate that were separated by iodixanol gradient ultracentrifugation. Black arrows point to CVs, white arrows point to virions. Adapted from [53].*

virions (white arrow). It was found by analyzing proteome with mass spectrometry that the samples contained capsid proteins of three tomato viruses [53].
