**3. Increased intracellular Ca2+ content of human RBCs results in an activation of the scramblase**

The PS exposure on the outer membrane leaflet of RBCs was studied for LPAtreated cells using fluorescence microscopy. It has been shown that the number of annexin V-positive cells (i.e. cells showing PS exposure) after LPA treatment in the presence of Ca2+ is about 35% [30].

Experiments have also been carried out after treating the RBCs with LPA or the PKC-activator PMA but in the absence of extracellular Ca2+ (presence of 1 mM EGTA). In the case of LPA, we did not find significant differences between treatment and control conditions (without activation) regarding PS exposure on human RBCs. Only in the case of PMA treatment, about 50% of RBCs showed PS exposure in the absence of Ca2+ (compared to about 80% in the presence of Ca2+). The data obtained for control conditions in the presence of Ca2+ were not significantly different from the data of the control conditions in the absence of Ca2+ [30].

It is evident that for stimulation with the phorbol ester PMA, there is no correlation between the number of cells showing an elevated Ca2+ content and the induced PS exposure, because it is known that PMA activates PKCα also in the absence of Ca2+.

Furthermore we like to state that the threshold for the forward scatter in the flow cytometry measurements was set in a way to make sure that only the events based on the size of intact cells were counted. This was of importance since we realised the formation of micro-vesicles after treatment with LPA as well as with PMA, which is in agreement with findings reported before [41, 42]. Calpain, a Ca2+-dependent

**21**

*Red Blood Cells Actively Contribute to Blood Coagulation and Thrombus Formation*

[43–45] and therefore could be involved in the vesiculation process.

when the scramblase and the PKCα are inhibited simultaneously [34].

ferent mechanisms could be responsible for the PS exposure on RBCs:

proteolytic enzyme, cleaves spectrin and actin, leading to cytoskeleton breakdown

The findings that Ca2+ content and PS exposure do not correlate in all cells were further supported by confocal fluorescence microscopy, where double-labelling experiments (for intracellular Ca2+ and PS in the outer membrane leaflet) have been carried out. The results of the LPA treatment showed that most of the cells have an elevated Ca2+ content and depict a PS exposure. However, some cells are displaying an increased Ca2+ level, but no PS exposure, whereas other cells externalise PS

In the case of PMA treatment, PS exposure without high Ca2+ content is more pronounced, which is not surprising since PMA is an artificial compound, which activates conventional PKCs without the otherwise necessary presence of Ca2+.

The percentage of RBCs showing PS exposure after activation with LPA or PMA is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 [34]. It is also significantly reduced after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced

Based on our experiments, it seems reasonable to assume that at least three dif-

• The first mechanism is related to the Ca2+-stimulated scramblase activation (and flippase inhibition) [4, 8, 10, 11, 47]. This effect involves the activation of

• The second mechanism involves the PKCα, which can be directly activated by PMA. It has been reported that PKCα is involved in the PS exposure in human RBCs [25, 26]. In addition, it has been shown that LPA activates in human RBCs both the PKCα (Ca2+-dependent) and the PKCζ (Ca2+-independent) [22]. Whether there is a direct activation of the scramblase in human RBCs by PKCα and/or PKCζ remains to be proofed. The more pronounced effect of PS exposure observed after treatment of RBCs with PMA as compared to LPA can be explained by assuming that PMA activates all available PKCα, whereas LPA stimulation triggers a signalling cascade [46] resulting only in partial activa-

the ω-agatoxin-TK-sensitive, CaV2.1-like (P/Q-type) Ca2+ channel [33].

• The third mechanism is the enhanced lipid flop caused by LPA [30]. This

have been carried out where the physiological solution was replaced by a solution containing 150 mM KCl plus 2.5 mM NaCl instead of 145 mM NaCl plus 7.5 mM KCl. Under these conditions of a high extracellular KCl concentration, an opening of the

of the PS exposure in the presence of LPA is caused by the volume decrease.

scope. The obtained results are presented in **Figure 3**.

mechanism is the only one in sheep RBCs suggesting the absence of scramblase

To consider a possible effect of cell volume alteration on PS exposure, experiments

panying cell shrinkage. Based on this strategy, it has been found that a substantial part

It remains to be elucidated why, in the experiments reported above, some cells are displaying an increased Ca2+ level, but no PS exposure, whereas other cells externalise PS without having a higher Ca2+ content. In that context, we carried out single-labelling experiments (fluo-4 for intracellular Ca2+ and annexin V-FITC for exposed PS) and focussed on the shape of RBCs using also a fluorescence micro-

channel (Gardos channel) does not result in a KCl loss and accom-

*DOI: http://dx.doi.org/10.5772/intechopen.86152*

without having a higher Ca2+ content.

tion of the PKCα pool.

activity in these cells [30].

Ca2+-dependent K+

#### *Red Blood Cells Actively Contribute to Blood Coagulation and Thrombus Formation DOI: http://dx.doi.org/10.5772/intechopen.86152*

proteolytic enzyme, cleaves spectrin and actin, leading to cytoskeleton breakdown [43–45] and therefore could be involved in the vesiculation process.

The findings that Ca2+ content and PS exposure do not correlate in all cells were further supported by confocal fluorescence microscopy, where double-labelling experiments (for intracellular Ca2+ and PS in the outer membrane leaflet) have been carried out. The results of the LPA treatment showed that most of the cells have an elevated Ca2+ content and depict a PS exposure. However, some cells are displaying an increased Ca2+ level, but no PS exposure, whereas other cells externalise PS without having a higher Ca2+ content.

In the case of PMA treatment, PS exposure without high Ca2+ content is more pronounced, which is not surprising since PMA is an artificial compound, which activates conventional PKCs without the otherwise necessary presence of Ca2+.

The percentage of RBCs showing PS exposure after activation with LPA or PMA is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 [34]. It is also significantly reduced after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously [34].

Based on our experiments, it seems reasonable to assume that at least three different mechanisms could be responsible for the PS exposure on RBCs:


To consider a possible effect of cell volume alteration on PS exposure, experiments have been carried out where the physiological solution was replaced by a solution containing 150 mM KCl plus 2.5 mM NaCl instead of 145 mM NaCl plus 7.5 mM KCl. Under these conditions of a high extracellular KCl concentration, an opening of the Ca2+-dependent K+ channel (Gardos channel) does not result in a KCl loss and accompanying cell shrinkage. Based on this strategy, it has been found that a substantial part of the PS exposure in the presence of LPA is caused by the volume decrease.

It remains to be elucidated why, in the experiments reported above, some cells are displaying an increased Ca2+ level, but no PS exposure, whereas other cells externalise PS without having a higher Ca2+ content. In that context, we carried out single-labelling experiments (fluo-4 for intracellular Ca2+ and annexin V-FITC for exposed PS) and focussed on the shape of RBCs using also a fluorescence microscope. The obtained results are presented in **Figure 3**.

*Erythrocyte*

**Figure 2.**

*Hertz et al. [40].*

**3. Increased intracellular Ca2+ content of human RBCs results in an** 

*on the outer leaflet of the membrane; activation of the Gardos channel followed by the efflux of K<sup>+</sup>*

*H2O; and consecutive cell shrinkage. All these processes may lead to an increased RBC aggregation and/or accelerated RBC clearance, which is impaired when patients are splenectomised. This figure is modified from* 

ent from the data of the control conditions in the absence of Ca2+ [30].

It is evident that for stimulation with the phorbol ester PMA, there is no correlation between the number of cells showing an elevated Ca2+ content and the induced PS exposure, because it is known that PMA activates PKCα also in the

Furthermore we like to state that the threshold for the forward scatter in the flow cytometry measurements was set in a way to make sure that only the events based on the size of intact cells were counted. This was of importance since we realised the formation of micro-vesicles after treatment with LPA as well as with PMA, which is in agreement with findings reported before [41, 42]. Calpain, a Ca2+-dependent

The PS exposure on the outer membrane leaflet of RBCs was studied for LPAtreated cells using fluorescence microscopy. It has been shown that the number of annexin V-positive cells (i.e. cells showing PS exposure) after LPA treatment in the

*Proposed mechanisms leading to increased intracellular Ca2+ levels in diseased RBCs. Alternative or cumulating Ca2+ entry pathways are highlighted with grey background: increased abundance of NMDA-receptors (NMDAR), e.g. in sickle cell disease; altered activity of Piezo1, e.g. in hereditary xerocytosis; increased activity of the Gardos channel, e.g. in Gardos Channelopathy; or unspecified Ca2+ transport mechanisms. Additionally, ineffective extrusion of Ca2+ due to disruption of ATP pools fuelling the plasma membrane Ca2+ ATPase (PMCA) can contribute. Several downstream processes follow Ca2+ overload in RBCs, e.g. activation of calmodulin by formation of the Ca2+-calmodulin complex (Ca-CaM) and activation of calpain, thereby loosening the cytoskeletal structure; activation of the scramblase (Scr) leading to exposure of phosphatidylserine* 

*, Cl<sup>−</sup> and* 

Experiments have also been carried out after treating the RBCs with LPA or the PKC-activator PMA but in the absence of extracellular Ca2+ (presence of 1 mM EGTA). In the case of LPA, we did not find significant differences between treatment and control conditions (without activation) regarding PS exposure on human RBCs. Only in the case of PMA treatment, about 50% of RBCs showed PS exposure in the absence of Ca2+ (compared to about 80% in the presence of Ca2+). The data obtained for control conditions in the presence of Ca2+ were not significantly differ-

**activation of the scramblase**

presence of Ca2+ is about 35% [30].

**20**

absence of Ca2+.

#### **Figure 3.**

*Human RBC shapes after 2.5 μM LPA stimulation. Transmitted light (left) and fluorescence images (right) of RBCs. (A) Intracellular Ca2+ content detected using fluo-4, (B) PS exposure detecting using annexin V-FITC. (C, D) Flow cytometric analysis of responding cells after LPA stimulation in different solutions correlate with predominant cell shapes. Predominantly discocytes, physiological solution (black); predominantly echinocytes, low ionic strength solution + DIDS (red) and physiological solution + DIDS (orange); predominantly stomatocytes or discocytes, low ionic strength solution (blue); physiological solution at pH 5.6 (violet).*

In **Figure 3Aa** one can see different cell shapes in the transmitted light image for LPA stimulation. However, the corresponding fluorescence image (**Figure 3Ab**) does not show differences of the increased Ca2+ content comparing RBCs with different shapes. The situation changes when analysing the PS exposure of the RBCs with different shapes (**Figure 3B**). Again, RBCs with different shapes can be seen (**Figure 3Ba**), but in the case of PS exposure, the cell shape seems to play a significant role. Some echinocytes can be seen in the transmitted light images (red circles in **Figure 3Ba**). Almost all of them lack PS exposure (corresponding fluorescence image in **Figure 3Bb**). Only one echinocyte showing PS exposure can be observed. The explanation of such an effect could be based on the bilayer couple hypothesis. If an echinocyte is formed, the outer membrane leaflet is already extended compared with the inner membrane leaflet. It means that any process of externalisation of a membrane lipid located mainly in the inner membrane leaflet is hindered. In addition we carried out experiments where we transferred the RBCs to different solutions as shown in

**Figure 3C and D**. The RBC shapes, which predominantly occur in these solutions, are also indicated in **Figure 3**. Again, the PS exposure is lowest in solution where one can expect mostly echinocytes although the Ca2+ content is increased (red curves in **Figure 3**). The PS exposure of RBCs can be small also in solution where cells predominantly occur with the shape of discocytes or stomatocytes, however, in this case only in solutions where the increase of the intracellular Ca2+ content is prevented, e.g. physiological solution of low pH (5.6, see violet curves in **Figure 3**).

Furthermore, we carried out double-labelling experiments (X-Rhod-1 for intracellular Ca2+ and annexin V-FITC for exposed PS) and investigated the RBCs using a fluorescence microscope. The obtained results are presented in **Figure 4**. One RBC (indicated with white arrow in all panels of **Figure 4**) shows an increased intracellular Ca2+ content, but the cell suddenly disappeared from the transmitted light image

**23**

**Figure 4.**

*Red Blood Cells Actively Contribute to Blood Coagulation and Thrombus Formation*

(**Figure 4Ab**). However, PS exposure of this cell still can be seen (**Figure 4Bb**). It can

*RBCs were stimulated with lysophosphatidic acid, and panels (A) depict images 1 min after the start of the stimulation and panels (B) 1.5 min after the beginning of the stimulation. Panel (A) shows wide-field images and panel (B) confocal sections of the fluorescent biomarkers X-Rhod-1 (a Ca2+ indicator) and FITC-labelled annexin V (for PS detection). Panel (C) is an overlay of panels (A) and (B). The white arrow points to a cell* 

A different behaviour of RBCs depending on the age of the cell has been controversially discussed. Therefore, we performed additional short-time incubation experiments, comparable to the experiments carried out by Nguyen et al. [30], with RBCs separated in five fractions with different cell age according to the method of Lutz et al. [47]. The intracellular Ca2+ content and PS exposure at the outer membrane leaflet of human RBCs with different age have been investigated using, e.g. LPA stimulation. Here we present a reanalysis of these already published data [48]. Interestingly, the percentage of RBCs showing increased Ca2+ content and PS

be assumed that this RBC lysed, i.e. we see the remaining membrane structure

(a ghost) and the Ca2+-sensitive fluorescent dye diffused out of the cell.

*that lysed after capturing images (a), i.e. in images (b) it's a ghost.*

*DOI: http://dx.doi.org/10.5772/intechopen.86152*

*Red Blood Cells Actively Contribute to Blood Coagulation and Thrombus Formation DOI: http://dx.doi.org/10.5772/intechopen.86152*

#### **Figure 4.**

*Erythrocyte*

**Figure 3.**

In **Figure 3Aa** one can see different cell shapes in the transmitted light image for LPA stimulation. However, the corresponding fluorescence image (**Figure 3Ab**) does not show differences of the increased Ca2+ content comparing RBCs with different shapes. The situation changes when analysing the PS exposure of the RBCs with different shapes (**Figure 3B**). Again, RBCs with different shapes can be seen (**Figure 3Ba**), but in the case of PS exposure, the cell shape seems to play a significant role. Some echinocytes can be seen in the transmitted light images (red circles in **Figure 3Ba**). Almost all of them lack PS exposure (corresponding fluorescence image in **Figure 3Bb**). Only one echinocyte showing PS exposure can be observed. The explanation of such an effect could be based on the bilayer couple hypothesis. If an echinocyte is formed, the outer membrane leaflet is already extended compared with the inner membrane leaflet. It means that any process of externalisation of a membrane lipid located mainly in the inner membrane leaflet is hindered. In addition we carried out experiments where we

*Human RBC shapes after 2.5 μM LPA stimulation. Transmitted light (left) and fluorescence images (right) of RBCs. (A) Intracellular Ca2+ content detected using fluo-4, (B) PS exposure detecting using annexin V-FITC. (C, D) Flow cytometric analysis of responding cells after LPA stimulation in different solutions correlate with predominant cell shapes. Predominantly discocytes, physiological solution (black); predominantly echinocytes, low ionic strength solution + DIDS (red) and physiological solution + DIDS (orange); predominantly stomatocytes or discocytes, low ionic strength solution (blue); physiological solution at pH 5.6 (violet).*

**Figure 3C and D**. The RBC shapes, which predominantly occur in these solutions, are also indicated in **Figure 3**. Again, the PS exposure is lowest in solution where one can expect mostly echinocytes although the Ca2+ content is increased (red curves in **Figure 3**). The PS exposure of RBCs can be small also in solution where cells predominantly occur with the shape of discocytes or stomatocytes, however, in this case only in solutions where the increase of the intracellular Ca2+ content is prevented, e.g. physi-

Furthermore, we carried out double-labelling experiments (X-Rhod-1 for intracellular Ca2+ and annexin V-FITC for exposed PS) and investigated the RBCs using a fluorescence microscope. The obtained results are presented in **Figure 4**. One RBC (indicated with white arrow in all panels of **Figure 4**) shows an increased intracellular Ca2+ content, but the cell suddenly disappeared from the transmitted light image

transferred the RBCs to different solutions as shown in

ological solution of low pH (5.6, see violet curves in **Figure 3**).

**22**

*RBCs were stimulated with lysophosphatidic acid, and panels (A) depict images 1 min after the start of the stimulation and panels (B) 1.5 min after the beginning of the stimulation. Panel (A) shows wide-field images and panel (B) confocal sections of the fluorescent biomarkers X-Rhod-1 (a Ca2+ indicator) and FITC-labelled annexin V (for PS detection). Panel (C) is an overlay of panels (A) and (B). The white arrow points to a cell that lysed after capturing images (a), i.e. in images (b) it's a ghost.*

(**Figure 4Ab**). However, PS exposure of this cell still can be seen (**Figure 4Bb**). It can be assumed that this RBC lysed, i.e. we see the remaining membrane structure (a ghost) and the Ca2+-sensitive fluorescent dye diffused out of the cell.

A different behaviour of RBCs depending on the age of the cell has been controversially discussed. Therefore, we performed additional short-time incubation experiments, comparable to the experiments carried out by Nguyen et al. [30], with RBCs separated in five fractions with different cell age according to the method of Lutz et al. [47]. The intracellular Ca2+ content and PS exposure at the outer membrane leaflet of human RBCs with different age have been investigated using, e.g. LPA stimulation. Here we present a reanalysis of these already published data [48]. Interestingly, the percentage of RBCs showing increased Ca2+ content and PS

**Figure 5.**

*Reanalysis of data initially presented in [48]. In the original publication, only two fractions were compared with each other, while here we followed the approach to plot (and analyse) the measured effect in dependence of the cell age. (A) presents the situation under control conditions (without stimulation) and (B) the after 15 min stimulation with LPA (p < 0.5 is marked with \*).*

exposure depict a linear behaviour in dependence of cell age with a very good regression, R2 of 0.94 and 0.92, respectively, as outlined in **Figure 5A**. However, the slope of this linear regression failed to be significantly different from zero.

After stimulation of the RBCs with LPA, the situation is even more complex. **Figure 5B** depicts the situation after 15 min of LPA stimulation. While the Ca2+ concentration seems to relate inversely proportional to RBC age, PS-positive cells show a rather quadratic dependence on cell age. This is in contradiction to earlier investigations we performed on mouse RBCs [31]. Although we cannot completely resolve the situation part of the explanation might be caused in the detection technique: While microscopy is a rather gentle approach, in flow cytometry the cells under investigation experience high pressure and significant shear forces [60]. Therefore a significant number of high Ca2+ cells that are more fragile may lyse in the flow cytometer and hence are excluded from the detection.

Furthermore it is worthwhile to mention that under in vivo conditions, cells with a permanent high Ca2+ content and/or PS exposure are removed from the circulation, mostly in the spleen, while after long in vitro incubation time (48 h), old RBCs responded with higher increase of intracellular Ca2+ content as well as higher PS exposure compared to young RBCs [48, 49].

The intracellular Ca2+ content and the PS exposure at the outer membrane leaflet have been investigated for human RBCs also in physiological solutions prepared with oxygen-enriched water (in comparison to normal physiological solution). This was a study for a company (futomat®) producing equipment for the production of oxygen-enriched water for drinking. It has been found that Ca2+ content and the PS exposure are not changed significantly in oxygen-enriched water. However, one interesting effect was found when the RBCs were treated with LPA. Under such conditions the PS exposure was significantly reduced in futomat® water compared to normal physiological solution. It remains open and requires clinical studies to see whether there is a relevant effect in human beings, meaning a positive effect preventing thrombus formation.
