**2. Stimulation of intracellular Ca2+ increase in human RBCs**

PGE2 and LPA are local mediators released by platelets after their activation within the coagulation cascade. PGE2 can be even released by the RBCs themselves under conditions of mechanical stress [29]. We were able to show that the addition of both mediators to suspensions of human RBCs leads to an increase of the intracellular free Ca2+ concentration in these cells. In the case of PGE2 (0.1 nM), 45% of the RBCs responded with increased Ca2+ content; in the case of LPA (5 μM), nearly all cells reacted [17, 30] but still showed a strong heterogeneity in the time course and the intensity of the response [31].

The identity of the Ca2+ entry was proposed to be channel-mediated; on the one hand, the CaV2.1-channel [32] and on the other hand the non-selective voltage dependent cation channel [17] were suggested. While the activation of CaV2.1 was linked to protein kinase C (PKC) activity [33], we could show that CaV2.1 and PKC belong to different signalling branches [18].

However, in a series of papers utilising fluorescence microscopy and flow cytometry, we could link PKCα activity and Ca2+ entry [30, 34].

The above described effect is probably mediated by a specific pathway rather than a non-specific leak since the Ca2+ entry shows a clear dose-response relationship towards the LPA concentration [35]. For a recent review about Ca2+ channels in human RBCs, see [36].

Another known port for Ca2+ entry that is likely to have some pathophysiological relevance is the NMDA-receptor. Among the RBCs, NMDA-receptors were first identified in rat RBCs [37] but later also in human precursor cells as well as in circulating RBCs [38]. NMDA-receptors are cation channels permeable to Ca2+ and can be activated by homocysteine and homocysteic acid [39].

To extend on this, **Figure 2** provides an overview of the current knowledge about Ca2+ entry and the major cellular consequences in different pathophysiological conditions.

*Erythrocyte*

**18**

mended [12].

*from [2], and (B) is reproduced from [3].*

**Figure 1.**

and transport the phospholipids against their respective concentration gradients. The structure of the flippase has been published recently [7]. The scramblase is ATP-independent and activated by an increasing intracellular Ca2+ concentration in human red blood cells (RBCs) [8]. The scramblase has been identified recently as a member of the TMEM16 protein family, and the crystal structure was published [9]. The activity of the three proteins is shown in **Figure 1B**. PS exposure on the outer leaflet of the cell membrane has been described as a marker for apoptosis in nucleated cells [5]. Although the programmed cell death of RBCs is still under discussion, these cells show some signs of apoptosis such as PS exposure, membrane blebbing and vesicle formation [10]. This process was denominated eryptosis by Lang et al. [11]. However, the use of this term is very controversial and finally not recom-

*(A) The asymmetric distribution of phospholipids in the human red cell membrane. Abbreviations: SM, sphingomyelin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. (B) Transporter-controlled exchange of phospholipids between both lipid leaflets of the cell membrane. Unidirectional phospholipid transport by flippase is directed inwards, whereas floppase promotes outward directed transport. Both transporters are ATP-dependent and frequently move phospholipids against their respective concentration gradients. For example, aminophospholipid translocase (flippase) rapidly shuttles PS and PE from outer to inner leaflet, while ABCC1 (floppase) moves both choline phospholipids and aminophospholipids more slowly towards the outer leaflet. The concerted action of both transporters is thought to create a dynamic asymmetric steady state, in which the outer monolayer is rich in choline phospholipids, whereas aminophospholipids predominantly occupy the inner leaflet. Bidirectional phospholipid transport is catalysed by a scramblase, the activation of which may occur following Ca2+ increase. Since scramblase activity moves all major phospholipid classes back and forth between the two leaflets, it promotes the collapse of membrane phospholipid asymmetry with appearance of PS at the cells' outer surface. Panel (A) is modified* 

#### **Figure 2.**

*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 on the outer leaflet of the membrane; activation of the Gardos channel followed by the efflux of K<sup>+</sup> , Cl<sup>−</sup> and 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 Hertz et al. [40].*
