Red Blood Cells Actively Contribute to Blood Coagulation and Thrombus Formation

*Ingolf Bernhardt, Mauro C. Wesseling, Duc Bach Nguyen and Lars Kaestner*

## **Abstract**

The chapter describes the likely molecular mechanisms leading to the aggregation of human red blood cells (RBCs) under conditions of physiological coagulation when prostaglandin E2 (PGE2) or lysophosphatidic acid (LPA) is released from activated platelets and under pathophysiological conditions, in particular thrombi formation in sickle cell disease when patients are in a vaso-occlusive crisis. In both scenarios cation channels are activated. This leads to an increase of the free intracellular Ca2+ concentration resulting in an activation of the lipid scramblase, which in turn mediates a movement of phosphatidylserine (PS) from the inner to the outer membrane leaflet. In addition, the increased Ca2+ concentration leads to the activation of the Gardos channel. Experiments suggesting this mechanism have been performed with fluorescence microscopy, flow cytometry as well as single-cell force spectroscopy. The Ca2+-triggered RBC aggregation force has been identified to be close to 100 pN, a value large enough to play a significant role during thrombus formation or in pathological situations.

**Keywords:** red blood cells, intracellular Ca2+ concentration, phosphatidylserine, cation channels, lipid scramblase, thrombus formation

#### **1. Introduction**

It is well known that phospholipids are asymmetrically distributed in the cell membrane of most, if not all, living cells. Sphingomyelin (SM) and phosphatidylcholine (PC) are located predominantly in the outer leaflet of the membrane bilayer, while phosphatidylserine (PS) and phosphatidylethanolamine (PE) are located mostly in the inner leaflet [1] as depicted in **Figure 1A**. **Figure 1B** shows the possible movements of phospholipids from one leaflet of the membrane to the other one (for explanation see text below).

The distribution of the membrane phospholipids is realised by three main proteins: flippase [4], floppase and scramblase [2, 5, 6]. The flippase (also named aminophospholipid translocase (APLT)) transports relatively quickly (in some minutes) PS, and a bit slower PE, from the outer to the inner membrane leaflet. The floppase transports PC and SM in the opposite direction, i.e. from the inner to the outer membrane leaflet [2]. The flippase and floppase are ATP-dependent

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*Erythrocyte*

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#### **Figure 1.**

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

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 recommended [12].

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cal conditions.

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

the PS exposure on RBCs [22, 25, 26]. A further discussion follows below.

any PC, and the outer layer consists exclusively of SM [27, 28].

and the intensity of the response [31].

human RBCs, see [36].

belong to different signalling branches [18].

etry, we could link PKCα activity and Ca2+ entry [30, 34].

can be activated by homocysteine and homocysteic acid [39].

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

Based on findings showing a correlation between decreased haematocrit and longer bleeding times [13] and experiments of Andrews and Low [14], an active role of RBCs in thrombus formation has been proposed [14]. Kaestner et al. suggested a more detailed signalling cascade based on Ca2+ uptake via a non-selective cation channel that could be activated by prostaglandin E2 (PGE2) and/or lysophosphatidic acid (LPA) [15–17]. Recent considerations suggest more complicated signalling cascades to be involved [18, 19], including the participation of the mechanosensitive

Although it is known that LPA induces PS exposure on the outer membrane leaflet in RBCs, there are conflicting reports about the mechanism. While Chung et al. [22] claim that it is a totally Ca2+-independent process, we showed that Ca2+ alone is sufficient to induce PS exposure in human RBCs [23]. Woon et al. found that an increase of the intracellular Ca2+ level in RBCs results in the exposure of PS to the outer membrane leaflet due to the activation of the scramblase and inhibition of the flippase [24]. Protein kinase Cα (PKCα) has been also described to be involved in

An interesting model for lipid studies we consider below is sheep RBCs since it is known that these cells have a completely different phospholipid distribution in the membrane. Like in human RBCs, PS and PE are distributed in the inner membrane leaflet. In contrast, the sheep RBC (like all bovine RBC) membrane does not contain

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

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

However, in a series of papers utilising fluorescence microscopy and flow cytom-

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

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

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

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

channel Piezo1 [20, 21].

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

Based on findings showing a correlation between decreased haematocrit and longer bleeding times [13] and experiments of Andrews and Low [14], an active role of RBCs in thrombus formation has been proposed [14]. Kaestner et al. suggested a more detailed signalling cascade based on Ca2+ uptake via a non-selective cation channel that could be activated by prostaglandin E2 (PGE2) and/or lysophosphatidic acid (LPA) [15–17]. Recent considerations suggest more complicated signalling cascades to be involved [18, 19], including the participation of the mechanosensitive channel Piezo1 [20, 21].

Although it is known that LPA induces PS exposure on the outer membrane leaflet in RBCs, there are conflicting reports about the mechanism. While Chung et al. [22] claim that it is a totally Ca2+-independent process, we showed that Ca2+ alone is sufficient to induce PS exposure in human RBCs [23]. Woon et al. found that an increase of the intracellular Ca2+ level in RBCs results in the exposure of PS to the outer membrane leaflet due to the activation of the scramblase and inhibition of the flippase [24]. Protein kinase Cα (PKCα) has been also described to be involved in the PS exposure on RBCs [22, 25, 26]. A further discussion follows below.

An interesting model for lipid studies we consider below is sheep RBCs since it is known that these cells have a completely different phospholipid distribution in the membrane. Like in human RBCs, PS and PE are distributed in the inner membrane leaflet. In contrast, the sheep RBC (like all bovine RBC) membrane does not contain any PC, and the outer layer consists exclusively of SM [27, 28].
