**4. Active role of RBCs in blood coagulation**

When the endothelium of blood vessels is damaged, platelets become activated and transport PS to their external membrane surface [50]. After activation, the exposed PS provides a catalytic surface for the formation of active enzyme-substrate

**25**

*panel (C) is a reprint from [60].*

**Figure 6.**

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

complexes of the coagulation cascade, especially for the tenase and prothrombinase complexes [51]. Under these circumstances exposed PS provides a procoagulant surface and is, in general, needed as a response to injury. Therefore, the mechanism of PS exposure has to occur with a relative high transport rate of the lipids. Platelets treated with a Ca2+ ionophore show a phospholipid scrambling rate of 78 × 103

second [51]. Human RBCs also show the mechanism of PS exposure after increased intracellular Ca2+ content (see above) and are able to adhere to endothelial cells under pathophysiological conditions [22, 52–54]. In addition, exposed PS is sought to serve as a signalling component for macrophages to eliminate old or damaged RBCs from the circulation [55–58]. Since PS-exposing RBCs can adhere to the vascular wall, which may lead to disturbance of the microcirculation [59], the elimination of these cells is a very important mechanism. However, compared to platelets, RBCs

*(A) shows a sketch of the working principle of single-cell force spectroscopy (SCFS). A cell is bound to a cantilever and is brought into contact with another cell at the surface. During the approach and withdrawal of the cell, the deflection is monitored and gives direct information about the adhesion force between the cells. (B) shows a sketch of the working principle of the optical tweezer measurements. Two RBCs are trapped in the foci of two laser beams and are brought into contact. By measuring the deflection of the cells out of the centre of the laser foci, one can determine the adhesion force between the cells. (C) shows a force vs. distance curve derived from the SSFS measurements. A weak interaction of approximately 20 pN can be observed that is mainly due to an artefact of the measurement (see original paper). This 20 pN is the lower limit that one can measure using this type of cell with this technique. (D) shows a force calibration of one RBC in an optical trap. It can be observed that with the given laser power, the trap is only linear up to forces of 15 pN, i.e. this is the upper limit that can be measured with this technique on these types of cells. This figure is reproduced with kind permission from Elsevier. (A) is a reprint from [23], panels (B) and (D) are reprints from [35] and* 

per

per second) [51].

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

have a lower phospholipid scrambling rate (0.45 × 103

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

complexes of the coagulation cascade, especially for the tenase and prothrombinase complexes [51]. Under these circumstances exposed PS provides a procoagulant surface and is, in general, needed as a response to injury. Therefore, the mechanism of PS exposure has to occur with a relative high transport rate of the lipids. Platelets treated with a Ca2+ ionophore show a phospholipid scrambling rate of 78 × 103 per second [51]. Human RBCs also show the mechanism of PS exposure after increased intracellular Ca2+ content (see above) and are able to adhere to endothelial cells under pathophysiological conditions [22, 52–54]. In addition, exposed PS is sought to serve as a signalling component for macrophages to eliminate old or damaged RBCs from the circulation [55–58]. Since PS-exposing RBCs can adhere to the vascular wall, which may lead to disturbance of the microcirculation [59], the elimination of these cells is a very important mechanism. However, compared to platelets, RBCs have a lower phospholipid scrambling rate (0.45 × 103 per second) [51].

#### **Figure 6.**

*Erythrocyte*

regression, R2

*15 min stimulation with LPA (p < 0.5 is marked with \*).*

**Figure 5.**

exposure depict a linear behaviour in dependence of cell age with a very good

*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* 

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

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

The intracellular Ca2+ content and the PS exposure at the outer membrane leaflet

When the endothelium of blood vessels is damaged, platelets become activated and transport PS to their external membrane surface [50]. After activation, the exposed PS provides a catalytic surface for the formation of active enzyme-substrate

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

slope of this linear regression failed to be significantly different from zero.

the flow cytometer and hence are excluded from the detection.

higher PS exposure compared to young RBCs [48, 49].

**4. Active role of RBCs in blood coagulation**

preventing thrombus formation.

of 0.94 and 0.92, respectively, as outlined in **Figure 5A**. However, the

**24**

*(A) shows a sketch of the working principle of single-cell force spectroscopy (SCFS). A cell is bound to a cantilever and is brought into contact with another cell at the surface. During the approach and withdrawal of the cell, the deflection is monitored and gives direct information about the adhesion force between the cells. (B) shows a sketch of the working principle of the optical tweezer measurements. Two RBCs are trapped in the foci of two laser beams and are brought into contact. By measuring the deflection of the cells out of the centre of the laser foci, one can determine the adhesion force between the cells. (C) shows a force vs. distance curve derived from the SSFS measurements. A weak interaction of approximately 20 pN can be observed that is mainly due to an artefact of the measurement (see original paper). This 20 pN is the lower limit that one can measure using this type of cell with this technique. (D) shows a force calibration of one RBC in an optical trap. It can be observed that with the given laser power, the trap is only linear up to forces of 15 pN, i.e. this is the upper limit that can be measured with this technique on these types of cells. This figure is reproduced with kind permission from Elsevier. (A) is a reprint from [23], panels (B) and (D) are reprints from [35] and panel (C) is a reprint from [60].*

Even more important was the demonstration that human RBCs play an active role in clot formation [23]. This is lacking in medical textbooks, where one can find statements claiming that RBCs only become part of clots because they are so abundant in the circulation. First demonstration of increased interacting forces between two RBCs when the intracellular free Ca2+ concentration is increased was performed using non-invasive holographic optical tweezers [23]. In addition, using single-cell force spectroscopy, it has been shown that the upper force limit for Ca2+ triggered adhesion of the RBCs was approximately 100 pN, a value large enough to be of significance during clot formation or in pathological situations [23]. **Figure 6** summarises the in vitro force measurements performed.

#### **5. Active participation of RBCs in thrombotic events**

There are numerous indications for the active participation of RBCs in the induction of thrombotic events. The first example we like to mention is the occurrence of thrombotic complications in anaemic patients that experienced a splenectomy. Numerous hereditary anaemic disorders such as spherocytosis, stomatocytosis or elliptocytosis are associated with distorted RBCs, which are preferentially removed in the spleen. Therefore, splenectomy is believed to improve the anaemic symptoms because cells cannot be removed in the spleen. In principle, this concept works out but with the severe side effect that some patients suffer from thrombotic events. Since the 'maintenance' of the RBCs in the spleen is missing, it is likely that the RBCs are the major cause for the thrombotic events. In patients diagnosed with hereditary xerocytosis, mostly related to mutations in the Piezo1 channel, thrombotic complications were regularly reported after these patients underwent splenectomy [61], whereas patients diagnosed for 'Gardos Channelopathy', even after splenectomy, thrombotic events were not observed [62].

An even more prominent example is sickle cell disease associated with vasoocclusive pain crisis as the major and most severe symptom of the patients. Since the mutation associated with sickle cell disease is in the haemoglobin, it seems obvious that also the symptoms of the disease are associated with RBCs. The common belief is that vaso-occlusive pain crises in sickle cell disease patients are caused by the crystallisation of the mutated haemoglobin under deoxygenation conditions. While the sickle formation under deoxygenation at stasis is undoubted, it's not clear if the same shape change happens in vivo. However, although it is clear that deoxygenated RBCs of sickle cell disease patients have an impaired deformability, the link to the vaso-occlusive crises must be a bit more complicated because deoxygenation happens continuously as deoxygenated RBCs are continuously passing the circulation and vaso-occlusive pain crises happen only sporadically and are so far unpredictable. A possible explanation is the activity of the NMDA-receptors (see above) that are activated by homocysteine and homocysteic acid, which are markers for inflammation in the blood plasma [63].

Such the above described mechanisms triggered by intracellular Ca2+ increase are likely to happen also during vaso-occlusive crises in sickle cell disease patients. A first clinical pilot study on sickle cell disease patients using memantine, a drug blocking the NMDA-receptor (and approved to treat Alzheimer disease), showed very promising results both in the support of the mechanism we sketch and in the patients showing a lower number and less severe vaso-occlusive pain crises [64, 65].

Furthermore, it is well known that in RBCs of sickle cell disease patients, the Gardos channel activity is increased [66], which is an indicator for an increased Ca2+ since the Gardos channel is a Ca2+-activated K+ channel. However, a clinical trial testing senicapoc, a Gardos channel inhibitor, failed because vaso-occlusive crises were not improved [66]. Since senicapoc addresses the Gardos channel and

**27**

provided the original work is properly cited.

Saarland University, Saarbrücken, Germany

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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

\*Address all correspondence to: i.bernhardt@mx.uni-saarland.de

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

not the upstream increase in Ca2+ that causes all the effects described above, the

Additionally, we like to mention and discuss another aspect: The process we describe, in particular the Ca2+-triggered aggregation to initiate thrombus formation, takes some time [23], and an argument is that the time required is too long that aggregation happens between the fast-moving RBCs in the circulation. In this context, we like to mention the hydrodynamic clustering [67], which is perfectly reversible but provides the cellular interaction time since the lifetime of the hydro-

It seems obvious that RBC participation in blood coagulation and thrombus formation is more than an accidental trapping in the process. In this chapter we summarised indications, evidences and proofs for active participation of RBCs in blood clotting and thrombus formation. However, this concept so far did not make it into haematological text books and standard medical education. With this book chapter, we like to make a little contribution to better explain and propagate this concept. Although we face severe experimental and clinical evidence for the active participation of RBCs in blood coagulation and thrombus formation, there is a demand for further research on the regulation and manipulation of this aspect in the coagulation sometimes also referred to as RBC hypercoagulation. We are looking forward to

the next years of investigations in coagulation and thrombosis research.

The authors don't declare a conflict of interests.

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

dynamic clusters can be in the range of several seconds.

failure of senicapoc is explainable.

**6. Conclusions and outlook**

**Conflict of interest**

**Author details**

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

not the upstream increase in Ca2+ that causes all the effects described above, the failure of senicapoc is explainable.

Additionally, we like to mention and discuss another aspect: The process we describe, in particular the Ca2+-triggered aggregation to initiate thrombus formation, takes some time [23], and an argument is that the time required is too long that aggregation happens between the fast-moving RBCs in the circulation. In this context, we like to mention the hydrodynamic clustering [67], which is perfectly reversible but provides the cellular interaction time since the lifetime of the hydrodynamic clusters can be in the range of several seconds.
