**6. Conclusions and outlook**

*Erythrocyte*

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**

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

summarises the in vitro force measurements performed.

Ca2+ since the Gardos channel is a Ca2+-activated K+

trial testing senicapoc, a Gardos channel inhibitor, failed because vaso-occlusive crises were not improved [66]. Since senicapoc addresses the Gardos channel and

channel. However, a clinical

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

**26**

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.

## **Conflict of interest**

The authors don't declare a conflict of interests.

#### **Author details**

Ingolf Bernhardt\*, Mauro C. Wesseling, Duc Bach Nguyen and Lars Kaestner Saarland University, Saarbrücken, Germany

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

© 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, provided the original work is properly cited.
