## 3 Medical Gas Plasma as a Hemostatic Agent
However, NO' can additionally react with free oxygenated hemoglobin (Hb) to form Met-Hb and nitrate as a major pathway for NO' elimination in our body [81], which reduces the bioavailability of the latter [82]. In addition to related signal transduction processes in the platelet, this restricts the contribution of NO' to the onset of gas plasma coagulation. Still, the direct effect of gas plasma on platelet activation can only account for a fraction of hemostatic activity observed, which is reasoned with regard to the proportionate biomass among the total hematocrit [24] (Figure 1). In the course of studying the side effects of medical gas plasma treatment, lysis of blood cells was investigated early already, although cell types seemed to be affected to different degrees [83]. Likewise, [PERSON] and colleagues described membrane formation from lysed erythrocytes as a second mechanism of action, but differences were found depending on the plasma source used [84]. However, in this case, blood coagulation was only traced to the coagulant membrane formation, and platelets' importance was not further discussed. In 2017, platelets were suggested being the primary mediators of gas plasma-initiated hemostasis, which was demonstrated by using clopidogrel anticoagulation, an inhibitor of platelet aggregation [8]. Strikingly, it was found that platelet activation's central trigger could be attributed to hemolysis in response to gas plasma exposure [24]. This was demonstrated because the coagulating effect was enhanced in whole blood (WB) compared to platelet-rich plasma (PRP). Furthermore, the blood coagulation could be mimicked by adding equal concentrations of an isotonic hemolysis to WB. Hemolysis as a mediator of platelet activation has been addressed and discussed before in the pathogenesis of vaso-occlusive diseases [85]. During hemolysis, various molecules and DAMPs (damage-associated molecular patterns) are being released, such as hemoglobin, heme, and the nucleotides ADP and ATP [85, 86]. The latter can act directly at platelet receptors, thereby activating platelets [87]. In line with this, inhibition of gas plasma coagulation by clopidogrel, whose active metabolite inhibits the ADP receptor P\({}_{2}\)Y\({}_{12}\), was described previously [8, 88]. P\({}_{2}\)Y\({}_{12}\) is a G\({}_{i}\)-protein coupled receptor whose \(\beta/\gamma\) subunit can initiate PI3K/Akt/p38 signaling upon activation [47, 50] and was later identified as a central pathway. By using Ly294002, a PI3K inhibitor, a decrease in gas plasma-mediated platelet activation by 56% was observed, equaling the predetermined percentage attributed to hemolysis [89]. Using inhibitors of different isoforms of PLC, the contribution of Gq-mediated signaling upon gas plasma exposure could be excluded [43]. In patients with sickle cell disease, free hemoglobin could lead to platelet activation associated with hyperpolarization of the mitochondrial membrane and increased intracellular ROS production. This has been attributed to inhibiting complex V respiratory chain activity [90]. A similar mode of action could be suggested, as hyperpolarization of mitochondrial membrane potential has been observed upon gas plasma exposure [24] (Figure 4). Studies in various pre-clinical models successfully demonstrated the potential of medical gas plasmas as a putative hemostatic agent for clinical application. Contrary to suggestions made in the beginning, direct effects of gas plasma treatment on either cellular or plasma hemostasis as critical drivers of blood coagulation were less observed. Instead, platelet activation could be traced back to ROS/RNS-induced hemolysis, resulting in the local release of hemostatic DAMPs. This directly contrasts with currently applied electrocauterization or argon plasma coagulation that achieves blood hemostasis by tissue carbonization at the injured side.