## 3 Medical Gas Plasma as a Hemostatic Agent  
Blood coagulation by medical gas plasma was first described more than 10 years ago [17]. Since then, many studies have emphasized the pro-coagulant potential of medical gas plasmas on blood from healthy donors. Moreover, sufficient hemostasis has been achieved in vivo on mouse saphenous veins [64], rat and mouse livers [8; 65; 66], and rabbit spleens [67], as well as acute skin wounds of mice and pigs [68; 69] (Table A1). Initially, the clot-promoting effect was attributed to the provision of Ca2+, which initiates platelet activation and enables versatile platelet shape changes [70]. This was suggested as ethylene diamine tetraacetic acid (EDTAA)-anticoagulated blood samples, which exhibit decreased free Ca2+ concentration due to chelation, showed increased platelet activation upon medical gas plasma treatment compared to heparin-anticoagulated blood [71]. However, the role of free Ca2+ was further refuted by subsequent studies observing that Ca2+ fluctuations only occur at longer treatment times and thus cannot contribute to the early processes of blood coagulation since visible coagulation processes occur before that [72]. At the same time, dominating effects of temperature and electric field effects were negated, which distinguished gas plasma-mediated blood coagulation clearly from clinically employed argon plasma coagulation (hot plasma) or electrocauterization. Conversely, the suggestion was that medical gas plasma instead interferes in plasmatic hemostasis by direct cleavage of fibrinogen as the aggregate formation of a fibrinogen solution could be observed after gas plasma exposure. In addition, histopathological  
Figure 3: Simplified scheme of blood coagulation upon vessel injury. Created with bioreactor.com (accessed on 1 February 2022). mation was noted in samples obtained after bleeding control using medical gas plasmas in C57 BL/6 mice, and protein aggregation was further investigated. However, scanning electron microscopy (SEM) and macroscopic examination of platelet-rich plasma (PRP) already revealed a pro-coagulant effect on platelets directly, though the underlying mechanisms remained unclear [65, 68]. In other biomedical research areas, including wound healing or oncology, gas plasma-mediated effects have been mainly linked to the variety of ROS/RNS being generated simultaneously [20]. Along those lines, it seemed evident that ROS/RNS also play a significant role in gas plasma-mediated platelet activation. In most in vitro gas plasma applications using gas plasma jets, a dominating role of long-lived species such as hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) [73] and hypochlorous acid (HOCI) [74] is evident. Hence, it seemed plausible that addition of these agents would recapitulate the hemostatic action of gas plasma treatment. Surprisingly, this was not the case, independent of the concentration that was added [24]. Vice versa, scavenging of H\({}_{2}\)O\({}_{2}\) via catalase also failed to protect from gas plasma-mediated hemostasis. In general, due to their high reactivity, the influence of short-lived ROS is challenging to control. The influence of superoxide anions (O\({}_{2}\)-) in thrombine-mediated platelet activation has been documented [75, 76], but scavenging thereof using superoxide dismutase (SOD) could not reduce gas plasma-mediated platelet activation. In the same view, increased platelet aggregation has been observed when exposing PRP to gaseous ozone [77]. NO' is abundantly generated by gas plasma jets [78], being known for its pleiotropic functions in hematology [79], and its role concerning plasma-mediated effects has been discussed [55, 57, 80]. Diffusion of NO' across the platelet membrane enables platelet activation via sGC, PKG, and p38 activation initially [47, 56, 57] but acts inhibitory as blood coagulation progresses [57].