Ankaferd hemostat: from molecules to medicine

Ankaferd hemostat (ABS; Ankaferd Blood Stopper®, İstanbul, Turkey) is a hemostatic agent having an impact on red blood cell–fibrinogen interactions. The hemostatic effect of ABS depends upon the quick promotion of a protein network, particularly fibrinogen gamma, in relation to the erythrocyte aggregation. The entire physiological process involves ABS-induced formation of the protein network by vital erythrocyte aggregation. Vital erythrocyte aggregation occurs with the spectrine, ankyrin, and actin proteins on the membrane of the red blood cells. ABS notably affects cell metabolism and cell cycle mechanisms. Meanwhile, ABS has antiproliferative effects on cancer cells. The aim of this review is to assess molecular basis of ABS as a hemostatic drug. The literature search on ABS was performed in PubMed, Web of Science (SCI expanded), and Scopus with particular focus on the studies of molecular basis of ABS, in vivo research, case series, and controlled randomized clinical studies. Current perspective for the utilization of ABS is to provide hemostasis with accelerating wound healing. Future controlled trials are needed to elucidate the pleiotropic clinical effects of ABS such as antineoplastic, antiinflammatory, antiinfective, antifungal, and antioxidative effects.

outcome was the upregulation of tumor suppressor proteins UCHL1 and RPL5. In particular, RPL5 directly activates the p53 apoptotic pathway and causes apoptosis [11]. Protein-protein interaction networks are important interactions in understanding cellular processes such as metabolism, signal transduction, and drug resistance. The interactions of the identified proteins were mapped as depicted in Figure 1. These proteins were linked and worked together to protect the Caco-2 cells against the effect of ABS as depicted in Figure 2 [11].

The effects of ABS on transcription factors
The effects of ABS on transcription factors and erythrocyte protein profile in HUVEC endothelium were investigated. In one study, ABS was shown to be highly effective not only outside the cells but also inside the cell at low doses (5 μL) and could affect many molecular mechanisms in endothelial cells. The level of the activity of the transcription factors (AP2, AR, CREATF1, CREB, E2F1-5, E2F6, EGR, GATA, HNF1, ISRE, Myc-Max, NF1, NF-κB, p53, PPAR, SMAD2/3, SP1, TRE/AP1, and YY1) were significantly increased in response to ABS. Those transcription factors regulate a wide variety of biological functions, including hemostasis, infection, cellular proliferation, and inflammation [12]. GATA regulates erythroid differentiation and promotes the production of erythroid proteins such as spectin. In another study, GATA activity was significantly increased after ABS administration [12]. ABS importantly increased the level of activity of the following transcription factors; AP2, AR, CRE-ATF1, CREB, E2F1-5, E2F6, EGR, ISRE, Myc-Max, NF1, NF-κB, p53, PPAR, SMAD2/3, SP1, TRE/AP1, and YY1. Those regulator molecules affect different steps of cellular proliferation, such as cell cycle regulation, signal transduction, angiogenesis, apoptosis, inflammation, acute phase reaction, immunity, and several metabolic molecular pathways [12].

The effects of ABS on the cellular biology
The antineoplastic effect of ABS was preliminarily defined in cancer cell lines [13,14]. In a study, a dose-dependent inhibition of cell proliferation and a significant reduction in the survival of SAOS-2 cells were observed after ABS administration [13]. In another study, the antineoplastic effects of ABS on colon cancer cells were also defined. Following the addition of ABS to the culture medium, the inhibition of cellular reproduction, and loss in the viabilities of human colon CaCo-2 cells were observed [14]. Mumcuoglu et al. demonstrated that, depending on the concentration and duration of the application, ABS could cause apoptosis via regulating PAR1 and p53independent p21 involvement in apoptosis stimulation within leukemia cells [15]. Likewise, Akalın et al. showed that the antiproliferative effects of ABS on lymphoid neoplastic cells (B-CLL and RAJI tumor cell lines). First, the inflation of the hematopoietic tumor cells was observed with the addition of ABS solution. The inflation and proliferation continued on B-CLL cells at day 3 and produced aggregation islands [16]. Turk et al. also demonstrated that the most resistant cell type was SK-MEL-10 and the least resistant neoplastic cell type was A2058. The anticancer effect of ABS was also evident on light microscopic images of untreated M307 primary cells [17]. Another study revealed that ABS induces DNA damage, apoptosis, and cytotoxic activity via generating reactive oxygen species in melanoma cell lines [18]. One study investigated the biological activity of ABS-derived iron on iron-regulated genes during iron-deficiency anemia. The results showed that ABS-derived ironinfluenced transcriptions of iron-regulated marker genes, including divalent metal transporter (Dmt1), transferrin receptor (TfR), ankyrin repeat domain 37 (Ankrd37), and hepcidin (Hamp). ABS might have an ability to reduce levels of iron deficiency anemia [19].
Nanotechnological approaches were applied to further develop ABS drug [21,22]. Using the classical ABS solution to create active hemostasis during partial nephrectomy could not be so efficient due to the insufficient contact surface between the ABS hemostatic liquid agent and the bleeding area. In order to expand the contact surface, Huri et al. generated a chimeric hemostatic agent, ABS Coagulation proteins (Factors V, VII, VIII, IX, X, XI, and XIII), prothrombin time (PT) and activated partial thromboplastin time (aPTT) were normal during ABS administration. However, it was observed that thrombin time (TT) was prolonged due to fibrinogen gamma [1]. On the contrary, a recent hemorheological study showed that ABS has antierythrocyte aggregation effect. ABS inhibits pathological aggregation of red blood cells. Antithrombotic clinical effects of ABS could be ascribed to the paradoxal antierythrocyte aggregation actions of the drug [23]. . In another animal study, the effectiveness of ABS was compared to several conventional antihemorrhagic methods. Hemostasis was successfully achieved via ABS application. Moreover, warm ischemia times decreased by ABS [31]. In another study, the rats underwent femoral vein puncture. One subgroup was treated with ABS tampon or spray and the other control group was left untreated. After two weeks, each group underwent partial tissue excision from the same femoral region, brain, heart, kidney, and liver. ABS was observed to stop the bleeding. There were no histopathological changes at the tissue level and no pathological effects in other organ tissues under light microscope [32]. Beyazit et al. investigated the macroscopic and microscopic changes in the cervical mucosa and immunohistochemical staining for IL-1 β in response to ABS treatment in a chemically induced cervicitis model of rats. The study showed that ABS had significant protective effects on vascular congestion and cervical erosion [33].

Clinical usage of ABS
ABS has hemostatic, antithrombotic, antiinfective, antineoplastic, and wound healing effects. ABS has also been employed in controlled clinical trials as depicted in Table.

The usage of Ankaferd Hemostat (ABS) in bleeding
Clinical studies have demonstrated that ABS can be safely and effectively used in surgical and dental procedures in patients with normal and abnormal hemostasis. Huri  Another study showed that the local use of ABS decreases bleeding significantly during the operation. Therefore, transfusion requirements of erythrocyte suspension and

The usage of ABS in wound healing
Functional proteomic analyses had defined antithrombin and prohemostatic activities of ABS which are related to fibrinogen gamma chain and prothrombin [58]. ABS can improve the wound-healing process via providing inhibition of extra cellular matrix-degrading enzymes during wound repair. Moreover, ABS enhanced the stimulated migration of 3T3 fibroblasts to an artificial wounded area [59]. Plant extracts in ABS have been reported to inhibit various enzymes. The extracts of T. vulgaris were shown to inhibit collagenase and elastase inhibition by 25% and 17%, respectively, while hyaluronidase was shown to be inhibited 100% [60]. Additionally, another study showed that ursolic acid extracted from U. dioica exhibited elastase and collagenase inhibition activity of 24.5% and 16.2%, respectively [61]. Topal et al. showed that the wound contraction in the ABS and silver sulphadiazine groups was significantly higher than in the control group on days 14, 21, and 28 and suggested that ABS could be successfully used for burn wound healing besides silver sulphadiazine [62]. ABS may be useful in the treatment of acute burn lesions. It was observed that the wounds healed rapidly with the topical application of ABS onto the burn lesions [63]. In one study, the effect of ABS on the cut surface of the pancreatic duct as well as the pancreatic remnant was a safe procedure that prevents the formation of pancreatic fistula without causing adverse side effects such as pancreatitis [64]. Radiation-induced esophageal ulcer in a patient with breast cancer was completely healed with topical application [65]. It has been shown that ABS has a protective effect against intestinal damage in an experimental rat necrotizing enterocolitis model due to its antioxidant, antiinflammatory, and antiapoptotic characteristics [66]. In another study, intravaginal ABS injection caused a significant decrease in the number of inflammatory cells in the cervical mucosa and ABS had significant protective effects on cervical erosion [33]. In another animal study, it was observed that ABS administration improved uterine fibrosis and histopathological inflammation by lowering the level of inflammatory markers such as IL-1 and IL-6 [67].

The usage of ABS in infections
Vitis vinifera, one of the components of ABS has potential prebiotic effects on modulating the gut microbiota composition and generating SCFAs that contribute to the improvements of host health [68]. ABS was evaluated on 102 clinical isolates from both gram-negative and grampositive bacteria and four standard strains, including MRSA ATCC 43300, MSSA ATCC 25923, P. aeruginosa ATCC 27853 and E. coli ATCC 35218. The study showed that ABS was significantly active against all of the bacteria investigated [69]. Another study disclosed that ABS is highly effective against several gram-negative and gram-positive bacteria including frequent foodborne microorganisms [70]. Akkoc et al., using agar well diffusion test, found that ABS has a high antifungal effect against Zygosaccharomyces bailii, C albicans, Aspergillus flavus, and Aspergillus parasiticus [71]. In another study, ABS reported that when applied directly to Candida species, there were changes in growth conditions [72]. The efficiency of ABS on H. pylori was also demonstrated. Possible anti-H. pylori effect of ABS will broaden the therapeutic spectrum of the drug in GI lesions as peptic ulcer disease (PUD) and lymphoid tissue (MALT) lymphomagenesis [73]. In vitro activity of ABS against M. Tuberculosis isolates was evaluated. It was concluded that ABS solution used topically was active against tuberculosis bacilli in vitro. Therefore, ABS might be used as a supportive agent in combination with anti-tuberculosis drugs during debridement of multidrug resistant M. tuberculosis osteomyelitis and lymphadenitis [74]. In one study, the efficacy of ABS with hydatid cysts was compared with other antiseptic agents. It can be said that ABS is an effective antiseptic agent for percutaneous interventions to treat hydatid cysts [75]. In a study, the efficacy of ABS in prophylaxis and oral mucositis treatment in patients receiving chemotherapy in childhood was evaluated. ABS was thought to be effective in prophylaxis and treatment of oral mucositis secondary to chemotherapy in childhood cancers [76].

Toxicity profile of ABS
Quantitative analysis of heavy metals in the ABS sample revealed that there were no Pb, Cd, Hg, and As in ABS. Chromatographic analysis of pesticide analysis in ABS sample revealed that ABS does not contain pesticides.
In the analysis of mycotoxin detection using the HPLC method in the ABS drug, ABS was revealed not to contain mycotoxins (i.e. Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, and Aflatoxin G2; total aflatoxins are not present inside ABS). GMO analyses of the detection of genetically altered organisms in the ABS sample represented that no plasmid extraction was performed and ABS did not have GMO treatment during the preparation process. Dioxin analyses in the ABS sample showed that ABS does not contain toxic dioxin and dioxin-like chemical compounds. Thus, the available toxicological results have shown the safety of ABS [77].

Intratumoral ABS for controlling the growth of solid tumors and hepatic metastases
In a previous study, the antineoplastic effects of ABS on myeloma cell line, in vitro and plasmocytoma development in Balb/c mice were investigated by intraperitoneal preterm injection in vitro. The cytotoxicity of ABS against the MM cell lines was determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide-dye reduction assay [78]. It was shown that chemopreventive effects of ABS in 7,12-dimethylbenz[a]anthracene (DMBA) induced oral epithelial dysplasia. Histological studies have shown that the buccal pouches of animals treated with DMBA alone showed up serious dysplasia while only moderate or no dysplasia were noticed in DMBA+ABS group [79]. ABS has been used as an embolizing agent in renal and splenic arteries by Medical Interventional Radiology for medical nephrectomy and splenectomy purposes [80,81]. Under fluoroscopic guidance, 2 mL of ABS mixed with 2 mL of nonionic contrast agent (Iopromide) was slowly injected until a resistance to injection secondary to embolization was observed. Slow flow and stagnation were observed with fluoroscopy. Approximately 3.5-4 mL of the mixture was used for embolization [80,81]. In one study, ABS was used instead of alcohol for hepatic embolization in mice. There was a statistically significant difference in the development of necrosis in ABS-treated rats compared to ethanol and saline group [82]. ABS has antineoplastic effects in colon cancer [11]. In colon cancer liver metastases and hepatocellular cancer patients, tumor ablation treatment could be applied via interventional radiology techniques in future clinical practice of ABS. In the current technique: alcohol, N-butyl-2-cyanoacrylate, and caustic substances are used as tumor embolizers. In clinical practice in colon cancer liver metastases and hepatocellular cancer patients, ABS could be used for the palliative, adjuvant, neo-adjuvant, or supportive use via interventional radiology techniques for the management of solid tumors. This hypothesis should be tested in future studies.

Conclusion
ABS acts as a hemostatic agent in various clinical hemorrhages and has many pleiotropic effects. The hemostatic effect of ABS depends upon the quick promotion of a protein network, especially fibrinogen gamma, in relation to the erythrocyte aggregation. The entire physiological process involves ABS-induced formation of the protein network by vital erythrocyte aggregation. Vital erythrocyte aggregation occurs with the spectrine, ankyrin, and actin proteins on the membrane of the red blood cells. ABS notably affects cell metabolism and cell cycle mechanism. ABS has antiproliferative effects on cancer cells. The expanding spectrum of ABS includes antiinfective, antineoplastic, and woundhealing properties. Current perspective for using ABS is to provide hemostasis and accelerating wound healing. Future controlled trials are needed to show the pleiotropic effects of ABS such as antineoplastic, antithrombotic, antiinflammatory, antiinfective, antifungal, and antioxidative effects.