PEGylated Recombinant Aplysia punctata Ink Toxin Depletes Arginine and Lysine and Inhibits the Growth of Tumor Xenografts.

In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

Upregulation of IL4-induced gene 1 enzyme by B2 cells during melanoma progression impairs their antitumor properties.

B cells present in human cutaneous melanoma have been associated with protective or detrimental effects on disease progression according to their phenotype. By using the RET model of spontaneous melanoma and adoptive transfer of B16 melanoma cells, we show that immature and follicular B2 (B2-FO) cells exert a protective effect on melanoma progression by promoting the generation of effector memory T cells and limiting the recruitment of polymorphonuclear myeloid-derived suppressor cells. Unfortunately, this beneficial effect progressively wanes as a consequence of enhanced expression of the IL4-induced gene 1 (IL4I1) enzyme by immature B cells and B2-FO cells. Endogenous IL4I1 selectively decreases CXCR5 expression in splenic immature B cells, subverting their trafficking to primary tumors and enhancing the production of IL-10 by B2 cells, thereby promoting an immunosuppressive microenvironment. Accordingly, B2 cells from RET IL4I1KO mice more efficiently controlled B16 melanoma growth than B2 cells from IL4I1-competent RET mice. Collectively, immature B cells and B2-FO cells are key actors in the control of melanoma growth, but their mobility and functions are differently impaired by IL4I1 overexpression during melanoma progression. Thus, our present data strongly urge us to associate an IL4I1 antagonist with current immunotherapy to improve the treatment of metastatic melanoma.

Oxidative stress induced by Pollonein-LAAO, a new L-amino acid oxidase from Bothrops moojeni venom, prompts prostate tumor spheroid cell death and impairs the cellular invasion process in vitro.

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.

Action mechanism of snake venom l-amino acid oxidase and its double-edged sword effect on cancer treatment: Role of pannexin 1-mediated interleukin-6 expression.

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

Cluster of resistance-inducing genes in MCF-7 cells by estrogen, insulin, methotrexate and tamoxifen extracted via NMF.

Breast cancer has become a leading cause of death for women as the economy has grown and the number of women in the labor force has increased. Several biomarkers with diagnostic, prognostic, and therapeutic implications for breast cancer have been identified in studies, leading to therapeutic advances. Resistance, on the other hand, is one of clinical practice's limitations. In this paper, we use Nonnegative Matrix Factorization to automatically extract two gene signatures from gene expression profiles of wild-type and resistance MCF-7 cells, which were then investigated further using pathways analysis and proved useful in relating resistance pathways to breast cancer regardless of the stimulus that caused it. A few extracted genes (including MAOA, IL4I1, RRM2, DUT, NME4, and SUMO3) represent new elements in the functional network for resistance in MCF-7 ER+ breast cancer. As a result of this research, a better understanding of how resistance occurs or the pathways that contribute to it may allow more effective therapies to be developed.

IL4I1 enhances PD-L1 expression through JAK/STAT signaling pathway in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the major type of lung cancer and is one of the deadliest cancers worldwide. IL4I1, as a gene associated with unsatisfactory prognosis, is involved in tumor immune escape, but its immune regulatory mechanism in LUAD is limited. Bioinformatics analysis was applied to analyze the differentially expressed mRNAs and enriched signaling pathways in LUAD tissue. Quantitative real-time polymerase chain reaction (qRT-PCR) was manipulated to test IL4I1 expression. We carried out several methods to examine cell functions: CCK-8 to measure LUAD cell proliferation; flow cytometry to determine cell apoptosis; Western blot to assess the expression of JAK/STAT pathway-related proteins and PD-L1; T cell cytotoxicity assay to evaluate the effect of IL4I1 on the immune escape of LUAD cells. Through bioinformatics analysis, IL4I1 was verified to be highly expressed in LUAD tissue, participate in the modulation of JAK/STAT signaling pathway, and be positively associated with CD274 (PD-L1) expression. Cell function experiments indicated that silencing IL4I1 notably repressed LUAD cell proliferation and induced apoptosis. IL4I1 silence would block JAK/STAT signaling pathway, but this effect could be reversed by RO8191 activator treatment. Moreover, IL4I1 silence suppressed PD-L1 expression and facilitated T cell cytotoxicity, while its inhibitory impact on PD-L1 expression and immune escape of LUAD cells could be reversed by atezolizumab treatment. Overall, we confirmed that IL4I1 promoted the malignant cell behaviors and immune escape of LUAD through JAK/STAT signaling pathway. IL4I1 has the potential to be a diagnostic biomarker for LUAD.

Comparative Venom Proteomics of Iranian, Macrovipera lebetina cernovi, and Cypriot, Macrovipera lebetina lebetina, Giant Vipers.

Envenoming by Macrovipera lebetina subspecies causes severe life-threatening difficulties for people living in North Africa and the Middle East. To better understand the pathophysiology of envenoming and improve patient management, knowledge about the venom components of the subspecies is essential. Here, the venom proteomes of Macrovipera lebetina lebetina from Cyprus and Macrovipera lebetina cernovi from Iran were characterized using RP-HPLC separation of the crude venom proteins, SDS-PAGE of fractionated proteins, and LC-MS/MS of peptides obtained from in-gel tryptic digestion of protein bands. Moreover, we also used high-resolution shot-gun proteomics to gain more reliable identification, where the whole venom proteomes were subjected directly to in-solution digestion before LC-HR-MS/MS. The data revealed that both venoms consisted of at least 18 protein families, of which snake venom Zn2+-dependent metalloprotease (SVMP), serine protease, disintegrin, phospholipase A2, C-type lectin-like, and L-amino acid oxidase, together accounted for more than 80% of the venoms' protein contents. Although the two viper venoms shared mostly similar protein classes, the relative occurrences of these toxins were different in each snake subspecies. For instance, P-I class of SVMP toxins were found to be more abundant than P-III class in the venoms of M. l. cernovi compared to M. l. lebetina, which gives hints at a more potent myonecrotic effect and minor systemic hemorrhage following envenoming by M. l. cernovi than M. l. lebetina. Moreover, single-shot proteomics also revealed many proteins with low abundance (<1%) within the venoms, such as aminopeptidase, hyaluronidase, glutaminyl-peptide cyclotransferase, cystatin, phospholipase B, and vascular endothelial growth factor. Our study extends the in-depth understanding of the venom complexity of M. lebetina subspecies, particularly regarding toxin families associated with envenoming pathogenesis and those hard-detected protein classes expressed in trace amounts.

The anti-ovarian carcinoma activity of L-amino acid oxidase from Crotalus adamanteus venom in vivo and in vitro.

Snake venom L-Amino-acid oxidase (svLAAO) has become a critical research target in molecular biology and medical science since its widespread presence and diverse biological roles, including antitumor application. Our research confirmed that Crotalus adamanteus (C. adamanteus) venom LAAO exhibited potential anti-ovarian cancer activity both in vivo and in vitro. C. adamanteus venom LAAO significantly reduced viability of ovarian cancer cells and caused morphological changes that preceded cell death. The results of molecular biology experiments showed that C. adamanteus venom LAAO caused expression changes of genes related to apoptotic pathways either intrinsically or extrinsically in ovarian cancer cells. Animal experiments and histological analysis also proved that C. adamanteus venom LAAO could effectively inhibit the tissue damage caused by ovarian cancer, and animals treated with C. adamanteus venom LAAO showed higher survival time. Catalase blocked the major apoptosis induction of C. adamanteus venom LAAO on ovarian cancer cells, suggesting that the cytotoxicity of C. adamanteus venom LAAO on ovarian cancer cells was mainly mediated by H2O2. These results infer that C. adamanteus venom LAAO will have some advantages in new drug research and antitumor drug development in future.

Pharmacological Investigation of CC-LAAO, an L-Amino Acid Oxidase from Cerastes cerastes Snake Venom.

Snake venom proteins, which are responsible for deadly snakebite envenomation, induce severe injuries including neurotoxicity, myotoxicity, cardiotoxicity, hemorrhage, and the disruption of blood homeostasis. Yet, many snake-venom proteins have been developed as potential drugs for treating human diseases due to their pharmacological effects. In this study, we evaluated the use of, an L-amino acid oxidase isolated from Cerastes cerastes snake venom CC-LAAO, as a potential anti-glioblastoma drug, by investigating its in vivo and in vitro pharmacological effects. Our results showed that acute exposure to CC-LAAO at 1 and 2.5 µg/mL does not induce significant toxicity on vital organs, as indicated by the murine blood parameters including aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) activities, and creatinine levels. The histopathological examination demonstrated that only at high concentrations did CC-LAAO induce inflammation and necrosis in several organs of the test subjects. Interestingly, when tested on human glioblastoma U87 cells, CC-LAAO induced a dose-dependent apoptotic effect through the H2O2 generated during the enzymatic reaction. Taken altogether, our data indicated that low concentration of CC-LAAO may be safe and may have potential in the development of anti-glioblastoma agents.

Preparative Biocatalytic Synthesis of α-Ketoglutaramate.

α-Ketoglutaramate (KGM) is an underexamined metabolite of L-glutamine in the metabolic pathway of glutaminase II of α-ketoglutarate formation. Presumably, KGM may be a biomarker of hepatic encephalopathy and other hyperammonemic diseases. This metabolite is a substrate for the ω-amidase enzyme and is used to determine its activity in the study of the biochemistry of various types of cancer. However, the commercial unavailability of KGM hinders its widespread use. Methods for the preparative synthesis of KGM are known, but they either do not provide the proper yield or proper purity of the target product. In this work, a detailed description of the procedures is given that allows the production of KGM with a purity above 97% and a yield of the target product above 75% using L-amino acid oxidase from C. adamanteus as a catalyst of L-glutamine conversion. KGM can be obtained both in the form of a highly concentrated aqueous solution and in the form of crystals of sodium salt. The developed methods can be used both for scaling up the synthesis of KGM and for creating economical biocatalytic technologies for the production of other highly purified preparations.

Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis.

Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease.

Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1+CD274(PD-L1)+IDO1+ macrophages, which accumulated in the tumor periphery in a T cell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory T cell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states.

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.

Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.

Proteomic and toxinological characterization of Peruvian pitviper Bothrops brazili ("jergón shushupe"), venom.

Bothrops brazili is a pitviper from Amazonian region, responsible for many accidents in Peru. Despite its relevance, its venom has not been extensively characterized. In the present work, Bothrops brazili venom (BbV) components were analyzed by RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Approximately 37 proteins were identified, belonging to 7 families. Snake venom metalloproteinases (SVMPs) were the most abundant proteins of the venom (33.05%), followed by snake venom serine proteinases (SVSPs, 26.11%), phospholipases A2 (PLA2, 25.57%), snake C-type lectins (CTLs, 9.61%), L-aminoacid oxidase (LAAO, 3.80%), cystein-rich secretory proteins (CRISP, 1.67%) and Bradykinin-potentiating peptide (BPP, 0.20%). In vitro enzymatic activities of BbV showed high levels of SVMP activity and reduced Hyal activity in comparison with other bothropic venoms. Furthermore, BbV reduced VERO cells viability. ELISA and Western Blotting showed that both Peruvian and Brazilian bothropic antivenoms were able to recognize BbV components. This work provides an overview of BbV venom content and indicates a potential efficiency of Peruvian and Brazilian antivenoms to treat accidents with this species.

Methylated derivatives of l-tyrosine in reaction catalyzed by l-amino acid oxidase: isotope and inhibitory effects.

l-Amino acid oxidase (LAAO) is widely distributed in nature and shows important biological activity. It induces cell apoptosis and has antibacterial properties. This study was designed to investigate the effect of methyl substituent on its activity as methylated derivatives of l-tyrosine, labelled with short-lived B+ emitters, have been used in oncological diagnostics. To study isotope effects in the oxidative deamination of O-methyl-l-tyrosine, the deuterated isotopomer, i.e. O-methyl-[2-2H]-l-tyrosine, was synthesized by isotope exchange, catalyzed enzymatically by tryptophanase. Isotope effects were determined using the spectrophotometric non-competitive method. The values of isotope effects indicate that the α-C-H bond cleavage occurs in the rate determining step of the investigated reaction and α-hydrogen plays a role in the substrate binding process at the enzyme active site. The inhibitory effect on LAAO activity was studied with α-methyl-l-tyrosine and N-methyl-l-tyrosine. The mode of inhibition was determined based on Lineweavear-Burk plots intersections. α-Methyl-l-tyrosine has been found a mixed type inhibitor of the investigated enzyme, whereas N-methyl-l-tyrosine is a non-competitive inhibitor of LAAO.

Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and l-amino acid oxidase.

The phospholipase A2 (PLA2) and l-amino acid oxidase (LAAO) are two major enzymes found in the venoms from most snake species. These enzymes have been structurally and functionally characterised for their pharmacological activities. Both PLA2 and LAAO from different venoms demonstrate considerable cytotoxic effects on cancer cells via induction of apoptosis, cell cycle arrest and suppression of proliferation. These enzymes produce more pronounced cytotoxic effects in cancer cells than normal cells, thus they can be potential sources as chemotherapeutic agents. It is proposed that PLA2 and LAAO contribute to an elevated oxidative stress due to their catalytic actions, for instance, the ability of PLA2 to produce reactive oxygen species during lipolysis and formation of H2O2 from LAAO catalytic activity which consequently lead to cell death. Nonetheless, the cell-death signalling pathways associated with exposure to these enzymatic toxins are not fully elucidated yet. Here in this review, we will discuss the cytotoxic effects of PLA2 and LAAO in relationship to their catalytic mechanisms and the underlying mechanisms of cytotoxic actions.

Interleukin 4-Induced Gene 1 as an Emerging Regulator of B-Cell Biology and its Role in Cutaneous Melanoma.

Interleukin 4 (IL4)-induced gene 1 (IL4I1) is an oxidase that degrades l-phenylalanine into phenylpyruvate, hydrogen peroxide, and ammonia. In contrast to other amino acid catabolic enzymes (i.e., indoleamine 2,3-dioxygenase and inducible nitric oxide synthase), IL4I1 is expressed not only in an intracellular form but also an active secreted form. Although about 20 yr ago IL4I1 was identified in murine B cells in response to IL4, we only recently established its key role in controlling B-cell receptor-mediated signaling during murine B-cell ontogeny and responses in physiological settings. Genetic IL4I1 invalidation increases the number of tumor-associated B cells and delays development of spontaneous metastatic melanoma in mice that are transgenic for the RET oncogene, without impairing tumor-specific antibody response. Although no consensus exists on phenotype and functions of melanoma-associated B cells, our results in RET mice argue for a protective role, with IL4I1 dampening this benefit. However, regulation of IL4I1 expression in innate-like and conventional B-cell subsets and its impact on B-cell properties are incompletely known, in particular, in cancer settings. This review aims to summarize our present knowledge of B cells in human and murine melanoma and address emerging questions about the impact of IL4I1 on B-cell functions in physiological and cancer settings. We note that during melanoma progression, IL4I1 may selectively be expressed by regulatory B cells and/or indirectly promote B-cell-mediated immunosuppression.

Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities.

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.