Monocyte NLRP3 inflammasome and interleukin-1ß activation modulated by alpha-1 antitrypsin therapy in deficient individuals.

INTRODUCTION: Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1ß secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS: Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1ß secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS: C3d acting via CR3 induces NLRP3 and pro-IL-1ß production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1ß secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1ß (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION: These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, µ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent µ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 µg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.

Targeting of Glycosaminoglycans in Genetic and Inflammatory Airway Disease.

In the lung, glycosaminoglycans (GAGs) are dispersed in the extracellular matrix (ECM) occupying the interstitial space between the capillary endothelium and the alveolar epithelium, in the sub-epithelial tissue and in airway secretions. In addition to playing key structural roles, GAGs contribute to a number of physiologic processes ranging from cell differentiation, cell adhesion and wound healing. Cytokine and chemokine-GAG interactions are also involved in presentation of inflammatory molecules to respective receptors leading to immune cell migration and airway infiltration. More recently, pathophysiological roles of GAGs have been described. This review aims to discuss the biological roles and molecular interactions of GAGs, and their impact in the pathology of chronic airway diseases, such as cystic fibrosis and chronic obstructive pulmonary disease. Moreover, the role of GAGs in respiratory disease has been heightened by the current COVID-19 pandemic. This review underlines the essential need for continued research aimed at exploring the contribution of GAGs in the development of inflammation, to provide a better understanding of their biological impact, as well as leads in the development of new therapeutic agents.

A Review of Alpha-1 Antitrypsin Binding Partners for Immune Regulation and Potential Therapeutic Application.

Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.

A simple, cost-effective, and rapid separation process for the isolation of anticoagulant active fraction from the fruit extract of Momordica charantia: Characterization of bioactive components and anticoagulant mechanism of active fraction in a mouse model.

A simple, rapid, and cost-effective process for the separation of an active anticoagulant fraction from the aqueous fruit extract of Momordica charantia by using rice husk as adsorbed is described. The in vitro anticoagulant activity of active anticoagulant fraction was comparable to commercial anticoagulants heparin and warfarin. Phytochemical analysis revealed the presence of alkaloids, flavonoids, and phytols in the active anticoagulant fraction, nevertheless; it was devoid of glycosides, triterpenoids, tannins, saponins, steroids, and carbohydrates. By gas chromatography with mass spectrometry analysis, decanoic acid, 1,2,3-propanetriyl ester (22.3%), dodecanoic acid, 1,2,3-propanetriyl ester-d5 (17.3%), dodecenoic acid, 1,2,3-propanetriyl ester (12.5%), and 4-B-methylandrostane 2,3-diol-1,17-dione (11.4%) were identified as the most abundant constituents of active anticoagulant fraction. Presence of αß-fibrinogenase enzyme was identified by biochemical assay but not by liquid chromatography with tandem mass spectrometry analysis suggesting presence of a novel protease enzyme in this fraction. The active anticoagulant fraction demonstrated biding to fibrinogen but not to thrombin or Factor Xa, inhibited the collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, noncytotoxic to mammalian cells, showed in vivo plasma defibrinogenation and anticoagulant activities, and inhibited k-carrageen-induced thrombus formation in the tails of mice. Therefore, active anticoagulant fraction (an herbal drug) may find therapeutic application for the prevention and/or treatment of hyperfibrinogenemia/thrombosis-associated cardiovascular disorders.

First Report of Plant-Derived ß-Sitosterol with Antithrombotic, in Vivo Anticoagulant, and Thrombus-Preventing Activities in a Mouse Model.

Inhibitors of thrombin, a key enzyme in the blood coagulation cascade, are of great interest because of their selective specificity and effectiveness in anticoagulation therapy against cardiovascular disorders. The natural soybean phytosterol, ß-sitosterol (BSS) demonstrated anticoagulant activity by dose-dependent inhibition of thrombin in an uncompetitive manner with a Ki value of 0.267 µM as well as by partial inhibition of thrombin-catalyzed platelet aggregation with a half-maximal inhibitory concentration (IC50) value of 10.45 ± 2.88 µM against platelet-rich plasma and 9.2 ± 1.2 µM against washed platelets. An in silico study indicated binding of BSS to thrombin, which was experimentally verified by spectrofluorometric and isothermal calorimetric analyses. Under in vitro conditions, BSS demonstrated thrombolytic activity by activating plasminogen, albeit it is devoid of protease (fibrinogenolytic) activity. BSS was noncytotoxic to mammalian cells, nonhemolytic, demonstrated its in vivo anticoagulant activity when administered orally, and inhibited k-carrageen-induced thrombus formation in the tails of mice. Our results suggest that dietary supplementation of BSS may help to prevent thrombosis-associated cardiovascular disorders.

The vacuolar anti-Pseudomonal activity of neutrophil primary granule peptidyl-arginine deiminase enzymes.

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa (P. aeruginosa) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro, bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro, PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.

Studies on in vivo antithrombotic activity of quercetin, a natural flavonoid isolated from a traditional medicinal plant, African eggplant (Solanum indicum).

ETHNOPHARMACOLOGICAL RELEVANCE: Every year, cardiovascular diseases (CVDs) account for about 17.9 million deaths, making them the primary cause of both morbidity and mortality. Conventional drugs, which are often prescribed to treat cardiovascular diseases, are costly and have adverse effects. Consequently, dietary modifications and other medications are needed. Traditional use of Solanum indicum as cardiotonic to treat hypertension and anticoagulant potency has been reported but poorly evaluated scientifically. AIM OF THE STUDY: This study investigated the in vivo anticoagulant activity and mechanism of anticoagulation of quercetin (QC), a bioactive compound isolated from S. indicum (SI) hydroethanolic fruit extract. MATERIALS AND METHODS: Bioassay-guided fractionation (anticoagulant activity) extracted QC from hydroethanolic SI extract. QC was extensively characterized biochemically and pharmacologically. The interaction between QC and thrombin was investigated using spectrofluorometric and isothermal calorimetric methods. Cytotoxicity, antiplatelet, and thrombolytic studies were carried out in vitro. The Swiss albino mice were used to assess the in vivo, anticoagulant, and antithrombotic activities of QC. RESULTS: QC exhibits anticoagulant activity via (i) uncompetitive inhibition of thrombin but not FXa with a Ki value of 33.11 ± 4.2 µM and (ii) a partial inhibition of thrombin-catalyzed platelet aggregation with an IC50 value of 13.2 ± 1.2 µM. The experimental validation of the in silico study's prediction of QC's binding to thrombin was confirmed by spectrofluorometric and isothermal calorimetric analyses. QC was nontoxic to mammalian, non-hemolytic cells and demonstrated thrombolytic activity by activating plasminogen. QC demonstrated in vivo anticoagulant efficacy, preventing k-carrageen-induced thrombus formation in mice's tails. In the acute circulatory stasis paradigm in mice, QC reduces thromboxane B2 (TXB2) and endothelin-1 (ET-1) while increasing nitric oxide synthase (eNOS) and 6-keto prostaglandin F1α (6-keto-PGF1 α). CONCLUSION: Effective in vivo anticoagulant and antithrombotic properties of S. indicum's bioactive component QC point to the plant's potential use as a herbal anticoagulant medication for preventing and treating cardiovascular diseases linked to thrombosis.

C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

Characterization of active anticoagulant fraction and a fibrin(ogen)olytic serine protease from leaves of Clerodendrum colebrookianum, a traditional ethno-medicinal plant used to reduce hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular diseases are the major cause of mortality and morbidity, causing over 17.9 million deaths a year worldwide. Currently used therapy is often having side effects and expensive, dietary interventions and alternative medicines are required. Clerodendrum colebrookianum has been used to treat cardiac hypertension but anticoagulant potency was not evaluated. AIM OF THE STUDY: To characterize an active anticoagulant fraction (AAFCC) and a 30 kDa fibrin(ogen)olytic serine protease (clerofibrase) isolated from aqueous leave extract of C. colebrookianum. MATERIALS AND METHODS: AAFCC/clerofibrase was subjected to extensive biochemical and pharmacological characterization including LC-MS/MS, amino acid compositional and GC-MS analyses. Interaction between clerofibrase with fibrinogen was studied by spectrofluorometric analysis. In vitro thrombolytic, antiplatelet and cytotoxicity assay were performed. In vivo toxicity, anticoagulant, defibrinogen and antithrombotic activities were determined on Swiss albino mice. RESULTS: The in vitro anticoagulant activity of AAFCC was found to be superior to heparin and clerofibrase and comparable to Nattokinase and warfarin. The proteomics and amino acid composition analyses suggest that clerofibrase is a previously uncharacterized novel plant protease capable of degrading the -αß chains of fibrinogen/fibrin. AAFCC/clerofibrase exerts their anticoagulant action via fibrinogenolytic activity and partially by antiplatelet activity albeit they have no effect on thrombin and FXa inhibition. The spectrofluorometric analysis revealed the binding of clerofibrase to fibrinogen but not to thrombin and FXa. The phytochemical constituents and bioactive components of AAFCC were characterized by biochemical, and GC-MS analyses. The AAFCC and clerofibrase inhibited collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, and non-cytotoxic to mammalian cells. The AAFCC showed and dose-dependent in vivo plasma defibrinogenating and anticoagulant activities and inhibited k-carrageen-induced thrombus formation in the tails of mice. CONCLUSION: The potent in vivo anticoagulant and antithrombotic effects of AAFCC suggests its pharmacological significance as herbal anticoagulant drug for the prevention and/or treatment of hyperfibrinogenemia- and thrombosis associated cardiovascular disorders.

Anticoagulant mechanism, pharmacological activity, and assessment of preclinical safety of a novel fibrin(ogen)olytic serine protease from leaves of Leucas indica.

The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fibrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purified from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme's novel characteristic. Lunathrombase is an αß-fibrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofluorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fibrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125-0.5 mg/kg) in vivo anticoagulant and plasma defibrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological effects in treated animals.

The N-terminal-truncated recombinant fibrin(ogen)olytic serine protease improves its functional property, demonstrates in vivo anticoagulant and plasma defibrinogenation activity as well as pre-clinical safety in rodent model.

An N-terminal truncated fibrino(geno)lytic serine protease gene encoding a ~42kDa protein from Bacillus cereus strain AB01 was produced by error prone PCR, cloned into pET19b vector, and expressed in E5 coli BL21 DE3 cells. The deletion of 24 amino acid residues from N-terminal of wild-type Bacifrinase improves the catalytic activity of [Bacifrinase (ΔN24)]. The anticoagulant potency of [Bacifrinase (ΔN24)] was comparable to Nattokinase and Warfarin and results showed that its anticoagulant action is contributed by progressive defibrinogenation and antiplatelet activities. Nonetheless, at the tested concentration of 2.0µM [Bacifrinase (ΔN24)] did not show in vitro cytotoxicity or chromosomal aberrations on human embryonic kidney cells-293 (HEK-293) and human peripheral blood lymphocytes (HPBL) cells. [Bacifrinase (ΔN24)], at a dose of 2mg/kg, did not show toxicity, adverse pharmacological effects, tissue necrosis or hemorrhagic effect after 72h of its administration in Swiss albino mice. However, at the tested doses of 0.125 to 0.5mg/kg, it demonstrated significant in anticoagulant effect as well as defibrinogenation after 6h of administration in mice. We propose that [Bacifrinase (ΔN24)] may serve as prototype for the development of potent drug to prevent hyperfibrinogenemia related disorders.

Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 µmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells, bactericidal or hemolytic activity. The future therapeutic application of NnPLA2-I for treatment and prevention of cardiovascular disorders is therefore suggested.

Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B.

In this study, biochemical and pharmacological characterization of Brevithrombolase, a fibrinolytic serine protease purified from Brevibacillus brevis strain FF02B has been reported. An assessment of its thrombolytic potency has also been made. The molecular mass of this monomeric protease was determined as 55 kDa, and 56043 Da, respectively, by SDS-PAGE and MALDI-TOF-MS. In the analytical studies, the N-terminal sequence of Brevithrombolase was found to be blocked; however, the peptide mass fingerprinting and amino acid composition analyses demonstrated the similarity of Brevithrombolase with endopeptidases in possessing serine in their catalytic triad. This finding was confirmed by the observation that the serine protease inhibitors decrease the catalytic (fibrinolytic) activity of Brevithrombolase. The secondary structure of Brevithrombolase was composed of 30.6% alpha helix and 69.4% random coil. Brevithrombolase showed the Km and Vmax values towards the chromogenic substrate for plasmin at 0.39 mM and 14.3 µmol/min, respectively. Brevithrombolase demonstrated optimum fibrinolytic activity at pH 7.4 and 37 °C, and showed marginal hydrolytic activity towards globulin, casein and fibrinogen. The anticoagulant potency of Brevithrombolase was comparable to the low molecular mass heparin/antithrombin-III and warfarin. Among the three enzymes-Brevithrombolase, plasmin and streptokinase-the fibrinolytic activity and in vitro thrombolytic potency of Brevithrombolase was found to be superior. The RP-HPLC and SDS-PAGE analyses suggested a similar pattern of fibrin degradation by Brevithrombolase and plasmin, indicating that former enzyme is a plasmin-like fibrinolytic serine protease. Brevithrombolase did not show in vitro cytotoxicity on HT29 and HeLa cells or hemolytic activity. At a dose of 10 mg/kg, Brevithrombolase did not exhibit lethality or toxicity on Wistar strain albino rats. Brevithrombolase did not inhibit factor Xa, and its mechanism of anticoagulant action was associated with the enzymatic cleavage of thrombin. The combined properties of Brevithrombolase indicate its therapeutic potential in peptide-based cardiovascular drug development.