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

Abstract

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 αβ-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.

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

“…Modifications of the histidine, cysteine, and serine residues of protease enzyme(s) of AAF were made by preincubating AAF (at a final concentration of 2.0 μg/mL) with 4‐bromophenacyl bromide ( p BPB), iodoacetamide (IAA), phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT), tosyl phenylalanyl chloromethyl ketone (TPCK), tosyl phenylalanyl chloromethyl ketone (TLCK), and EDTA at final inhibitor concentrations of 2.0 mM (for PMSF) and 5.0 mM (for the others) at room temperature for 30 min and then biochemically assaying protease activity with comparisons to the appropriate controls [20]. In another set of experiments, AAF (2.0 μg/mL) was preincubated with α 2 ‐macroglobulin or antiplasmin (3.0 μM) for 60 min at 37˚C and the fibrin(ogen)olytic activity was then determined as described above.…”

“…Fibrinogen zymography of AAF was performed according to Majumdar et al [17]. Fibrinogen was incubated with 1 μg/mL Nattokinase or AAF or1XPBS, pH 7.4 (control) for different time intervals (5, 15, and 30 min) at 37 • C and the fibrinogen degradation pattern by AAF and Nattokinase was also evaluated by RP-HPLC analysis [20]. All experiments were done in triplicate.…”

“…For each experiment, a control was used where the protease was incubated with an equivalent volume of assay buffer. The activity of the control was taken as 100% and the experimental values were determined by comparison [20].…”

Effects of microwave combined with ultrasound treatment on the pasteurization and nutritional properties of bottle gourd (Lagenaria siceraria) juice

“…The protein content was estimated by Bradford's method using the manufacturer's protocol (Sigma Aldrich, USA). The described protocol was also followed for amino acid compositional analysis (Gogoi et al., 2018; Majumdar et al., 2014). Raw, conventional, and microwave‐treated BGJ protein (500 µg) were hydrolyzed at 110°C for 24 hr under vacuum using 6 N HCl.…”

Anticoagulant and antiplatelet activities of novel serine protease purified from seeds of Cucumis maderaspatensis

“…Leucas indica is a traditional medicine used to reduce nasal congestion and asthma. The leaf extracts of this plant are reported to possess anticoagulant fibrinogenolytic property due to expression of serine protease (Gogoi et al 2018). Cucumis maderaspatensis, L belongs to the family Cucurbitaceae which is consumed as vegetable (Gaikwad et al 2015) in south Indian subcontinent (Fig.…”