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.
Highlights
Cardiovascular diseases (CVDs) such as myocardial infarction, stroke, deep-vein thrombosis, and pulmonary embolism are major causes of mortality worldwide[1,2]
Herbs containing antithrombotic activities have been suggested to act as medicinal plants that could lead to the discovery of novel therapeutic agents for treating thrombosis-associated diseases[19,20,21,22,23]
Lunathrombase is a major fibrinogenolytic protease purified from the leaves of L. indica
Summary
Cardiovascular diseases (CVDs) such as myocardial infarction, stroke, deep-vein thrombosis, and pulmonary embolism are major causes of mortality worldwide[1,2]. The prothrombinase complex eventually converts inactive prothrombin to active thrombin for the conversion of soluble fibrinogen into insoluble fibrin polymer (clot), which is degraded by plasmin[4,6] Any disruption in this delicate balance leads to thrombosis and/or hemorrhage that results in disseminated intravascular coagulopathy (DIC), which poses a clinical challenge for treatment. To the best of our knowledge, this is the first report on the biochemical and pharmacological characterization, and elucidation of the anticoagulant mechanism of a fibrin(ogen)olytic serine protease purified from the aqueous leaf extract of L. indica This plant-derived fibrinogenolytic serine protease demonstrated dual inhibition of thrombin and FXa, and did not show in vivo toxicity in experimental animals which has never before been demonstrated for any protease, and the finding suggests its therapeutic application as an anticoagulant, antithrombotic drug
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