A serine protease inhibitor obtained of Juquiri (Mimosa regnelli Benth.) seeds with anti-inflammatory, anticoagulant and heparin adjuvant activities
Thrombosis; Neutrophil; Macrophage; Trypsin; Inflammation; Coagulation.
Hemostasis is the event that mammals use to block the loss of blood and promote injury repair, and failure by overeating contributes to the spread of inflammatory and thrombotic factors. Atherosclerosis is an example of such diseases, and heparin (Hep) is a treatment drug. Patients with antithrombin deficiency have resistance to the anticoagulant effects of Hep, so researches for novel homeostatic molecules that contribute to clinical treatments are necessary. Plant protease inhibitors have been effective in controlling biological processes, such as coagulation and inflammation. The present work aimed isolate a trypsin inhibitor from the Juquiri (ITJ) seed and evaluate its activity against serine proteases, and its anticoagulant and anti-inflammatory potentials. ITJ was isolated by affinity and ion exchange chromatography, presenting two major protein bands of 11.9 and 19.2 kDa on SDS-PAGE, and was able to inhibit trypsin and chymotrypsin. In coagulation, ITJ prolonged partial thromboplastin time (APTT) at concentrations greater than 2 μg/100 μL plasma, without prolonging prothrombin time (PT). Associated with Hep, ITJ was able to enhance the anticoagulant effect of the drug in both tests. Without toxicity to human erythrocytes, ITJ reduced 40% of elastase concentrations released by neutrophils induced by platelet activating factor (PAF). ITJ at concentrations between 20-80 μg/mL stimulated TNF production by macrophages (RAW 264.7) in culture without provoking other inflammatory stimuli. LPS-activated macrophages had a reduction in NO, IL-6 and TNF productions when treated with ITJ at 20-160 μg/mL concentrations. These data point to ITJ as promising in the treatment of thrombotic and inflammatory diseases and have been effective as heparin adjuvant and to promote the reduction of neutrophil inflammatory stimuli by MAPK-dependent pathways and of NF-κB transcription factor pathways in macrophages.