Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and Clot-Dissolving Properties.

In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant, antiplatelet, and clot-dissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation and ion-exchange column chromatography techniques. The purity of the enzyme was evaluated by SDS-PAGE, RP-HPLC, and MALDI-TOF. FSCP was observed as a single band of approximately 160 kDa in SDS-PAGE under reducing and non-reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDI-TOF spectrometry. The CD spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no beta-sheet structures. FSCP hydrolyzed both casein and gelatin with a specific activity of 3.5 and 4.2 unit/mg min respectively. The proteolytic activity of FSCP was completely abolished by iodoacetic acid (IAA), suggesting FSCP is a cysteine protease. The pH optimum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong anticoagulant effect in both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) by extending the clotting time from 222 to 1100 s and from 256 to 1210 s, respectively. FSCP degraded human fibrinogen and fibrin clots. The products of fibrinogen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets triggered by ADP, epinephrine, thrombin, collagen, arachidonic acid, and platelet activating factor (PAF). FSCP was found to be nontoxic as it did not damage the membrane of red blood cells (RBCs) and did not induce hemorrhage and edema in experimental mice.

Protective Effect of Tamarind Seed Coat Ethanol Extract on Eryptosis Induced by Oxidative Stress.

Suicidal erythrocyte death, or eryptosis, is the key event in eliciting anemia in numerous pathological conditions, including diabetes, chronic kidney disease, cancer, sepsis, etc. Oxidative stress is an important trigger in the acceleration of erythrocyte loss via eryptosis and an underlying mechanism of anemia emergence in the above pathologies. Therefore, there is an increasing demand for identification of antioxidants and anti-eryptotic agents for the management of stress-related ailments. Here, we demonstrated the antioxidant and anti-eryptotic properties of the tamarind seed coat ethanol extract (TSCEE) against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress and eryptosis. The presence of probable secondary metabolites in the TSCEE extract was investigated by RP-HPLC. Active groups present in the TSCEE were studied by the Fourier-transform infrared spectroscopy. Cyclic voltammetric studies confirmed the antioxidant potential of TSCEE. The protective effect of TSCEE on red blood cells was confirmed by assessing various eryptotic markers, such as reactive oxygen species generation, intracellular calcium levels, and phosphatidylserine exposure. TSCEE reduced lipid peroxidation and protein carbonyl content and restored the levels of glutathione, antioxidant enzymes, and enzymes involved in glutathione replenishment. In conclusion, TSCEE was found to exhibit multiple therapeutic properties, which makes it a promising agent for treating oxidative stress-induced eryptosis and subsequent anemia in various pathologies.