Thrombolytic along with anti-platelet activity of crinumin, a protein constituent of Crinum asiaticum.

Several anticoagulants, anti-platelet and thrombolytic medications are used for the treatment of thrombotic disorders. Anti-coagulants and anti-platelet agents prevent the formation of blood clots but do not dissolve existing clots, whereas thrombolytic agents are able to dissolve a clot but emboli can form even after successful treatment. Thus, none of them provide a permanent and complete solution. In this regard a single molecule that could both dissolve the clot and prevent the formation of new clots would be useful in the treatment of thrombotic diseases. Crinumin, a stable and active (in many adverse conditions) serine protease, shows plasmin-like fibrinolytic activity and inhibits platelet aggregation and P-selectin exposure, as established by photography, phase contrast microscopy, whole blood optical Lumi-aggregometry and flow cytometry. Crinumin could be an efficient and inexpensive therapeutic agent for the treatment and prevention of thromboembolic diseases.

Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity.

Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure-function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, α = γ = 90, ß = 114.19°. The Matthews coefficient was 2.81 Å(3) Da(-1), corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress.

Structural functional and folding scenario of an anti platelet and thrombolytic enzyme crinumin.

A folding pattern, conformational stability and therapeutic role of a protein helps in developing a suitable drug. Crinumin, a thrombolytic and anti platelet agent, has been studied for its functional and conformational properties by equilibrium unfolding methods. The crinumin belongs to α+ß class of protein and exhibits a non native structure and two molten globule states at different conditions. Two domains in the molecular structure of the protein with altered stability are present that unfold sequentially. The enzyme maintains activity as well as structural integrity even in adverse conditions. These observations provide an understanding of protein folding as well as facilitate the development of a potential drug.

Biochemical and spectroscopic characterization of a novel metalloprotease, cotinifolin from an antiviral plant shrub: Euphorbia cotinifolia.

A high molecular mass novel metalloprotease, cotinifolin is purified from the latex of Euphorbia cotinifolia by a combination of anion exchange and hydrophobic interaction chromatography. The nonglycosylated enzyme has a molecular mass of 79.76 kDa (ESI-MS) and the isoelectric point of the enzyme is pH 7.7. Cotinifolin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity. The K(m) value of the enzyme was found to be 20 µM with azocasein. The enzyme is not prone to autolysis even at very low concentrations. Polyclonal antibodies specific to enzyme was raised and immunodiffusion reveals that the enzyme has unique antigenic determinants. Maximum caseinolytic activity of cotinifolin is observed in the range of pH 7.0-8.0 and temperature of 50 °C. Using 0.2 mL of 1 mM solution of each metal ion, the purified protease was inhibited slightly by Ba²âº and Mn²âº, moderately by Mg²âº, Ca²âº and Cs²âº and significantly by Zn²âº, Cu²âº and Co²âº. On the other hand, substantial activation in caseinolytic activity was achieved by Ni²âº. The enzyme activity was also inhibited by EDTA and o-phenanthroline but not by any other protease inhibitors. Perturbation studies by temperature, pH, and chaotrophs of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Spectroscopic studies reveal that cotinifolin has secondary structural features with α/ß type with approximately 9% of α-helicity. Easy availability and simple purification procedure makes the enzyme a good system for biophysical study, biotechnological and industrial applications.