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Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.

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The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.

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Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4-and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4-and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.