cellulose and Hemorrhage

A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation.

Topics: Animals; Bothrops; Caseins; Catalysis; Chromatography, Ion Exchange; Crotalid Venoms; DEAE-Cellulose; Edema; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Hemorrhage; Metalloproteases; Mice; Muscle, Skeletal; Muscular Diseases; Necrosis

Binding of heparin to DEAE-cellulose membranes may reduce bioavailable heparin, thus increasing the amount of heparin needed for anticoagulation during dialysis. To test this hypothesis, blood loss and coagulation were evaluated during dialysis with DEAE-cellulose and polysulphone membranes. The heparin dose required to effect a given increase in the baseline recalcified activated clotting time (RACT) was determined using a pharmacokinetic model. Blood remaining in the dialyser post-dialysis (RBV) was measured by red cell lysis and haemoglobinometry. Plasma thrombin-antithrombin III complex (TAT) was used to assess activation of the coagulation system. RBV and changes in TAT were determined in two crossover studies. Firstly, DEAE-cellulose membranes were used at doses of heparin calculated to increase baseline RACT by 12.5% and 25%. Secondly, DEAE-cellulose and polysulphone membranes were compared at a heparin dose calculated to increase baseline RACT by 15%. RBV for DEAE-cellulose membranes was independent of the dose of heparin and did not differ from that found for polysulphone membranes. TAT concentrations increased during dialysis; however, there was no difference between the two membranes. These results show that DEAE-cellulose membranes do not require increased heparin to avoid dialyser-associated blood loss.

Topics: Adult; Aged; Antithrombin III; DEAE-Cellulose; Dose-Response Relationship, Drug; Female; Hemorrhage; Heparin; Humans; Male; Membranes, Artificial; Middle Aged; Peptide Hydrolases; Renal Dialysis; Whole Blood Coagulation Time

Topics: Agkistrodon; Animals; Biochemical Phenomena; Biochemistry; Chromatography; Crotalid Venoms; Cysteine; DEAE-Cellulose; Edetic Acid; Electrophoresis; Hemorrhage; Peptide Hydrolases; Rabbits; Research; Snake Venoms; Snakes; Ultracentrifugation; Venoms