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Coagulopathy is one of the major complications following envenomations by crotalid and viperid snakes. The present study was undertaken to examine the effect of a hemorrhagic metalloproteinase in Bothrops jararaca venom, jararafibrase I (JF I), on the development of coagulopathy using rat snakebite model. Coagulation parameters were monitored after subcutaneous injection of B. jararaca crude venom, JF I-neutralized venom and purified JF I in rats. Crude venom induced unclottable blood and fibrinogen consumption, while JF I-neutralized venom and purified JF I did not induce coagulopathy. Plasma venom antigen level of rats given JF I-neutralized venom was lower than that of rats given crude venom. We conclude that venom hemorrhagic metalloproteinases play an important role in the development of coagulopathy through rapid spreading of venom coagulation components from the injected area into systemic circulation.

Jararafibrase I is a hemorrhagic metalloproteinase purified from Bothrops jararaca venom, which induces local hemorrhage by degrading the basement membrane components. The present study was undertaken to investigate the inhibition of jararafibrase I by human and rat serum proteinase inhibitors. The proteolytic activity of jararafibrase I was completely inhibited by human and rat sera. In particular, rat serum displayed a greater inhibitory capacity. The inhibitory capacities of both sera were dependent on α -macroglobulins. SDS-PAGE analysis revealed that jararafibrase I formed complexes with α -macroglobulins that were present in normal sera. The proteolytic activity of jararafibrase I was completely inhibited by α 1-macroglobulin and murinoglobulin in rat serum, and by human α 2-macroglobulin. The inhibition molar ratios of α -macroglobulin/jararafibrase I were 1.5 for rat α 1-macroglobulin and human α 2-macroglobulin, and 2.4 for rat murinoglobulin. SDS-PAGE under reducing conditions demonstrated that the bait region of human α 2-macroglobulin and rat murinoglobulin was cleaved by jararafibrase I. The bait region cleavage sites were identified as being situated at the 696 Arg– 697 Leu peptide bond in human α 2-macroglobulin, and at the 686 Ala– 687 Val peptide bond in rat murinoglobulin.

M. Tanigawa , M. Maruyama , M. Sugiki , K. Shimaya , K. Anai and H. Mihara . Clearance and distribution of a haemorrhagic factor purified from Bothrops jararaca venom in mice. Toxicon 32, 583–593, 1994.—We previously purified two fibrinolytic/haemorrhagic enzymes (jararafibrase-I and II) from Bothrops jararaca venom. In the present study, the clearance, organ distribution and local absorption rate were examined in mice using 125 I-labelled jararafibrase-I. Following intravenous injection of 125 I-labelled jararafibrase-I, a complex was rapidly formed with the plasma protein and the radioactivity quickly disappeared from the circulation with a half-life of about 3 min for the initial part of the curve. The highest level of the radioactivity (59.5%) was seen in the liver at 5 min after dosing, and the next highest level of radioactivity (14.4%) was seen in the kidney at 60 min after dosing. At 60 min after dosing, 36.8% of the total injected radioactivity was seen in the contents of the small intestine, and 11.4% of the total injected radioactivity was seen in the contents of the large intestine at 120 min after dosing. It is assumed that the jararafibrase-I was metabolized mainly in the liver, to small mol. wt products, and excreted in the intestine via the bile duct. Also, a small amount of jararafibrase-I appeared to be metabolized in the kidney. Following subcutaneous injection, a high-dose group revealed a low local absorption rate. The low local absorption rate was apparently due to a diminished blood flow caused by subcutaneous haemorrhage.

To examine the effect of Bothrops jararaca venom and its major hemorrhagic metalloproteinase, jararafibrase I (JF I), on vascular endothelial cells, B. jararaca crude venom and JF I were infused intravenously into rabbits. The degree of endothelial cell injury was estimated from the plasma level of soluble thrombomodulin (TM). The fibrinogen level, prothrombin time (PT), JF I antigen level and macroglobulin activity of the plasma were also measured. The TM level was not increased even by a large quantity of JF I, while the crude venom caused an increase in TM level suggesting the occurrence of endothelial cell injury. No alterations of fibrinogen level and PT were noted with a high amount of JF I, and no systemic bleeding was observed. Macroglobulin, which is the main inhibitor of metalloproteinase in rabbit plasma, was not significantly reduced despite a high dose of JF I. The elevation of TM level in the rabbit plasma after infusion of crude venom was totally suppressed by pretreatment with heparin. These findings suggest that the endothelial cell injury caused by B. jararaca venom is not due to the hemorrhagic metalloproteinase but to the coagulating factors in the venom. Plasma macroglobulin appears to be efficient enough to neutralize the circulating hemorrhagic metalloproteinases inoculated by B. jararaca.

SummaryFollowing wounding, the surrounding fibroblasts migrate towards the clotted blood in the wounded space to form granulation tissue resulting in wound repair. One of the most abundant proteins in the wound is hemoglobin (Hb). The aim of the present study was to examine the effect of Hb on fibroblasts in producing components of the plasminogen-plasmin system which play an important role in wound healing. Human Hb Ao added to cultures of human fibroblasts elicited a dose-dependent increase in fibrinolytic activity. ELISA demonstrated an increased fibrinolytic activity due to increased urokinase-type plasminogen activator (uPA). An increase in tissue-type PA was also detected, while the type-1 PA inhibitor level remained unaffected. Globin showed a similar effect, while hemin and protoporphyrin IX exerted no effect. The influence of Hb was quenched when haptoglobin was added. Although northern blot analysis revealed no difference in uPA transcripts between stimulated and non-stimulated cells, immunopretipitation experiments confirmed an increased uPA synthesis in Hb- and globin-treated cells, suggesting that enhanced expression is achieved through translational regulation. These findings suggest a potential role for globin in modulating cellular functions during the process of wound healing.

Urokinase-type plasminogen activator (uPA) activates plasminogen to plasmin, which is involved in the degradation of the vascular basement membrane and extracellular matrix. The present study was undertaken to examine the effects of several hemorrhagic metalloproteinases, jararafibrase (JF) I, II, III and IV, purified from Bothrops jararaca venom, on the single-chain zymogen form of uPA (scuPA). Activation of scuPA by JF I IV was estimated using a synthetic substrate for uPA (S-2444). Only JF I activated the scuPA in a time- and dose-dependent manner. SDS-PAGE analysis revealed that, after incubation with JF I, the intensity of the 55 kDa band of scuPA decreased concomitantly with increases in the intensity of the major two bands at 32 and 22 kDa under reduced and non-reduced conditions. The 32 kDa band demonstrated fibrinolytic activity in fibrin-zymographic studies. Amino-acid-sequence analysis revealed that JF I cleaved the position of 143Glu-144Leu in scuPA, indicating that JF I formed low molecular weight scuPA. From these results, it seems possible that activation of scuPA by JF I could be responsible in part for the local hemorrhage and tissue damage that are frequently observed in human victims of B. jararaca envenoming.