Masahiko Sugiki

Broad substrate specificity of snake venom fibrinolytic enzymes: Possible role in haemorrhage

We found previously that two fibrinolytic enzymes (jararafibrases I and II) purified from Bothrops jararaca venom displayed a haemorrhagic activity. To elucidate the mechanisms involved and the role of the enzymatic activity in haemorrhage, the enzymatic properties of the purified enzymes were examined. The substrate specificity of the enzymes was determined using type I collagen, type IV collagen, gelatin, laminin and fibronectin as substrates. The enzymes degraded type IV collagen, gelatin, laminin and fibronectin into smaller fragments, but degraded type I collagen only partially in a non-specific manner. The specific activities of jararafibrase I for type IV collagen and gelatin were 172 ± 5 units/mg protein and 1315 ± 177 units/mg protein, respectively. The specific activities of jararafibrase II for type IV collagen and gelatin were9.2 ± 0.6 units/mg protein and 143 ± 15 units/mg protein, respectively. It was evident that the enzymes had rather broad substrate specificities and degraded basement membrane components including type IV collagen. The number of type IV collagen units of bacterial collagenase which gave the minimal haemorrhagic dose was 191.4, while the numbers of type IV collagenase units of jararafibrases I and II which gave the minimal haemorrhagic dose were 1.5 and 0.25, respectively. It is suggested that the broad substrate specificity of the enzymes is essential for inducing haemorrhage with a single enzyme.

Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom

Two fibrinolytic enzymes, jararafibrase I and jararafibrase II, were purified from Bothrops jararaca venom. The purified jararafibrase I and jararafibrase II ran as single protein bands on analytical polyacrylamide gel electrophoresis and had mol. wts of 47,000 +/- 2000 and 21,400 +/- 500, respectively, by SDS-polyacrylamide gel electrophoresis. The isoelectric points of jararafibrase I and jararafibrase II were 4.6 and 6.5, respectively. The specific activities of jararafibrase I and jararafibrase II were 2.2 units/mg protein and 6.3 units/mg protein, respectively. Both enzymes exhibited no detectable plasminogen activating activity. The activity of the enzymes was completely inhibited by 1,10-phenanthroline and ethylenediaminetetraacetate, suggesting that both enzymes were metalloproteinases. Jararafibrase I and jararafibrase II had single-chain protein compositions, and the amino acid sequence up to the 49th amino acid from the NH2-terminal of jararafibrase II was: Leu-Pro-Glu-His-Gln-Arg-Tyr-Ile-Glu-Leu-Phe-Ile-Val-Val-Asp-His-Gly-Met- Phe-Met-Lys-Tyr-Asn-Gly-Asn-Ser-Asp-Lys-Ile-Arg-Arg-Arg-Ile-His-Gln- Met-Val-Asn-Ile-Met-Lys-X-Ala-Tyr-Arg-Tyr-Leu-Tyr-Ile-(X = not confirmed).

Neutralization of a snake venom hemorrhagic metalloproteinase prevents coagulopathy after subcutaneous injection of Bothrops jararaca venom in rats

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.

Modulation of the receptor for urokinase-type plasminogen activator in macrophage-like U937 cells by inflammatory mediators.

Macrophages in the tissues have been shown to express receptor for urokinase-type plasminogen activator (uPAR) on their cell surface which plays an important role in cell invasion and attachment. We examined the effects of inflammatory mediators on the expression of uPAR employing U937 cells which have monocyte/ macrophage-like characteristics. U937 cells were incubated with various mediators such as interleukins (IL), tumor necrosis factors (TNF), dexamethasone, thrombin, fibrin fragment D, bradykinin, complement C5a, and components of the extracellular matrix. The uPAR expression on the cell surface was then analyzed by radio-ligand binding assay using125I-scuPA. The strongest enhancement of uPAR was observed in the cells stimulated by TNFα and TNFβ. IL-1β, IL-6, and C5a also increased the uPA binding sites with various patterns of affinity change. Dexamethasone decreased the uPA binding sites without changing the affinity. Fibrin fragment D and IL-3 reduced the affinity without changing the number of receptors. These findings suggest that the expression of uPAR in inflammatory cells could be modulated by various inflammatory mediators.

Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues

The distribution and localization of acid stable trypsin inhibitor (ASTI) in normal and malignant human tissues from various organs were examined using immunohistochemical techniques that used goat antibody raised against highly purified ASTI from human urine. Tissues were assessed as positive only when they were stained by both the biotin‐avidin‐peroxidase complex system and biotin‐streptavidin‐β‐galactosidase complex system, and the staining was abolished by absorption with purified ASTI. Under normal conditions, ASTI immunoreactivity was observed in only a few organs. Positive tissues for ASTI immunoreactivity included the kidney proximal tubules, glial cells of the cerebrum, fibrillar structures of the lamina propria of the stomach and colon, and bronchial epithelial cells. No ASTI immunoreactivity was observed in the cardiovascular system, reproductive system, or other tissues examined. As is not the case for normal tissues, ASTI immunoreactivity was found to be widely distributed in malignant tumors. Staining was observed in the extracellular space, i.e., in the stroma of the tumor and in connective tissues around the tumor invasion, whereas no ASTI immunoreactivity was detected in the malignant cells. Considering the identity of the first 36 NH2‐terminal residues of ASTI purified from plasma or urine with a recently reported endothelial cell growth factor, the present findings suggest that ASTI could play an important role, not limited to its function as a protease inhibitor, in the invasive growth of malignant neoplasms.

Inhibition of a snake venom hemorrhagic metalloproteinase by human and ratα-macroglobulins

Abstract 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.

Clearance and distribution of acid-stable trypsin inhibitor (ASTI)

The clearance, organ distribution and metabolic pathway of the acid-stable trypsin inhibitor (ASTI) were studied in mice using 125I-labeled urinary trypsin inhibitor (UTI), the most typical ASTI in the urine. Following intravenous injection of 125I-UTI, the radioactivity disappeared rapidly from the circulation with a half-life of 4 min for the initial part of the curve. Gel filtration of plasma samples revealed that the rapid disappearance of the radioactivity was due to elimination of free inhibitor from the plasma. 125I-UTI was cleared primarily in the kidney. Gel filtration of urine samples showed that part of the radioactivity in the urine appeared at the same elution volume as 125I-UTI in the plasma, indicating that the origin of UTI was ASTI in the plasma.

Vascular endothelial cell injury induced by Bothrops jararaca venom; non-significance of hemorrhagic metalloproteinase

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.

Enhancement of plasma fibrinolysis in vitro by jararhagin, the main haemorrhagic metalloproteinase in Bothrops jararaca venom.

Jararhagin, a haemorrhagic metalloproteinase from Bothrops jararaca venom, plays an important role in systemic as well as local haemorrhage. In this study, the effect of jararhagin on the fibrinolytic system was investigated. The fibrinolytic activity of various kinds of animal plasmas was measured by the fibrin plate method. No activity was detected in plasma alone. However, after mixing plasma with jararhagin, strong fibrinolytic activity was recorded in guinea-pig, horse, dog, rabbit and human plasmas. The mechanism of the increase of firbinolytic activity by jararhagin was studied further in guinea-pig plasma. Fibrin-zymographic studies indicated that jararhagin increased tissue-type plasminogen activator (tPA) activity by the dissociation of a complex of tPA with type 1 plasminogen activator inhibitor (PAI-1). alpha 2-Plasmin inhibitor (alpha 2-PI) activity in the plasma was measured using a synthetic chromogenic substrate method after incubation with jararhagin. The alpha 2-PI activity in the plasma decreased in both time-dependent and dose-dependent manners. These in vitro results suggest that, in some animal plasmas, jararhagin increases plasma fibrinolytic activity by causing dissociation of the tPA/PAI-1 complex and by the inactivation of alpha 2-PI. It is possible that this direct action of jararhagin on the enhancement of plasma fibrinolytic activity may contribute to the aetiology of systemic haemorrhage frequently observed in human victims of B. jararaca envenoming.

N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom

We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells.