0041-0101/90 $3.00 + .00 Pergamon Press pic

Toxico/l Vol. 28, No 7, pp. 837-846, 1990. Printed in Great Britam.

PATHOGENESIS OF HEMORRHAGE INDUCED BY BILITOXIN, A HEMORRHAGIC TOXIN ISOLATED FROM THE VENOM OF THE COMMON CANTIL (AGKISTRODON BILINEATUS BILINEATUS) CHARLOTTE L. OWNBY, 1 TOSHIAKI NIKA,2 KUNIKO IMAI 2 and HISAYOSHI SUGlliARA2 'Department of Physiological Sciences, Oklahoma State University, Stillwater, OK, 74078, U.S.A., and iDepartment of Microbiology, Faculty of Pharmacy, Meijo University, Tenpaku-ku, Nagoya, Japan

(Accepted for puhlication 20 December 1989)

C, L. OWNBY, T. NIKAI, K. IMAI and H. SUGIHARA. Pathogenesis of hemorrhage induced by bilitoxin, a hemorrhagic toxin isolated from the venom of the common cantil (Agkistrodon bilineatus bilineatus). Toxicon 28, 837-846, 1990.-A-The pathogenesis of hemorrhage induced by the i.m. injection of the hemorrbagic toxin, bilitoxin, was studied using light and electron microscopy.i' White mice were injected with sublethal doses of the toxin, and tissue samples were obtained at 5 and 30 min, and 1, 3 and 24 hr after the injection. There was a good correlation between amount of toxin injected and amount of hemorrhage observed. Microscopically, hemorrhage was visible in all parts of the connective tissue surrounding muscle cells just 5 min after injection and fibrin was present both intravascularly and extravascularly. At later time periods the hemorrhage was more extensive and there was more fibrin. Many vessels were plugged with platelets. At 30 min after the injection, muscle cells appeared to be damaged having either delta lesions or disrupted myofibriis. Electron microscopy revealed damaged capillaries with ruptured endfthelial cells, disrupted basal lamina and intact intercellular junctions. Thus, this hemorrhagic toxin acts rapidly to disrupt the capillary endothelium with ut damaging the intercellular junctions, and it also appears to damage skeletal muscle celIS'

INTRODUCTION

LOCAL hemorrhage is one of the most obvious symptoms of venom poisoning by snakes in the subfamily Crotalinae. Over the last 20 years progress has been made in the isolation and biochemical characterization of hemorrhagic toxins from these snake venoms (BJARNASON and Fox, 1988; OWNBY, 1990), especially from venoms of members of the genus Crotalus. Several hemorrhagic toxins have been isolated from venoms of members of the genus Agkistrodon. At least six hemorrhagic toxins have been isolated from the venom of Agkistrodon acutus and at least four from the venom of A. halys blomhoffii (BJARNASON and Fox, 1988; OWNBY, 1990). The common cantil (A. bilineatus bilineatus) is a member of the genus Agkistrodon found from Southern Mexico to Nicaragua. Recently, IMAI et al. (1989) reported the isolation of bilitoxin, a hemorrhagic toxin from this venom. Bilitoxin 837

838

C. L. OWNBY

el

al.

has a mol. wt of 48,000 and an isoelectric point of 4.2. It is a metalloproteinase which hydrolyzes the oxidized B chain of insulin as well as the Ar:x and BfJ chains of fibrinogen. Although many hemorrhagic toxins have been well-characterized biochemically, relatively little is known about the pathogenesis of hemorrhage induced by these toxins. There are two possible ways in which hemorrhage can occur (OWNBY, 1982). Tn hemorrhage 'per diapedesis', the intercellular junctions between capillary endothelial cells widen allowing blood to escape through them into the connective tissue space. In hemorrhage 'per rhexis', the capillary endothelial cells undergo lysis leading to large intracellular gaps, i.e. within the endothelial cells, through which blood escapes. OWNBY et al. (1978) reported that the pathogenesis of hemorrhage induced by three hemorrhagic toxins (a, b, and e) from Crotalus atrox venom consisted of hemorrhage per rhexis. OWNBY and GEREN (1987) reported that hemorrhagic proteinase IV from C. h. horridlls venom also caused hemorrhage per rhexis. There have been no reports on the pathogenesis of hemorrhage induced by any of the toxins isolated from venoms of members of the genus Agkistrodon. The present paper describes the pathogenesis of hemorrhage induced by bilitoxin, a hemorrhagic toxin from the venom of the common cantil, A. b. bilineatlls.

MATERIALS AND METHODS Bilitoxin was isolated from venom of the common cantil (A. b. bilineatlls) purchased from the Miami Serpentarium (Salt Lake City, UT) as described by IMAI et al. (1989). Briefly, crude venom was separated by column chromatography using Sephadex G-75 (0.01 M Tris-HCI buffer, pH 7.2 containing 0.01 M NaCI), followed by DEAE-Sephacel (linear gradient of D.Ot to 0.5 M NaCI in 0.01 M Tris-HCI buffer, pH 7.2), and finally Sephadex G-100 (5 mM Tris-HCI buffer, pH 7.2). Homogeneity of pcak 2 from the Sephadex. G-IOO column was demonstrated by obtaining one band in SDS-PAGE on 8.5% gels and isoelectric focusing. Mice (CDI, Charles River) were injected i.m. in the lateral aspect of the right thigh (biceps femoris muscle) with varying volumes (0.05 ml per 25 g mouse) of a 1.2 Jlg/mt solution to give a dose of 0.03 J1g/g body wcight of mouse. The toxin was diluted with 0.85% physiologic saline, and control mice were injected with physiologic saline alone in the same manner as the experimental mice. The animals were killed by cervical dislocation at cither 5 or 30 min, I, 3 or 24 hr after injection. Muscle tissue was taken from the medial aspect of the thigh (gracilis and semimembranous muscles) to avoid sampling regions damaged by the needle. Twenty-four mice weighing 25-30 g were used in two separate experiments. Muscle tissue was fix.ed initially in 2% cacodylate buffered glutaraldehyde (pH 7.4) for 2 hr at room temperature, fixed secondarily in 2% osmium tetroxide, rinsed in deionized water, and thcn dehydrated in a graded series of ethyl alcohol solutions before embedding in Polybed (Polysciences, Warrington, PAl. Thick \ I JIm) sections were taken from the polymerized blocks using an MT-6000 ultramicrotome with glass knives and were stained with toluidine blue for light microscopic studies. Selected areas were thcn sectioned (70-90 nm) with an MT-6000 ultramicrotome using a diamond knife and stained with mcthanolic uranyl acetate and lead citrate. The thin sections were observed and photographed with a lEOL 100 ex TEMSCAN electron microscope at an accelerating voltage of 80 kV.

RESULTS

There was no hemorrhage or myonecrosis in any of the tissue from control mice. Electron microscopy showed that blood vessels and skeletal muscle cells had normal fine structure. Gross examination of the thighs injected with bilitoxin revealed severe hemorrhage of a very dark red color over the thigh at 5 min after injection, severe hemorrhage which covered the thigh and leg at 30 min, 1 and 3 hr, and extensive hemorrhage over the thigh, leg and part of the abdomen at 24 hr. Also at 24 hr after injection, the muscle tissue was hard rather than soft and pliable as in control mice.

Pathogenesis of Hemorrhage by Bilitoxin

839

FIG. 1. LIGHT MICROGRAPH OF SKELETAL MUSCLE 3 HR AFTER INJECTION OF BILlTOXIN (0.03 IJ,g/g). M, skeletal muscle cells; note presence of numerous erythrocytes (E) in the connective tissue; blood vessels are occluded with erythrocytes and platelets (arrow). (Bar = 25 Jlm.) FIG. 2. LIGHT MICROGRAPH OF SKELETAL MUSCLE 3 HR AFTER INJECTION OF BlLlTOXIN (0.03 Ilg/g). Note large delta lesions in several muscle cells (arrows). Nerve (N) is apparently normal. (Bar = 25 11m.)

Light microscopic examination of the muscle tissue showed that hemorrhage occurred very soon after injection. Hemorrhage was visible in all parts of the connective tissue at 5 min after injection. At all time periods after injection, capillaries and other small blood vessels were occluded with erythrocytes and platelets (Fig. 1). There were many erythrocytes in the connective tissue spaces, and a flocculent material which appeared to be plasma was observed (Fig. 1). At 5 min after injection the muscle cells looked normal, but at 30 min some had delta lesions. At 3 hr cells with delta lesions were common (Fig. 2), and some muscle cells had disorganized myofibrils (Fig. 3).

840

C. L OWNBY et al.

FIG. 3. LIGHT MIC'ROGRAI'H OF SKELETAL MUSCLE 3 HR AFTER INJECTION OF DIUTOXIN (0.03 /lg/g). M. normal muscle cells; note damaged skeletal muscle cells with disorganized myofibrils (arrows). (Bar"" 25 JIm.) FIG. 4. ELECTRON MICROGRAPH OF CAPILLARY 5 MIN AFTER INJECTION 01' BILlJOXIN (0.03 JIg/g). Note the large numbers of platelets occluding the lumen (P) some of which have lost their granules; note discontinuity of capillary wall (arrows). (Bar = 1 llm.)

Pathogenesis of Hemorrhage by BiJitoxin

FIG. 5.

ELECTRON MICROGRAPH OF PORTION OF A SKELETAL MUSCLE CELL TO SHOW DELTA LESION 30 MIN AFTER INJECTION OF IlILITOXIN (0.03 fl.g/g).

Arrows indicate extent of delta lesion; arrowhead indicates remnant of plasma membrane; CT, connective tissue of the endomysium; LE, lysed erythrocyte. (Bar = 111m.) FlO. 6. ELECTRON MICROGRAPH OF A PORTION OF A CAPILLARY I HR AFTER INJECTION OF I3JLITOXIN (0.03 j1.g/g). Note rupturc of endothelial cell betwecn arrows and also intact intcrcellular junction (J). (Bar = 1 j1.m.)

841

842

C. L. OWNBY et al.

Electron microscopic examination of the tissue showed extensive damage to blood vessels even at 5 min after injection of bilitoxin; Fig. 4 is representative of this type of damage. Damaged capillaries had ruptured endothelial cells, disrupted basal lamina, but intact intercellular junctions. The lumen of these damaged capillaries were occluded with aggregations of platelets, most of which had lost their granules except for some platelets near the center of the lumen. Fibrin was present both intravascularly and extravascularly. Blood vessels at 30 min after injection appeared similar to those at 5 min, but at 30 min some muscle cells were also damaged. Figure 5 is an electron micrograph of a muscle cell with a delta lesion showing lysis of the plasma membrane and the presence of a triangular area devoid of myofilaments beneath the membrane remnants. At the later time periods of 1, 3 and 24 hr, the changes in the blood vessels were essentially the same as described above except that there appeared to be more fibrin, both within the vessel walls and in the connective tissue. Vessels with the morphology of the one shown in Fig. 6 demonstrate that although the endothelial cells of the vessel are disrupted, the intercellular junctions remain intact. The increased amount of intravascular fibrin can be seen in Fig. 7 at 1 hr after injection. An increase in extravascular fibrin as well as disruption of the endothelial cell can be seen in Fig. 8 at 3 hr after injection. DISCUSSION

Bilitoxin induces hemorrhage per rhexis, and in this respect, it is similar to the hemorrhagic toxins from the venoms of C. atrox (OWNBY et al., 1978) and C. h. horridus (OWNBY and GEREN, 1987). Like these other hemorrhagic toxins, bilitoxin is a metalloenzyme which is capabl.e of hydrolyzing the oxidized B chain of insulin. Bilitoxin hydrolyzes several peptide bonds including Ala(l4)-Leu(15) which is also hydrolyzed by hemorrhagic proteinase IV (HP IV) from C. h. horridus venom, by hemorrhagic toxin c and hemorrhagic toxin d (isotoxins), hemorrhagic toxin e (HTe) and hemorrhagic toxin f from C. atrox venom and hemorrhagic toxins HT-l, HT-2 and HT-3 from C. r. ruber venom (MORI et al., 1987). The pathogenesis of hemorrhage induced by HP IV and HTe have been studied, and both cause hemorrhage per rhexis. Of the toxins isolated from venoms of snakes in the genus Agkistrodon, bilitoxin is chemically most similar to the Ac3-proteinase isolated from A. acutus venom (SUGIHARA et al., 1979; YAGIHASHI et al., 1986). Unfortunately, there are no data on the pathogenesis of hemorrhage induced by this toxin. Other hemorrhagic toxins isolated so far from venoms of snakes in this genus are from the venom of A. acutus (NIKAI et al., 1977; SUGIHARA et al., 1978, 1979; Xu et al., 1981; MORI et 01., 1984; ZHANG et al., 1984) and A. halys blomhoffii (OMORI et al., 1964; OSHIMA et 01., 1968a,b, 1972; NIKAI et al., 1986), but again, there are no data on the pathogenesis of hemorrhage induced by any of these toxins. Bilitoxin also resembles bothropasin, a hemorrhagic toxin isolated from the venom of Bothrops jararaca. Bothropasin has a mol. wt of 48,000 (MANDELBAUM et al., 1982), the same as bilitoxin, and it hydrolyzes four of the same peptide bonds of the oxidized B chain of insulin. Again, there are no data on the pathogenesis of hemorrhage induced by bothropasin. If information on the pathogenesis of hemorrhage is taken only from reports in which the homogeneity of the toxin is not in question, we are left with only two investigations, i.e. that of OWNBY et al. (1978) concerning three hemorrhagic toxins (HTa, HTb and HTe) from C. atrox venom and that of OWNBY and GEREN (1987) concerning hemorrhagic proteinase IV (HPIV) from C. h./lOrridus venom. In both of these studies, the patho-

843

Pathogenesis of Hemorrhage by Bilitoxin

FIG.

7.

ELECTRON MICROGRAPH OF CAPILLARY

1 HR

AFTER INJECTION OF HlLlTOXIN

(0.03 jJ.gjg).

M, portion of skeletal muscle cell; CT, connective tissue of the endomysium; note lack of continuity of the capillary wall with only a small piece of the endothelial cell remaining intact (arrow). Also note presence of large amounts of fibrin (F). (Bar = 1 JIm.) FIG.

8.

ELECTRON MICROGRAPH

or

A PORTION

or

A CAPILLARY

(0.03 JLg/g).

3 HR

AFTER INJECTION

or

BILlTOXIN

M, portion of a skeletal muscle cell; note discontinuity of endothelial eell (arrows) and large amount of fibrin (F) in the connective tissue around the capillary. (Bar = I pm.)

844

C.

L. OWNBY et al.

genesis of hemorrhage was determined to be per rhexis in which endothelial cells of affected capillaries undergo changes such as swelling and blebbing which eventually lead to the destruction of the cell integrity. There is also evidence for disruption of the basal lamina beneath the capillary endothelium. In all cases, platelet aggregations within damaged vessels were observed, and in the case of HP N, intravascular and extravascular fibrin deposition was also observed. The pathogenesis of hemorrhage induced by bilitoxin is basically the same as reported for these other toxins. The exact biochemical mechanism of hemorrhage per rhexis induced by these toxins is still not clear. One possibility is that these toxins hydrolyze the basal lamina resulting in lysis of the endothelium as has been suggested by OHSAKA (1979) and BJARNASON and Fox (1988). There is no question that they are capable of hydrolyzing basal lamina proteins (BJARNASON and Fox, 1988) in vitro, but this does not mean that they do this in vivo nor does it exclude the possibility that they could also act directly on the endothelial cells at the level of the plasma membrane. There is evidence from electron microscopic studies that these toxins act on endothelial cells to disrupt volume control and hydrolyze the plasma membrane (OWNBY et at., 1978; OWNBY and GEREN, 1987). Another question which needs to be answered is just how much of the basal lamina must be destroyed in order for the endothelium to lose its integrity. In the previous studies on purified hemorrhagic toxins, even severely damaged capillaries had large portions of their basal lamina intact. In studies on other epithelia, even after complete removal of the basal lamina with elastase, the naked epithelial cells remain connected, were viable and still formed a tube (KOEFOED, 1987). In a separate study, after digestion of the basal lamina, the epithelial cells were dissociated but still viable (LEVINSON and BRADLEY, 1984). Several hemorrhagic toxins also cause myonecrosis in addition to hemorrhage. In tissue taken 3 hr after the injection of HTb from C. atrox venom, there were necrotic skeletal muscle cells (OWNBY et al., 1978). Viriditoxin, isolated from the venom of the prairie rattlesnake (c. v. viridis), was reported to cause hemorrhage as well as myonecrosis (FABIANO and Tu, 1981). Additional work on viriditoxin by GLEASON et al. (1983) showed that myonecrosis did not occur until 12 hr after the injection of the toxin, and these investigators concluded that myonecrosis was a secondary consequence of ischemia. KOMORI and SUGIHARA (1988) isolated a hemorrhagic factor from the venom of the aspic viper (Vipera aspis aspis) which caused an increase in creatine kinase levels 30 min after Lm. injection and produced degeneration of skeletal muscle cells 48 hr after injection. The increase in creatine kinase levels does indicate damage to muscle cells as early as 30 min after injection, but the histological data indicate muscle damage at 48 hr but not at 1 hr. Bilitoxin also causes myonecrosis, but apparently much more rapidly. There were delta lesions in skeletal muscle cells within 30 min after injection, indicating a very rapid lytic effect on the cells. Delta lesions were also present in some cells 3 hr after injection, but other muscle cells were in a more advanced pathologic state in which the cells consisted of an amorphous mass surrounded and invaded by phagocytic cells. The presence of cells in this more advanced state of necrosis indicates that the initial injury might have occurred much earlier than 3 hr. This is supported by the work of OWNBY and COLBERG (1988) which showed that cells initially in different pathologic states all reach a common pathologic state of necrosis. In the cases of HTb and viriditoxin, myonecrosis might be explained as a result of ischemic conditions induced by extensive hemorrhage in the tissue, but with bilitoxin, it seems that the myonecrosis must be a direct effect of the toxin on the muscle cells since there were delta lesions within 30 min after injection before hemorrhage could lead to ischemic conditions in the tissue.

Pathogenesis of Hemorrhage by Bilitoxin

845

AcknolVledgements-The authors thank DENISE REx and JANICE PENNINGTON of the OSU Electron Microscope Laboratory for processing and sectioning the tissue; and ED JOHNSON, TERRY COLBERO and SANDRA REISBECK for critically reading the manuscript. This study was supported by Public Health Service Grant 5 ROI AI26923 from the National Institute of AUergy and Infectious Diseases. CLO was the recipient of a Research Career Development Award (KG4 AlOO474) from this Institute during this study. This is publication number 89-Q28 from the College of Veterinary Medicine, Oklahoma State University.

REFERENCES BJARNASON, J. B. and Fox, J. W. (1988) Hemorrhagic toxins from snake venoms. Toxin Rev. 7, 121-209. FABIANO, R. J. and Tu, A. T. (1981) Purification and biochemical study of viriditoxin, tissue damaging toxin, from prairie rattlesnake venom. Biochemistry 20, 21-27. GLEASON, M. L., ODELL, G. V. and OWNBY, C. L. (\983) Isolation and biological activity of viriditoxin and a viriditoxin variant from Crotalus viridis viridis venoms. Toxin Rev. 2, 235-265. IMAI, K., NIKAI, T., SUGIHARA, H. and OWNBY, C. L. (1989) Hemorrhagic toxin from the venom of Agkistrodoll bilineatus (common cantil). Int. J. Biochem. 21, 667-673. KoEFoED, B. M. (1987) The ability of an epithelium to survive removal of the basal lamina by enzymes: fine structure and content of sodium and potassium of the midgut epithelium of the larva of Tenebrio molitor after withdrawal of the basal lamina by elastase. Tissue and Cell 19, 65-70. KOMORI, Y. and SUGIHARA, H. (1988) Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera asp is aspis (aspic viper). Int. J. Biochem. 20, 1417-1423. LEVINSON, G. and BRADLEY, T. J. (1984) Removal of insect basal laminae using elastase. Tissue and Cell 16, 367-375. MANDELBAUM, F. R., REICHEL, A. P. and ASSAKURA, T. (1982) Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca Uararaca). TOXICOIl 20, 955-972. MoRI, N., NIKAI, T. and SUGIHARA, H. (1984) Purification of proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon acutlts). Toxicoll 22, 451-461. MoRI, N., NIKAI, T., SUGIHARA, H. and Tu, A. T. (1987) Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom. Arch. Biochem. Biophys. 253, 108-121. NIKAI, T., GOURI, E., KISHlDA, M., SUGIHARA, H., MoRI, N. and Tu, A. T. (1986) Reevaluation of hemorrhagic toxin, HR-!, from Agkistrodon halys blomhoffii venom: proof of proteolytic enzyme. Int. J. Biochem. 18, !O3-108. NIKAI, T., SUGIHARA, H. and TANAKA, T. (1977) Enzymochemical studies on snake venoms. II. Purification of lethal protein ACI-proteinase in the venom of Agkis/rodon acutus. J. Pharm. Soc. Japan 97, 507-514. OHSAKA, A. (1979) Hemorrhagic, necrotizing and edema-forming effects of snake venoms. In: Handbook of Experimental Pharmacology, pp. 480-546 (BORN, G., FARAH A., HERKEN, H. and WELCH, A. D., Eds). Berlin: Springer. OMORI, T., IWANAGA, S. and SUZUKI, T. (l964) The relationship between the hemorrhagic and lethal activities of Japanese Mamushi (Agkistrodon halys blomhoffiO venom. Toxicoll 2, 1-4. OSHIMA, G., IWANAGA, S. and SUZUKI, T. (1968a) Studies on snake venoms. XVIII. An improved method for purification of the proteinase b from the venom of Agkistrodon halys blomhoffii and its physiochemical properties. J. Biochem. 64, 215-225. OSHIMA, G., MATSUO, Y., FWANAGA, S. and SUZUKI, T. (l968b) Studies on snake venoms. XIX. Purification and some physiochemical propcrties of proteinase a and c from the venom of Agkislrodon halys hlomhoffii. J. Biochem. 64, 227-238. OSHIMA, G., OMORI-SATOH, T., IWANAGA, S. and SUZUKI, T. (1972) Studies on snake venom hemorrhagic factor I (HR-I) in the venom of Agkistrodon halys blomhoffii. Its purification and biological properties. J. Biochem. 72, 1483-1494. OWNBY, C. L. (1982) Pathology of rattlesnake envenomation. In: Rattlesnake Venoms: Their Actions and Treatment, pp. 163-209 (Tu, A. T, Ed.). New York: Marcel Dekker. OWNBY, C. L. (1990) Locally acting agents: myotoxins, hemorrhagic toxins and dermonecrotic factors. In: Handbook on Toxinology (MEBS, D. and SHIER, T., Eds). New York: Marcel Dekker (In Press). OWNBY, C. L. and COLBERG, T. R. (1988) Classification of myonecrosis induced by snake venoms: venoms from the prairie rattlesnake (Crotalus viridis viridis), western diamondback rattlesnake (Crotalus atrox) and the Tndian cobra (Naja lloJa naja). Toxicon 26,459-474. OWNBY, C. L. and GEREN, C. R. (1987) Pathogenesis of hemorrhage induced by hemorrhagic proteinase TV from timber rattlesnake (Crotalus horridus horridus) venom. Toxicon 25, 517-526. OWNBY, C. L., BJARNASON, J. and Tu, A. T. (1978) Hemorrhagic toxins from rattlesnake (Crotalus atrox) venom. Am. J. Pathol. 93, 201-210. SUGIHARA, H., NIKAI, T., KAWAGUSHI, T. and TANAKA, T. (1978) Enzymochemieal studies on snake venoms. TV. Purification of lethal protein Ac2-proteinase in the venom of Agkistrodon aeu/lls. J. Pharm. Soc. Japall 98, 1523-1529.

846

C. L.

OWNBY el 01.

H., NIKAI, T., UMEDA, H. and TANAKA, T. (1979) Enzymochemical studies on snake venoms. V. Purification of lethal protein Ac3-proteinase in the venom of Agkislrodoll aell/us. J. Pharm. Soc. Japan 99, 1161-1167. Xu, X., WANG, C., LIU, J. and Lu, Z. (1981) Purification and characterization of hemorrhagic components from Agkistrodon OCUlUS (hundred pace snake) venom. Toxicon 19, 633-644. YAGlHASHl, S., NlKAI, T., MaRl, N., KISHHIDA, M. and SUGIHARA, H. (1986) Characterization of Ac3-proteinase from the venom of Agkistrodon OCUlIlS (hundred pace snake). Int. J. Biochem. 18, 885-892. ZHANG, J., CHEN, Z., HE, Y. and Xu, X. (1984) Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodoll aeutlls) venom. Toxicoll 22, 931-935. SUGIHARA,

Pathogenesis of hemorrhage induced by bilitoxin, a hemorrhagic toxin isolated from the venom of the common cantil (Agkistrodon bilineatus bilineatus).

The pathogenesis of hemorrhage induced by the i.m. injection of the hemorrhagic toxin, bilitoxin, was studied using light and electron microscopy. Whi...
4MB Sizes 0 Downloads 0 Views