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The spurting of erythrocytes through junctions of the vascular endothelium treated with snake venom
MICROVASCULAR
RESEARCH,
(1975)
10,208-213
BRIEF COMMUNICATIONS The Spurting of Erythrocytes Through Vascular Endothelium Treated with A.
K. SUZUKI,
OHSAKA,
AND
Junctions of the Snake Venom
M. OHASHI
The 2nd Department of Bacteriology, Department of Chemistry, and the 1st Department of Bacteriology, National Institute of Health, Shinagawa-ku, Tokyo 141, Japan Received February &I975 The vascular endothelium treated with the venom of Trimeresurus flavoviridis, a crotalid, was investigated by electron microscopy. The results demonstrated that the erythrocytes, changing their shape as does the amoeba, spurt through opened junctions of the endothelial cell lining, the adjacent basement membrane being disrupted to permit eventual extravasation of the erythrocytes. A small number of platelets gathers at the site of hemorrhage but they show no signzof viscous metamorphosis. These results, together with our recent findings that highly purified venom hemorrhagic principles can disrupt the isolated basement membrane in an enzymatic mode of action and also can inhibit the in vitro aggregation of platelets, do allow us to visualize the physiological mechanisms involved in hemorrhage.
INTRODUCTION Hemorrhage is one of the most striking manifestations following envenomation from bite by crotalid and viperid snakes (Boquet, 1964; Ohsaka et al., 1966; JimenezPorras, 1968). In 1970 we succeeded in isolating two immunologically distinct hemorrhagic principles, HRl and HR2 (a and b), from the venom of TrimeresurusJlavoviridis, a crotalid (Omori-Satoh and Ohsaka, 1970; Takahashi and Ohsaka, 1970; Ohsaka et al., 1971b). By using these venom hemorrhagic principles and also crude venom as an analytical tool, we have attempted an experimental approach to elucidating the physiological mechanisms involved in hemorrhage (Ohsaka et al., 1971a; Ohsaka, Just, and Habermann, 1973a,b; Ohsaka, 1973; Tsuchiya et al., 1974; Yamanaka et al., 1974; Ohsaka, 1975; Ishida et al., 1975). Cinematographic recording revealed that the erythrocytes ooze out one by one through the pinpoint holes formed in true capillaries exposed to venom, but not from along the capillaries (Ohsaka et al., 1971a, Tsuchiya et al., 1974). For hemorrhage to occur, the erythrocytes must cross both the endothelial cell lining and the basement membrane which constitute the vessel wall (Ohsaka et al., 1971a; Ohsaka, 1973; Ohsaka, 1975). Recently we demonstrated the ability of the venom hemorrhagic principles to disrupt the isolated basement membrane by an enzymatic mode of action, releasing
proteins
and carbohydrates
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
(Ohsaka 208
et al., 1973a,b).
Electron
microscopic
BRIEF
COMMUNICATIONS
209
examination done by us of the vascular endothelial cells treated with venom suggested that the erythrocytes spurt through a junction of the lining endothelial cells (Tsuchiya et al., 1974). This paper presents the electron microscopic observations demonstrating the spurting of erythrocytes through opened junctions of the vascular endothelium exposed to snake venom. MATERIALS
AND METHODS
Male rats of Wistar strain, weighing approximately 150 g, were anesthetized by intraperitoneal injection with sodium pentobarbital in a dose of 50 mg/kg body wt. A middle incision was made on a shaved portion of abdominal skin, and the mesentery was pulled out of the peritoneal cavity onto a glass plate. To keep it humid, the tissue was partially covered with pieces of cotton soaked with a 1% gelatin-Ringer solution. A 0.25 % venom (Batch No. 64A) in a 1% gelatin-Ringer solution was topically applied to the mesentery under light microscopic observation. After 4-5 min when hemorrhage began to occur, the mesenteric tissue was fixed in situ by dripping 1 % 0~0, in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature onto the tissue for 3 min. The tissue was excised and cut into small pieces and then fixed for 4.5 hr in the above fixative at 4”. After fixation, the pieces were dehydrated in ascending concentrations of ethanol and propylene oxide and were embedded in Epon (Luft, 1961). Fine sections, approximately 400-700 A thick were cut on a LKB 8800 ultramicrotome and collected on copper grids. The sections were double stained first with an aqueous solution of 2% uranyl acetate for 1.5 hr and then with 0.2 ‘A lead citrate for 10 min. Grids were examined in a Hitachi HUl I-B electron microscope at 75 kV. RESULTS
AND
DISCUSSION
It is well known that the vessel wall of the muscle capillary consists of an endothelial cell lining and the basement membrane surrounding it (Fawcett, 1963). For hemorrhage to occur, the erythrocytes must cross these two barriers. Figure la shows an electron micrograph of the vascular endothelial cells of the rat’s mesentery treated with TrimeresurusJlavoviridis venom. An erythrocyte in the lumen is just penetrating into a junction (about 0.2 pm wide) by forming a protrusion like a pseudopodium of the amoeba; the adjacent basement membrane has partially been disrupted. Figure lb shows an electron micrograph of another section from the same tissue. An erythrocyte is spurting out from the vessel through an opened junction (about 0.7 pm wide) and the basement membrane has extensively been destroyed. From these observations we conclude that the erythrocyte, changing its shape as does the amoeba, spurts through an opened junction of the endothelial cell lining, the adjacent basement membrane being disrupted to permit the eventual extravasation of erythrocytes. Our observations are different from those of McKay et al. (1970) that the erthrocytes traverse the cytoplasm of the endothelial cells. While we examined the vascular endothelial cells of the rat’s mesentery directly exposed to crotalid venom, McKay et al. (1970) injected Vipera palestime venom hemorrhagin subcutaneously into rabbit and
210
BRIEF
COMMUNICATIONS
z c-4
‘s!soyd.romajaw sno+h aq$ o) padola~ap )ou s! (7) uawnl m1nXeh aq~ u! (a) )a[ale[d aye ‘u!eZv .paA‘o.wap i((aaya]xa s! (~8) auwq -urau~ JuaLLIaseqaq) : (LKJ) sl[X pz[aq)opua ay) 30 (f) UOyXUI[ pCmd0 u1?q%IO.IqJ [asah aq$ u10.13In0 8ugmds s! (2) alhOJq$bJa uv ‘LLIOU~A ayXS 0) amsodxa lal3v U!UI s ‘[assaA poolq ya)uasam B 30 uoyas pmpn@kro~ ‘q 1 ‘+X-J .s!soqdlouwlaur snoX!a aq) 01 padoIaAap IOU s! (6) )a[aw[d aqL .paldnls!p bIpz!$Jed SF (~8) aumqwaur luamassq $uaMpo aq$ :~qaorue aql30 wmpodopnasd E ay!g uo!snl]old e %!LII.IO~ dq (~3) slIa3 Iyaqlopua aql30 (1) uo!mm[ t? oy %U!W.IJ -auad lsnf s! (7) uaumf .ryn3st?A aql II! (a) alho.rqln’la uv ‘cuouaA ayr?us 01 amsodxa lal3e U!LLIg ‘[ass% poolq g!.rawasaw B 30 uo!vas ~xI!~~I~!~uo~ ‘e[ .ord
212
BRIEF
COMMUNICATIONS
investigated the vascular effects at the site of injection. It is possible, therefore, that the discrepancy may be due to the different experimental models used by both groups. It remains to be answered how the junction opens. In this connection, however, it is worth mentioning the findings of Ishida et al. (1975) that the purified hemorrhagic principles induce release of mediators such as histamine, serotonin, and some other substances from various isolated organs and tissues. Such mediators, if released in uivo, may well open the junction of the endothelial cells to facilitate spurting of the erythrocytes across the endothelium (Ohsaka, 1973; Ohsaka, 1975). Figure la and lb also indicate that a small number of platelets gathered at the site of hemorrhage but they showed no sign of viscous metamorphosis, in exact accordance with our previous findings (Tsuchiya et al., 1974). These results, together with the reported ability of the venom hemorrhagic principles to inhibit the in vitro aggregation of platelets (Yamanaka et al., 1974), well explain the seldom formation of white thrombus (platelet aggregate) at the site of venom-induced hemorrhage (Ohsaka et al., 1971a; Tsuchiya et al., 1974). The present results, together with our previous findings (Ohsaka et al., 1971a; Ohsaka, 1973; Ohsaka et al., 1973a,b; Tsuchiya et al., 1974; Yamanaka et al., 1974; Ohsaka, 1975; Ishida et al., 1975), do allow us to visualize the physiological mechanisms involved in hemorrhage. The mechanism of hemorrhage as revealed by the use of venom hemorrhagic principle has recently been reviewed (Ohsaka, 1973 ; Ohsaka, 1975). REFERENCES BOQUET, P. (1964). Venins de serpents (lere Partie). Physio-pathologie de l’envenimation et proprietes biologiques des venins. T&con 2, 541. FAWCE~, D. W. (1963). Comparative observations on the fine structure of blood capillaries. In “The Peripheral Blood Vessels” (J. L. Orbison and D. E. Smith, eds.), pp. 17-44. Williams and Wilkins, Baltimore. ISHIDA, Y., YAMASHITA, S., OHSAKA, A., TAKAHASHI, T., AND OMORI-SATOH, T. (1975). Pharmacological studies of the hemorrhagic principles isolated from the venom of Trimeresurusflauoviridis, a crotalid. In “Animal, Plant and Microbial Toxins” (A. OHSAKA, K. HAYASHI AND Y. SAWAI, eds.), Plenum Publishing Company, London, in press. JIM~NEZ-PORRAS, J. M. (1968). Pharmacology of peptides and proteins in snake venoms. Ann. Rev. Pharmacol. 8,299-318. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MCKAY, D. G., MOROZ, C., DE VRIES, A., CSAVOSSY, I., AND CRUSE, V. (1970). The action of hemorrhagin and phospholipase derived from Viperapalestinae venom on the microcirculation. Lab. Invest. 22,387-399. OHSAKA, A. (1973). An approach to the mechanism of hemorrhage: Hemorrhagic principles isolated from snake venom as a useful analytical tool (text in Japanese). Seitui no Kaguku (Life Science) 24, 26&293. OHSAKA, A. (1975). An approach to the physiological mechanisms involved in hemorrhage: Snake venom hemorrhagic principles as a useful analytical tool. In “Animal, Plant and Microbial Toxins” (A. OHSAKA, K. HAYASHI, AND Y. SAWAI, eds.), Plenum Publishing Company, London, in press. OHSAKA, A., OMORI-SATOH, T., KONDO, H., KONDO, S., AND MURATA, R. (1966). Biochemical and pathological aspects of hemorrhagic principles in snake venom with special reference to Habu (TrimeresurusJlavoviridis) venom. Mem. Inst. Butantan 33, 193-205. OHSAKA, A., OHASHI, M., TSUCHIYA, M., KAMISAKA, Y., AND FUJISHIRO, Y. (1971a). Action of Trimeresurusflauouiridis venom on the microcirculatory system of rat; dynamic aspects as revealed by cinephotomicrographic recording. Japan. J. Med. Sci. Biol. 24, 34-39.
BRIEF
COMMUNICATIONS
213
A., TAKAHASHI, T., OMORI-SATOH, T., AND MURATA, R. (1971 b). Purification and characterization of the hemorrhagic principles in the venom of Trimeresurusflavoviridis. In “Toxins of Animal and Plant Origin” (A. de Vries and E. Kochva, eds.), Vol. 1, pp. 369-399. Gordon and Breach, London. OHSAKA, A., JUST, M., AND HABERMANN, E. (1973a). Action of snake venom hemorrhagic principles on isolated glomerular basement membrane. Biochim. Biophys. Acta 323,415428. OHSAKA, A., JUST, M., AND HABERMANN, E. (1973b). Action of snake venom hemorrhagic principles on isolated glomerular basement membrane. In “Animal and Plant Toxins” (E. Kaiser, ed.), pp. 93-97. Wilhelm Goldmann Verlag, Miinchen. OMORI-SATOH, T., AND OHSAKA, A. (1970). Purification and some properties of hemorrhagic principle 1 in the venom of Trimeresurusf7avoviridis. Biochim. Biophys. Acta 207,432L444. TAKAHASHI, T., AND OHSAKA, A. (1970). Purification and some properties of two hemorrhagic principles (HR2a and HR2b) in the venom of Trimeresurusjfavoviridis; complete separation of the principles from proteolytic activity. Biochim. Biophys. Acta 207,65-75. TSUCHIYA, M., OHSHIO, C., OHASHI, M., OHSAKA, A., SUZUKI, K., AND FUIISHIRO, Y. (1974). Cinemarographic and electron microscopic analyses of the hemorrhage induced by the main hemorrhagic principle, HRl, isolated from the venom of Trimeresurusflavoviridis. In “Platelets, Thrombosis, and Inhibitors” (P. Didisheim, T. Shimamoto, and H. Yamazaki, eds.), pp. 43946. F. K. Schattauer Verlag, Stuttgart. YAMANAKA, M., MATSUDA, M., ISOBE, J., OHSAKA, A., TAKAHASHI, T., AND OMORI-SATOH, T. (1974). Effect of purified hemorrhagic principles from Habu snake venom on platelet aggregation. In “Platelets, Thrombosis, and Inhibitors” (P. Didisheim, T. Shimamoto, and H. Yamazaki, eds.), pp. 335-344. F. K. Schattauer Verlag, Stuttgart. OHSAKA,
RESEARCH,
(1975)
10,208-213
BRIEF COMMUNICATIONS The Spurting of Erythrocytes Through Vascular Endothelium Treated with A.
K. SUZUKI,
OHSAKA,
AND
Junctions of the Snake Venom
M. OHASHI
The 2nd Department of Bacteriology, Department of Chemistry, and the 1st Department of Bacteriology, National Institute of Health, Shinagawa-ku, Tokyo 141, Japan Received February &I975 The vascular endothelium treated with the venom of Trimeresurus flavoviridis, a crotalid, was investigated by electron microscopy. The results demonstrated that the erythrocytes, changing their shape as does the amoeba, spurt through opened junctions of the endothelial cell lining, the adjacent basement membrane being disrupted to permit eventual extravasation of the erythrocytes. A small number of platelets gathers at the site of hemorrhage but they show no signzof viscous metamorphosis. These results, together with our recent findings that highly purified venom hemorrhagic principles can disrupt the isolated basement membrane in an enzymatic mode of action and also can inhibit the in vitro aggregation of platelets, do allow us to visualize the physiological mechanisms involved in hemorrhage.
INTRODUCTION Hemorrhage is one of the most striking manifestations following envenomation from bite by crotalid and viperid snakes (Boquet, 1964; Ohsaka et al., 1966; JimenezPorras, 1968). In 1970 we succeeded in isolating two immunologically distinct hemorrhagic principles, HRl and HR2 (a and b), from the venom of TrimeresurusJlavoviridis, a crotalid (Omori-Satoh and Ohsaka, 1970; Takahashi and Ohsaka, 1970; Ohsaka et al., 1971b). By using these venom hemorrhagic principles and also crude venom as an analytical tool, we have attempted an experimental approach to elucidating the physiological mechanisms involved in hemorrhage (Ohsaka et al., 1971a; Ohsaka, Just, and Habermann, 1973a,b; Ohsaka, 1973; Tsuchiya et al., 1974; Yamanaka et al., 1974; Ohsaka, 1975; Ishida et al., 1975). Cinematographic recording revealed that the erythrocytes ooze out one by one through the pinpoint holes formed in true capillaries exposed to venom, but not from along the capillaries (Ohsaka et al., 1971a, Tsuchiya et al., 1974). For hemorrhage to occur, the erythrocytes must cross both the endothelial cell lining and the basement membrane which constitute the vessel wall (Ohsaka et al., 1971a; Ohsaka, 1973; Ohsaka, 1975). Recently we demonstrated the ability of the venom hemorrhagic principles to disrupt the isolated basement membrane by an enzymatic mode of action, releasing
proteins
and carbohydrates
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
(Ohsaka 208
et al., 1973a,b).
Electron
microscopic
BRIEF
COMMUNICATIONS
209
examination done by us of the vascular endothelial cells treated with venom suggested that the erythrocytes spurt through a junction of the lining endothelial cells (Tsuchiya et al., 1974). This paper presents the electron microscopic observations demonstrating the spurting of erythrocytes through opened junctions of the vascular endothelium exposed to snake venom. MATERIALS
AND METHODS
Male rats of Wistar strain, weighing approximately 150 g, were anesthetized by intraperitoneal injection with sodium pentobarbital in a dose of 50 mg/kg body wt. A middle incision was made on a shaved portion of abdominal skin, and the mesentery was pulled out of the peritoneal cavity onto a glass plate. To keep it humid, the tissue was partially covered with pieces of cotton soaked with a 1% gelatin-Ringer solution. A 0.25 % venom (Batch No. 64A) in a 1% gelatin-Ringer solution was topically applied to the mesentery under light microscopic observation. After 4-5 min when hemorrhage began to occur, the mesenteric tissue was fixed in situ by dripping 1 % 0~0, in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature onto the tissue for 3 min. The tissue was excised and cut into small pieces and then fixed for 4.5 hr in the above fixative at 4”. After fixation, the pieces were dehydrated in ascending concentrations of ethanol and propylene oxide and were embedded in Epon (Luft, 1961). Fine sections, approximately 400-700 A thick were cut on a LKB 8800 ultramicrotome and collected on copper grids. The sections were double stained first with an aqueous solution of 2% uranyl acetate for 1.5 hr and then with 0.2 ‘A lead citrate for 10 min. Grids were examined in a Hitachi HUl I-B electron microscope at 75 kV. RESULTS
AND
DISCUSSION
It is well known that the vessel wall of the muscle capillary consists of an endothelial cell lining and the basement membrane surrounding it (Fawcett, 1963). For hemorrhage to occur, the erythrocytes must cross these two barriers. Figure la shows an electron micrograph of the vascular endothelial cells of the rat’s mesentery treated with TrimeresurusJlavoviridis venom. An erythrocyte in the lumen is just penetrating into a junction (about 0.2 pm wide) by forming a protrusion like a pseudopodium of the amoeba; the adjacent basement membrane has partially been disrupted. Figure lb shows an electron micrograph of another section from the same tissue. An erythrocyte is spurting out from the vessel through an opened junction (about 0.7 pm wide) and the basement membrane has extensively been destroyed. From these observations we conclude that the erythrocyte, changing its shape as does the amoeba, spurts through an opened junction of the endothelial cell lining, the adjacent basement membrane being disrupted to permit the eventual extravasation of erythrocytes. Our observations are different from those of McKay et al. (1970) that the erthrocytes traverse the cytoplasm of the endothelial cells. While we examined the vascular endothelial cells of the rat’s mesentery directly exposed to crotalid venom, McKay et al. (1970) injected Vipera palestime venom hemorrhagin subcutaneously into rabbit and
210
BRIEF
COMMUNICATIONS
z c-4
‘s!soyd.romajaw sno+h aq$ o) padola~ap )ou s! (7) uawnl m1nXeh aq~ u! (a) )a[ale[d aye ‘u!eZv .paA‘o.wap i((aaya]xa s! (~8) auwq -urau~ JuaLLIaseqaq) : (LKJ) sl[X pz[aq)opua ay) 30 (f) UOyXUI[ pCmd0 u1?q%IO.IqJ [asah aq$ u10.13In0 8ugmds s! (2) alhOJq$bJa uv ‘LLIOU~A ayXS 0) amsodxa lal3v U!UI s ‘[assaA poolq ya)uasam B 30 uoyas pmpn@kro~ ‘q 1 ‘+X-J .s!soqdlouwlaur snoX!a aq) 01 padoIaAap IOU s! (6) )a[aw[d aqL .paldnls!p bIpz!$Jed SF (~8) aumqwaur luamassq $uaMpo aq$ :~qaorue aql30 wmpodopnasd E ay!g uo!snl]old e %!LII.IO~ dq (~3) slIa3 Iyaqlopua aql30 (1) uo!mm[ t? oy %U!W.IJ -auad lsnf s! (7) uaumf .ryn3st?A aql II! (a) alho.rqln’la uv ‘cuouaA ayr?us 01 amsodxa lal3e U!LLIg ‘[ass% poolq g!.rawasaw B 30 uo!vas ~xI!~~I~!~uo~ ‘e[ .ord
212
BRIEF
COMMUNICATIONS
investigated the vascular effects at the site of injection. It is possible, therefore, that the discrepancy may be due to the different experimental models used by both groups. It remains to be answered how the junction opens. In this connection, however, it is worth mentioning the findings of Ishida et al. (1975) that the purified hemorrhagic principles induce release of mediators such as histamine, serotonin, and some other substances from various isolated organs and tissues. Such mediators, if released in uivo, may well open the junction of the endothelial cells to facilitate spurting of the erythrocytes across the endothelium (Ohsaka, 1973; Ohsaka, 1975). Figure la and lb also indicate that a small number of platelets gathered at the site of hemorrhage but they showed no sign of viscous metamorphosis, in exact accordance with our previous findings (Tsuchiya et al., 1974). These results, together with the reported ability of the venom hemorrhagic principles to inhibit the in vitro aggregation of platelets (Yamanaka et al., 1974), well explain the seldom formation of white thrombus (platelet aggregate) at the site of venom-induced hemorrhage (Ohsaka et al., 1971a; Tsuchiya et al., 1974). The present results, together with our previous findings (Ohsaka et al., 1971a; Ohsaka, 1973; Ohsaka et al., 1973a,b; Tsuchiya et al., 1974; Yamanaka et al., 1974; Ohsaka, 1975; Ishida et al., 1975), do allow us to visualize the physiological mechanisms involved in hemorrhage. The mechanism of hemorrhage as revealed by the use of venom hemorrhagic principle has recently been reviewed (Ohsaka, 1973 ; Ohsaka, 1975). REFERENCES BOQUET, P. (1964). Venins de serpents (lere Partie). Physio-pathologie de l’envenimation et proprietes biologiques des venins. T&con 2, 541. FAWCE~, D. W. (1963). Comparative observations on the fine structure of blood capillaries. In “The Peripheral Blood Vessels” (J. L. Orbison and D. E. Smith, eds.), pp. 17-44. Williams and Wilkins, Baltimore. ISHIDA, Y., YAMASHITA, S., OHSAKA, A., TAKAHASHI, T., AND OMORI-SATOH, T. (1975). Pharmacological studies of the hemorrhagic principles isolated from the venom of Trimeresurusflauoviridis, a crotalid. In “Animal, Plant and Microbial Toxins” (A. OHSAKA, K. HAYASHI AND Y. SAWAI, eds.), Plenum Publishing Company, London, in press. JIM~NEZ-PORRAS, J. M. (1968). Pharmacology of peptides and proteins in snake venoms. Ann. Rev. Pharmacol. 8,299-318. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MCKAY, D. G., MOROZ, C., DE VRIES, A., CSAVOSSY, I., AND CRUSE, V. (1970). The action of hemorrhagin and phospholipase derived from Viperapalestinae venom on the microcirculation. Lab. Invest. 22,387-399. OHSAKA, A. (1973). An approach to the mechanism of hemorrhage: Hemorrhagic principles isolated from snake venom as a useful analytical tool (text in Japanese). Seitui no Kaguku (Life Science) 24, 26&293. OHSAKA, A. (1975). An approach to the physiological mechanisms involved in hemorrhage: Snake venom hemorrhagic principles as a useful analytical tool. In “Animal, Plant and Microbial Toxins” (A. OHSAKA, K. HAYASHI, AND Y. SAWAI, eds.), Plenum Publishing Company, London, in press. OHSAKA, A., OMORI-SATOH, T., KONDO, H., KONDO, S., AND MURATA, R. (1966). Biochemical and pathological aspects of hemorrhagic principles in snake venom with special reference to Habu (TrimeresurusJlavoviridis) venom. Mem. Inst. Butantan 33, 193-205. OHSAKA, A., OHASHI, M., TSUCHIYA, M., KAMISAKA, Y., AND FUJISHIRO, Y. (1971a). Action of Trimeresurusflauouiridis venom on the microcirculatory system of rat; dynamic aspects as revealed by cinephotomicrographic recording. Japan. J. Med. Sci. Biol. 24, 34-39.
BRIEF
COMMUNICATIONS
213
A., TAKAHASHI, T., OMORI-SATOH, T., AND MURATA, R. (1971 b). Purification and characterization of the hemorrhagic principles in the venom of Trimeresurusflavoviridis. In “Toxins of Animal and Plant Origin” (A. de Vries and E. Kochva, eds.), Vol. 1, pp. 369-399. Gordon and Breach, London. OHSAKA, A., JUST, M., AND HABERMANN, E. (1973a). Action of snake venom hemorrhagic principles on isolated glomerular basement membrane. Biochim. Biophys. Acta 323,415428. OHSAKA, A., JUST, M., AND HABERMANN, E. (1973b). Action of snake venom hemorrhagic principles on isolated glomerular basement membrane. In “Animal and Plant Toxins” (E. Kaiser, ed.), pp. 93-97. Wilhelm Goldmann Verlag, Miinchen. OMORI-SATOH, T., AND OHSAKA, A. (1970). Purification and some properties of hemorrhagic principle 1 in the venom of Trimeresurusf7avoviridis. Biochim. Biophys. Acta 207,432L444. TAKAHASHI, T., AND OHSAKA, A. (1970). Purification and some properties of two hemorrhagic principles (HR2a and HR2b) in the venom of Trimeresurusjfavoviridis; complete separation of the principles from proteolytic activity. Biochim. Biophys. Acta 207,65-75. TSUCHIYA, M., OHSHIO, C., OHASHI, M., OHSAKA, A., SUZUKI, K., AND FUIISHIRO, Y. (1974). Cinemarographic and electron microscopic analyses of the hemorrhage induced by the main hemorrhagic principle, HRl, isolated from the venom of Trimeresurusflavoviridis. In “Platelets, Thrombosis, and Inhibitors” (P. Didisheim, T. Shimamoto, and H. Yamazaki, eds.), pp. 43946. F. K. Schattauer Verlag, Stuttgart. YAMANAKA, M., MATSUDA, M., ISOBE, J., OHSAKA, A., TAKAHASHI, T., AND OMORI-SATOH, T. (1974). Effect of purified hemorrhagic principles from Habu snake venom on platelet aggregation. In “Platelets, Thrombosis, and Inhibitors” (P. Didisheim, T. Shimamoto, and H. Yamazaki, eds.), pp. 335-344. F. K. Schattauer Verlag, Stuttgart. OHSAKA,