Toxicon, Vol. 16, pp. 333-341.

0041-0101178[0701-.0333502.0010

© Pergamon Press Ltd 1978. Printed in Great Britain.

AGGREGATION OF PLATELETS IN THE MESENTERIC MICROCIRCULATION OF THE RAT INDUCED BY a-TOXIN (PHOSPHOLIPASE C) O F C L O S T R I D I U M

PERFRINGENS AKIRA OHSAKA,* MASAHARU TSUCHIYA,t CHIKARA OSHIO,t MAMORU MIYAIRI,~" KENJ! SUZUKI~ a n d YOSHIO YAMAKAWA* *The 2nd Department of Bacteriology and ~:Department of Medical Technology, National Institute of Health, Shinagawa-ku, Tokyo 141 and tDepartment of Internal Medicine, Faculty of Medicine, Keio University, Shinjuku-ku, Tokyo 160, Japan

(Accepted for publication 20 September 1977) A. OHSAKA,M. TSUCHIYA,C. OSHIO,M. MIYAIRI,K. SuzuKI and Y. YAMAKAWA.Aggregation of platelets in the mesenteric microcirculation of the rat induced by ¢t-toxin (phospholipase C) of Clostridium perfringens. Toxicon 16, 333-341, 1978.--We have investigated the effects of purified ct-toxin (phospholipase C) of Clostridium perfringens (YAMAKAWA and OHSAKA, 1977) on the behavior of cellular components in the microcirculation, using cinematography on a microscopic level and electron microscopy. We demonstrated that after topical application of a-toxin to the mesentery of the rat, rolling of leucocytes along the vessel wall and sticking of leucocytes to the vessel wall occurred in venules but not in arterioles. Some of the leucocytes remained attached to the vessel walk Thrombi were formed frequently in venules and capillaries, and at a later stage, in arterioles. With time, thrombi increased in number and size, leading eventually to stasis of the blood stream. Thrombi were also observed frequently in the mesenteric microcirculation when toxin was injected into the jugular vein of the rat. The experiments with adenosine pretreatment followed by topical application of a-toxin to the mesentery suggested that the formation of thrombi induced by this toxin does not involve the mediation of ADP. Electron-microscopic examination confirmed the formation of thrombi consisting solely of platelets. It was concluded that thrombosis must be involved as an early step in the pathogenesis of necrosis caused by a-toxin. The death of the animals injected intravenously with a-toxin may be due, at least in part, to thrombosis. It is possible that thrombosis induced by or-toxin may be one of the factors involved in the causation of toxemia often manifested in the late stage of gas gangrene. INTRODUCTION

perfringens type A is known to contain lethal, hemolytic and necrotic activities (GLENNY et al., 1933; vmq HEYNINGEN, 1950) and plays an important role in the pathogenesis of gas gangrene (IsPOLA~OVSKAVA, 1971; Ixo et al., 1978). a-Toxin was identified as phospholipase C (phosphatidylcholine: choline phosphohydrolase, E. C. 3.1.4.3.), which catalyzes hydrolysis of lecithin into phosphorylcholine and 1,2-diglyceride

R-TOXIN o f CIostridium

(MACFARLANE a n d KNIGHT, 1941).

To date, little is known about the effects of a-toxin on the general microcirculation. KozLov and ISPOLAa'OVSKAVA(1972) examined the effects of a partially purified preparation of ~-toxin on the mesenteric microcirculation by light microscopy. In addition to various biological effects, they also described aggregation of platelets. ELDER and MILES (1957) reported an increase in vascular permeability induced in guinea-pig skin by an impure preparation of a-toxin; similar observations were reported by STARK and DUNCAN (1972) 333

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an d HABERMANN (1960). Since this m i c r o - o r g a n i s m is k n o w n to p r o d u ce various toxins an d enzymes (VAN HEYNINGEN, 1950), definitive experiments are required to determine wh et h er or n o t a - t o x i n itself is responsible for these biological effects. R e c e n t l y we succeeded in isolating a - to x i n f r o m C. perfringens in a physico-chemically h o m o g e n e o u s state by two alternative procedures (YAMAKAWA et al., 1976), namely, affinity c h r o m a t o g r a p h y on agarose-linked egg-yolk lipoprotein (TAKAHASHI et al., 1974) or ion-exchange c h r o m a t o g r a p h y (YAMAKAWA an d OHSAKA, 1977). Th e availability of such highly purified p r e p a r a t i o n s o f a - to x i n (phospholipase C) p r o m p t e d us to investigate the effects o f this toxin on the general microcirculation. W e d e m o n s t r a t e d that the highly purified e - t o x i n caused increased vascular permeability (SUGAHARAet al., 1977). T o determine the specific effects o f a - t o x i n on microcirculation, we studied the effects o f the purified a - t o x i n on the m e s e n t e r i c m i c r o c i r c u l a t i o n o f rats by c i n e m a t o g r a p h y on a microscopic level a n d also electron microscopy. T h e present p a p e r describes the results o f our experiments. These d e m o n s t r a t e d the ability o f a-toxin to induce the f o r m a t i o n o f t h r o m b i (platelet aggregates) in the mesenteric microcirculation. T h e present findings are submitted as a step t o w a r d the u n d e r s t a n d i n g o f the pathogenesis o f gas gangrene. MATERIALS AND METHODS Chemicals Adenosine was purchased from Sigma Chemical Co., St. Louis, Missouri, U.S.A. ; bovine serum albumin (BSA) from Armour Pharmaceutical Co., Eastbourne, Sussex, England. National Standard Gas Gangrene (C. perfringens) antitoxin containing anti-a-toxin titer of 20 i.u./ml was produced by National Institute of Health, Japan. All other chemicals were of analytical reagent grade.

Estimation of Protein Protein contents were estimated by measuring absorbancy at 280 nm with a 1 cm cell in a Hitachi type EPU-2A spectrophotometer. A factor of 1.0 was used to convert A280nm to mg of protein per ml. Determination of phospholipase C activity of et-toxin For the routine assay of phospholipase C activity, the antitoxin-binding power in Lv units was determined as described by MURATAet al., (1965). One Lv unit was defined as the amount of phospholipase C (s-toxin) neutralized by exactly one International Unit of the National Standard Gas Gangrene (C. perfringens) antitoxin. Purification of eL-toxin (phospholipase C) ct-Toxin was highly purified by successive chromatography on CM-Sephadex, DEAE-Sephadex and Sephadex G-100 (YAMAKAWAand OHSAI~A,1977). The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line ir immunodiffusion with the National Standard Gas Gangrene (C. petfringens) antitoxin, indicating the high degree of purity of the preparation. The purified u-toxin was concentrated by precipitation with ammonium sulfate at 60 per cent saturation and stored at -20°C until used. When used, the preparation contained 280 Lv or 1.7 × 104 mouse i.v. LDs0 per mg of protein. Microscopy of the effects of eL-toxin on the mesenteric microcirculation Male rats of the Wistar strain, weighing 150-200 g, were anesthesized by intraperitoneal injection with Nembutal (sodium pentobarbital) in doses of 50 mg per kg body weight. A midline incision was made on the shaved abdominal skin under local anesthesia with 0,5 per cent Xylocalne (lidocaine hydrochloride). The mesentery was pulled out of the peritoneal cavity onto a glass plate. To keep it moist, the tissue exposed over the glass plate was partly covered with pieces of cotton soaked with a Ringer's solution containing 1 per cent gelatin. Instruments for microscopic observation were set up in a chamber maintained at 37°C. Prior to the application of each sample, the microcirculation in the tissue was carefully inspected microscopically to ensure that there were no abnormalities (TstrcHIVA et al., 1969). The following three types of experiments were conducted. (1) A half ml of a-toxin containing 100 Lv or 6 × 108 mouse t.v. LO~0per ml of 0.1 M Tris-HCI buffer, pH 7.5 in 0.1 M NaC1 was topically applied to the mesentery through an injector under light-microscopic observation. The animals survived through the experimental period (usually 30 rain) despite receiving a larger dose of toxin. The reason for this may be that only a small part of the toxin applied was absorbed into the circulation. Fourteen rats including two control animals given the buffer

z

FL

l(c)

AND

CD).

FiG. I(E) AND (F). FIG. 1. FORMATION OF THROMB1 IN MICROCIRCULATION t31;"RAT'S MESENTERY EXPOSED TO {/.-TOXIN. A, before application of u-toxin; B, 56 sec after topical application of 0.5 ml of a-toxin ( 100 Lv/ml); C, 3 rain 20 sec after application; D, 3 rain 26 sec after application ; E, 3 rain 27 sec after application; F, 5 rain 10 sec after application. The arrows indicate the thrombi. Scale: one division equals 25 Bin. Photographed with Olympus C-35A camera on Kodak Tri-X pan film.

FIG. 2. ELECTRON-MICROGRAPH OF LONGITUDINAL SECTION OF A MESENTERIC BLOOD VESSEL (VENULE), 4 rain AFTER EXPOSURE TO 0"5 m l OF O.-TOXIN, CONTAINING 100 Lv/ml AS IN FIG. 1.

The mesenteric tissues were fixed in situ by dripping 1 per cent OsO4 in 0,1 M sodium cacodylate buffer (pH 7.4) at room temperature onto the tissue for 3 min. The tissues were excised and cut into small pieces and then fixed for 1-5 hr in the above fixative at 4°C. The pieces were dehydrated and embedded in Epon, and the fine sections were stained with 2 per cent uranyl acetate in 50 per cent ethanol and an aqueous solution of 0.2 per cent lead citrate. The electronmicrograph demonstrates the formation of thrombi consisting solely of platelets in the microcirculation. Some degeneration of endothelial cells adjacent to the thrombi is also seen. No leucocytes are demonstrable. E, erythrocyte; En, endothelial cells; P, platelet,

C. perfringens a-Toxin-Induced Thrombosis

339

alone were used for this experiment. (2) In some experiments, a-toxin (0-5 ml, 100 Lv/ml in 0-1 M NaCI) was topically applied to the mesenteric microcirculation of two rats which had been injected i.v. with adenosine in doses of 240 lag per animal. (3) In some other experiments, 0.2 ml of a-toxin containing 1 Lv or 60 mouse I.v. LOs0 per ml of 0"1 per cent BSA in the buffer as above was administered into the jugular vein. This amount of toxin corresponds roughly to 1 i.v. LD~0 for a rat. Six rats including three control animals injected with BSA alone were used for this experiment The changes of the mesenteric microcirculation following the topical i.v. administration of a-toxin were observed in vivo microscopically and recorded at various intervals of time by a 35 mm still camera (Olympus C-35A, Kodak Tri-X pan film), a 16 mm movie camera (Boauliau R-16, Kodak Ektachrome Film Type 7241) or a videocamera (Ikegami MK-309 Color Videocamera connected with Victor Video Cassette Recording Model CR-6300 Recorder and lkegami TM20-7A Color TV Monitor). Electron microscopy

The mesenteric tissues of additional two rats exposed to a-toxin (0"5 ml, 100 Lv/ml in 0-1 M NaCI) for 4 min were fixed in situ by dripping 1 per cent OsO4 in 0-1 M sodium cacodylate buffer (pH 7.4) at room temperature onto the tissue for 3 min. The tissues were excised and cut into small pieces (approximately 8mm ~) and then fixed for 1'5 hr in the above fixative at 4°C. After fixation, the pieces were dehydrated in increasing concentrations of ethanol and propylene oxide and embedded in Epon (Lurr, 1961). Fine sections, approximately 400-500 A thick were cut on a LKB 8800 ultramicrotome and collected on copper grids. The sections were double-stained first with 2 per cent uranyl acetate in 50 per cent ethanol for 1-5 hr and then with an aqueous solution of 0"2 per cent lead citrate for 10 min. The grids were examined under a Hitachi H-500 electron microscope at 50 KV. RESULTS AND DISCUSSION To d e t e r m i n e the specific effects o f a - t o x i n on the general microcirculation, we microscopically e x a m i n e d the mesenteric m i c r o c i r c u l a t i o n o f living rats e x p o s e d to a-toxin. This m e t h o d e n a b l e d us to observe where a n d h o w the events h a p p e n in a m i c r o c i r c u l a t o r y unit, which includes preferential channels, true capillaries a n d a r t e r i o - v e n o u s a n a s t o m o s e s , all existing between the arteriole a n d the venule (ZwEIFACH,1961). We d e m o n s t r a t e d t h a t a b o u t 1-1.5 min after the topical a p p l i c a t i o n o f a - t o x i n (0.5 mi, 100 Lv/ml in 0" I M NaC1) to the mesentery o f the rat, rolling o f leucocytes a l o n g the vessel wall, a n d sticking o f leucocytes to the vessel wall occurred in venules b u t not in arterioles, with s o m e o f the leucocytes r e m a i n i n g a t t a c h e d to the vessel wall. T h r o m b i (platelet aggregates) were f o r m e d frequently in venules a n d capillaries, a n d at a later stage, in arterioles, which h a d been e x p o s e d to a - t o x i n (Fig. 1 A - E ) . As time elapsed, t h r o m b i increased in n u m b e r a n d grew in size, leading eventually to stasis o f the b l o o d stream (Fig. 1 F). The same results as a b o v e were o b t a i n e d consistently with 12 animals, while two control animals treated in the same w a y except toxin a p p l i c a t i o n showed no such events. These results were in a g r e e m e n t with some o f the earlier observations which KOZLOV a n d ISPOLATOVSKAYA (1972) m a d e when dealing with a p a r t i a l l y purified p r e p a r a t i o n o f a-toxin. T h r o m b i were also observed frequently in the mesenteric m i c r o c i r c u l a t i o n 0.5-2.5 min after the administ r a t i o n o f toxin (0.2 ml, 1 Lv/ml in 0.1 per cent BSA) into the j u g u l a r vein o f the rat. The results o b t a i n e d with three experimental animals were consistently the same; three c o n t r o l animals a d m i n i s t e r e d 0-1 p e r cent B S A alone showed no aggregation o f platelets. E l e c t r o n - m i c r o s c o p i c e x a m i n a t i o n (Fig. 2) confirmed the f o r m a t i o n o f t h r o m b i consisting solely o f platelets in the microcirculation exposed to a-toxin. This was a c c o m p a n i e d by some degeneration o f vascular endothelial cells a d j a c e n t to the t h r o m b i . These results were confirmed with specimens from a n o t h e r rat. In the e l e c t r o n m i c r o g r a p h (Fig. 2) no leucocytes are d e m o n s t r a b l e . It remains to be answered whether the sticking a n d attachm e n t o f leucocytes to the vessel wall occurring in venules triggered the aggregation o f 01atelets. It is well k n o w n t h a t A D P induces the aggregation o f platelets (BORN, 1962; O'BR1EN, 1962 ; MUSTARD a n d PACKHAM, 1970; RODMAN, 1973) a n d t h a t this aggregation is inhibited by c o m p o u n d s such as adenosine, which have structural similarities to A D P (MUSTARD

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a n d PACKHAM, 1970). To d e t e r m i n e whether o r n o t a - t o x i n - i n d u c e d f o r m a t i o n o f t h r o m b i is m e d i a t e d b y A D P , a - t o x i n (0.5 ml, 100 L v / m l in 0.1 M NaC1) was topically a p p l i e d to the mesenteric m i c r o c i r c u l a t i o n o f t w o rats which h a d been injected i.v. with. a d e n o s i n e in doses o f 240 lag per animal. As with. the c o n t r o l animals m e n t i o n e d above, the rolling a n d sticking o f leucocytes a n d the f o r m a t i o n o f t h r o m b i were observed in venules, starting f r o m a b o u t 1.5-2.0 rain after toxin application, suggesting t h a t the f o r m a tion o f t h r o m b i (platelet aggregates) induced b y a - t o x i n does n o t involve the m e d i a t i o n o f A D P . This suggestion is further s u p p o r t e d b y our recent experiments which d e m o n strated the ability o f the purified a - t o x i n to induce the in vitro aggregation o f platelets (SUGAHARA et al., 1976). The aggrega*ion started after a time lag, the length o f which d e p e n d e d on c o n c e n t r a t i o n o f toxin. T h e reciprocal o f this time lag was directly p r o p o r t i o n a l to toxin c o n c e n t r a t i o n . The in vitro aggregation o f platelets induced by a - t o x i n was irreversible, indicating an obvious difference f r o m the A D P - i n d u c e d aggregation o f platelets (SUGAHARA et al,, 1976) which is k n o w n to be reversible (BORN, 1962; O'BRIEN, 1962). F r o m these results we c o n c l u d e t h a t t h r o m b o s i s ( f o r m a t i o n o f platelet aggregates) m u s t be involved as an early, i m p o r t a n t step in the pathogenesis o f necrosis caused by a-toxin. T h e d e a t h o f the animals injected i.v. with a - t o x i n m a y be due, at least in part, to t h r o m b o s i s . I t is quite possible t h a t t h r o m b o s i s i n d u c e d b y a - t o x i n m a y be one o f the factors involved in the c a u s a t i o n o f t o x e m i a often manifested in the late stage o f gas gangrene. REFERENCES BORN, G. V. R. (1962) Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194, 927. ELDER,J. M. and MILES,A. A. (1957) The action of the lethal toxins of gas-gangrene clostridia on capillary permeability. J. Path. Bact. 74, 133. GLENNY, A. T., BARR, M., LLEWELLYN-JONES,M., DALLING,T. and Ross, H. E. (1933) Multiple toxins produced by some organisms of the CI. welchii group. J. Path. Bact. 37, 53. HABERMANN,E. (1960). Zur Toxikologie und Pharmakologie des Gasbrandgiftes (Clostridium welchii Typ A) und seiner Komponenten. Arch. exp. Path. Pharmak. 238, 502. ISPOLATOVSKAYA,M. V. (1971) Type A Clostridium perfringens toxin. In: Microbial Toxins, Vol. IIA, Bacterial Protein Toxins, p. 109, (KADIS,S., MONTIE, T, C. and AJL, S. J., Eds.). New York: Academic Press. ITO, A., KAMEYAMA,S. and MURATA,R. (1978) Role of toxins of Clostridium perfringens type A in the experimental gas gangrene. In: Actual Data on the Biology and Pathology of Anaerobic Bacteria, (BITTNER, J. et al., Eds.). Medical Publishing House. To be published. KozLov, V. I. and ISPOLATOVSKAVA,M. V. (1972) The effect of CI. perfringens type A toxin and of its lethal lecithinase factor on the microcirculation. Biul. Eksp. Biol. Med. 73, 22 (text in Russian). LUFT, J. H. (1961) Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409. MACFARLANE,M. G. and K~GrIT, B. C. J. G. (1941) The biochemistry of bacterial toxins---l. The lecithinase activity of CI. welchii toxins. Biochem. J. 35, 884. MURATA, R., YAMAMOTO,A., SODA, S. and ITO, A. (1965) Nutritional requirements of Clostridium perfringens PB6K for alpha toxin production. Jap. J. reed. Sci. Biol. 18, 189. Mus'rAR~, J. F. and PACKHAM,M. A. (1970) Factors influencing platelet function: adhesion, release and aggregation. Pharm. Rev. 22, 97. O'BRIE~, J. R. (1962) Platelet aggregation. Part I. Some effects of the adenosine phosphates, thrombin, and cocaine upon platelet adhesiveness. J. clin. Path. 15, 446: Part II. Some results from a new method of study. Ibid. 15, 452. OHSAKA,A., SUGAHARA,T., TAKA;aAS/aI,T. and YAMAVA,S. Effects of a-toxin (phospholipase C) of Clostridium perfringens on microcirculation. In Toxins: Animal, Plant, Microbial, (RoSE, BERG, P., Ed.). Oxford: Pergamon Press. To be published. RODMAN, N. F. (1973) Thrombosis. In: The Inflammatory Process, Vol. II, 2nd Ed., p. 363, (ZwHVACH, B. W., GRANT, L. and McCLUSKEY, R. T., Eds.). New York: Academic Press. STARK, R. L. and DUNCAN,C. L. (1972) Transient increase in capillary permeability induced by Clostridium perfringens type A enterotoxin. Infect. Immunity 5, 147. SUGAHARA,T., TAKAHASm,T., Y'AMAVA,S. and OHSAKA,A. (1976) In vitro aggregation of platelets induced by a-toxin (phospholipase C) of Clostridium perfringens. Jap. J. reed. Sci. Biol. 29, 255.

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SUGAHARA, T., TAKAHASHI, T., YAMAYA, S. and OHSAKA, A. (1977) Vascular permeability increase by a-toxin (phospholipase C) of Clostridium perfringens. Toxicon 15, 81. TAKAHASH1, T., SUGAHARA,T. and OHSAKA, A. (1974) Purification of Clostridium perfringens phospholipase C (a-toxin) by affinity chromatography on agarose-linked egg-yolk lipoprotein. Biochim. biophys. Acta 351, 155. TSUCmYA, M., FtlZlSHIRO, Y. and SHISHIDO,T. (1969) Observation vitale de la thrombog6ndse. In: Atherogenesis, International Congress Series No. 201, p. 145, (SmMAMOTO,T. and NUMANO,F., Eds.). Amsterdam : Excerpta Medica Foundation. YAMAKAWA,Y., TAr~AHASHI,T., SUOAHARA,T. and OHSAKA, A. (1976) Purification and characterization of a-toxin (phospholipase C) of Clostridium perfringens. In : Animal, Plant, and Microbial Toxins, Vol. 1, Biochemistry, p. 409, (OHSAKA,A., HAVASm, K. and SAWAI, Y., Eds.). New York: Plenum Press. YAMA~AWA, Y. and OHSAI