British Journal of Haematology. 1992, 82, 142-1 50

Cytotoxicity of activated platelets to autologous red blood cells MARIKOOKADA,TAKESHIK O D A M A . * AKIOT O M I N A G AKAZUNORI , KON, TERUTAKA S A G A W AAND SAYAKAU T S U M Department I~ of Clinical Pathology, Ehime College of Health Science, Tobe-cho, Iyo-gun, Ehime 791-21, *Tokushima Laboratory. Otsuka Pharmaceutical Co. Ltd, Kagasuno, Kawauchi-cho, Tokushima 771-01, and tDepartment of Microbiology, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime 791-02, Japan

Received 21 February 1992: acceptedfor publication 27 April 1992

Summary. Gel-filtered human platelets exerted lytic activity on autologous red blood cells (RBC) when they were coincubated at 3 7°C with platelet-activating agents, such as thrombin, collagen, ADP. LPS or PMA in the absence of plasma. Lysis of activated platelets themselves did not occur during the incubation period examined. Morphological observations showed that RBC exposed to thrombin-activated platelets were fragmented and/or transformed into spherocytes. This haemolytic reaction by thrombin-activated platelets did not occur at 4"C, or in the presence of agents which inhibited glycolysis or elevated intracellular levels of CAMP, indicating that energy-dependent and CAMP-regulated platelet metabolism was required for this reaction. When platelets and RBC were incubated in the same vessel, but were prevented from coming into direct cell to cell contact

by means of a membrane barrier, their cytotoxicity was reduced but not eliminated completely. No cytotoxic activity against RBC was detected in platelet-free supernatants obtained by centrifugation after activation of platelets with thrombin. On the contrary, activated and washed platelets retained the activity. These observations suggested that the cytotoxic activity was carried by some diffusible and easily inactivated factors, which were continuously produced and liberated from activated platelets. Cyclo-oxygenaseinhibitors inhibited the haemolytic activity of thrombin-activated platelets, suggesting a role for some products of platelet-cyclooxygenase pathway in platelet-mediated haemolysis. These results provide the first evidence for a direct role of activated platelets in mediation of RBC-damage in the absence of any plasma factors.

Platelets play important roles not only in haemostasis but also in a variety of immunological and inflammatory events (Henson & Ginsberg, 1981: Oxholm & Winther, 1986). The indication that platelets may be one of the primary cytotoxic effector cells comes from the following observations. Mouse platelets mediate lysis of antibody-coated sheep RBC involving complement (Soper et al. 1982: Slezak et al. 1987). Human and rat platelets kill Schistosonia mansoni larvae in an IgE-, a lymphokine- or a C-reactive protein-dependent manner (Joseph et al, 1983: bout et al. 1986: Pancre et al, 1987). Human platelets are cytotoxic against certain adherent human tumour cell Lines (Ibeleet al. 1985). certain leukaemic cell lines (Sagawa eta!, 1990).and Toroplasmagondii(Yong et a!, 1991) in the absence of antibodies. Furthermore, some lines of evidence indicate that platelets exert cytotoxic effect not only on foreign cells but also on endothelial cells both in vivo (Jergensen et nl, 1970; Lough & Moore, 1975: Fujimoto et al. 1985) and in vitro (Jergensen et al. 1986: Kishi & Numano, 1989).when activated by agonists.

It is well known that haemolysis and fragmentation of RBC are observed under various pathological conditions where activation of platelets occurs (Wintrobe, 1981).However, little has been known about how activated platelets can influence RBC. In this study, we demonstrate that cytotoxicity against RBC is induced in platelets after activation with various agonists and that the cytotoxic activity resides in the cell body of activated platelets but not in materials released from granules. The cytoxicity was evaluated by measuring haemoglobin release and by examining RBC morphology. Our results may provide some clues to understand the role of platelets in mediation of RBC-damage during intravascular activation of platelets. MATERIALS A N D METHODS

Materials. Acetylsalicylicacid, ADP, bovine serum albumin (BSA)(globulin and fatty acid free), catalase, dibutyryl CAMP (dbcAMP), indomethacin, phenylmethylsulfonyl fluoride, phorbol 12-myristate 13-acetate (PMA), prostaglandin E l , soybean trypsin inhibitor, superoxide dismutase (SOD), N-ptosyl-L-phenylalanine chloromethyl ketone, thrombin from

Correspondence: Dr Mariko Okada. Department of Clinical Pathology, Fhime College of Health Science, 543 Tako-oda. Tobe-cho. Iyogun. Ehime 791-21. Japan.

142

Platelet-mediated Haemolysis human plasma with activity of approximately 3000 NIH units per mg protein were obtained from Sigma Chemical Co. (St Louis): collagen was obtained from Hormon-Chemie Miinchen GMBH Corp. (Miinchen); lipopolysaccharide(LPS) derived from S. typhosa 0901 was obtained from Difco Labs (Michigan);C1-inhibitor was purified from human plasma as previously described (Okada & Utsumi, 1989). All other chemicals were of analytical grade. Solutions. Medium I: 145 mM NaCI, 4 mM KCI, 1mM MgCI2, 0-5 mM sodium phosphate, 0.1%(w/v) glucose. 0.1% (w/v) BSA, and 5 mM PIPES, pH 6.8. Medium 11: 145 mM NaCI, 4 mM KCI, 1mM MgC12,0.5 mbi sodium phosphate, 0.1%(w/v) glucose, 0.1%BSA, and 5 mM HEPES, pH 7.4. Medium 111: Medium I1 containing 2.5 mM CaC12.Medium IV: Medium I1 without glucose and BSA. VBS: 145 mM NaCl and 0.5 mM sodium barbital, pH 7.4 (Wiedmer & Sim, 1985; Sagawa et al, 1990).Medium 111without glucose was used when the effect of 2-deoxy-~-glucosewas examined. When the effect of water insoluble agents was examined, the following medium was used Medium III containing 0.1% ethanol for PMA and prostaglandin E l ; Medium 111 containing 0.25% DMSO for acetylsalicylic acid and indomethacin. These agents were dissolved in organic solvent at high concentration and were diluted in Medium 111 before use. Preparation ofpIatefets and RBC. Gel-filtered platelets were prepared from freshly drawn healthy human blood with 1/6 volume of citrate anticoagulant (8 g/l citric acid, 22 g/l trisodium citrate, 24.5 g/l glucose, pH 4.5) according to the method of Wiedmer & Sim (1985) with slight modifications as described previously (Sagawa et al, 1990). Briefly, anticoagulated blood (52.5 ml) was centrifuged at 250 g for 20 min. The platelet-rich plasma (PRP)was carefully removed and an additional 1/5 volume of citrate anticoagulant was added to the PRP. The PRP was centrifuged at 700 g for 10 min and the platelet pellet was suspended in 0.5 ml Medium I. This suspension was applied to a column ( l o x 200 mm) of Sepharose CL-2B (Pharmacia AS Biotech, Uppsala) equilibrated in Medium I. The peak cell fraction eluting in the void was collected and was resuspended in 0.5-1 ml Medium I1 ( 3 x 1Oy/ml).These preparation procedures were performed at 2 5-30°C throughout in plastic tubes. Contamination of RBC in these preparations was less than 0.1%.Leucocytes and mononuclear cells were not detectable. RBC from the same blood as used for platelets preparation were incubated in VBS containing 10 mM EDTA at 3 7°C for 30 min and then washed three times with the same buffer. These RBC were washed twice with and resuspended in Medium 111. Only antiglobulin test negative RBC were used for experiments. Cytotoxicity assays. Platelets at various concentrations (120 x 1O8/mI)and RBC at a concentration of 5 x 107/mlwere incubated at 37'C or 4°C for 0-5 h in Medium 111 in the presence or absence of stimulating agents in 1 . 5 ml polypropylene tubes. Plate1et:RBC was 20: 1,except where indicated otherwise. Cytotoxicity of platelets against RBC was evaluated by haemolysis and by examining transformation of RBC. For haemolytic assay, the reaction mixtures were centrifuged at 8000 g for 5 min at 2 5°C and the optical density (OD) of the

143

supernatant at 413 nm was measured. Per cent specific haemolysis ( y) was calculated as follows: y = OD (sample) -OD (spontaneous)/OD (100%)-OD (spontaneous) x 100, where OD (sample), OD (spontaneous) and OD (100%)were obtained from RBC samples incubated with platelets in the presence or absence of thrombin, incubated with medium alone and lysed by 0.01%Triton X-100, respectively. For examination of transformation of RBC, the reaction mixtures were fixed with 0.75% glutaraldehyde in Medium IV and observed under an optical microscope. Per cent transformation (number of spherocytes and spherocytes with blebs as shown in Fig 3/total number of RBC counted x 100) was calculated. At least 200 RBC were counted per assay. Co-cultivation of RBC and platelets without direct cell to cell contact. Platelets and RBC were incubated in the same vessel, but were prevented from coming into direct cell to cell contact by means of a membrane barrier as follows. 200 yl of platelets (5 x 109/ml)or medium alone in the presence of thrombin (2 U/ml) were placed in a small culture chamber whose bottom was made by a nitrocellulose membrane filter with 0.45 pm pore size (Millicell-HA, Millipore, Bedford), and then the chamber was placed in a culture plate well containing 200 pl of 5 x 107/mlRBC. The culture was incubated for 4 h at 3 7°C in a humidified chamber before examination of RBC damages. Supernatants versus cell body comparison as to the localization of platelets cytotoxicity. 100 p1 of platelets (1x 10y/ml) in Medium I1were activated with thrombin at 2 U/ml for 10 min at 37°C. After incubation, the pellet and supernatant fractions were separated by centrifugation at 2000 g for 10 min at 25°C. The pellet fraction was washed once with and resuspended in Medium 111. To the supernatant fraction, CaCI2 was added to make 2 . 5 m ~ Both . fractions were assayed for their cytotoxicity against RBC (5 x lo6)in a total volume of 100 pl Medium m. Measurement of lactate dehydrogenase activity as an indicator ofplatelets lysis. Lactate dehydrogenase (LDH) activity of the supernatants obtained after incubation of platelets at 1x 1OY/mlin the presence or absence of thrombin at 2 U/ml at 3 7°C for various times was quantitated by measurement of NAD according to the method of Bergmeyer (1974). Per cent LDH release was calculated as follows: % release=(LDH activity of the sample supernatant/LDH activity of the supernatant obtained after lysis of platelets with 0.04% Triton X-100) x 100. Observations ojRBC morphology. After fixation with 0.75% glutaraldehyde in Medium IV,the specimens were post-fixed with 2% Os04 in Medium IV at 4°C for 1h and observed with a scanning electron microscope (Hitachi HS-500) as described previously (Sagawa et al. 1990). For continuous observation of transformation of RBC by an optical microscope equipped with a video monitoring system, platelets, RBC and thrombin were mixed in a polypropylene tube, and immediately after mixing, the reaction mixture was injected into a siliconized glass microcell (100 pn in depth) set in a water jacket, which was kept at 37°C and placed on the stage of the microscope.

144

Mariko Okada et a1 Table I. Donor to donor variability of haemolytic activity of platelets.

A % haemolysis*

Donor

c

0

2

0

82.8f7.8 (3)t 78.5 11.2 (4) 72.1 f 5 . 0 ( 3 ) 70.8 f 5 . 6 (5) 61.8f8.8 ( 3 ) 57.9f27.5 ( 3 ) 4 3 . 6 f 9 . 5(3) 40.7f8.3 ( 3 ) 3 5 . 5 (1) 1 7 6 (1)

1 2 3 4 5 6 7

8

*

8 9

L

10

Time ( h )

LB

* % haemolysis induced by platelets (1 x 1OY/ml)stimulated with 2 U/ml thrombin at a plate1et:RBC of 20: 1 was measured after 5 h. Values are means+ SD of separate experiments except in nos. 9 and 10. and the numbers in parentheses are the number of experiments repeated.

''0

0

H~~OIs YSI

50

100 I

Med I urn

(

0 0

Coilagen

0,

1 10 ThroTbin ( U /m I )

001

01

100

Fig 1. Haemolysis of autologous RBC by thrombin-stimulated platelets. (A) Kinetics of haemolysis. Platelets ( 1 x 109/ml)and RBC ( 5 x 107/ml)were incubated at 37°C ( 0 . 0)or 4OC ( 0 . m) in the presence ( 0 ,0 )or absence (17. m) of 2 U/ml of thrombin for various times. (B) Dependence of extent of haemolysis on the concentration of platelets. Platelets at various concentrations and RW ( 5 x 10'lml) were incubated for 5 h at 37°C in the presence ( 0 )or absence (17)of 2 U/ml of thrombin. (C) Dependence of extent of haemolysis on the concentration of thrombin. RBC (5 x 107/ml) were incubated alone ( A ) or with platelets (1 x 1UY/ml)( 0 ) in the presence of various concentrations of thrombin for 5 h at 37OC. After incubation, reaction mixtures were centrifuged at 8000 g for 5 min at 25°C. % haemolysis was calculated as described in Materials and Methods. Values are means of triplicate determinations.

Thrombin 2 U!m. )

(80pg;rni

IPS (

2 mghi )

0ioioEthanol

i

t+

Fig 2. Platelet-mediated haemolysis induced by various plateletactivating agents. RBC ( 5 x 107/ml)alone (closed column) or with platelets (1 x 109/ml)(open column) were incubated for 5 h at 3 7°C in presence or absence of various platelet-activating agents at the concentrations indicated. The activity of thrombin, ADP. collagen, and LPS was examined in Medium 111, and that ofPMA was tested in Medium III containing 0.1%ethanol. Values are meansfSD of triplicate determinations.

Platelet-mediated Haemolysis RESULTS

Platelet-mediated haemolysis of autologous RBC Unstimulated human platelets did not express appreciable levels of haemolytic activity even in an assay as long as 5 h. However, as illustrated in Figs 1A and lB, which present data from a typical experiment, platelets activated with thrombin had significant haemolytic activity on autologous RBC at 3 7OC. The haemolysis occurred in a thrombin-dose dependent manner (Fig 1C). In most experiments, the optimum thrombin concentration was around 2 U/ml. Thrombin alone at the concentrations used here did not induce haemolysis in the absence of platelets. In a series of 35 experiments, the median per cent haemolysis induced by platelets activated with 2 U/ml of thrombin at a plate1et:RBC of 20:l was 55.4% at 5 h (range 17.5-85.0%). No haemolysis was observed at 4°C (Fig 1A). We observed a considerable variability in haemolytic activity of thrombinactivated platelets depending on the donor of the blood samples (Table I). The ability of platelets to lyse RBC was not restricted to platelets activated with thrombin. As shown in Fig 2, activation with collagen, ADP, LPS or PMA also induced significant haemolytic activity of platelets. As with thrombin, none of these agents alone induced haemolysis without platelets. Transformation of RBC induced by thrombin-activated platelets Damage to RBC induced by platelets could also be shown by examination with a scanning electron microscope. After incubation with platelets and thrombin (2 U/ml) at 3 7°C for 4

145

h, RBC had undergone morphological transformation, and had become spherocytes with or without blebs (Figs 3B and 3C), or fragmented microspherocytes (Fig 3D). Not only RBC directly attached to aggregated platelets (Fig 3B) but also free RBC (Figs 3C and 3D) showed transformed morphology. No such transformation was observed in the RBC sample incubated with either platelets alone (Fig 3A) or thrombin alone (data not shown). Transformation of RBC exposed to thrombin-activated platelets was observed continuously by an optical microscope using a video monitoring system (Fig 4). After a lag period of about 1 h, a majority of RBC began to form blebs on their surface ( 7 4 min), and they were transformed from biconcave shapes into spherocytes (86-94 min). Quantitative assessments of the extent of transformation and haemolysis (Fig 5, data from a representative experiment) show that haemolysis occurred about 2 h after transformation and that not all the intracellular haemoglobin is released from damaged RBC. even at the time when all RBC had been frag mented and/or transformed into spherocytes. This means that damaged RBC somehow retain their integrity for some time.

Platelet-lytic activity of thrombin-activated platelets In order to know whether thrombin-activation results in the lysis of platelets themselves, release of LDH was measured. During incubation of plateIets with thrombin at 2 U/ml over 6 h at 37"C, less than 5% LDH release into the supernatant was observed (data not shown).

Fig 3. Transformationof RBC induced by thrombin-activatedplatelets.RBC and platelets were incubated in the absence (A)or presence (B, C. D) of 2 U/ml of thrombin for 4 h at 37'C. After incubation,the reaction mixtures were pre-fixed with 0.75% glutaraldehyde and post-fixed with 2% OsO4 and observations were made with a scanning electron microscope (Hitachi HS 500). Spherocytes with one or two blebs on their surface (arrows)and microspherocytes(arrowheads)were observed in RBC samples incubated with platelets in the presence of thrombin. Magnification: x 660 (A), x 1600 (B). x 4400 (C, D).

146

Muriko Okada et al

Fig 4. Time course of transformation of RBC induced by thrombin-activated platelets. Platelets and RBC in the presence of thrombin were incubated in a microcell at 37°C under an optical microscope equipped with a video monitoring system. Each frame is identified by the time (in minutes) after the initiation of incubation. Transformation of RBC identified by the numbers 1-5 is shown. At 60 min. most of RBC show biconcave shapes. At 74 min. RBC No. 1 begins to form a bleb on its surface. The shape of this RBC becomes spherocytic (86-94 min). Bar 10 pm.

Eflect of separation ofplatelets from RBC by n nienibrane barrier on the cytotoxicity Separation of activated platelets from RBC by a membrane barrier in a tissue culture well prevented haemolysis almost completely even when the concentration of platelets was raised to 5 x IOy/ml. In this experiment. however. the cytotoxicity of activated platelets was clearly demonstrated by examining transformation of RBC (Fig 6). No platelet was found in the compartment. which originally contained RBC. when examined after the incubation.

The supernatant fraction obtained after 2 h incubation of platelets with thrombin at 3 7°C also had no cytotoxic activity (data not shown). On the contrary, the 2000 g pellet of activated platelets, which had lost the granular contents almost completely when examined by transmission electron microscopy (data not shown), retained significant cytotoxic activity. The supernatant had no enhancing effect on the activity of the pellet fraction when mixed with it. No activity was detected in both supernatant (data not shown) and pellet fractions from unstimulated platelets.

l~calitationof cytotoxic activity As illustrated in Fig 7 , no cytotoxic activity against RBC was detected in the 2000 g supernatant fraction obtained after activation of platelets with 2 U/ml of thrombin for 10 min.

Eflects of metabolic inhibitors, cyclo-oxygenuse inhibitors or scavengers of reactive oxygen intermediates (ROI) on the haemolytic activity of thrombin-activated platelets Activation dependent changes in platelets are known to

-

0

9L

Sarr i er

"lo Transforma!ion 50

100

Thrombin barrier Thrombin[ barrier

$ 0 0

2

Time ( h )

4

Fig 5. Different kinetics of haemolysis and transformation of RBC induced by thrombin-activated platelets. Aliquots of the reaction mixtures of platelets. RBC and thrombin were fixed with 0.750/, glutaraldehyde at various incubation times and the shapes of RBC were observed with an optical microscope. All the remaining reaction mixtures were centrifuged and the OD of the supernatant was measured. Per cent transformation ( 0 )and % haemolysis ( 0 ) were calculated. Values are means of triplicate determinations.

b

Fig 6. Effect of separation of RBC and platelets by a membrane barrier on platelet-mediated cytotoxicity against RBC. Platelets ( 5 x 109/ml) with thrombin in a culture plate chamber with a nitrocellulose membrane filter (0.45 pm pore size) and RBC ( 5 x 10'/ml) in a 24well culture plate were co-incubated for 4 h at 37°C. Positive control wells contained platelets, RBC and thrombin without a membrane barrier and negative control wells contained thrombin alone in the upper chamber with a membrane barrier. Transformation of RBC was examined after incubation. Values are means+ SD of triplicate determinations. The extent of haemoIysis induced by thrombinactivated platelets in the presence of the membrane barrier was 0.2 +O.O%. and that in the absence of the barrier was 19.9+0.6%.

% Transformation

0

I

pellet

unstirnula ted PL

too

[ peile t

Fig 7. Cytotoxic activity of platelet fractions. Platelet fractions were obtained from 1 x lo8 platelets by centrifugation at 2000 g for 10 min at 2 5°C. Pellet fractions of thrombin-activated or unstimulated platelets, platelet-free supernatant obtained from thrombin activated platelets, or thrombin-activated platelets pellet reconstituted with the supernatant were incubated for 5 h at 37OC with RBC (5 x lo6)in a volume of 100 pl. Per cent transformation of RBC was measured. Values are means fSD of triplicate determinations.

require ATP as energy source and are controlled by intracellular levels of CAMP(Holmsen et al, 1982; Krishnamurthi et ul, 1984; Kroll et al, 1988;McCrae et al, 1990). We therefore investigated whether the haemolytic activity of thrombinactivated platelets also required ATP and was controlled by

Platelet-mediated Haemolysis

intracellular levels of CAMP. When platelets were preincubated with 2-deoxy-~-glucose to deplete glucose in platelets, or with prostaglandin E l or dbcAMP to elevate the intracellular level of CAMP, and these agents were present during the entire incubation period, haemolytic activity of platelets induced by thrombin activation was significantly inhibited (Table 11, Ekp. A). These agents had also inhibitory effect on the haemolytic activity of pre-activated platelets, which had been treated with thrombin in the absence of these metabolic inhibitors (Table 11, Exp. B). In order to explore whether arachidonic acid metabolites (Hamberg et al, 1975; Smith et al, 1976: Gerrard et al, 1977) or ROI (Marcus et al, 1977: Worner, 1981), which are produced and liberated from activated platelets, play any role in this plateletmediated haemolytic reaction, effect of cyclo-oxygenase inhibitors, SOD and catalase was examined (Table 11). Cyclooxygenase inhibitors acetylsalicyclic acid and indomethacin showed significant inhibitory effect on haemolysis not only when the inhibitors were included in the reaction mixtures during the entire incubation period, but even when they were added to pre-activated platelets. Intact SOD appeared to inhibit the platelet-mediated haemolysis. However, an equal level of inhibition by heat-inactivated SOD was observed, indicating that such inhibition was not caused by the specific activity of SOD as a scavenger of ROI but was caused by some unknown non-specific effects. Catalase had no effect. All

Table 11. Effect of various agents on haemolytic activity of platelets. EXP. B t

Exp. A*

Agent

Concn.

Pre-incubation time (min)

% inhibition of haemolysisg

% inhibition of haemolysisg

2-deoxy-D-glucose

5 m ~ 30

90.9f2.4 (3)s

9 6 . 8 f 0 . 6 (3)s

Prostaglandin E l dbcAMP

1P M

3 10

73.5f8.8 (3) 91.5f2.4 (3)

ND 88.7f4.0(3)

10 10 0 0 0

47.7f2-9 (3) 62.8f13.9 (5) 32.0f8.5 (5) 45.9f 10.4 (3) 9.0&17.7(3)

ND 76.2f16.5 (3) ND ND ND

Acetylsalicylic acid Indomethacin SOD Heat-inactivated SOD Catalase

1mM 1mM 5 0 p ~

10560U/ml 10560 U/ml 1040U/ml

14 7

*In Exp. A, platelets (2 x 109/ml) were pre-incubated in a control medium or in a medium containing the respective agent at 3 7°C for the time indicated, and then thrombin and RBC were added to the platelets-suspension to make 1x 109/mlplatelets, 2 U / d thrombin and 5 x 107/ml RBC. t In Exp. B, platelets activated with 2 U/ml thrombin at 37OC for 5 min in Medium I1 were centrifuged and washed once with fresh medium. These pre-activated platelets were incubated with RBC in the presence or absence of the respective agent at 37OC. In both Exp. A and Ekp. B, haemolysis was measured 5 h after the initiation of the incubation of RBC with platelets. $The inhibitory effect of these agents was expressed as % inhibition (% I) of haemolysis calculated as follows: % I = % haemolysis (medium control) - % haemolysis (agent)/% haemolysis (medium control)/ x 100,where % haemolysis (medium control) and % haemolysis (agent) are the values of % haemolysis obtained from samples incubated in the control medium and in the medium containing the agent, respectively. §Values are meansfSD of separate experiments, and the numbers in parentheses are the number of experiments repeated. N D not done.

148

Mariko Okada et a2

these results indicate that haemolytic activity of activated platelets requires ATP and is dependent on CAMP-regulated platelet metabolism. and that some product of cyclo-oxygenase pathway other than ROI may play a role in the haemolytic reaction. Effect of esterase inhibitors on the platelet haemolytic activity Esterases have been implicated in the cytotoxic reactions mediated by other cytotoxic effector cells (Adams, 1980; Pasternack et al. 1986; Pontrenoli et al. 1986: Young et al, 1986; Masson & Tschopp, 1987) and in tumoricidal activity of platelets (Sagawa et a!. 1990). However. C1 inhibitor, soybean trypsin inhibitor, N-p-tosyl-L-phenylalanine chloromethyl ketone, or phenylmethylsulphonyl fluoride had no inhibitory effect on the haemolytic activity of thrombinactivated platelets (data not shown),indicating that esterases are not involved in platelet-mediated haemolysis as effector molecules.

DISCUSSION The results presented here show that platelets exert cytotoxicity in vitro against autologous RBC in an activation dependent manner. All our experiments described so far were carried out under conditions free of plasma or serum factors and target autologous RBC had not been pre-sensitized with added antibody or autoantibody. since all RBC samples used here were negative in antiglobulin test. This is in contrast to the mouse platelet-induced antibody-dependent cellmediated cytotoxicity against sheep RBC reported by Soper et al (1982) and Slezak et a1 (1987), which required both antibody and complement component C3. It has been reported that mouse platelets can adhere to immune complexes only if complement components are bound (Henson & Ginsberg. 1981). Thus, in their studies. IgG antibody and complement component bound to target sheep RBC might play roles not only as ligands for receptors on platelets but also as agonists which activate platelets to become cytotoxic. Separation of platelets from RBC by a membrane barrier reduced the cytotoxicity but did not eliminate it completely. As we could not find any platelets in the lower culture well which originally contained RBC, the possibility that the platelets in the upper chamber passed through the membrane and came directly into contact with RBC can be ignored. Another possibility is that platelets. although they cannot pass the membrane, might project pseudopoda through the pores of the membrane and contact directly with RBC to exert cytotoxicity. This possibility, however, can also be ignored because the plate was incubated without shaking. and, moreover. the distance from the bottom of the upper chamber to that of the lower culture well was more than 700 pm, which is far longer than the usual length of pseudopoda of thrombin-activated platelets (Hattori et (11. 1969). thus the probability of direct contact between RBC and the platelet pseudopoda will be negligible. We believe that it is most probable that activated platelets produce and liberate cytotoxic effector molecules which are small enough to diffuse through the membrane barrier. Platelet-free supernatant

obtained after activation of platelets with thrombin contains various soluble and diffusible materials secreted from dense bodies, such as serotonin and ATP, both of which have been reported to be cytotoxic against certain cells (Charo et al, 1977; Kishi & Numano, 1989: DiVirgilio et al, 1990) and from r-granules (Gogstad et al, 1982) as well as products which are synthesized de novo and liberated by activated platelets (Hamberg et al, 1975; Smith et al, 1976; Gerrard et al. 1977).However, this fraction had no cytotoxic activity. In addition, the supernatant fraction obtained after more prolonged (2 h) incubation of platelets with thrombin also had no cytotoxic activity. On the contrary, the pellet fraction of thrombin activated platelets retained the cytotoxic activity. The most likely explanation for all these results is that the cytotoxicity is mediated by some soluble and diffusible factor(s) which are continuously liberated from the cell body of activated platelets. but which are so unstable that they can be inactivated during centrifugation at 25°C or diffusion through the membrane barrier. The possibility that soluble cytoplasmic materials of platelets exert the cytotoxicity can be ignored because no lysis of platelets occurred during the incubation of platelets with thrombin. Another possibility that granular contents retained in the cell body of activated platelets are gradually secreted during the incubation and exert cytotoxicity seems improbable, if not completely impossible. because most of the contents of granules in activated platelets have been lost. The most probable possibility is that some material synthesized and liberated by activated platelets carry the cytotoxic activity. The electron microscopic observations also support the conclusion that RBC damage is mediated by some soluble factors, because free KBC as well as RBC directly attached to aggregated platelets were damaged. The observations that the induction of the haernolytic activity of platelets was prevented at 4% and that compounds which inhibit glycolysis or raise the intracellular level of CAMP prevent the haemolytic activity of pre-activated platelets suggest that the production and liberation of cytotoxic factors is energy-dependent and regulated by the intracellular level of CAMP. When platelets are activated, the arachidonate metabolic pathways, which have been reported to be energy- and CAMP-dependent (Holmsen et al. 1982; Krishnamurthi et al, 1984), are activated and various bioactive products including ROI are produced and liberated (Hamberg et al. 1975: Smith et al. 1976; Gerrard et al, 1977: Marcus et al, 1977: Worner, 1981). Our data obtained by using cyclo-oxygenase inhibitors and scavengers of ROI suggest that some product of cyclo-oxygenase pathway other than ROT may be important in the cytotoxicity of activated platelets against RBC. It is very suggestive that a major product of cyclo-oxygenase pathway of platelets, thromboxane A2, is unstable with a half-life of about 30 s at pH 7.4 and 37°C (Hamberg et al, 1975). Yong et a1 (1991) have suggested the importance of thromboxane A2 in antibody independent platelet-mediated cytotoxicity against Toxoplasma gondii. Although esterases have been implicated in the cytotoxicity mediated by other cytotoxic effector cells (Adams. 1980:Pontrenolietal, 1986;Pasternacket al, 1986: Young et a!. 1986; Masson & Tschopp, 1987).and in plateletmediated tumour cell killing (Sagawa et al, 1990).we found

Platelet-mediated Haemolysis that esterases are not involved as effectors in the plateletmediated haemolysis. Morphological observations demonstrated that plateletmediated damage of RBC resulted in formation of one or two blebs on the surface of RBC. followed by fragmentation and morphological transformation into microspherocytes. In various diseases characterized by a thrombo-occlusive process in the microvasculature, haemolysis and fragmentation of RBC have been observed clinically (Wintrobe. 1981). These phenomena can also be induced experimentally by injections of endotoxin or thrombin in rabbits (Brain & Hourihane, 1967: Brain et al, 1967). It has been reported that the RBC fragments produced in the microvasculature are taking the form of crescents, helmets, triangles, and microspherocytes (Wintrobe, 1981). Bull & Kuhn (1970) have proposed that RBC fragmentation was induced by forcing RBC through the fibrin meshwork deposited in the vessel. In contrast to their studies, our studies were performed under conditions where no plasma was present and no force was applied. Out data therefore provide the first evidence for a direct role of activated platelets in mediation of haemolysis and fragmentation of RBC in the absence of fibrin meshwork or stress. Although only spherocytes with or without blebs or microspherocytes were produced under the experimental conditions described here, our findings suggest that, besides the mechanical fragmentation of RBC by the fibrin meshwork, which may produce irregular shaped RBC fragments, activated platelet-mediated direct damage of RBC could be one of the mechanisms of haemolysis and fragmentation of RBC associated with local or systemic coagulation process in small vessels. The factor(s) that cause these damages in RBC appear to be unstable. Thus for activated platelets to produce damaged RBC in vivo, an adequate number of platelets and sufficientstasis would be needed to allow the accumulation of the cytotoxic factors. Such conditions could exist in the microvasculature under certain pathological conditions. ACKNOWLEDGMENTS We are grateful to Drs Shigeru Fujita and Yoshinobu Sugino for helpful discussions. This work was supported in part by Grant-in-Aid from the Ministry of Education, Science and Culture of Japan (No. 01571242). REFERENCES Adams, D.O. (1980) Effector mechanisms of cytolytically activated macrophages. I. Secretion of neutral proteases and effect of protease inhibitors. Journal of Immunology, 124, 286-292. Bergmeyer, H.U. (1974) Lactate dehydrogenase. Methods of Enzymatic Analysis, Vol. 2 (ed. by H. U. Bergmeyer). pp. 574-589. Academic Press, New York. Bout, D., Joseph, M., Pontet, M., Vorng, H.. D e s k , D. & Capron, A. (1986) Rat resistance to schistosomiasis: platelet-mediated cytotoxicity induced by C-reactive protein. Science, 231, 153-156 Brain, M.C., Esterly,J.R. &Beck,E.A. (1967) Intravascular haemolysis with e2perimentally produced vascular thrombi. British Journal of Haernatology. 13, 868-890. Brain, M.C. & Hourihane, D.OB. (1967) Microangiopathic haemolytic anaemia: the occurrence of haemolysis in experimentally

149

produced vascular disease. British Journal of Haematology. 13, 13 5-142. Bull, B.S. & Kuhn. I.N. (1970) The production of schistocytes by fibrin strands (a scanning electron microscope study). Blood. 35, 104-1 1 1. Charo, I.F., Feinman, R.D. & Detwiler, T.C. (1977) Interrelations of platelet aggregation and secretion. Journal of Clinical Investigation. 60, 866-873. Di Vugilio, F., Pmo. P.. Zanovello,P., Bronte, V. & Collavo. D. (1990) Extracellular ATP as a possible mediator of cell-mediated cytotoxicity. Immunology Today, 11, 274-277. Fujimoto, T., Suzuki, H., Tanoue. K., Fukushima, Y . & Yamazaki, H. (1 98 5) Cerebrovascular injuries induced by activation of platelets in vivo. Stroke, 16, 245-250. Gerrard, J.M.,Peller, J.D.,Krick,T.P.&White,J.G. (1977)CyclicAMP and platelet prostaglandin synthesis. Prostaglandins, 14, 39-50. Gogstad. G.O., Hagen, I.. Korsmo, R. & Solum. N.O. (1982)Evidence for release of soluble, but not of membrane-integrated, proteins from human platelet a-granules. Biochimica et Biophysica Acta, 702.81-89. Hamberg, M.. Svensson. J. & Samuelsson. B. (1975)Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides. Proceedings of the National Academy of Sciences of the United States of America, 72, 2994-2998. Hattori, A.. Tokunaga, J., Fujita, T. & Matsuoka. M. (1969)Scanning electron microscopic observations on human blood platelets and their alterations induced by thrombin. Archivum Histologicum Japonicum, 31, 37-54. Henson. P.M. & Ginsberg. M.H. (1981) Immunological reactions of platelets. Platelets in Biology and Pathology. Vol 2. (ed. by J. L. Gordon), pp. 2 65-308. Elsevier/North-Holland Biomedical Press, Amsterdam. Holmsen, H., Kaplan, K.L. & Dangelmaier, C.A. (1982) Differential energy requirements for platelet responses. Biochemical Journal, 208,9-18. Ibele, G.M.. Kay, N.E., Johnson, G.J. &Jacob, H.S. (1985) Human platelets exert cytotoxic effects on tumor cells. Blood, 65, 12521255. Joseph, M., Auriault. C., Capron, A., Vorng, H. & Viens, P. (1983)A new function for platelets: IgE-dependent killing of schistosomes. Nature, 303, 810-812. Jergensen, L., Grethe, A.G., Larsen, T., Kinlough-Rathbone. R.L. & Mustard, J.F. (1986) Injury to cultured endothelial cells by thrombin-stimulated platelets. Laboratory Investigation, 54, 4084 1 5. Jergensen, L., Hovig, T.. Rowsell, H.C. & Mustard, J.F. (1970) Adenosine diphosphate-induced platelet aggregation and vascular injury in swine and rabbits. American Journal of Pathology, 61, 161-176. Kishi. Y. & Numano, F. (1989)In vitro study of vascular endothelial injury by activated platelets and its prevention. Atherosclerosis, 76, 95-101. Krishnamurthi. S., Westwick,J. & Kakkar, V.V. (1984)Regulation of human platelet activation. Analysis of cyclooxygenase and cyclic AMP-dependent pathways. Biochemical Pharmacology, 33, 302 53035. Kroll, M.H., Zavoico, G.B. & Schafer, A.I. (1988) Control of platelet protein kinase C activation by cyclic AMP. Biochimica et Biophysica A c ~ u ,970, 61-67. Lough, J. &Moore,S. (1975) Endothelial injury induced by thrombin or thrombi. Laboratory Investigation, 33, 130-135. Marcus, A.J.. Silk, S.T., Safier. L.B. & Ul1man.H.L. (1977)Superoxide production and reducing activity in human platelets. Journal of Clinical Investigation, 59, 149-158.

150

Mariko Okada et al

Masson. D. & Tschopp. J. (1987) A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell. 49, 679-685. McCrae. K.R., Shattil. S.J. & Cines, D.B. ( 1 9 9 0 ) Platelet activation induces increased Fcy receptor expression. Journal of Immunology. 144, 3920-3927. Okada. M. & Utsumi. S. (19 8 9 ) Role for the third constant domain of the IgG H chain in activation of complement in the presence of C1 inhibitor. Journal of ~mmunologg.142, 195-201. Oxholm. P. & Winther. K. (1986) Thrombocyte involvement in immune inflammatory reactions. Allergg. 4 1 , 1- 10. Pancre, V.. Joseph, M.. Mazingue. C.. Wietzerbin. A,. Capron. A. & Auriault, C. (1987) Induction of platelet cytotoxic functions by lymphokines: role of interferon-;.. Journal of Immunology. 138, 4490-4495. Pasternack. M.S., Vervet. C.R.. Liu. M.A. & Eisen. H.N. ( I 9 8 6 ) Serine esterase in cytolytic T lymphocytes. Nature. 322. 740-743. Pontrenoli. S.. Melloni. E.. Michetti, M., Sacco. 0.. Sparatore. B.. Salamino. F.. Damiani. G. & Horecker. B.L. ( 1 9 8 6 )Cytolytic effects of neutrophils: role for a membrane-bound neutral protease. Proceedings ofthe Yutioml Acndem!j ofSciences ofthe United States of America. 83. 3685-1689. Sagawa. S . . Kodama. T.. Tominaga. A. & Okada. M. ( 1990) Human platelets effectively kill K-562 cells. a chronic myelogenic leukemia cell line, in vitro. fapanese JournalofCancer Reseurch. 81.449-4 5 3 . Slezak. S.. Symer. D.E. & Shin. H.S. ( 1 9 8 7 ) Platelet-mediated

cytotoxicity. Role of antibody and C3. and localization of the cytotoxic system in membranes. Journal of Experimental Medicine, 166,489-505. Smith, J.B.. Ingerman, C.M. & Silver. M.J. (1976) Formation of prostaglandin D2 during endoperoxide-induced platelet aggregation. Thrombosis Research, 9, 413-418. Soper, W.D.. Bartlett. S.P. & Winn. H.J. (1982) Lysis of antibodycoated cells by platelets. Journal o/ Experimental Medicine, 156, 12 10-1 2 2 1. Wiedmer. T. & Sim. P.J. (1 98 5) M e e t of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential. Journal of Biological Chemistry, 260, 80148019. Wintrobe. M.M. (1981) The red cell fragmentation syndromes. Clinical Haernatology, 8th edn (ed. by M. M. Wintrobe), pp. 958977. Lea & Febiger. Philadelphia. Worner. P. (19 8 1 ) Arachidonic acid-induced chemiluminescence of human platelets: contribution of the prostaglandin and lipoxygenase pathways. Thrombosis and Huemostasis. 46, 584-589. Yong. E.C.. Chi, E.Y.. Fritsche, T.R. & Henderson, W.R., Jr (1991) Human platelet-mediated cytotoxicity against Toxoplasrna gondii: role of thromboxane. JournalofExperimental Medicine, 1 7 3 , 6 5 7 8 . Young, J.D.-E..Leong. L.G.. Liu. C.-C., Damiano. A.. Wall. D.A. & Cohn. Z.A. (1986) Isolation and characterization of a serine esterase from cytolytic T cell granules. Cell. 4 7 , 183-194.