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Free radical-mediated platelet activation by hemoglobin released from red blood cells
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 299, No. 2, December, pp. 220-224, 1992
Free Radical-Mediated Platelet Activation by Hemoglobin Released from Red Blood Cells’ Luigi Iuliano,“,’ Francesco Violi,* Jens Z. Pedersen,t Domenico Pratic&* Giuseppe Rotilio,? and Francesco Balsano* *Institute University
1st Clinical Medicine, University of Rome “La Sapienza, ” 00185 Rome; and tDepartment of Rome “Tor Vergata, ” 00173 Rome, Italy
of
of Biology,
Received May 1, 1992, and in revised form July 29, 1992
It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptorbased, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation Of platelets. (0 1992AcademicPress.1nc.
Platelets play a pivotal role in the mechanism of thrombosis which accounts for a major part of the complications in cardiovascular diseases, the primary cause of death in Western populations. Thrombotic events almost always occur during disturbed blood flow at sites of vascular damage (1) and the importance of plateleterythrocyte interactions have long been known from the predominance of these two cell t,ypes in the composition of thrombi (2-4), and from in. vitro studies (5-7). Since the classical work of Born (5,6) the activation of platelets by erythrocytes has been explained by release of ADP, but evidence in favor of this idea is scarce. The initial events leading to platelet aggregation are not well eluci’ This work was supported by The Andrea Cesalpino Foundation by the CNR Special Project FATMA. ’ To whom correspondence should be addressed.
220
dated, but work from this and other laboratories has demonstrated that platelets are activated through mechanisms involving oxygen free radicals (8-12). Recent studies have been facilitated by the use of platelets primed by preexposure to minute, nonaggregating concentrations of arachidonic acid or collagen (13-16). Primed platelets can be activated by very low (300 IIM) concentrations of H,O,; such levels are easily reached physiologically during inflammatory stimulation of neutrophils (17). Furthermore platelets themselves generate superoxide anions which may act as activator after dismutation to H202 (13). This system of reactive oxygen species operating during the activation process constitutes a possible stage for platelet interactions with other cells. We here demonstrate that submicromolar concentrations of hemoglobin released from damaged red blood cells can induce platelet aggregation. The mechanism of activation does not include ADP, but involves platelet-generated HzOz and a hitherto unknown free radical intermediate species. MATERIALS
AND
METHODS
Materials. Arachidonic acid, ADP, and apyrase were obtained from Sigma; catalase from Calbiochem; deoxyribose and DMP03 from Aldrich; collagen from Menarini; all other chemicals were from Merck. Arachidonic acid stock solutions were prepared in absolute ethanol under nitrogen in the dark and were kept at -20°C. OxyferroHb was obtained from fresh human blood (18). Methemoglobin was prepared by oxidizing oxyferro-hemoglobin with sodium nitrite, followed by purification on Sephadex G-25 (18). Hemoglobin concentrations were determined spectrophotometrically (19), final concentrations were 1 1M unless otherwise stated. Ascorbate freshly prepared in high purity water was used at 60 PM. Carbon monoxide treatment was made by exposing erythrocytes, supernatants, or pure hemoglobin solutions to a gentle flux of pure CO gas for 5 min. Ascorbate and solutions containing iron were prepared in high purity water shortly before the experiments.
and 3 Abbreviations hemoglobin.
used: DMPO, 5,5-dimethyl-l-pyrroline-N-oxide;
0003.9861/92
Hb,
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
RADICAL-MEDIATED
PLATELET
ACTIVATION
u
C
c
1 f
0
1
2
0
1
Time
2
1 I
b I 1
1 2
di
I 3
(min)
FIG. 1.
Representative tracings of platelet aggregation induced by erythmcytes, erythrocyte supernatant, or hemoglobin. A, platelets challenged with erythrocytes at various erythrocyte to platelet ratios (a, 1:2O; h, 1:40; c, 1%)). with CO-treated erythrocytes (d, erythrocyte concentration as in trace a), or without additions (e). B, platelets activated by supernatant of stirred erythrocytes (a), supernat.ant plus apyrase (h), or CO-treated supernatant (c). C, platelets activated by oxyferroHb (a), methemoglobin (b), methemoglobin plus ascorbate (c), or CO-treated hemoglobin (d). Aggregation curves are shown displayed vertically to avoid overlay.
Isolation oj c&s. Platelets, prepared from human blood as previously described (I:~), were used at a concentration of 2.5 X 10’ cells/ml in Hank’s balanced salt solution, calcium and magnesium free, containing 10% (vol/vol) autologous platelet poor plasma. Erythrocytes from human hlood were washed three times and resuspended in isotonic saline solution (1 vol packed red cells to I vol saline solution). Supernatants of 1 ml erythrocytes (50% hematocrit) stirred at 1000 rpm for 3 min were ohtained by renrriftlgation; 10 ~1 was used to induce aggregation. I’latc~kt aggwgntirm expf,rimFnfs. Aggregation was measured photometrically according to Born (20) using a single channel aggregometer (13). Samples of 250 yl or 750 ~1 platelet suspension were preincubated for 5 min and measured at 37°C under continuous stirring. Platelets were primed with nonaggregating concentrations (lo-20 pM) of arachidonic acid (13); identical results were obtained using collagen (0.1-0.2 pg/ml) as primer. Agonists were added within 1 min after priming. The apyrase concentration used was 50 pg/ml; this concentrat,ion was able to block completely the aggregation inducedhy 70 pM ADP (not shown). FSH ,L 172~nsLIrcmcrl,ts. Samples were prepared exactly as in aggregation measurements hut with the addition of 100 mM DMPO. At different reaction times, 40. to 50.~1 aliquots were drawn into glass capillaries, sealed, and measured at room temperature with a Bruker ESP300 instrument (10 mW power at 9.83 GHz, 1 G modulation, 100 G scanning in 5 s, eight scans accumulated). The assignment of the DMPO-OH signal was confirmed by control experiments made under the same conditions. Platelet aggregation was blocked by the spin trap DMPO; this inhibition could he overcome by large concentrations of agonists (collagen or arachidonic acid). Radical formation could also he observed when the DMPO was added half-way through the aggregation process. Thromhoxane A, was measured Qunntificafion ofplate~el products. by a radioimmunoassay (12); dense granule content release was monitored by evaluating the percentage of radiolabeled serotonin secreted (16).
RESULTS
AND
DISCUSSION
Primed platelets were activated rapidly in the presence of very low concentrations of erythrocytes (Fig. lA), ap-
221
BY HEMOGLOBIN
proximately 3000 cells/gl, corresponding to an erythrocyte:platelet ratio of 1530. The effect could be attributed to release of cytosol from ruptured cells because identical results were observed using the supernatant of stirred erythrocytes (Fig. 1B). The activation was not due to ADP release since the use of apyrase or creatine/creatine kinase to eliminate ADP did not influence the aggregation process (Fig. lB), in agreement with a recent report on platelet-erythrocyte interactions (21). Pretreat,ment of erythrocytes with carbon monoxide completely prevented erythrocyte-dependent platelet activation, suggesting a role for hemoglobin (Hb) (Figs. lA, 1B). Consistently, submicromolar concentrations of Hb were found to mimick the effect of erythrocytes and cytosol (Fig. lc). The mechanism of Hb-induced platelet activation was related to the redox-state of the heme-iron as demonstrated by: (i) only oxyferroHb was effective, methemoglobin was not; (ii) methemoglobin became effective in the presence of reductants like ascorbate or glutathione; (iii) CO pretreatment of Hb completely prevented the activation of platelets. Hb-induced platelet activation was identical to that induced by common agonists, as reflected in thromboxane A, production and serotonin release (Table I). It was concluded from these results that the long known effect of erythrocytes on
TABLE Effect,
1
of Hemoglobin, Free Iron, and EDTA-Bound on Primed Platelets LT (c/o)
Control HbFe” HhFe”’ HbFe”’ t ascorbate HhFe” + catalase HhFe”’ t ascorhate + cat.alase HbFe” + deoxvribose” Fe”’ 1 ~UU FeL+ Fe2+ + catalase FeL+ + deoxyribose” EDTA-Fe”’ t ascorhate FDTA-Fe”’ 2 + asrorhate + deoxyribose” EDTA-Fe”’ + ascorhate + catalase
TxA,
(rig/ml)
Iron
5HT
(?h)
15 54 _+4 <5 51 & 3 <5
<1 .?l+-5
<3 46 i :3 <:i 44 +- 4 <3
<5
<3 <:1 <3 43 IL 3 <3 <3 49 i 3
<5
<1
4
15
t3
Note. Activation of platelets was measured as light transmission (LT), thromhoxane production (TxA,), and dense granule content release (5. HT). Control, primed platelets (see Fig. 1 for details). OxyferroHh or methemoglobin was used at a concentration of 1 pM; Fez+ and EDTAFe”’ were 25 PM, unless otherwise stated; ascorbate, 60 FM; catalase, 0.1 mg/ml; deoxyrihose, 5 mM. Data represent means t SD of five separate experiments. ’ similar results were seen with 5 mM mannitol.
222
IULIANO
a
I 3480
1 3520
I 3500 Magnetic
Field
IGI
FIG. 2.
ESR spectra of spin-trapped radicals formed during platelet, activation. (a) Platelets primed with collagen or arachidonic acid at subthreshold levels (0.1-0.2 pg/ml and lo-20 FM, respectively). (b) Primed platelets after addition of 9 PM Fe”+. The DMPO-OH’ signal (0) was seen immediately and reached a maximum within a few minutes, followed by a fast decay; the six-line signal (0) appeared after the first 2-3 min and increased steadily during a lo-min period. (c) Primed platelets after addition of 2 fiM oxyferroHb. The six-line signal appeared immediately and increased with time. The DMPO-OK signal was never observed with Hb, not even when 5 times higher Hb concentrations were used. (d) Same as “co but including 0.6 mg/ml catalase. All spectra shown were recorded at a reaction time of 5 min.
ET AL.
to activate primed platelets is one order of magnitude less than the concentration of free iron needed: 0.5 PM Hb compared to 10 ELM Fe ‘+ . Thus the radical generating system seems to be more complicated than the simple reaction [ 11. The amount of radical formed increased with the Hb concentration to reach a plateau at approximately 2 PM Hb, the same concentration that gave the highest level of platelet aggregation (Fig. 3). No further increase in the size of the radical ESR signal was observed in the range 2-10 PM Hb. In the presence of catalase it was not possible to detect any radical formation (Fig. 2d); the same result was obtained when 5 mM mannitol or deoxyribose was added to the samples (results not shown). Although Hb has been reported to react with HzOz in a Fenton-like manner (24), recent studies have demonstrated the formation of a particular ferry1 state in Hb and other heme-proteins (25-27): Hb(Fe”)02
+ HzOz + Hb(Fe’“)-OH
Fe”’ + H,O, + Fe”+ + OH- + OH
[2]
What is the origin of the sextet ESR signal, assuming that reaction [2] takes place? In the case of methemoglobin, reaction with Hz02 produces a ferry1 radical state with a protein radical formed close to the heme group, probably at a tyrosine residue (25): Hb(Fe”‘)
platelet aggregation is caused by a Hb-mediated redox process. The effects of erythrocytes, oxyferro-Hb, and methemoglobin/ascorbate were blocked completely by catalase, thereby demonstrating a fundamental role for plateletgenerated HzOz in the activation (Table I). Aggregation was also inhibited by free radical scavengers such as deoxyribose and mannitol; taken together these results pointed to a metal-catalyzed formation of radicals from peroxides, like the Fenton reaction (22):
+ OH- + 0,
+ Hz02 + Hb’(Fe’“)-OH
However, methemoglobin gation, and acetylation
+ H20
]31
did not induce platelet aggreof the tyrosine residues of
[II
In accordance with this model, free Fe’+ or EDTA-Fe” was able to activate primed platelets in a process inhibited by catalase and deoxyribose (Table I). The production of hydroxyl radicals was confirmed by electron spin resonance spectroscopy measurements, using the spin trap DMPO to detect the characteristic four-line DMPO-OH adduct signal, together with an unassigned six-line signal, when primed platelets were exposed to Fe’+ (Fig. 2b). The DMPO-OH’ signal gave the expected hyperfine splitting, aN = C$ = 14.9 G (23). However, ESR measurements during activation of platelets with Hb did not reveal OH formation; only the six-line signal was observed (Fig. 2~). Furthermore, the lowest concentration of Hb sufficient
I i.0
I 0.5
1.0 oxyferroHb
1.5
2.0
2.5
3.00
(PM)
3. Dose-dependence of Hb-induced aggregation (A) and free radical formation (0) by human platelets primed with arachidonic acid. Experimental conditions as in Figs. 1 and 2. Data represent means * SD of five separate measurements. The percentage of aggregation was determinated from light transmission curves. Radical formation was measured as the average of the peak-to-peak heights of the six lines in the spectrum, after exclusion of high frequency noise by Fourier transform data analysis (34)
FIG.
RADICAL-MEDIATED
PLATELET
C
I -30
I -20
-10
Magnetic
I 0
Field
I l 20
l lO
J l 30
K4
FIG. 4. Computer simulations of the observed radical ESR spectra. (a) Simulation of the DMPO-OH’ adduct, using aN = 14.9 G and a; = 14.9 G. (b) Simulation of the six-line radical signal, using aN = 15.9 G and a; ~ 29.0 G. (c) Simulation fitting the spectrum in Fig. Zb, composed of 74.3”; DMPO-OH’ and 25.7% six-line radical signal. All simulations were based on a 1.5-G linewidth.
oxyferroHb did not prevent induction of platelet aggregation (not shown). In addition, the spin-trapped ferry1 radical gives a broad, partially anisotropic four-line ESR spectrum, very different from that observed here (28). The sharp sextet signal could be simulated with the coupling constants uN = 15.9 G and CL; = 23.0 G (Fig. 4); these values are typical for a small alkyl radical, whereas alkoxyl and peroxyl radicals give smaller P-hydrogen couplings (29, 30). Interestingly the same radical has been observed during lipid peroxidation experiments (31); it was originally identified as an ethoxy adduct, but this assignment was later shown to be erroneous (29). Preliminary experiments showed that formation of the six-line radical during platelet activation was proportional to the arachidonic acid concentration, but unequivocal assignment of this radical has not yet been achieved. In any case, this radical is derived from the primed platelets and is not observed with unprimed platelets or in samples containing only HzO, and oxyhemoglobin. It should be noted that the radical is also seen with collagen-primed platelets, thus excluding the possibility of an artifact caused by the ethanol introduced with arachidonic acid priming. The correlation between the amount of radical formed and the degree of platelet aggregation (Fig. 3) and the inhibition by specific radical scavengers (Table I) leave little doubt that free radicals participate in Hb-induced platelet activation. This finding supports our previous hypothesis (12-16) that the initial events of platelet activation proceed through a sequence of extracellular reactions including reactive oxygen species and free radicals,
ACTIVATION
BY HEMOGLOBIN
223
which eventually lead to stimulation of the arachidonic acid cascade. The fact that all the components involved exist at submicromolar concentrations shows that these reactions must be highly specific. The radicals clearly act as signal transmitters in the activation process, corresponding to a function as extracellular second messengers. The molecular mechanism of the reaction between Hb and HzOz is very complex and has not been solved in detail (25-27). A similar reaction can be expected with myoglobin; upon reaction with low levels of H,02 myoglobin has recently been show to form an “activated” oxidase state (32). Irrespective of the reaction mechanism, the results presented here suggest that platelet activation by erythrocytes occurs through a hemoglobin-mediated reaction, independent of ADP release. This may give new insight into the mechanism of thrombosis, particularly in clinical conditions where red cells have a key role such as polycythemias (33), and offer new therapeutic approaches, as the use of antioxidants, to prevent cardiovascular diseases. REFERENCES 1. Badimon, L., and Badimon, ,J. .J. (1989) J. Clin. Inuest. 84, 11X41144. 2. Falk, E. (1985) Circulation 71, 699-708. 3. Davies, M. ,J., and Thomas, A. C. (1986) N. En&. J. Med. 310, 11:37-l 140. 4. Ross, R., Masuda, J., Raines, E. W., Gown. A. M., Katsuda, S., Sasahara, M., Malden, L. T., Masuko, H., and Sate, H. (1990) Science 248, 1009-1012. 5. Born, G. V. R., and Wehmeier, A. (1979) Nature 282, 212-213. 6. Born, G. V. R., Hergqvist, D., and Afors, K.-E. (1976) Nature 259, 238-236. 7. Violi. F., Ghiselli, A., Alessandri, C., Frattaroli, S., Iuliano, L., and Balsano, F. (1985) N. En&. J. Med. 313, 1091&1092. 8. Marcus, A. .J., Silk, S. T., Safier, L. B., and IJllman, H. L. (1977) J. C’lin. Inucst. 59, 149-158. 9. Handin, It. I., Karabin, R., and Boxer, G. ,J. (1977) J. Clin. Inuest. 59,959s965. 10. Salvemini, D., De Nucci, G., Sneddon, J. M., and Vane, J. R. (1989) Br. J. Pharmacol. 97, 1145-1150. 11. Salvemini, D., De Nucci, G., and Vane, J. R. (1991) Thromb. Haemostnsis 65, 421-424. 12. Violi, F., Ghiselli, A., Iuliano, I,., Alessandri, C., Cordova, C., and Balsano, F. (1988) Naemostasis 18, 91-98. 13. Iuliano, L., Pratich, D., Ghiselli, A., Bonavita, M. S., and Violi, F. (1991) Arch. Riochem. Biophys. 289, 180-183. 14. Pratico, D., Iuliano, L., Ghiselli, A., Alessandri, C., and Violi, F. (1991) Haenostasis 21, 1699174. 15. Iuliano, L., Pratico, D., Bonavita, M. S., and Violi, F. (1992) Platelets 2, 87 90. 16. Pratico, D.. Iuliano, L., Pulcinelli, F. M., Bonavita, M. S., Gazzaniga, P. P., and Violi, F. (1992) J. Lab. Clin. Med. 119, 364-370. 17. Test, S. T., and Weiss, S. J. (1984) J. Biol. Chem. 259, 399-405. 18. Spagnuolo, C., Rinelli, P., Coletta, M., Chiancone, E., and Ascoli, F. (1987) Biochim. Biophys. Acta 911, 59-65. 19. Winterbourn, C. C. (1985) in CRC Handbook of Methods for Oxygen Free Radical Research (Greenwald, R. A., Ed.), pp. 137-141, CRC Press, Boca Raton, FL.
224
IULIANO
20. Born, G. V. R., and Cross, M. J. (1963) J. Physiol. 168, 178-195. 21. Marcus, A.
G. R. (1987) Free Radical
Biol.
Med.
3, 259-303.
23. Halliwell, B., and Gutteridge, G. M. C. (1989) Free Radicals in Biology and Medicine, 2nd ed., Oxford Univ. Press (Clarendon), London/New York. 24. Sadrzadeh, S. M. H., Grat’, E., Panter, S. S., Hallaway, Eaton, J. W. (1984) J. Riol. (‘hem. 259, 14354-14356.
P. E., and
25. La Mar, G. N., de Ropp, ,J. S., Latos-Grazynski, L., Balch, A. L.,
265,
19453-
ET AL. 27. Bielski, B. H. d. (1991) Free Radical Res. Commun. 12-13, 469477. 28. Davies, M. J. (1990) Free Radical Res. Commun. 10, 361-370. 29. Janzen, E. G. (1980) in Free Radicals in Biology, (Pryor, W. A., Ed.), Vol. 4, pp. 115-154, Academic Press, New York. 30. Davies, M. J., and Slater, T. F. (1986) Biochem. J. 240, 789-795. 31. Lai, C.-S., and Piette, I,. H. (1977) Biochem. Biophys. Res. Commun. 78,51-62. 32. Osawa, Y., and Korzekwa, K. (1991) Proc. N&l. Acad. Sci. USA 88, 7081b7085. ~(3. Smith, B. I>., and La Celle, P. L. (1982) in Progress in Hemostasis and Thrombosis (Spaet, T. H., Ed.), Vol. 6, pp. 179-201, Grune and Stratton, New York. 27, 34. Pedersen, .J. Z., Musci, G., and Rotilio, G. (1988) Biochemistry 8534N3536.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 299, No. 2, December, pp. 220-224, 1992
Free Radical-Mediated Platelet Activation by Hemoglobin Released from Red Blood Cells’ Luigi Iuliano,“,’ Francesco Violi,* Jens Z. Pedersen,t Domenico Pratic&* Giuseppe Rotilio,? and Francesco Balsano* *Institute University
1st Clinical Medicine, University of Rome “La Sapienza, ” 00185 Rome; and tDepartment of Rome “Tor Vergata, ” 00173 Rome, Italy
of
of Biology,
Received May 1, 1992, and in revised form July 29, 1992
It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptorbased, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation Of platelets. (0 1992AcademicPress.1nc.
Platelets play a pivotal role in the mechanism of thrombosis which accounts for a major part of the complications in cardiovascular diseases, the primary cause of death in Western populations. Thrombotic events almost always occur during disturbed blood flow at sites of vascular damage (1) and the importance of plateleterythrocyte interactions have long been known from the predominance of these two cell t,ypes in the composition of thrombi (2-4), and from in. vitro studies (5-7). Since the classical work of Born (5,6) the activation of platelets by erythrocytes has been explained by release of ADP, but evidence in favor of this idea is scarce. The initial events leading to platelet aggregation are not well eluci’ This work was supported by The Andrea Cesalpino Foundation by the CNR Special Project FATMA. ’ To whom correspondence should be addressed.
220
dated, but work from this and other laboratories has demonstrated that platelets are activated through mechanisms involving oxygen free radicals (8-12). Recent studies have been facilitated by the use of platelets primed by preexposure to minute, nonaggregating concentrations of arachidonic acid or collagen (13-16). Primed platelets can be activated by very low (300 IIM) concentrations of H,O,; such levels are easily reached physiologically during inflammatory stimulation of neutrophils (17). Furthermore platelets themselves generate superoxide anions which may act as activator after dismutation to H202 (13). This system of reactive oxygen species operating during the activation process constitutes a possible stage for platelet interactions with other cells. We here demonstrate that submicromolar concentrations of hemoglobin released from damaged red blood cells can induce platelet aggregation. The mechanism of activation does not include ADP, but involves platelet-generated HzOz and a hitherto unknown free radical intermediate species. MATERIALS
AND
METHODS
Materials. Arachidonic acid, ADP, and apyrase were obtained from Sigma; catalase from Calbiochem; deoxyribose and DMP03 from Aldrich; collagen from Menarini; all other chemicals were from Merck. Arachidonic acid stock solutions were prepared in absolute ethanol under nitrogen in the dark and were kept at -20°C. OxyferroHb was obtained from fresh human blood (18). Methemoglobin was prepared by oxidizing oxyferro-hemoglobin with sodium nitrite, followed by purification on Sephadex G-25 (18). Hemoglobin concentrations were determined spectrophotometrically (19), final concentrations were 1 1M unless otherwise stated. Ascorbate freshly prepared in high purity water was used at 60 PM. Carbon monoxide treatment was made by exposing erythrocytes, supernatants, or pure hemoglobin solutions to a gentle flux of pure CO gas for 5 min. Ascorbate and solutions containing iron were prepared in high purity water shortly before the experiments.
and 3 Abbreviations hemoglobin.
used: DMPO, 5,5-dimethyl-l-pyrroline-N-oxide;
0003.9861/92
Hb,
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
RADICAL-MEDIATED
PLATELET
ACTIVATION
u
C
c
1 f
0
1
2
0
1
Time
2
1 I
b I 1
1 2
di
I 3
(min)
FIG. 1.
Representative tracings of platelet aggregation induced by erythmcytes, erythrocyte supernatant, or hemoglobin. A, platelets challenged with erythrocytes at various erythrocyte to platelet ratios (a, 1:2O; h, 1:40; c, 1%)). with CO-treated erythrocytes (d, erythrocyte concentration as in trace a), or without additions (e). B, platelets activated by supernatant of stirred erythrocytes (a), supernat.ant plus apyrase (h), or CO-treated supernatant (c). C, platelets activated by oxyferroHb (a), methemoglobin (b), methemoglobin plus ascorbate (c), or CO-treated hemoglobin (d). Aggregation curves are shown displayed vertically to avoid overlay.
Isolation oj c&s. Platelets, prepared from human blood as previously described (I:~), were used at a concentration of 2.5 X 10’ cells/ml in Hank’s balanced salt solution, calcium and magnesium free, containing 10% (vol/vol) autologous platelet poor plasma. Erythrocytes from human hlood were washed three times and resuspended in isotonic saline solution (1 vol packed red cells to I vol saline solution). Supernatants of 1 ml erythrocytes (50% hematocrit) stirred at 1000 rpm for 3 min were ohtained by renrriftlgation; 10 ~1 was used to induce aggregation. I’latc~kt aggwgntirm expf,rimFnfs. Aggregation was measured photometrically according to Born (20) using a single channel aggregometer (13). Samples of 250 yl or 750 ~1 platelet suspension were preincubated for 5 min and measured at 37°C under continuous stirring. Platelets were primed with nonaggregating concentrations (lo-20 pM) of arachidonic acid (13); identical results were obtained using collagen (0.1-0.2 pg/ml) as primer. Agonists were added within 1 min after priming. The apyrase concentration used was 50 pg/ml; this concentrat,ion was able to block completely the aggregation inducedhy 70 pM ADP (not shown). FSH ,L 172~nsLIrcmcrl,ts. Samples were prepared exactly as in aggregation measurements hut with the addition of 100 mM DMPO. At different reaction times, 40. to 50.~1 aliquots were drawn into glass capillaries, sealed, and measured at room temperature with a Bruker ESP300 instrument (10 mW power at 9.83 GHz, 1 G modulation, 100 G scanning in 5 s, eight scans accumulated). The assignment of the DMPO-OH signal was confirmed by control experiments made under the same conditions. Platelet aggregation was blocked by the spin trap DMPO; this inhibition could he overcome by large concentrations of agonists (collagen or arachidonic acid). Radical formation could also he observed when the DMPO was added half-way through the aggregation process. Thromhoxane A, was measured Qunntificafion ofplate~el products. by a radioimmunoassay (12); dense granule content release was monitored by evaluating the percentage of radiolabeled serotonin secreted (16).
RESULTS
AND
DISCUSSION
Primed platelets were activated rapidly in the presence of very low concentrations of erythrocytes (Fig. lA), ap-
221
BY HEMOGLOBIN
proximately 3000 cells/gl, corresponding to an erythrocyte:platelet ratio of 1530. The effect could be attributed to release of cytosol from ruptured cells because identical results were observed using the supernatant of stirred erythrocytes (Fig. 1B). The activation was not due to ADP release since the use of apyrase or creatine/creatine kinase to eliminate ADP did not influence the aggregation process (Fig. lB), in agreement with a recent report on platelet-erythrocyte interactions (21). Pretreat,ment of erythrocytes with carbon monoxide completely prevented erythrocyte-dependent platelet activation, suggesting a role for hemoglobin (Hb) (Figs. lA, 1B). Consistently, submicromolar concentrations of Hb were found to mimick the effect of erythrocytes and cytosol (Fig. lc). The mechanism of Hb-induced platelet activation was related to the redox-state of the heme-iron as demonstrated by: (i) only oxyferroHb was effective, methemoglobin was not; (ii) methemoglobin became effective in the presence of reductants like ascorbate or glutathione; (iii) CO pretreatment of Hb completely prevented the activation of platelets. Hb-induced platelet activation was identical to that induced by common agonists, as reflected in thromboxane A, production and serotonin release (Table I). It was concluded from these results that the long known effect of erythrocytes on
TABLE Effect,
1
of Hemoglobin, Free Iron, and EDTA-Bound on Primed Platelets LT (c/o)
Control HbFe” HhFe”’ HbFe”’ t ascorbate HhFe” + catalase HhFe”’ t ascorhate + cat.alase HbFe” + deoxvribose” Fe”’ 1 ~UU FeL+ Fe2+ + catalase FeL+ + deoxyribose” EDTA-Fe”’ t ascorhate FDTA-Fe”’ 2 + asrorhate + deoxyribose” EDTA-Fe”’ + ascorhate + catalase
TxA,
(rig/ml)
Iron
5HT
(?h)
15 54 _+4 <5 51 & 3 <5
<1 .?l+-5
<3 46 i :3 <:i 44 +- 4 <3
<5
<3 <:1 <3 43 IL 3 <3 <3 49 i 3
<5
<1
4
15
t3
Note. Activation of platelets was measured as light transmission (LT), thromhoxane production (TxA,), and dense granule content release (5. HT). Control, primed platelets (see Fig. 1 for details). OxyferroHh or methemoglobin was used at a concentration of 1 pM; Fez+ and EDTAFe”’ were 25 PM, unless otherwise stated; ascorbate, 60 FM; catalase, 0.1 mg/ml; deoxyrihose, 5 mM. Data represent means t SD of five separate experiments. ’ similar results were seen with 5 mM mannitol.
222
IULIANO
a
I 3480
1 3520
I 3500 Magnetic
Field
IGI
FIG. 2.
ESR spectra of spin-trapped radicals formed during platelet, activation. (a) Platelets primed with collagen or arachidonic acid at subthreshold levels (0.1-0.2 pg/ml and lo-20 FM, respectively). (b) Primed platelets after addition of 9 PM Fe”+. The DMPO-OH’ signal (0) was seen immediately and reached a maximum within a few minutes, followed by a fast decay; the six-line signal (0) appeared after the first 2-3 min and increased steadily during a lo-min period. (c) Primed platelets after addition of 2 fiM oxyferroHb. The six-line signal appeared immediately and increased with time. The DMPO-OK signal was never observed with Hb, not even when 5 times higher Hb concentrations were used. (d) Same as “co but including 0.6 mg/ml catalase. All spectra shown were recorded at a reaction time of 5 min.
ET AL.
to activate primed platelets is one order of magnitude less than the concentration of free iron needed: 0.5 PM Hb compared to 10 ELM Fe ‘+ . Thus the radical generating system seems to be more complicated than the simple reaction [ 11. The amount of radical formed increased with the Hb concentration to reach a plateau at approximately 2 PM Hb, the same concentration that gave the highest level of platelet aggregation (Fig. 3). No further increase in the size of the radical ESR signal was observed in the range 2-10 PM Hb. In the presence of catalase it was not possible to detect any radical formation (Fig. 2d); the same result was obtained when 5 mM mannitol or deoxyribose was added to the samples (results not shown). Although Hb has been reported to react with HzOz in a Fenton-like manner (24), recent studies have demonstrated the formation of a particular ferry1 state in Hb and other heme-proteins (25-27): Hb(Fe”)02
+ HzOz + Hb(Fe’“)-OH
Fe”’ + H,O, + Fe”+ + OH- + OH
[2]
What is the origin of the sextet ESR signal, assuming that reaction [2] takes place? In the case of methemoglobin, reaction with Hz02 produces a ferry1 radical state with a protein radical formed close to the heme group, probably at a tyrosine residue (25): Hb(Fe”‘)
platelet aggregation is caused by a Hb-mediated redox process. The effects of erythrocytes, oxyferro-Hb, and methemoglobin/ascorbate were blocked completely by catalase, thereby demonstrating a fundamental role for plateletgenerated HzOz in the activation (Table I). Aggregation was also inhibited by free radical scavengers such as deoxyribose and mannitol; taken together these results pointed to a metal-catalyzed formation of radicals from peroxides, like the Fenton reaction (22):
+ OH- + 0,
+ Hz02 + Hb’(Fe’“)-OH
However, methemoglobin gation, and acetylation
+ H20
]31
did not induce platelet aggreof the tyrosine residues of
[II
In accordance with this model, free Fe’+ or EDTA-Fe” was able to activate primed platelets in a process inhibited by catalase and deoxyribose (Table I). The production of hydroxyl radicals was confirmed by electron spin resonance spectroscopy measurements, using the spin trap DMPO to detect the characteristic four-line DMPO-OH adduct signal, together with an unassigned six-line signal, when primed platelets were exposed to Fe’+ (Fig. 2b). The DMPO-OH’ signal gave the expected hyperfine splitting, aN = C$ = 14.9 G (23). However, ESR measurements during activation of platelets with Hb did not reveal OH formation; only the six-line signal was observed (Fig. 2~). Furthermore, the lowest concentration of Hb sufficient
I i.0
I 0.5
1.0 oxyferroHb
1.5
2.0
2.5
3.00
(PM)
3. Dose-dependence of Hb-induced aggregation (A) and free radical formation (0) by human platelets primed with arachidonic acid. Experimental conditions as in Figs. 1 and 2. Data represent means * SD of five separate measurements. The percentage of aggregation was determinated from light transmission curves. Radical formation was measured as the average of the peak-to-peak heights of the six lines in the spectrum, after exclusion of high frequency noise by Fourier transform data analysis (34)
FIG.
RADICAL-MEDIATED
PLATELET
C
I -30
I -20
-10
Magnetic
I 0
Field
I l 20
l lO
J l 30
K4
FIG. 4. Computer simulations of the observed radical ESR spectra. (a) Simulation of the DMPO-OH’ adduct, using aN = 14.9 G and a; = 14.9 G. (b) Simulation of the six-line radical signal, using aN = 15.9 G and a; ~ 29.0 G. (c) Simulation fitting the spectrum in Fig. Zb, composed of 74.3”; DMPO-OH’ and 25.7% six-line radical signal. All simulations were based on a 1.5-G linewidth.
oxyferroHb did not prevent induction of platelet aggregation (not shown). In addition, the spin-trapped ferry1 radical gives a broad, partially anisotropic four-line ESR spectrum, very different from that observed here (28). The sharp sextet signal could be simulated with the coupling constants uN = 15.9 G and CL; = 23.0 G (Fig. 4); these values are typical for a small alkyl radical, whereas alkoxyl and peroxyl radicals give smaller P-hydrogen couplings (29, 30). Interestingly the same radical has been observed during lipid peroxidation experiments (31); it was originally identified as an ethoxy adduct, but this assignment was later shown to be erroneous (29). Preliminary experiments showed that formation of the six-line radical during platelet activation was proportional to the arachidonic acid concentration, but unequivocal assignment of this radical has not yet been achieved. In any case, this radical is derived from the primed platelets and is not observed with unprimed platelets or in samples containing only HzO, and oxyhemoglobin. It should be noted that the radical is also seen with collagen-primed platelets, thus excluding the possibility of an artifact caused by the ethanol introduced with arachidonic acid priming. The correlation between the amount of radical formed and the degree of platelet aggregation (Fig. 3) and the inhibition by specific radical scavengers (Table I) leave little doubt that free radicals participate in Hb-induced platelet activation. This finding supports our previous hypothesis (12-16) that the initial events of platelet activation proceed through a sequence of extracellular reactions including reactive oxygen species and free radicals,
ACTIVATION
BY HEMOGLOBIN
223
which eventually lead to stimulation of the arachidonic acid cascade. The fact that all the components involved exist at submicromolar concentrations shows that these reactions must be highly specific. The radicals clearly act as signal transmitters in the activation process, corresponding to a function as extracellular second messengers. The molecular mechanism of the reaction between Hb and HzOz is very complex and has not been solved in detail (25-27). A similar reaction can be expected with myoglobin; upon reaction with low levels of H,02 myoglobin has recently been show to form an “activated” oxidase state (32). Irrespective of the reaction mechanism, the results presented here suggest that platelet activation by erythrocytes occurs through a hemoglobin-mediated reaction, independent of ADP release. This may give new insight into the mechanism of thrombosis, particularly in clinical conditions where red cells have a key role such as polycythemias (33), and offer new therapeutic approaches, as the use of antioxidants, to prevent cardiovascular diseases. REFERENCES 1. Badimon, L., and Badimon, ,J. .J. (1989) J. Clin. Inuest. 84, 11X41144. 2. Falk, E. (1985) Circulation 71, 699-708. 3. Davies, M. ,J., and Thomas, A. C. (1986) N. En&. J. Med. 310, 11:37-l 140. 4. Ross, R., Masuda, J., Raines, E. W., Gown. A. M., Katsuda, S., Sasahara, M., Malden, L. T., Masuko, H., and Sate, H. (1990) Science 248, 1009-1012. 5. Born, G. V. R., and Wehmeier, A. (1979) Nature 282, 212-213. 6. Born, G. V. R., Hergqvist, D., and Afors, K.-E. (1976) Nature 259, 238-236. 7. Violi. F., Ghiselli, A., Alessandri, C., Frattaroli, S., Iuliano, L., and Balsano, F. (1985) N. En&. J. Med. 313, 1091&1092. 8. Marcus, A. .J., Silk, S. T., Safier, L. B., and IJllman, H. L. (1977) J. C’lin. Inucst. 59, 149-158. 9. Handin, It. I., Karabin, R., and Boxer, G. ,J. (1977) J. Clin. Inuest. 59,959s965. 10. Salvemini, D., De Nucci, G., Sneddon, J. M., and Vane, J. R. (1989) Br. J. Pharmacol. 97, 1145-1150. 11. Salvemini, D., De Nucci, G., and Vane, J. R. (1991) Thromb. Haemostnsis 65, 421-424. 12. Violi, F., Ghiselli, A., Iuliano, I,., Alessandri, C., Cordova, C., and Balsano, F. (1988) Naemostasis 18, 91-98. 13. Iuliano, L., Pratich, D., Ghiselli, A., Bonavita, M. S., and Violi, F. (1991) Arch. Riochem. Biophys. 289, 180-183. 14. Pratico, D., Iuliano, L., Ghiselli, A., Alessandri, C., and Violi, F. (1991) Haenostasis 21, 1699174. 15. Iuliano, L., Pratico, D., Bonavita, M. S., and Violi, F. (1992) Platelets 2, 87 90. 16. Pratico, D.. Iuliano, L., Pulcinelli, F. M., Bonavita, M. S., Gazzaniga, P. P., and Violi, F. (1992) J. Lab. Clin. Med. 119, 364-370. 17. Test, S. T., and Weiss, S. J. (1984) J. Biol. Chem. 259, 399-405. 18. Spagnuolo, C., Rinelli, P., Coletta, M., Chiancone, E., and Ascoli, F. (1987) Biochim. Biophys. Acta 911, 59-65. 19. Winterbourn, C. C. (1985) in CRC Handbook of Methods for Oxygen Free Radical Research (Greenwald, R. A., Ed.), pp. 137-141, CRC Press, Boca Raton, FL.
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