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Platelet aggregation after inhibition of ADP release
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Short Communications P L A T E L E T A G G R E G A T I O N A F T E R I N H I B I T I O N OF ADP R E L E A S E
Y. H. ABDULLA Department of Pathology, Guy's Hospital Medical School, London, S.E. l (Great Britain)
(Received May 3Oth, 1968)
SUMMARY Atractyloside was nto the ambient fluid, aggregation b y collagen, some doubt on the view gation mechanisms.
shown to inhibit the i n vitro release of ADP from platelets but its addition did not prevent or even reduce platelet adrenalin and 5-hydroxytryptamine. This observation casts that ADP release is the final common pathway of all aggre-
K e y words: Platelet aggregation - - Cell m e m b r a n e - - A D P release - - Collagen - - A d r e n olin -- 5-Hydroxytryptamine -- Atractyloside
INTRODUCTION I t is commonly accepted that thrombocyte aggregation induced b y a variety of dissimilar compounds is due to release of adenosine 5'-diphosphate (ADP) into the ambient fluid; the released ADP then causes aggregation 1-6. The basis for this assumption is impressive; aggregation b y whatever agent is accompanied b y release of ADP, and this compound is known to be a powerful aggregating agent. The causal connection, however, has never been tested stringently. Atractyloside inhibits both entry and exit of adenine nucleotides across the mitochondriaI membrane 7-12. If atractylate should have similar effect on the platelet membrane it would be possible directly to test whether ADP release is a necessary and sufficient prerequisite for the induction of aggregation. METHODS The preparation of citrated platelet-rich plasma and the spectrophotometric detection of aggregation were carried out as described in a recent publication b y BORN AND HUME 13. J. Atheroscler. Res., 1968, 8:855-858
856
SHORT COMMUNICATIONS
Platelet aggregation was studied at 20~ in samples of platelet-rich plasma with the following additions: (1) 0.2 ml collagen suspension 14, (2) 100 # M atractyloside + 0.2 ml collagen suspension, (3) 5/~M adrenalin, (4) 100/~M atractyloside + 5 #M adrenalin, (5) 5/~M 5-hydroxytryptamine, (6) 100/~M atractyloside + 5/~M 5-hydroxytryptamine (5HT), (7) 1 #M ADP, (8) 100/zM atractyloside + 1 # M ADP. After complete recording of aggregation, the samples were centrifuged at 700 • g for 10 rain at 4~ A sample of platelet-rich plasma without any additions was included as a control. ADP was estimated in the platelet-free plasma by ADAM'S method 15. RESULTS
Aggregation induced by any of the specific aggregating agents tried, remained unaltered (or even increased) after addition of atractylate (Fig. 1). The effect of atractyloside on ADP release is summarised in Table 1. These results are representative
TABLE
1
EFFECT OF ATRACTYLOSIDE ON A D P AND ADRENALIN
RELEASE INDUCED BY COLLAGEN, 5-HYDROXYTRYPTAMINE
A D P in supernatant (m#moles[ml)
XVithout a t r a c t y l o s i d e \Vith atractyloside
collagen
adrenalin
5HT
A DP
no additions
16 1
13 0.8
10 0.8
0.9 1.6
0.8 1.0
of at least ten consecutive studies. Addition of atraetyloside either 30 rain or immediately before incubation with the aggregating agent did not alter the results. DISCUSSION
The results show that collagen, adrenalin and 5-hydroxytryptamine do induce aggregation in the absence of ADP release into the ambient plasma. Apparently atractyloside inhibits release of ADP without affecting aggregation. The drug, however, did not prevent aggregation due to extraneous added ADP. This means that added ADP need not enter the platelet to cause aggregation. The relation between aggregation by ADP and that induced by other aggregating j . Atheroscler. Res., 1968, 8 : 8 5 5 - 8 5 8
SHORT
857
COMMUNICATIONS
(a)
OPTICAL DENSITY 1,5 1,/,
A0P
t.Z. tO-
ADP + Atracty[ate
0.8" 0.O" 0.t.
L
TIME (minutes)
OPTICAL 0ENSITY 1.6.
(b)
1.1,.
1.2. Cottagen
1,00.8.
CoLtageh § Atractytate 0.5 O,
1
2
3
OPTICAL 0ENSITY 1,6.
~
TIME (m;nutes)
5
(c) 5HT
1./,,,
5 HT * Atr
].,Z84 Adr
1.0, Adr + A i r 0,8 0.6 0.~
i
i
i
L
g
TIME (minutes)
Fig. 1. Effect of atractyloside on aggregation of platelets by (a) ADP, (b) collagen, (c) adrenalin and 5'-hydroxytryptamine. agents can be viewed in two different ways. If we still want to retain A D P as the point at which the two mechanisms converge, it is possible to postulate that although atractyloside prevents translocation of A D P across the membrane, it m a y still allow endogenous A D P to attach to specific sites in the membrane without being released. However, the point at which these mechanisms converge m a y be b e y o n d the stage where A D P induces aggregation. The situation is then similar to stimulation of sarcolemma with an external electric stimulus or b y application of acetyl choline. ACKNOWLEDGEMENTS I am indebted to Prof. N. Siliprandi for a generous gift of atractyloside and to Prof. C. W. M. A d a m s for his guidance and encouragement. The work was supported b y the British Heart Foundation. J. Atheroscler. Res., 1968, 8:855-858
858
SHORT COMMUNICATIONS
REFERENCES 1 SPAET, T. H. AND M. B. ZUCKER, Mecbanism of platelet plug and role of adenosine triphosphate, Amer. J. Physiol., 1964, 206: 1267. 2 HOVlG, T., Release of platelet aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline "extract" of tendons, Thrombos. Diathes. haemorrh. (Stuttg.), 1963, 9: 264. a ZUCKER, M. B. AND J. BORELLI, Platelet clumping produced by connective tissue suspension and by collagen, Proc. Soc. exp. Biol. (N.Y.), 1962, 109: 779. 4 GLYNN, M. F., H. Z. MOVAT, E. A. MURPHY AND J. F. MUSTARD, Study of platelet adhesiveness and aggregation with latex particles, J. Lab. din. Med., 1965, 65: 179. 5 HASLAlVI,R. J., Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids, Nature (Loud.), 1964, 202: 765. 6 BORN, G. V. R., Mechanism of platelet aggregation and of its inhibition by adenosine derivatives, Fed. Proc., 1967, 26: 115. 7 CHAPPELL, J. B. AND A. R. CROFTS, Effect of atractylate and oligomycin on the behaviour of mitochondria towards adenine nucleotides, Biochem. J., 1965, 95: 707. 8 BRUNI, A., S. LOClANI AND A. R. CONTESSA, Inhibition by atractyloside of the binding of adenine nucleotides to rat liver mitochondria, Nature (Lond.), 1964, 201: 1219. 9 BRUNI, A. AND G. F. AZZONE, The sites of action of atractyloside and oligomycin in the mitochondrial energy transfer system, Biochim. biophys. Aeta, 1964, 93: 462. 10 PFAFF, E., M. KLINGENBERG AND H. W. HELDT, Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria, Biochim. biophys. Acta, 1965, 104: 312. 11 BRIELEY, G. AND R. L. O']3RIEN, Compartmentation of heart mitochondria, Part 2 (Mitochondrial adenine nucleotides and the action of atractyloside), J. biol. Chem., 1965, 240: 4532. 12 DU]~E, E. D. AND P. V. VIGNAIS, ]~change entre addnine-nucldotidesextra-et intramitochondriaux, Biochim. biophys. Acta, 1965, 107: 184. 13 BORN, G. ~V-.~R. AND M. HUME, Effects of numbers and sizes of platelet aggregates on the optical density of plasma, Nature (Loud.), 1967, 214: 1084. 14 CONSTANTINE, J. W., Aggregation and adhesion of rat platelets, Nature (Lond.), 1967, 214: 1084. 15 ADAM, H., Adenosine 5' diphosphate and adenosine 5' monophosphate. In: H.-U. BERGMYER (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, London, 1963, p. 573.
j . Atheroscler. Res., 1968, 8:855-858
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Short Communications P L A T E L E T A G G R E G A T I O N A F T E R I N H I B I T I O N OF ADP R E L E A S E
Y. H. ABDULLA Department of Pathology, Guy's Hospital Medical School, London, S.E. l (Great Britain)
(Received May 3Oth, 1968)
SUMMARY Atractyloside was nto the ambient fluid, aggregation b y collagen, some doubt on the view gation mechanisms.
shown to inhibit the i n vitro release of ADP from platelets but its addition did not prevent or even reduce platelet adrenalin and 5-hydroxytryptamine. This observation casts that ADP release is the final common pathway of all aggre-
K e y words: Platelet aggregation - - Cell m e m b r a n e - - A D P release - - Collagen - - A d r e n olin -- 5-Hydroxytryptamine -- Atractyloside
INTRODUCTION I t is commonly accepted that thrombocyte aggregation induced b y a variety of dissimilar compounds is due to release of adenosine 5'-diphosphate (ADP) into the ambient fluid; the released ADP then causes aggregation 1-6. The basis for this assumption is impressive; aggregation b y whatever agent is accompanied b y release of ADP, and this compound is known to be a powerful aggregating agent. The causal connection, however, has never been tested stringently. Atractyloside inhibits both entry and exit of adenine nucleotides across the mitochondriaI membrane 7-12. If atractylate should have similar effect on the platelet membrane it would be possible directly to test whether ADP release is a necessary and sufficient prerequisite for the induction of aggregation. METHODS The preparation of citrated platelet-rich plasma and the spectrophotometric detection of aggregation were carried out as described in a recent publication b y BORN AND HUME 13. J. Atheroscler. Res., 1968, 8:855-858
856
SHORT COMMUNICATIONS
Platelet aggregation was studied at 20~ in samples of platelet-rich plasma with the following additions: (1) 0.2 ml collagen suspension 14, (2) 100 # M atractyloside + 0.2 ml collagen suspension, (3) 5/~M adrenalin, (4) 100/~M atractyloside + 5 #M adrenalin, (5) 5/~M 5-hydroxytryptamine, (6) 100/~M atractyloside + 5/~M 5-hydroxytryptamine (5HT), (7) 1 #M ADP, (8) 100/zM atractyloside + 1 # M ADP. After complete recording of aggregation, the samples were centrifuged at 700 • g for 10 rain at 4~ A sample of platelet-rich plasma without any additions was included as a control. ADP was estimated in the platelet-free plasma by ADAM'S method 15. RESULTS
Aggregation induced by any of the specific aggregating agents tried, remained unaltered (or even increased) after addition of atractylate (Fig. 1). The effect of atractyloside on ADP release is summarised in Table 1. These results are representative
TABLE
1
EFFECT OF ATRACTYLOSIDE ON A D P AND ADRENALIN
RELEASE INDUCED BY COLLAGEN, 5-HYDROXYTRYPTAMINE
A D P in supernatant (m#moles[ml)
XVithout a t r a c t y l o s i d e \Vith atractyloside
collagen
adrenalin
5HT
A DP
no additions
16 1
13 0.8
10 0.8
0.9 1.6
0.8 1.0
of at least ten consecutive studies. Addition of atraetyloside either 30 rain or immediately before incubation with the aggregating agent did not alter the results. DISCUSSION
The results show that collagen, adrenalin and 5-hydroxytryptamine do induce aggregation in the absence of ADP release into the ambient plasma. Apparently atractyloside inhibits release of ADP without affecting aggregation. The drug, however, did not prevent aggregation due to extraneous added ADP. This means that added ADP need not enter the platelet to cause aggregation. The relation between aggregation by ADP and that induced by other aggregating j . Atheroscler. Res., 1968, 8 : 8 5 5 - 8 5 8
SHORT
857
COMMUNICATIONS
(a)
OPTICAL DENSITY 1,5 1,/,
A0P
t.Z. tO-
ADP + Atracty[ate
0.8" 0.O" 0.t.
L
TIME (minutes)
OPTICAL 0ENSITY 1.6.
(b)
1.1,.
1.2. Cottagen
1,00.8.
CoLtageh § Atractytate 0.5 O,
1
2
3
OPTICAL 0ENSITY 1,6.
~
TIME (m;nutes)
5
(c) 5HT
1./,,,
5 HT * Atr
].,Z84 Adr
1.0, Adr + A i r 0,8 0.6 0.~
i
i
i
L
g
TIME (minutes)
Fig. 1. Effect of atractyloside on aggregation of platelets by (a) ADP, (b) collagen, (c) adrenalin and 5'-hydroxytryptamine. agents can be viewed in two different ways. If we still want to retain A D P as the point at which the two mechanisms converge, it is possible to postulate that although atractyloside prevents translocation of A D P across the membrane, it m a y still allow endogenous A D P to attach to specific sites in the membrane without being released. However, the point at which these mechanisms converge m a y be b e y o n d the stage where A D P induces aggregation. The situation is then similar to stimulation of sarcolemma with an external electric stimulus or b y application of acetyl choline. ACKNOWLEDGEMENTS I am indebted to Prof. N. Siliprandi for a generous gift of atractyloside and to Prof. C. W. M. A d a m s for his guidance and encouragement. The work was supported b y the British Heart Foundation. J. Atheroscler. Res., 1968, 8:855-858
858
SHORT COMMUNICATIONS
REFERENCES 1 SPAET, T. H. AND M. B. ZUCKER, Mecbanism of platelet plug and role of adenosine triphosphate, Amer. J. Physiol., 1964, 206: 1267. 2 HOVlG, T., Release of platelet aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline "extract" of tendons, Thrombos. Diathes. haemorrh. (Stuttg.), 1963, 9: 264. a ZUCKER, M. B. AND J. BORELLI, Platelet clumping produced by connective tissue suspension and by collagen, Proc. Soc. exp. Biol. (N.Y.), 1962, 109: 779. 4 GLYNN, M. F., H. Z. MOVAT, E. A. MURPHY AND J. F. MUSTARD, Study of platelet adhesiveness and aggregation with latex particles, J. Lab. din. Med., 1965, 65: 179. 5 HASLAlVI,R. J., Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids, Nature (Loud.), 1964, 202: 765. 6 BORN, G. V. R., Mechanism of platelet aggregation and of its inhibition by adenosine derivatives, Fed. Proc., 1967, 26: 115. 7 CHAPPELL, J. B. AND A. R. CROFTS, Effect of atractylate and oligomycin on the behaviour of mitochondria towards adenine nucleotides, Biochem. J., 1965, 95: 707. 8 BRUNI, A., S. LOClANI AND A. R. CONTESSA, Inhibition by atractyloside of the binding of adenine nucleotides to rat liver mitochondria, Nature (Lond.), 1964, 201: 1219. 9 BRUNI, A. AND G. F. AZZONE, The sites of action of atractyloside and oligomycin in the mitochondrial energy transfer system, Biochim. biophys. Aeta, 1964, 93: 462. 10 PFAFF, E., M. KLINGENBERG AND H. W. HELDT, Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria, Biochim. biophys. Acta, 1965, 104: 312. 11 BRIELEY, G. AND R. L. O']3RIEN, Compartmentation of heart mitochondria, Part 2 (Mitochondrial adenine nucleotides and the action of atractyloside), J. biol. Chem., 1965, 240: 4532. 12 DU]~E, E. D. AND P. V. VIGNAIS, ]~change entre addnine-nucldotidesextra-et intramitochondriaux, Biochim. biophys. Acta, 1965, 107: 184. 13 BORN, G. ~V-.~R. AND M. HUME, Effects of numbers and sizes of platelet aggregates on the optical density of plasma, Nature (Loud.), 1967, 214: 1084. 14 CONSTANTINE, J. W., Aggregation and adhesion of rat platelets, Nature (Lond.), 1967, 214: 1084. 15 ADAM, H., Adenosine 5' diphosphate and adenosine 5' monophosphate. In: H.-U. BERGMYER (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, London, 1963, p. 573.
j . Atheroscler. Res., 1968, 8:855-858