Pergamon Press
Life Sciences Vol . 22, pp . 531-534 Printed in the U .S .A .
EFFECTS OF LYSOLECITHIN ON AGGREGATION OF HUMAN PLATELETS INDUCED BY ARACHIDONIC ACID AND A23187 ROLE OF PROSTAGLANDIN INTERMEDIARY METABOLISM B . A . Fiedel Department of Immunology Rush Medical College, Chicago, Illinois
60612, USA
(Received in final form December 19, 1977) Summary
Lysolecithin at non-cytotoxic concentrations (30-500 uM) was found capable of completely inhibiting the aggregation of human platelets induced by arachidonic acid in the absence o any effect upon total platelet production of malondialdehyde, an end-product of platelet prostaglandin intermediary metabolism, and to inhibit platelet aggregation stimulated y the calcium onophore, A23187 . As the induction of platelet aggregation by arachidonic acid is dependent upon an intact prostaglandin biosynthetic pathway while that of A23187 is not and since lysolecithin-induced inhibition of arachidonic acid-stimulated platelet aggregation was evident in the absence of an effect upon platelet malondialdehyde production, it is suggested that lysolecithin inhibits the platelet release reaction and irreversible aggregation by a mechanism separable from a major affect upon prostaglandin intermediary metabolism . The exposure of human platelets to various stimuli including ADP, epinephrine, collagen and thrombin results in the accumulation of arachidonic acid (AA) through the enzymatic cleavage of the phospholipid esterified fatty acid by phospholipase A2 (PLA2 ; 1-4) . This fatty acid has been shown to serve as a precursor for the generation of Prostaglandins G2 and HZ (PGG2/PGH2) through the action of the prostaglandin cyclo-oxygenase enzyme ; PGG2/PGH2 in turn serve as substrates for the generation of the platelet activating substance, thromboxane A2 (5) . It has recently been described that lysolecithin (lysophosphatidylcholine ; LPC), which is the lytic byproduct of PLA2 action (6), can inhibit both the platelet release reaction and irreversible aggregation induced by ADP, 5-hydroxytryptamine, collagen and epinephrine (7,8) . Since this is a property shared by many non-steroidal anti-inflammatory agents such as aspirin and indomethacin, which interfere with prostaglandin intermediary metabolism, it was deemed of interest to determine whether LPC might also be interfering with platelet activation by such a mechanism . This was undertaken by comparing the'effects of LPC upon platelet aggregation induced by AA, an agent whose platelet aggregating capacity is dependent upon an intact prostaglandin biosynthetic pathway (9) and by the ionophore A23187, an agent whose action is markedly independent of such a requirement (10) . Materials and Methods AA and A23187 were obtained and prepared as described (11) . LPC was obtained from Supelco, Bellefont, Pa . ; it was made soluble in chloroform ; methanol 531
532
Lyaolecithin and Platelet Aggregation
Vol . 22, No . 6, 1978
(1 :1) and aliquots for use in the assay were blown to dryness with nitrogen and resuspended in 0 .9% saline (8) . Platelets (as platelet-rich-plasma ; PRP) were obtained and aggregation recorded in response to AA and A23187 as decribed (11) . Total platelet malondialdehyde was quantitated using a thiobarbituric acid procedure (11,12) . All concentrations are given as final concentrations in the reaction vessel . Results and Discussion The addition of LPC to PRP resulted in the inhibition of platelet aggregation induced by AA . When 0 .3 mM AA was utilized, 30 uM LPC decreased the extent of aggregation (-20%) and 125 uM completely abrogated aggregation (Fi . la) . This inhibition was overcome by a greater stimulus (0 .6 mM AA ; Fig . l b? but was readily restored when LPC concentrations were increased to 250-500 uM . Concentrations of AA up to 1 mM did not overcome the inhibition of platelet aggregation induced by 500 uM LPC even when reactions were monitored up to 35 minutes post-stimulation . Quantitation of total platelet malondialdehyde, an end-product of platelet prostaglandin intermediary metabolism, did not reveal any significant differences between the AA-stimulated, LPC-inhibited and noninhibited PRP reaction mixtures suggesting that the inhibition of AA-induced platelet activation was not occurring via an obvious affect upon prostaglandin intermediary metabolism . This was supported by experiments utilizing the calcium ionophore A23187, an agent whose platelet activating properties are markedly independent of the prostaglandin biosynthetic pathway . With A23187 (2 uM), 30 uM LPC was marginally effective in inhibiting platelet aggregation in contrast to the inhibition observed with higher concentrations of LPC (125250 uM ; Fig . 2a), which were more or less completely inhibitory . Again, a greater stimulus (4 uM A23187 ; Fig . 2b) overcame the inhibition seen, but inhibition was readily restored in the presence of 250-500 uM LPC . The observed dose-dependent agonist-antagonist relationship argues against a cytotoxic effect by LPC and this wag ,confirmed with studies in which LPC-induced platelet damage was assessed by 51 Cr-release ; these latter results are in agreement with those of Joist et al (8) . The mechanism by which LPC causes inhibition of platelet aggregation remains obscure . The data presented herein indicates that LPC non-cytotoxically inhibits platelet aggregation induced by both AA and A23187 . AA is a direct precursor of the prostaglandin cyclic endoperoxides PGG2 and H2, which in the platelet are predominately converted to thromboxane A2 . Malondialdehyde is an end-product of this pathway and its quantitation is often used as an indicator of agents which interfere with prostaglandin intermediary metabolism (11,12) . The inhibition by LPC of platelet aggregation induced by AA did not result in an observable effect upon platelet production of malondialdehyde suggesting that LPC does not markedly affect prostaglandin metabolism and that this inhibitory activity must be occurring by some other mechanism . This was supported by data which indicated that LPC inhibited platelet aggregation stimulated by A23187, an agent whose mechanism of activation is predominantly independent of a requirement for an intact prostaglandin biosynthetic pathway . Thus, the inhibition of platelet function mediated by LPC would appear to be mechanistically distinct from the observed effects of certain prostaglandin metabolic inhibitors such as aspirin, indomethacin (5) and the acute phase reactant, Creactive protein (11) . Whether this effect is mediated by the ability of LPC to produce a membrane transition from the bimolecular leaflet to a localized micellular organization of membrane lipids or lipoproteins (8) such that platelet secretion and irreversible aggregation are critically affected remains to be determined .
Vol . 22, No . 6, 1978
Lpsolecithin and Platelet Aggregation
533
b
FIG . 1 Influence of LPC upon AA-induced aggregation of human platelets : a) AA = 0 .3 mM and b) AA = 0 .6 mM . LPC cencentrations are expressed as uM and are given at the terminus of each tracing . LPC was allowed to incubate with the platelets (3 min ; 37°C with stirring) prior to the addition of AA .
A23187(21.M) W U z Q H
j
A23187(41.M)
0 125
I MINUTE
250
z
a
500 W
Q W
FIG . 2 Influence of LPC upon A23187-induced aggregation of human platelets : a) A23187 = 2 uM and b) A23187 = 4 uM . LPC concentrations are expressed as uM and are given at the terminus of each tracin . LPC was allowed to incubate with the platelets (3 min ; 37°C with stirrin g prior to the addition of A23187 .
534
Lysolecithin and Platelet Aggrégation
Vol . 22, No . 6, 1978
Acknowledgement This work was supported by grants from the Chicago and Illinois Heart Association and the National Institutes of Health (AI 12870-03 and 5-SO7RR0514) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
A .L . WILLIS, P . DAVIDSON, P .W . RAMWELL, W .E . BROCKLEHURST, and B . SMITH, Prosta landins in Cellular Biology (P .W . Ramwell and B .B . Pharriss, tors New York, 1972, p . 7. M .J . SILVER, W . HOCH, J .J . KOCSIS, C .M . INGERMAN, and J .B . SMITH, Science 183 1085-1087 (1974) . }C3. MARCUS, H .L . WILLIAMS, and L .B . SAFIER, Lipid Res . 10 108-114 (1969) . J .B . SMITH, and A .L . WILLIS, Nature New Biol . 231 235-23T(1971) . M . HAMBERG, J . SVENSSON, and . B SAMUELSSOff , Froc . Nat . Acad . Sci . U .S .A . 72 2944-2998 (1975) . B .A . BRADLOW, and A .J . MARCUS, Proc . Soc . Exp . Biol . Med . 12 3 889-893 (1966) . E .M .M . BESTERMAN, and M .P .T . GILLET, Nature New Biol . 241 223-224 (1973) . ISHIZ A, and J .F . MUSTARD, J .H . JOIST, G . DOLEZEL, M .P . CUCUIANU, E .E . Blood 49 101-111 (1977) . ET-MWLMSTEN, M . HAMBERG, J . SVENSSON, and B . SAMUELSSON, Proc . Nat . Acad . Sci . U .S .A . 7 2 1446-1450 (1975) . H.R . RAO, and J .M . GERRARD, American J . Pathol . 77 135-149 J .G. Hl (1974) . B .A . FIEDEL, R .M . SIMPSON and H . GEWURZ, J . Immunol . 119 877-882 (1977) . J .B . SMITH, C .M . INGERMAN AND M .J . SILVER, _U_. T~Clin Med . 88 167-172 (1976) .
Life Sciences Vol . 22, pp . 531-534 Printed in the U .S .A .
EFFECTS OF LYSOLECITHIN ON AGGREGATION OF HUMAN PLATELETS INDUCED BY ARACHIDONIC ACID AND A23187 ROLE OF PROSTAGLANDIN INTERMEDIARY METABOLISM B . A . Fiedel Department of Immunology Rush Medical College, Chicago, Illinois
60612, USA
(Received in final form December 19, 1977) Summary
Lysolecithin at non-cytotoxic concentrations (30-500 uM) was found capable of completely inhibiting the aggregation of human platelets induced by arachidonic acid in the absence o any effect upon total platelet production of malondialdehyde, an end-product of platelet prostaglandin intermediary metabolism, and to inhibit platelet aggregation stimulated y the calcium onophore, A23187 . As the induction of platelet aggregation by arachidonic acid is dependent upon an intact prostaglandin biosynthetic pathway while that of A23187 is not and since lysolecithin-induced inhibition of arachidonic acid-stimulated platelet aggregation was evident in the absence of an effect upon platelet malondialdehyde production, it is suggested that lysolecithin inhibits the platelet release reaction and irreversible aggregation by a mechanism separable from a major affect upon prostaglandin intermediary metabolism . The exposure of human platelets to various stimuli including ADP, epinephrine, collagen and thrombin results in the accumulation of arachidonic acid (AA) through the enzymatic cleavage of the phospholipid esterified fatty acid by phospholipase A2 (PLA2 ; 1-4) . This fatty acid has been shown to serve as a precursor for the generation of Prostaglandins G2 and HZ (PGG2/PGH2) through the action of the prostaglandin cyclo-oxygenase enzyme ; PGG2/PGH2 in turn serve as substrates for the generation of the platelet activating substance, thromboxane A2 (5) . It has recently been described that lysolecithin (lysophosphatidylcholine ; LPC), which is the lytic byproduct of PLA2 action (6), can inhibit both the platelet release reaction and irreversible aggregation induced by ADP, 5-hydroxytryptamine, collagen and epinephrine (7,8) . Since this is a property shared by many non-steroidal anti-inflammatory agents such as aspirin and indomethacin, which interfere with prostaglandin intermediary metabolism, it was deemed of interest to determine whether LPC might also be interfering with platelet activation by such a mechanism . This was undertaken by comparing the'effects of LPC upon platelet aggregation induced by AA, an agent whose platelet aggregating capacity is dependent upon an intact prostaglandin biosynthetic pathway (9) and by the ionophore A23187, an agent whose action is markedly independent of such a requirement (10) . Materials and Methods AA and A23187 were obtained and prepared as described (11) . LPC was obtained from Supelco, Bellefont, Pa . ; it was made soluble in chloroform ; methanol 531
532
Lyaolecithin and Platelet Aggregation
Vol . 22, No . 6, 1978
(1 :1) and aliquots for use in the assay were blown to dryness with nitrogen and resuspended in 0 .9% saline (8) . Platelets (as platelet-rich-plasma ; PRP) were obtained and aggregation recorded in response to AA and A23187 as decribed (11) . Total platelet malondialdehyde was quantitated using a thiobarbituric acid procedure (11,12) . All concentrations are given as final concentrations in the reaction vessel . Results and Discussion The addition of LPC to PRP resulted in the inhibition of platelet aggregation induced by AA . When 0 .3 mM AA was utilized, 30 uM LPC decreased the extent of aggregation (-20%) and 125 uM completely abrogated aggregation (Fi . la) . This inhibition was overcome by a greater stimulus (0 .6 mM AA ; Fig . l b? but was readily restored when LPC concentrations were increased to 250-500 uM . Concentrations of AA up to 1 mM did not overcome the inhibition of platelet aggregation induced by 500 uM LPC even when reactions were monitored up to 35 minutes post-stimulation . Quantitation of total platelet malondialdehyde, an end-product of platelet prostaglandin intermediary metabolism, did not reveal any significant differences between the AA-stimulated, LPC-inhibited and noninhibited PRP reaction mixtures suggesting that the inhibition of AA-induced platelet activation was not occurring via an obvious affect upon prostaglandin intermediary metabolism . This was supported by experiments utilizing the calcium ionophore A23187, an agent whose platelet activating properties are markedly independent of the prostaglandin biosynthetic pathway . With A23187 (2 uM), 30 uM LPC was marginally effective in inhibiting platelet aggregation in contrast to the inhibition observed with higher concentrations of LPC (125250 uM ; Fig . 2a), which were more or less completely inhibitory . Again, a greater stimulus (4 uM A23187 ; Fig . 2b) overcame the inhibition seen, but inhibition was readily restored in the presence of 250-500 uM LPC . The observed dose-dependent agonist-antagonist relationship argues against a cytotoxic effect by LPC and this wag ,confirmed with studies in which LPC-induced platelet damage was assessed by 51 Cr-release ; these latter results are in agreement with those of Joist et al (8) . The mechanism by which LPC causes inhibition of platelet aggregation remains obscure . The data presented herein indicates that LPC non-cytotoxically inhibits platelet aggregation induced by both AA and A23187 . AA is a direct precursor of the prostaglandin cyclic endoperoxides PGG2 and H2, which in the platelet are predominately converted to thromboxane A2 . Malondialdehyde is an end-product of this pathway and its quantitation is often used as an indicator of agents which interfere with prostaglandin intermediary metabolism (11,12) . The inhibition by LPC of platelet aggregation induced by AA did not result in an observable effect upon platelet production of malondialdehyde suggesting that LPC does not markedly affect prostaglandin metabolism and that this inhibitory activity must be occurring by some other mechanism . This was supported by data which indicated that LPC inhibited platelet aggregation stimulated by A23187, an agent whose mechanism of activation is predominantly independent of a requirement for an intact prostaglandin biosynthetic pathway . Thus, the inhibition of platelet function mediated by LPC would appear to be mechanistically distinct from the observed effects of certain prostaglandin metabolic inhibitors such as aspirin, indomethacin (5) and the acute phase reactant, Creactive protein (11) . Whether this effect is mediated by the ability of LPC to produce a membrane transition from the bimolecular leaflet to a localized micellular organization of membrane lipids or lipoproteins (8) such that platelet secretion and irreversible aggregation are critically affected remains to be determined .
Vol . 22, No . 6, 1978
Lpsolecithin and Platelet Aggregation
533
b
FIG . 1 Influence of LPC upon AA-induced aggregation of human platelets : a) AA = 0 .3 mM and b) AA = 0 .6 mM . LPC cencentrations are expressed as uM and are given at the terminus of each tracing . LPC was allowed to incubate with the platelets (3 min ; 37°C with stirring) prior to the addition of AA .
A23187(21.M) W U z Q H
j
A23187(41.M)
0 125
I MINUTE
250
z
a
500 W
Q W
FIG . 2 Influence of LPC upon A23187-induced aggregation of human platelets : a) A23187 = 2 uM and b) A23187 = 4 uM . LPC concentrations are expressed as uM and are given at the terminus of each tracin . LPC was allowed to incubate with the platelets (3 min ; 37°C with stirrin g prior to the addition of A23187 .
534
Lysolecithin and Platelet Aggrégation
Vol . 22, No . 6, 1978
Acknowledgement This work was supported by grants from the Chicago and Illinois Heart Association and the National Institutes of Health (AI 12870-03 and 5-SO7RR0514) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
A .L . WILLIS, P . DAVIDSON, P .W . RAMWELL, W .E . BROCKLEHURST, and B . SMITH, Prosta landins in Cellular Biology (P .W . Ramwell and B .B . Pharriss, tors New York, 1972, p . 7. M .J . SILVER, W . HOCH, J .J . KOCSIS, C .M . INGERMAN, and J .B . SMITH, Science 183 1085-1087 (1974) . }C3. MARCUS, H .L . WILLIAMS, and L .B . SAFIER, Lipid Res . 10 108-114 (1969) . J .B . SMITH, and A .L . WILLIS, Nature New Biol . 231 235-23T(1971) . M . HAMBERG, J . SVENSSON, and . B SAMUELSSOff , Froc . Nat . Acad . Sci . U .S .A . 72 2944-2998 (1975) . B .A . BRADLOW, and A .J . MARCUS, Proc . Soc . Exp . Biol . Med . 12 3 889-893 (1966) . E .M .M . BESTERMAN, and M .P .T . GILLET, Nature New Biol . 241 223-224 (1973) . ISHIZ A, and J .F . MUSTARD, J .H . JOIST, G . DOLEZEL, M .P . CUCUIANU, E .E . Blood 49 101-111 (1977) . ET-MWLMSTEN, M . HAMBERG, J . SVENSSON, and B . SAMUELSSON, Proc . Nat . Acad . Sci . U .S .A . 7 2 1446-1450 (1975) . H.R . RAO, and J .M . GERRARD, American J . Pathol . 77 135-149 J .G. Hl (1974) . B .A . FIEDEL, R .M . SIMPSON and H . GEWURZ, J . Immunol . 119 877-882 (1977) . J .B . SMITH, C .M . INGERMAN AND M .J . SILVER, _U_. T~Clin Med . 88 167-172 (1976) .
