A NOVEL PATHWAY OF METABOLISM FOR ARACHIDONIC ACID IN HUMAN PLATELETS DANIEL DEYKIN, M.D., SUSAN RITTENHOUSE-SIMMONS, PH.D., FRANCIS A. RUSSELL BOSTON
Our laboratory is engaged in the study of regulatory mechanisms in hemostasis. As part of that interest we have been attracted to an examination of the role of lipids in platelet function. We have focused our attention primarily on an effort to characterize the pathways of phospholipid metabolism in human platelets and on changes that occur as platelets participate in hemostasis. A brief summary of the initial events in hemostasis will serve as a background for the reactions we are examining. The hemostatic mechanism is not continuously in operation. It is quiescent until it is activated by vascular injury sufficient to disrupt the endothelium at the site of injury. When flowing blood is exposed to subendothelial tissue, platelets adhere to the exposed collagen. There they undergo a series of reactions collectively termed the release reaction, during which platelet constituents including serotonin, calcium, and the nucleotide ADP are secreted. ADP causes platelets to become sticky and to aggregate. As they do so, they undergo the release reaction. Therefore, ADP serves as a chemical mediator which transforms and amplifies the mechanical stimulus initiated by vascular injury. Another amplification system also operates. As platelets adhere to collagen and as they undergo the release reaction, they form and liberate prostaglandin derivatives called thromboxanes, which are also powerful platelet aggregating agents. The exact role of each ofthe amplifying systems in hemostasis is not yet certain. However, the two are interrelated since drugs such as aspirin, which inhibits thromboxane formation, also inhibit ADP release and profoundly impair platelet function. Among the most important recent advances in our knowledge of platelet lipid metabolism has been the discovery by Samuelsson and his associates that platelets contain enzyme systems that introduce molecular oxygen into the polyunsaturated fatty acid, arachidonic acid, forming prostaglandins and thromboxanes, which are powerful promoters of platelet aggregation and vascular constriction. Arachidonic acid is cleaved from platelet phospholipids by hydrolytic enzymes called phospholipases. Oxygen is introduced by the enzyme cyclo-oxygenase, 206
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and the resulting product is then transformed either into thromboxanes, or, in a lesser amount, into the classic prostaglandins. Samuelsson has also described a second enzyme system, mediated by an enzyme called lipoxydase which forms another oxidation product of arachidonic acid given the abbreviated name of HETE. Goetzel has shown that this product is a powerful attractant for leukocytes. You will recognize of course that the requirement for phospholipase cleavage of arachidonic acid from phospholipids and for the transfer of that fatty acid to the oxygen-inserting enzymes is common to both pathways. The major point that underlies the rest of our presentation is that in platelets, as in other tissues, the primary control of prostaglandin synthesis, thromboxane formation, and HETE production lies not at the level of the specific enzymes that insert oxygen but rather at the level of the phospholipases. We have recently begun studying the effects of thrombin on platelet phospholipases in an attempt to understand the processes that control phospholipase activity. In our first study we incubated human platelets with tritiated arachidonic acid and exposed the platelets to thrombin. Thrombin caused the release of arachidonic acid, primarily from phosphatidycholine (PC) and phosphatidylinositol. We encountered an unexpected finding, however, in that thrombin caused a marked enhancement of label into a phospholipid which we had not previously encountered. Dr. Rittenhouse-Simmons set about the task of identifying the unknown lipid. She found that under the acidic conditions of our chromatography systems, the plasmalogen form of phosphatidylethanolamine, which I shall abbreviate as PPE, migrated to a position different from that of the diacyl form of phosphatidylethanolamine, which I shall abbreviate as DPE. It was PPE that was becoming selectively labeled when the platelets were aggregated by thrombin. To refresh your recall of phospholipid chemistry let me remind you that in the usual, or diacyl, form of phospholipids, fatty acids are linked to the 1- and 2- positions of the glycerol backbone by an ester bond. Plasmalogens contain fatty acids bound to the glycerol backbone by an ether bond. In the case of platelet PPE, the ether link exists at the 1- position and the 2- position is occupied almost exclusively by arachidonic acid in an ester link. We were intrigued by the heightened incorporation of arachidonic acid into PPE and we wished to determine whether it reflected accelerated synthesis of the entire molecule or an enhancement of the process of partial breakdown and reformation of the fatty acid bonds. We incubated platelets with 14C-labeled glycerol and with 3H-labeled arach-
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idonic acid. We reasoned that if thrombin promoted accelerated synthesis of the entire molecule then the incorporation of both glycerol and arachidonic acid would be stimulated. If thrombin promoted only turnover of the fatty acid portion of the molecule then there would be no enhancement of glycerol uptake. Indeed we found no stimulation of glycerol incorporation into PPE by thrombin. In other experiments we showed that the arachidonic acid that appeared in PPE was derived exclusively from platelet phospholipids, and not from plasma. Furthermore, only arachidonic acid was transferred to PPE. Therefore, the pathway we had uncovered is shown in Figure 1. When platelets are exposed to thrombin, phospholipases cleave arachidonic acid from the 2- position of PPE forming the lysoplasmalogen. Phospholipases also liberate arachidonic acid from other phospholipids, and that arachidonic acid is transferred to the lysoplasmalogen, regenerating the intact lipid. An interesting question is: what happens to the arachidonic acid liberated from the PPE? I shall come back to this question later. We next examined the relationship between the transfer of arachidonic acid to PPE and the release of arachidonic acid metabolic products from the platelet. Platelets were exposed to varying amounts of thrombin for varying amounts of time, resulting in a wide range of platelet activation. We found a direct, linear correlation between radioactivity transferred to PPE and the radioactivity released to the medium. Furthermore, the extrapolation of the data to the limiting case of zero release passed through the origin. We conclude from this experiment that there is a close relationship between the transfer of radioactivity to PPE and the delivery of arachidonic acid to the oxygen-inserting enzymes that lead to the release of arachidonic acid metabolites. Secondly our data indicate that the transfer of PPE occurs only as the platelet is activated, and to the degree the platelet is activated, suggesting a role for the transfer in platelet function. We turned next to a study of the influence of calcium on platelet phospholipases. In the absence of calcium both the rate and magnitude of the thrombin mediated reactions-the cleavage of arachidonic acid from PC and the transfer to PPE - were impaired. We were then curious to know whether an agent which directly mobilized platelet calcium stores, the ionophore A23187, could activate the phospholipases directly. When we exposed platelets to the ionophore in the absence of exogenous calcium we observed rapid hydrolysis of PC, prompt transfer to PPE, and release into the medium of transformed products of arachidonic acid, caused by direct mobilization of calcium from stores within the platelet. In further experiments we examined the energy requirements for the release of arachidonic acid from phospholipids. We prepared cells
ARACHIDONIC ACID IN HUMAN PLATELETS
209
PHOSPHOUPID |PtNs/4ohospCse A2
COOH
ARACHIDONIC ACID -O-C=C-R1 I2-0 Phosphohipase A
R2-O-
-0-C,C-R1
_*10
Acy Transferose-*
O-C=C-R1
AA-O{
O-P-Ethanolomine
O-P-Ethanolamine
PLASMALOGEN PE
LYSO-PLASMALOGEN PE
O-P-Ethanolamine
PLASMALOGEN PE
Fig. 1. The plasmalogen PE pathway.
depleted in metabolic ATP by incubating the platelets with 2- deoxyglucose and antimycin. We then examined the effect of energy depletion on thrombin or ionophore mediated phospholipase activity as reflected in the release of products of arachidonic acid metabolism into the medium. We found that in energy depleted cells thrombin did not activate phospholipases but ionophore did. We concluded that energy was required for thrombin to mobilize calcium (presumably by an energydependent contractile process), but that ionophore could strip calcium from tissue stores, prompting a flux of calcium that could activate
phospholipases directly. I have presented three pathways of arachidonic acid metabolism promoted by thrombin in human platelets. The first two - the cyclooxygenase and lipoxydase -lead to the release of specific metabolites of arachidonic acid from the platelet. What is the purpose of the third, the PPE pathway? I should like to speculate that the PPE pathway may serve as a shuttle mechanism for the transfer of arachidonic acid to one or both oxygenating enzymes of the platelet. As arachidonic acid is liberated from PPE it may be delivered directly to either cyclo-oxygenase or lipoxydase, themselves unregulated enzymes. If this speculation should be confirmed then the enzymes in this pathway-the phospholipases and transferases-could serve as potential sites for calcium-mediated regulation of normal platelet function and as potential sites of abnormalities in patients with hereditary and acquired disorders of platelet function. DISCUSSION DR. DANIEL N. MOHLER (Charlottesville): Enjoyed your paper very much. It's a whole new area of platelet metabolism opening up which ought to give us a lot of insights.
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DEYKIN ET AL.
Have you had a chance to look at any of the qualitative platelet defects, either inherited or acquired, to see how they affect this system? DR. DANIEL DEYKIN (Boston): We have looked at two patients, one of whom has a socalled release defect. His platelets function as though he had taken aspirin but he had not. We found a very striking decrease both in the phospholipases that cleave arachidonic acid from phosphatidylcholine and in the transferring mechanism to PPE. The rate is dramatically slow. We also have looked at a patient with hereditary storage pool defect and have found in him as well decrease in the transfer. DR. DANIEL N. MOHLER (Charlottesville): Have you looked at uremic patients? DR. DANIEL DEYKIN (Boston): No. We have not yet looked at uremic patients.
Our laboratory is engaged in the study of regulatory mechanisms in hemostasis. As part of that interest we have been attracted to an examination of the role of lipids in platelet function. We have focused our attention primarily on an effort to characterize the pathways of phospholipid metabolism in human platelets and on changes that occur as platelets participate in hemostasis. A brief summary of the initial events in hemostasis will serve as a background for the reactions we are examining. The hemostatic mechanism is not continuously in operation. It is quiescent until it is activated by vascular injury sufficient to disrupt the endothelium at the site of injury. When flowing blood is exposed to subendothelial tissue, platelets adhere to the exposed collagen. There they undergo a series of reactions collectively termed the release reaction, during which platelet constituents including serotonin, calcium, and the nucleotide ADP are secreted. ADP causes platelets to become sticky and to aggregate. As they do so, they undergo the release reaction. Therefore, ADP serves as a chemical mediator which transforms and amplifies the mechanical stimulus initiated by vascular injury. Another amplification system also operates. As platelets adhere to collagen and as they undergo the release reaction, they form and liberate prostaglandin derivatives called thromboxanes, which are also powerful platelet aggregating agents. The exact role of each ofthe amplifying systems in hemostasis is not yet certain. However, the two are interrelated since drugs such as aspirin, which inhibits thromboxane formation, also inhibit ADP release and profoundly impair platelet function. Among the most important recent advances in our knowledge of platelet lipid metabolism has been the discovery by Samuelsson and his associates that platelets contain enzyme systems that introduce molecular oxygen into the polyunsaturated fatty acid, arachidonic acid, forming prostaglandins and thromboxanes, which are powerful promoters of platelet aggregation and vascular constriction. Arachidonic acid is cleaved from platelet phospholipids by hydrolytic enzymes called phospholipases. Oxygen is introduced by the enzyme cyclo-oxygenase, 206
ARACHIDONIC ACID IN HUMAN PLATELETS
207
and the resulting product is then transformed either into thromboxanes, or, in a lesser amount, into the classic prostaglandins. Samuelsson has also described a second enzyme system, mediated by an enzyme called lipoxydase which forms another oxidation product of arachidonic acid given the abbreviated name of HETE. Goetzel has shown that this product is a powerful attractant for leukocytes. You will recognize of course that the requirement for phospholipase cleavage of arachidonic acid from phospholipids and for the transfer of that fatty acid to the oxygen-inserting enzymes is common to both pathways. The major point that underlies the rest of our presentation is that in platelets, as in other tissues, the primary control of prostaglandin synthesis, thromboxane formation, and HETE production lies not at the level of the specific enzymes that insert oxygen but rather at the level of the phospholipases. We have recently begun studying the effects of thrombin on platelet phospholipases in an attempt to understand the processes that control phospholipase activity. In our first study we incubated human platelets with tritiated arachidonic acid and exposed the platelets to thrombin. Thrombin caused the release of arachidonic acid, primarily from phosphatidycholine (PC) and phosphatidylinositol. We encountered an unexpected finding, however, in that thrombin caused a marked enhancement of label into a phospholipid which we had not previously encountered. Dr. Rittenhouse-Simmons set about the task of identifying the unknown lipid. She found that under the acidic conditions of our chromatography systems, the plasmalogen form of phosphatidylethanolamine, which I shall abbreviate as PPE, migrated to a position different from that of the diacyl form of phosphatidylethanolamine, which I shall abbreviate as DPE. It was PPE that was becoming selectively labeled when the platelets were aggregated by thrombin. To refresh your recall of phospholipid chemistry let me remind you that in the usual, or diacyl, form of phospholipids, fatty acids are linked to the 1- and 2- positions of the glycerol backbone by an ester bond. Plasmalogens contain fatty acids bound to the glycerol backbone by an ether bond. In the case of platelet PPE, the ether link exists at the 1- position and the 2- position is occupied almost exclusively by arachidonic acid in an ester link. We were intrigued by the heightened incorporation of arachidonic acid into PPE and we wished to determine whether it reflected accelerated synthesis of the entire molecule or an enhancement of the process of partial breakdown and reformation of the fatty acid bonds. We incubated platelets with 14C-labeled glycerol and with 3H-labeled arach-
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DEYKIN ET AL.
idonic acid. We reasoned that if thrombin promoted accelerated synthesis of the entire molecule then the incorporation of both glycerol and arachidonic acid would be stimulated. If thrombin promoted only turnover of the fatty acid portion of the molecule then there would be no enhancement of glycerol uptake. Indeed we found no stimulation of glycerol incorporation into PPE by thrombin. In other experiments we showed that the arachidonic acid that appeared in PPE was derived exclusively from platelet phospholipids, and not from plasma. Furthermore, only arachidonic acid was transferred to PPE. Therefore, the pathway we had uncovered is shown in Figure 1. When platelets are exposed to thrombin, phospholipases cleave arachidonic acid from the 2- position of PPE forming the lysoplasmalogen. Phospholipases also liberate arachidonic acid from other phospholipids, and that arachidonic acid is transferred to the lysoplasmalogen, regenerating the intact lipid. An interesting question is: what happens to the arachidonic acid liberated from the PPE? I shall come back to this question later. We next examined the relationship between the transfer of arachidonic acid to PPE and the release of arachidonic acid metabolic products from the platelet. Platelets were exposed to varying amounts of thrombin for varying amounts of time, resulting in a wide range of platelet activation. We found a direct, linear correlation between radioactivity transferred to PPE and the radioactivity released to the medium. Furthermore, the extrapolation of the data to the limiting case of zero release passed through the origin. We conclude from this experiment that there is a close relationship between the transfer of radioactivity to PPE and the delivery of arachidonic acid to the oxygen-inserting enzymes that lead to the release of arachidonic acid metabolites. Secondly our data indicate that the transfer of PPE occurs only as the platelet is activated, and to the degree the platelet is activated, suggesting a role for the transfer in platelet function. We turned next to a study of the influence of calcium on platelet phospholipases. In the absence of calcium both the rate and magnitude of the thrombin mediated reactions-the cleavage of arachidonic acid from PC and the transfer to PPE - were impaired. We were then curious to know whether an agent which directly mobilized platelet calcium stores, the ionophore A23187, could activate the phospholipases directly. When we exposed platelets to the ionophore in the absence of exogenous calcium we observed rapid hydrolysis of PC, prompt transfer to PPE, and release into the medium of transformed products of arachidonic acid, caused by direct mobilization of calcium from stores within the platelet. In further experiments we examined the energy requirements for the release of arachidonic acid from phospholipids. We prepared cells
ARACHIDONIC ACID IN HUMAN PLATELETS
209
PHOSPHOUPID |PtNs/4ohospCse A2
COOH
ARACHIDONIC ACID -O-C=C-R1 I2-0 Phosphohipase A
R2-O-
-0-C,C-R1
_*10
Acy Transferose-*
O-C=C-R1
AA-O{
O-P-Ethanolomine
O-P-Ethanolamine
PLASMALOGEN PE
LYSO-PLASMALOGEN PE
O-P-Ethanolamine
PLASMALOGEN PE
Fig. 1. The plasmalogen PE pathway.
depleted in metabolic ATP by incubating the platelets with 2- deoxyglucose and antimycin. We then examined the effect of energy depletion on thrombin or ionophore mediated phospholipase activity as reflected in the release of products of arachidonic acid metabolism into the medium. We found that in energy depleted cells thrombin did not activate phospholipases but ionophore did. We concluded that energy was required for thrombin to mobilize calcium (presumably by an energydependent contractile process), but that ionophore could strip calcium from tissue stores, prompting a flux of calcium that could activate
phospholipases directly. I have presented three pathways of arachidonic acid metabolism promoted by thrombin in human platelets. The first two - the cyclooxygenase and lipoxydase -lead to the release of specific metabolites of arachidonic acid from the platelet. What is the purpose of the third, the PPE pathway? I should like to speculate that the PPE pathway may serve as a shuttle mechanism for the transfer of arachidonic acid to one or both oxygenating enzymes of the platelet. As arachidonic acid is liberated from PPE it may be delivered directly to either cyclo-oxygenase or lipoxydase, themselves unregulated enzymes. If this speculation should be confirmed then the enzymes in this pathway-the phospholipases and transferases-could serve as potential sites for calcium-mediated regulation of normal platelet function and as potential sites of abnormalities in patients with hereditary and acquired disorders of platelet function. DISCUSSION DR. DANIEL N. MOHLER (Charlottesville): Enjoyed your paper very much. It's a whole new area of platelet metabolism opening up which ought to give us a lot of insights.
210
DEYKIN ET AL.
Have you had a chance to look at any of the qualitative platelet defects, either inherited or acquired, to see how they affect this system? DR. DANIEL DEYKIN (Boston): We have looked at two patients, one of whom has a socalled release defect. His platelets function as though he had taken aspirin but he had not. We found a very striking decrease both in the phospholipases that cleave arachidonic acid from phosphatidylcholine and in the transferring mechanism to PPE. The rate is dramatically slow. We also have looked at a patient with hereditary storage pool defect and have found in him as well decrease in the transfer. DR. DANIEL N. MOHLER (Charlottesville): Have you looked at uremic patients? DR. DANIEL DEYKIN (Boston): No. We have not yet looked at uremic patients.
