Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

(1) Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [¹⁴C]arachidonate/[³H]arachidonoylphosphatidylcholine or free [¹⁴C]oleate/[³H]oleoylphosphatidylcholine. Whereas [¹⁴C]arachidonate was incorporated at a 10–15 times higher rate than [¹⁴C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free ³H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A₂ hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [³H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [¹⁴C]choline-labelled dipalmitoyl- and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.     

Interference of transmethylation inhibitors with thromboxane synthesis in rat platelets

In order to determine whether a methylation reaction is involved in the platelet metabolism of arachidonic acid (AA), we investigated the effect of the transmethylase inhibitors 3-deazaadenosine (DZA) and L-homocysteine-thiolactone (Hcy) on the production of immunoreactive thromboxane (TX) B2 by rat platelets. Incubation for at least one hour of the plateletrich plasma with DZA and Hcy led to an inhibition of TX synthesis induced by collagen (5 μg.ml^?1). Platelets in plasma were then preincubated for 4 hours with DZA (10^?3M) in association with Hcy(5×10^?4M), washed, resuspended in buffer, and stimulated with 3 different activators. The formation of TXB2 in response to collagen (25 μg.ml^?1) was markedly reduced, whereas no inhibition occurred when AA (5×10^?6M) or the calcium ionophore A 23,187 (5×10^?6M) were used. In addition labelled AA was incorporated into the platelet phospholipids (PL). Its release induced by collagen (25 μg.ml^?1) was inhibited when platelets were preincubated with DZA and Hcy under the same experimental conditions. By contrast, the release of AA induced by A 23187 (10^?6M) was unaffected. This results strongly suggest the association of a methylation reaction with platelet activation, at a calcium-independent step of endogenous AA metabolism, before the cyclo-oxygenase level. Its precise biochemical nature remains to be determined.     

Vitamin E exerts antiaggregatory effects without inhibiting the enzymes of the arachidonic acid cascade in platelets

Vitamin E (α-tocopherol) and tocopherol acetate produced a slightly increased amount of thromboxane in treated compared to untreated platelets. In tocopherol acetate-treated platelets significantly more lipoxygenase products were produced. α-tocopherol induced an increased, but not significant, production of thromboxane B₂ during blood clotting. α-tocopherol was not found to affect platelet phospholipase activity as determined by its effect on the release of labelled arachidonic acid from platelet phospholipids by challenging the platelets with calcium ionophore A23,187. α-tocopherol potentiated the incorporation of labelled arachidonate in the platelet phospholipids. Inspite of having no effect on the arachidonic acid cascade in platelets, α-tocopherol inhibited aggregation induced by several aggregating agents including A23,187. Inhibition of aggregation may be explained by the ability of α-tocopherol to inhibit intracellular mobilization of sequestered calcium from the dense tubular system to the cytoplasm.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [3H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA-versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.     

Fatty acid exchange of phospholipids in membranes of rat heart

Incorporation of ¹⁴C fatty acids in phospholipids of plasma membranes and sarcoplasmic reticulum of rat heart was studied. Mainly phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were labelled. Our studies showed that the incorporation of unsaturated fatty acids (oleic and linoleic acid) was higher than for saturated fatty acids (palmitic and stearic acid). The range of uptake was between 0.2 and 2.2 nmol·mg⁻¹ protein·h⁻¹ and 0.5–7.4 nmol·μgatom⁻¹ P₁·h⁻¹, respectively. Uptake of activity in individual phospholipids (measured after separation on TLC) was calculated as percentage of total activity. Incorporation in phosphatidylcholine was higher than in phosphatidylethanolamine. Phosphatidylcholine showed an increasing sequence for the following fatty acids: C_18:0 < C_16:0 < C_18:0 < C_18:2. However, phosphatidylethanolamine showed a decreasing sequence for the incorporation of the same fatty acids. Labelling of PC was always greater than for PE, except for stearic acid which was better incorporated into phosphatidylethanolamine. Uptake of the same fatty acid into phospholipids of sarcoplasmic reticulum was always higher than uptake into plasma membranes. As incorporation of fatty acids bound to albumin was studied in isolated membranes of rat heart, the addition of ATP and CoASH was an absolute requirement.     

Group VIB Calcium-Independent Phospholipase A2 (iPLA2γ) Regulates Platelet Activation,Hemostasis and Thrombosis in Mice

In platelets, group IVA cytosolic phospholipase A₂ (cPLA₂α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A₂ and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA₂α-deficient mice have indicated that other PLA₂(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca²⁺-independent PLA₂ (iPLA₂γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A₂ production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA₂γ-null mice. Furthermore, mice lacking iPLA₂γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA₂γ is an additional, long-sought-after PLA₂ that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.     

Phosphatidylinositol-specific phospholipase-C of platelets: association with 1,2-diacyglycerol-kinase and inhibition by cyclic-AMP.

Horse platelets prelabeled with [¹⁴C]arachidonate (AA) rapidly degrade [¹⁴C]phosphatidylinositol (PI) to [¹⁴C]1,2-diacylglycerol (DG) upon treatment with deoxycholate (DOC). This phospholipase-C (PLC) activity is specific for PI since other phospholipids or neutral lipids are not affected. Although exogenous Ca²⁺ is not required for activity, EGTA or EDTA abolishes PI degradation. Addition of Mg²⁺ (1 mM) and ATP (1 mM) results in phosphorylation of the DG and production of phosphatidic acid (PA). Higher concentrations of DOC inhibit DG-kinase. These observations, together with the fact that different platelet agonists induce a rapid degradation of PI and production of PA, indicate that PLC and DG-kinase activities are intimately linked. Incubation of platelets with dibutyryl cyclic-AMP, cyclic AMP-phosphodiesterase inhibitors and pyridoxal-5′-phosphate, which prevent platelet aggregation, inhibits the DOC-dependent conversion of PI to DG. The activity of PLC may play a central role in mediating platelet function and aggregation.     

Mobilization of arachidonic acid from phosphatidylethanolamine fraction to phosphatidylcholine fraction in platelets

Rabbit platelets rapidly incorporated methyl groups of [³H] methionine to phosphatidylcholine (PC). Rabbit platelets also incorporated [³H]choline to PC, but the rate of incorporation was far lower than that of [³H]methionine. Further fractionation of labeled PC revealed that a considerable amount of arachidonyl PC was synthesized via the N-methylation pathway. Thrombin stimulation resulted in a release of arachidonic acid from PC, and not from phosphatidylethanolamine (PE). These observations suggest that the N-methylation pathway plays an important role in the intracellular mobilization of arachidonic acid from the PE fraction to the PC fraction, this fraction being more sensitive to the hydrolysis with phospholipase A₂ during platelet activation.     

Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release

We have investigated whether exposure of human platelets to elevated concentrations of linoleic acid, the principal dietary polyunsaturate, would influence platelet thromboxane A₂ release. Platelets were incubated with albumin-bound linoleic acid at 30°C for 24 h, with prostaglandin E₁ added to prevent aggregation. The linoleic acid supplemented platelets released, on averaged, 50% less thromboxane A₂ in response to stimulation with thrombin than corresponding control platelets. Other fatty acids were without appreciable effect. The inhibition of thrombin-stimulated thromboxane A₂ release was dependent on the time and temperature of incubation, as well as on the concentration of added linoleic acid. Supplementation increased the amount of linoleic acid in the platelet phospholipids, but the arachidonic acid content of the phospholipids was reduced. [1-¹⁴C]Linoleic acid was not converted to arachidonic acid by the platelets. Linoleic acid was released exclusively form the inositol phosphoglycerides when the enriched platelets were stimulated with thrombin. The linoleate-enriched platelets converted less [1-¹⁴C]arachidonic acid to all prostaglandin products, suggesting that the platelet cyclooxygenase was partially inhibited.     

Cyclic adenosine 3',5'-monophosphate and prostacyclin inhibit membrane phospholipase activity in platelets.

[¹⁴C]-Arachidonic acid is incorporated mainly into phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of horse platelet membranes. Treatment of washed platelets with thrombin leads to a rapid loss of radioactivity from these phospholipids. The liberated [¹⁴C]-arachidonate is immediately transformed into hydroxyacids and thromboxanes. Treatment with dibutyryl cyclic AMP, cyclic AMP phosphodiesterase inhibitors or prostacyclin, a newly discovered prostaglandin that stimulates platelet adenylate cyclase, prevents the action of thrombin on phospholipid break-down as well as on platelet aggregation. Dibutyryl cyclic AMP does not affect the metabolism of exogenous [¹⁴C]-arachidonic acid. Cyclic AMP may thus play a crucial role in the regulation of platelet phospholipase acitivity, and this could explain at least in part the inhibition of aggregation caused by substances which, like prostacyclin, raise the levels of cyclic AMP.     

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [14C]arachidonate/[3H]arachidonoylphosphatidylcholine or free [14C]oleate/[3H]oleoylphosphatidylcholine. Whereas [14C]arachidonate was incorporated at a 10-15-times higher rate than [14C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free 3H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A2 hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [3H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [14C]choline-labelled dipalmitoyl-and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.     

Metabolism of Glycerolipids in Plastidial and Extraplastidial Membranes of Etiolated Wheat Leaves: In Vivo and In Vitro Studies

Etiolated wheat leaves were fed with [l-¹⁴C]-acetate and chaseexperiments were performed in the dark or under light. In bothconditions, in extraplastidial membranes, phosphatidylglycerol(PG) and phosphatidylcholine (PC) were fatty acid donors, PGand PC providing palmitate and oleate respectively. The labelof linoleate increased only in phosphatidylethanolamine (PE).In etioplasts, PG was also a palmitate donor but PC, ratherpoor in labelled oleate, was an oleate acceptor, contrary towhat was observed in chloroplasts. The galactolipids and sulfoquinovosyldiacylglycerol(SL) remained poorly labelled. When isolated etioplasts were labelled in vitro, during thefirst two hours they incorporated the same amount of [l-¹⁴C]-acetatein their phospholipids, whether they were in the presence orin the absence of extraplastidial membranes. Afterwards, theaddition of a mitochondrial fraction enhanced the label of PG,mainly in palmitate, then in oleate, and to some extent, andonly under light in palmitoleate and linoleate. The mitochondrialfraction might be regarded here as a supplier of labelled precursorto he etioplasts which rapidly accumulated radioactivity inpalmitoyl-PG. In PC of isolated etioplasts, only palmitate wasfairly labelled. The deficiency in labelled oleoyl-PC in plastidsof dark-grown plants and of linoleoyl-PC in extraplastidialmembranes might be the reason for the delay in the labellingof unsaturated galactolipids. ¹ (Received March 9, 1987; Accepted August 21, 1987)     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

Asymmetric distribution of arachidonic acid in the plasma membrane of human platelets. A determination using purified phospholipases and a rapid method for membrane isolation.

1. Non-lytic degradation of human platelet phospholipids have been performed using a combination of bee venom phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) and Staphylococcus aureus sphingomyelinase C (sphingomyelin choline phosphohydrolase). Under these conditions, 25.4% of total phospholipds are degraded and 6.4% of total platelet arachidonic acid is released. 2. A new method for rapid isolation of platelet plasma membrane is described, based on the use of [3H]concanavalin A as a membrane marker and of self-generating gradients of Percoll. Plasma membranes are enriched 5.2 fold in lectin marker and 0.43 in N-acetyl-beta-D-glucosaminidase, the main contaminant. This method allows to estimate that 57% of the total cell phospholipids and 61% of the total arachidonic acid content are located in the plasma membrane. 3. The distribution of phospholipids and arachidonic acid between the two leaflets of the plasma membrane has been deduced by using these values and those obtained from non-lytic treatment of intact platelets by phospholipases. It is concluded that 45% of plasma membrane phospholipids, comprising 93% of sphingomyelin, 45% of phosphatidylcholine, 9% of phosphatidylserine, 16% of phosphatidylinositol and 20% of phosphatidylethanolamine form the outer half of the human platelet plasma membrane. The phospholipids appear to bear only 10% of the total membrane arachidonic acid.     

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E₂ and F_2α which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of ³H-arachidonic acid labelled phospholipids, releasing free ³H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases ³H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-³H]inositol labelled phospholipids. Phospholipase A₂ (PLA₂), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, ³H-arachidonic acid labelled phospholipid breakdown, and ³H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitve) whereas the formation of inositol phosphates from myo-[2-³H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA₂ and PLD, but not by activation of phosphatidylinositol-specific PLC. © 1993 Wiley-Liss, Inc.     

Oleic acid-induced Ca2+ mobilization in human platelets: is oleic acid an intracellular messenger?

The purpose of this study was to explore the effect of oleic acid (OA) on intracellular Ca²⁺ mobilization in human platelets. When applied extracellularly, OA produced a concentration dependent rise in cytosolic [Ca²⁺] [Ca²⁺]_cyt) when extracellular [Ca²⁺] ([Ca²⁺]_ext was zero (presence of EGTA), suggesting that OA caused an intracellular release of Ca²⁺. Intracellular Ca²⁺ release was directly proportional to entry of OA into platelets and OA entry was indirectly proportional to [Ca²⁺]_ext. In permeabilized platelets, OA caused the release of ⁴⁵Ca²⁺ from ATP dependent intracellular stores. Finally, our results show that thrombin stimulated the release of [³H]OA from platelet phospholipids. The saturated fatty acids stearic and palmitic acid did not stimulate an increase in [Ca²⁺]_cyt under these conditions, but the unsaturated fatty acid, linolenic acid produced effects similar to those of OA, suggesting specificity among fatty acids for effects on [Ca²⁺]_cyt. Taken together, our experiments suggest that OA which has been incorporated into platelet phospholipids was released intothe cytosol by thrombinstimulation. Our experiments also show that OA stimulates Ca²⁺ release from intracellular stores. These results support the hypothesis that OA may serve as an intracellular messenger in human platelets.     

Surface expression of fatty acid translocase (FAT/CD36) on platelets in myeloproliferative disorders and non-insulin dependent diabetes mellitus: Effect on arachidonic acid uptake

Increased platelet reactivity has been implicated in the vascular complications of myeloproliferative diseases and diabetes mellitus. The mechanisms of platelet hyperresponsiveness have not been fully explained. Expression of CD36 or fatty acid translocase (FAT) and its role in arachidonic acid (AA) uptake by platelets were examined in subjects with myeloproliferative disorders(MPD), those with non-insulin-dependent diabetes mellitus (NIDDM), and in normal, healthy, age-matched controls. Surface expression of CD36 on platelet membranes was increased in MPD (10.94 ± 0.76 pmol/mg protein) compared with normal controls (6.94 ± 0.48 pmol/mg protein), p < 0.001. Total platelet content of CD36 was also significantly higher (32.1 ± 0.61 pmol/mg protein, p < 0.01) compared with those in sex and age matched normal controls (25.7 ± 1.09 pmol/mg protein). In contrast, platelet surface expression of CD36 in NIDDM (6.5 ± 0.56 pmol/mg protein) was not significantly different from those of normal controls despite higher total content of CD36 (32.8 ± 1.2, pmol/mg protein, p < 0 .01). Intact MPD platelets bound significantly more arachidonic acid (AA) (1.53 ± 0.16 nmol/mg protein, p < 0.05), compared with controls (1.12 ± 0.07 nmol/mg protein) or NIDDM subjects (1.16 ± 0.16 nmol/mg protein). The capacity of MPD platelet membranes to bind ¹⁴C-AA was also increased (1.72 ± 0.25 nmol/ protein, p < 0.05) compared with that of controls (1.62 ± 0.05 nmol/protein) and of NIDDM (1.22 ± 0.08 nmol/protein). This is consistent with higher surface expression of CD36 in MPD platelets. Membrane fatty acid analysis indicated that the % of AA in platelet phospholipids was significantly lower in MPD (3.15 ± 0.81%) compared with the controls (5.62 ± 1.7%, p < 0.05. The AA content of diabetic platelets (4.82 ± 1.1%) was not significantly different from normal controls. In summary, both total and surface expression of CD36 are increased in MPD, consistent with an enhanced capacity for uptake of AA by platelets. Increased expression of CD36 in platelets may play a role in the vaso-occlusive manifestations of MPD.     

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A2 of varying pI values. The susceptibility of platelet phospholipids to basic phospholipase A2 of Naja nigricollis (pI 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244-258). The susceptibilities of platelet phospholipids to acidic phospholipase A2 of Naja naja atra (pI 5.2) and to neutral phospholipase A2 of Hemachatus haemachatus (pI 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: the PC and the PE domains exist on the platelet membrane surface; and the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.