Platelet Membrane Phospholipids Research Articles

Influence of subcutaneous injection of essential fatty acids on the stress-induced modifications of rat platelet aggregation and membrane lipid composition

Membrane lipids play an important role in the function of blood platelets but the mechanisms by which the lipid composition of the platelet membrane is adjusted remain unclear. It has been shown that stress and poly-unsaturated fatty acids modified the lipid composition of blood plasma and platelet lipids, but very little is known about the effect of stress and fatty acids on membrane platelet lipid composition. The purpose of the present investigation was to study the influence of the essential fatty acids: linoleic, linolenic and arachidonic acids on the composition of the platelet membrane lipids of rats assigned to heat and restraint stress. It was shown that injections of polyunsaturated fatty acids decrease or suppress the stress-induced increase in platelet aggregation, suppress the stress-induced modification of the composition of the platelet membrane lipids and modify the fatty acid composition of the platelet membrane phospholipids.

Loss of Platelet Membrane Phospholipid Asymmetry in Systemic Lupus Erythematosus: Association with Disease Activity and Hypercoagulable State.

Accelerated atherosclerosis (AE) not being explained by traditional risk factors, occurs frequently in systemic lupus erythematosus (SLE). Both SLE and AE share the condition of chronic inflammatory processes. Circulating, activated platelets, present in SLE patients, have been shown to exacerbate and promote AE in an animal model. Activated and/or apoptotic platelets lose membrane phopholipid asymmetry and expose negatively charged phosphatidylserine (PS), events that promote thrombin generation. In this context, it is well known that SLE serum has a strong apoptosis-inducing capacity on circulating blood cells. The aim of this work was to study the occurrence of apoptotic-like changes (PS exposure and loss of mitochondrial membrane potential [Δψ]) in circulating platelets in SLE patients and their relationship with an hipercoagulable state. Thirty patients with SLE (aged 15–72 years, mean age 38 years) and 30 healthy controls (aged 20–65 years, mean age 40 years) were studied. Patients fulfilled 4 revised criteria for SLE and disease activity was assessed by a modified SLE Disease Activity Index (MexSLEDAI). PS exposure was determined by flow cytometry (FC) by double labeling with FITC-labeled annexin V and PE-labeled anti-CD-61. The Δψ was studied with the J-aggregate-forming cation JC-1 and analyzed by FC. Tissue factor-dependent platelet procoagulant activity (pPCA) was determined in platelet membranes by conversion of factor X to Xa in the presence of FVIIa. Circulating microparticles were quantified through its PCA after being captured on a microtiter plate by annexin V and expressed as nM of PS. Soluble tissue factor (sTF) and thrombin anti-thrombin complexes (TAT) were measured by ELISA (American Diagnostica). The results are shown in the table:nAnnexin V (+) platelets (%)pPCA (Arbitrary U)MP (nM PS)Δψ (FL1/FL2)sTF (pg/ml)TAT μg/L) (Active SLE166.2±1.776±270.48±0.131.1±0.2148±174.3±4.9Non active SLE142.7±0.646±140.23±0.060.9±0.287±162.0±1.26Controls301.9±0.328±100.19±0.040.96±0.1107±131.4±1.6p ( Anova)*0.030.050.020.050.030.02* significance between active SLE and controlsThe results confirm a hypercoagulable state in SLE demonstrated by increased levels of TAT and sTF. PS exposure was significantly higher in active SLE patients than in healthy controls and decrease in mitochondrial Δψ suggests that apoptosis plays a role in this phenomenon. Our data also indicate that PS exposure in SLE platelets is associated with increased procoagulant activity of these cells and with higher levels of circulating MP. Moreover, PS exposure was positively correlated with levels of TAT (r: 0.55; p: 0.022; n:16). Taken together, our results suggest that loss of platelet membrane phospholipid asymmetry with PS externalization may constitute an important mechanism in the pathogenesis of the hypercoagulable state observed in SLE.

Nitric Oxide Production and Arachidonic Acid Metabolism in Platelet Membranes of Coronary Heart Disease Patients with and without Diabetes

Aim: To evaluate the levels of nitrite (NO^–₂) and nitrate (NO^–₃) ions and the incorporation of [³H]arachidonic acid (AA) into phospholipids of platelet membranes from coronary artery disease (CAD) patients with and without diabetes (NIDDM). Subjects and Methods: Eighteen CAD patients (group A), 18 CAD patients with NIDDM (group B), and 20 healthy controls (group C) without dyslipidemia, peripheral vascular disease and hypertension were included in the study. The groups were matched for age, sex and body mass index. The diagnosis of CAD was confirmed by coronary angiography. The nitric oxide end products (NOx), NO^–₂ plus NO^–₃ ions in platelet membranes, were determined using a spectrophotometric method based on the Griess reaction. The turnover of phospholipids was evaluated by incorporation of [³H]AA into platelet membrane phospholipids. Results: A significantly smaller amount of NOx ions was in the platelet membrane of groups A (40 ± 8 µmol/l) and B (29 ± 10 µmol/l) than C (57 ± 6 µmol/l), p < 0.001. Conversely a significantly greater amount of [³H]AA was incorporated into platelet phospholipids of group B patients (5,123 ± 1,637 dpm/mg) than groups A (3,159 ± 1,253 dpm/mg; p < 0.002) and C (1,621 ± 417 dpm/mg). An inverse correlation between [³H]AA incorporation and NOx levels was established: r = –0.76 (p < 0.05, n = 36) in CAD patients. Conclusions: Diabetes in CAD patients decreased the ability to produce platelet-derived NO and affects AA metabolism. This may result in higher platelet sensitivity to aggregating stimuli.

Cationic amphiphilic drugs and platelet phospholipase A 2 (cPLA 2)

The aim of the study was to verify and compare the effect of cationic amphiphilic drugs (CAD) from different pharmacological groups on activation of platelet phospholipase A 2 (PLA 2)—the essential enzyme of arachidonic pathway in blood platelets. β-Adrenoceptor-blocking (BAB) drugs inhibited platelet aggregation in the rank order of potency: propranolol>alprenolol>metipranolol>atenolol. The higher the inhibition of arachidonic acid (AA) liberation by BAB drugs, the higher the inhibition of aggregation. Similarly did the H 1-histamine antagonists bromadryl (BRO) and dithiaden (DIT) as well as the antimalarial chloroquine (CQ) show antiplatelet effect in vitro in the rank order of potency: DIT>BRO>CQ. Dose-dependent inhibition of aggregation was followed by the inhibition of AA liberation from membrane phospholipids of platelets stimulated either at the receptor site (thrombin) or by a stimulus bypassing membrane receptors (Ca 2+ ionophore A23187). The rank order potency for inhibition of stimulated 3H-AA liberation from membrane phospholipids was: (a) for BAB drugs: propranolol>alprenolol>metipranolol, (b) for other drugs: DIT>BRO>CQ. The investigated drugs' interference with stimulated liberation of AA showed nonspecific inhibition of platelet cytosolic PLA 2 (cPLA 2) by these drugs at intracellular level. The results revealed that besides the inhibition of cyclooxygenase pathway and receptors for adenosine diphosphate (ADP) and glycoproteins Gp II bIII a, the interaction of drugs with cPLA 2 may represent a further site for antiplatelet action.

Coagulation Factor Interaction with Platelets

Since the initial observation that bleeding is stopped by a platelet plug, it has been clear that platelets play an important role in hemostasis. Patients with very low platelet counts (thrombocytopenia) or with disfunctional platelets have a bleeding diathesis. With the further observation that the platelet plug becomes interwoven with fibrin, it has become clear that platelets interact with the coagulation proteins. In a review article in this issue of the journal, 1 Heemskerk et al. have summarized current information about the mechanisms by which platelets become activated. They have also examined current data on the interaction of coagulation factors with activated platelets. The purpose of this commentary is to highlight an area in which experts have differences of opinion: the question of whether platelets possess non-lipid binding proteins or receptors that bind procoagulant factors and coordinate assembly of the coagulation complexes. Heemskerk et al. have examined the mechanisms that lead to platelet activation with a particular emphasis on the changes that occur in phospholipid composition on the outer leaflet of the platelet membrane. The data cited in the review by Heemskerk et al. provides compelling evidence that changes in platelet membrane phospholipid composition are necessary for functional interaction of coagulation factors with platelets. The authors take the view that exposure of specific phospholipids on the outer leaflet of platelets is both necessary and sufficient to account for platelet procoagulant activity. To support this view, they cite work showing: that mutations of coagulation proteins which interfere with platelet binding also interfere with binding to phospholipid membranes; that blocking prothrombin binding to �

Antipsychotics and phospholipid metabolism in schizophrenia

To date numerous in-vivo (31)P-MRS and in-vitro studies in schizophrenic patients have been able to demonstrate changes in their membrane phospholipid metabolism, which might be relevant for the cause and the therapeutic responsiveness of this disorder. Thus far, however, only limited studies exist regarding the specificity of these findings for schizophrenia and the effect of antipsychotic medication. The present study examined the composition of membrane phospholipids in platelets of 67 neuroleptic-free schizophrenic patients compared to healthy and psychiatric controls. In a subsample of the schizophrenic patients we determined the effect of antipsychotic treatment on the phospholipid metabolism during six-months follow up. While untreated patients showed a decrease in major membrane phospholipid components, i.e. phosphatidylcholine and phosphatidylethanolamine, when compared to control subjects, as well as an increase in their breakdown-product lysophosphatidylcholine (LPC), there was a significant reduction in LPC during three weeks of pharmacotherapy with haloperidol. After six months treatment with different antipsychotics some divergent effects on phospholipid metabolism in schizophrenic patients could be demonstrated. While in the long-term course LPC remained decreased under continuous therapy with typical neuroleptics, patients being treated with the atypical drug zotepine showed an increase in LPC compared to their baseline level before therapy. Thus, specific mechanisms of the different antipsychotic therapies on phospholipid metabolism might serve to explain the divergent findings of (31)P-MRS in medicated patients.

POSSIBLE RELATIONSHIP BETWEEN ALTERED FATTY ACID COMPOSITION OF SERUM, PLATELETS, AND AORTA AND HYPERTENSION INDUCED BY SUGAR FEEDING IN RATS

To establish a relationship between alterations in fatty acid metabolism, induced by sugar ingestion, and hypertension, we analyzed fatty acid composition of serum, platelets and aorta in rats which had 30% of sugar in their drinking water for 18–20 weeks, and became hypertensive, hypertriglyceridemic and hyperinsulinemic. The fatty acid composition in sugar-fed as compared with that from control rats was as follows: in serum phospholipids, triglycerides and cholesterol ester fractions, palmitic, palmitoleic, oleic and cis-11-eicosadecaenoic acids were present in a higher proportion. In serum phospholipid fraction linoleic and arachidonic acids were decreased and a significant increase was observed in the proportion of dihomo-γ-linolenic acid. In the membrane phospholipids of platelets and aorta, higher proportions of palmitoleic and of oleic acids were observed. Differences in fatty acid composition of phospholipids between sugar-fed and control rats are consistent with altered membrane fluidity. Altered membrane function is a potential mechanism involved hypertension in rats.in sugar-induced

Reproducibility of in vivo measures of platelet membrane phospholipids in human subjects

Membrane phospholipid abnormalities in the brain neurons may be implicated in the pathophysiology of neuropsychiatric disorders. In the absence of methods to directly examine the levels of brain membrane phospholipids in vivo in human subjects, peripheral cells and platelets have been used as models in this field. We previously reported a method to determine the relative amounts of eight individual platelet membrane phospholipid classes using two-dimensional thin-layer chromatography, and scanning-laser densitometry ( Mallinger et al., 1993). Here we report the test/retest reproducibility of these platelet membrane phospholipid measures in healthy human subjects ( n=12) who were studied on two different occasions separated by a 3-week interval. The mean intra-subject coefficients of variation were 3.1–18%, and the intra-class correlation coefficients (ICCs) were 0.41–0.68. These findings are consistent with a low to moderate variability, and moderate reliability of these individual platelet membrane phospholipid measures over the period studied. When this method is applied for longitudinal studies of psychiatric populations, the degree of variability has to be considered in the interpretation of the results.

Platelet membrane phospholipids in euthymic bipolar disorder patients: are they affected by lithium treatment?

Background: Abnormalities in cell membrane processes and intracellular signal transduction pathways may be implicated in the pathophysiology of bipolar disorder. In this study, we attempted to investigate, in euthymic bipolar patients: 1) in vivo signal transduction abnormalities of the phosphatidylinositol pathway in platelets; and 2) possible in vivo effects of lithium treatment on platelet membrane phospholipids. Methods: We determined the relative absorbances of eight individual classes of platelet membrane phospholipids, using two-dimensional thin-layer chromatography in high-performance plates, followed by scanning laser densitometry, in a group of 10 lithium-treated euthymic bipolar patients and 11 normal controls. Results: The mean relative absorbance of phosphatidylinositol-4,5-bisphosphate (PIP 2) was lower in the patient group (0.29 ± 0.08% vs. 0.39 ± 0.12%; t = 2.35, df = 19, p = .03); no significant differences between patients and controls were found for the other phospholipids. Conclusions: This study provides in vivo evidence that bipolar patients on lithium treatment exhibit a decreased relative amount of PIP 2 in the platelet cell membranes compared to normal controls.

Effects of Brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets.

Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6 H)-tetrol) inhibited thrombin-,collagen- and ADP-induced aggregation of washed rat platelets. Thrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane A2 caused by thrombin, whereas it had no effect on the prostaglandin D2 formation. Brazilin inhibited [3H]-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase (PLA2) activity and [Ca2+]i elevation might be at least a part of antiplatelet mechanism of brazilin.

The role of ( E)-4-hydroxy-2-nonenal in platelet activation by low density lipoprotein and iron

( E)-4-Hydroxy-2-nonenal (HNE) is a highly reactive product of the oxidation of low density lipoprotein (LDL) which increases the platelet aggregation response to various agonists. HNE formation was increased during the enhanced platelet aggregation to thrombin, ADP, A23187 and epinephrine in the presence of LDL. The increase in platelet aggregation and HNE formation by LDL was inhibited by superoxide dismutase and catalase, suggesting superoxide and hydrogen peroxide produced by platelets during aggregation may be at least partly responsible. The responsiveness of platelets to LDL and the accompanying HNE formation was increased further in the presence of ferrous ion. The effect of ferrous ion on both platelet responses and HNE formation was decreased by superoxide dismutase, catalase and the antioxidants dipyridamole and probucol implicating platelet-derived free radicals. Ferrous ion caused an increase in the release of arachidonic acid from platelet membrane phospholipids in the presence of LDL which was probably caused by increased HNE production. The results suggest iron could increase platelet reactivity at sites of vascular injury by increasing HNE formation and promote the development of atherosclerotic lesions.

On the effect of 2-deuterium- and 2-methyl-eicosapentaenoic acid derivatives on triglycerides, peroxisomal β-oxidation and platelet aggregation in rats

A series of 2-substituted eicosapentaenoic acid (EPA) derivatives (as ethyl esters) have been synthesized and evaluated as hypolipidemic and antithrombotic agents in feeding experiments in rats. Repeated administration of purified 2-methyl-eicosapentaenoic acid and its deuterium analogues (all as ethyl esters) to rats resulted in a decrease in plasma triglycerides and high density lipoprotein cholesterol. The 2-methyl-EPA analogues were, apparently, four times more potent than EPA in inducing the triglyceride lowering effect. The 2-deuterium-2-methyl-EPA decreased plasma cholesterol level to ∼40%. A moderate enlargement of the liver was observed in 2-methyl-EPA treated rats. This was accompanied with an acute reduction in the liver content of triglycerides and a stimulation of peroxisomal β-oxidation and fatty acyl-CoA oxidase activity. The results suggest that the triglyceride-lowering effect of 2-methyl-EPA may be due to a reduced supply of fatty acids for hepatic triglyceride biosynthesis because of increased fatty acid oxidation. Platelet aggregation with ADP and A23187 was performed ex vivo in platelet-rich plasma, after administration of different doses of the EPA-derivatives for five days. EPA and 2,2-dideuterium EPA had no effect on ADP-induced aggregation, while 2-deuterium-, 2-methyl- and 2-deuterium-2-methyl EPA produced a biphasic effect, i.e. potentiation and inhibition at low (250 mg/day kg body weight) and higher doses (600–1300 mg/day kg body weight), respectively. A23187-induced platelet aggregation was affected in a similar way by feeding the 2-substituted EPA derivatives, except that 2-deuterium-2-methyl EPA had no effect relative to EPA itself and that the inhibition was far greater than that for ADP-induced aggregation (∼100% inhibition with 600 mg 2-methyl-EPA/day kg body weight). The ranking order of the EPA-derivatives to affect platelet aggregation and to cause hypolipidemia was different, suggesting different mechanisms. Our observations suggest that the effects of the EPA derivatives on platelet aggregation could be related to the degree of bulkiness around C 2 and that an asymmetric substitution at C 2 caused inhibition of platelet aggregation while a symmetric substitution did not. It is suggested that the bulky, asymmetric derivatives inhibit platelet aggregation by altering platelet membrane phospholipid packing.

Lipid peroxide overcomes the inability of platelet secretory phospholipase A2 to hydrolyze membrane phospholipids in rabbit platelets.

The present study investigated the effect of lipid peroxide on the ability of group IIA secretory phospholipase A2 (IIAsPLA2) to hydrolyze platelet membrane phospholipids. The treatment of rabbit platelets with tert-butyl hydroperoxide (BHP) and FeSO4 generated malondialdehyde, an index of lipid peroxidation, and slightly induced arachidonic acid liberation and lysophosphatidylcholine formation. Further addition of IIAsPLA2 purified from rabbit platelets synergistically enhanced the liberation and the formation induced by the oxidizing reagents, although the enzyme alone did not. When the IIAsPLA2 was pretreated with heparin, the enhancement was not observed. The combination of IIAsPLA2 with linoleic acid hydroperoxide and FeSO4 also caused synergistic arachidonic acid liberation. Furthermore, IIAsPLA2 enhanced thromboxane B2 generation and platelet aggregation induced by BHP and FeSO4. The synergistic aggregation was sensitive to indomethacin. With a membrane fraction as a substrate, IIAsPLA2 caused arachidonic acid liberation, which was enhanced in the presence of BHP and FeSO4. These results suggest that modification of membrane phospholipids by oxidizing reagents increases the accessibility of the membrane to platelet IIAsPLA2, and sequential enhancement of arachidonic acid liberation may contribute to the propagation of oxidative stress-induced cellular injury.

Effector Cell Protease Receptor-1, a Platelet Activation-dependent Membrane Protein, Regulates Prothrombinase-catalyzed Thrombin Generation

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.

Activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) in human platelets.

Blood platelets respond to the physiological agonist thrombin with shape change, aggregation and release of granular contents (1). Thrombin also evokes the rapid release of arachidonic acid esterified to platelet membrane phospholipids and thereby initiates the synthesis of prostaglandin endoperoxides, thromboxane A2 (TXA2) and other metabolites. The release of arachidonic acid from thrombin-stimulated platelets can be attributed largely to the action of PLA2 that specifically hydrolyzes phospholipids containing arachidonic acid (2,3). Platelet TXA2 generation represents a major pathway mediating both physiological and pathological platelet activities (4,5) and therefore the modulation of the involved PLA2 in platelets may play an important role in both haemostasis and thrombosis.

P.91 Fatty acid composition of platelet membrane phospholipids in critically ill patients after administration of two different fat emulsions

P.91 Fatty acid composition of platelet membrane phospholipids in critically ill patients after administration of two different fat emulsions

Effects of isomeric cis and trans eighteen carbon monounsaturated fatty acids on porcine platelet function

The effects of different positional cis and trans isomers (9, 11, 12 and 13) of C 18 monoenoic fatty acids on the response of porcine platelets to collagen and thrombin stimulation were determined. Cis isomers inhibited aggregation in response to 2 μg/ml or 5 μg/ml collagen and 0.1 U/ml of thrombin with almost equal effectiveness, with the exception of the 9- and 11-isomers which gave reduced inhibition of aggregation with thrombin and collagen stimulation respectively. All cis isomers inhibited TXA 2 formation (determined as TXB 2) elicited by collagen with almost equal effectiveness. Trans isomers were less effective than cis in inhibiting collagen induced aggregation particularly at the higher collagen concentrations. Inhibition of TXB 2 formation was less marked with trans isomers but a clear dissociation between the extent of inhibition of aggregation and TXB 2 formation was evident. Cis isomers also inhibited aggregation in response to 0.1 U/ml thrombin whereas trans isomers augmented aggregation but still reduced TXB 2 formation. Isomers did not elicit their effects through incorporation into platelet membrane phospholipids. Aggregation studies were carried out using the impedance method which is more sensitive than the optical method hitherto described, particularly for lipid studies.

Enhanced platelet thromboxane synthesis and reduced macrophage-dependent fibrinolytic activity related to oxidative stress in oral contraceptive-treated female rats

In previous studies conducted in rats and in women, we have shown that oral contraceptive (OC) administration induced a platelet hyperaggregation simultaneously with an increased platelet lipid biosynthesis which might be related to lipid peroxidation. In the present study, we specifically studied the arachidonic acid and the fibrinolytic pathways in relation to the fatty acid composition in female rats treated for 6 weeks with OC (ethinyl estradiol plus lynestrenol). We found that platelets of treated animals were not only hyper-responsive to thrombin and ADP, but also to sodium arachidonate. In addition, the results of the thrombin-induced release of labeled arachidonic acid pre-incorporated into platelet membrane phospholipids showed an increased biosynthesis of lipoxygenase and cyclooxygenase metabolites after OC treatment. These data indicated a stimulated platelet arachidonate metabolism in OC animals compared to controls which was further confirmed by the increased thrombin-induced production of thromboxane B 2 (TXB 2) as measured with a radioimmunoassay. The platelet thrombin-stimulated TXB 2 biosynthesis was inhibited in vitro in the presence of 500 μM aspirin and 1 mM vitamin E; the erythrocytes from OC animals compared with controls presented an enhanced in vitro susceptibility to free radical-induced hemolysis. These data indicated that a free radical mediated-process might occur. This hypothesis is confirmed by an increase of plasma lipid peroxidation parameters (conjugated dienes, lipid peroxides, thiobarbituric acid reactive substances). After OC-treatment, a decrease in plasma and platelet long chain polyunsaturated fatty acids, particularly (n-3), is in keeping with this idea. Furthermore, the results of the peritoneal macrophage-dependent fibrinolytic activity indicated that OC induced a drastic decrease in urokinase plasminogen activator activity which might further contribute to the platelet hyperactivity. Altogether these data suggest that besides the reported increase in clotting factors, platelet hyperactivity, possibly through a stimulated free radical-induced arachidonic acid metabolism, might be involved in the known high thrombogenic risk observed in OC users.

Arachidonic acid liberation induced by phosphatidic acid endogenously generated from membrane phospholipids in rabbit platelets

The action of phosphatidic acid generated from membrane phospholipids on phospholipase A 2 activation in rabbit platelets was investigated. When [ 3H]arachidonic acid-labelled platelets were treated with phorbol 12-myristate 13-acetate (PMA) and the membranes isolated from the cells incubated at 37°C with 50 μM CaCl 2 and 50 μM guanosine 5′- O-(3-thiotriphosphate) (GTPγS), both phosphatidic acid production and arachidonic acid liberation increased in PMA- and GTPγS-concentration-dependent manners. Ethanol dose-dependently inhibited these responses, accompanied by the formation of phosphatidylethanol. Since propranolol, an inhibitor of phosphatidic acid phosphohydrolase, had no influence on the production of phosphatidic acid, the arachidonic acid liberated does not appear to be derived from diacylglycerol which may be produced from phosphatidic acid through the action of this enzyme. In another approach, treatment of [ 3H]arachidonic acid-labelled membranes with phospholipase D from Streptomyces chromofuscus induced arachidonic acid liberation as well as phosphatidic acid formation in time- and dose-dependent manners. The former response was suppressed by p-bromophenacyl bromide, a phospholipase A 2 inhibitor. These results suggest that phosphatidic acid derived from membrane phospholipids potentiates phospholipase A 2 activation and contributes to the amplification of platelet activation.

Haemostatic investigations: The impact of new technologies

Blood loss following trauma is controlled by a complex series of interactions involving the vascular surface, blood platelets and ‘activated’ coagulation factors. Activation of the coagulation cascade involves a series of zymogen to enzyme transformations with each enzyme associating with a co-factor on a ‘surface’ such as negatively charged platelet membrane phospholipid to form reaction complexes. In physiological terms it is possible that this system is continuously switched on producing low levels of activated serine proteases which are continuously being neutralised by protease inhibitors and co-factor neutralising reactions which down-regulate blood clotting processes, thus maintaining patency of the vascular tree.