Changes in the erythrocyte in liver cirrhosis: role of cholesterol and plasma phospholipids

To investigate the role played by plasmatic lipids in the altered erythrocyte deformability observed in cirrhotic patients we studied 15 patients with liver cirrhosis (histologically diagnosed) and 10 healthy volunteers. Erythrocyte filtration time, plasmatic free and esterified cholesterol and phospholipids were measured in all subjects. The erythrocyte filtration time resulted to be significantly increased in cirrhotic patients (35' +/- 3, 35 M +/- SEM) when compared to control subjects (26' +/- 2, 53: M +/- SEM) (t = 2,078 p less than 0,05). This increase correlated in cirrhotic patients (but not in control subjects) with free/esterified cholesterol ratio (p less than 0,01) as well as free cholesterol/phospholipid ratio (p less than 0,001). Our results confirm that decreased erythrocyte deformability in cirrhotic patients which is accompanied by altered erythrocyte morphology is due, at least in part, to the altered lipids blood levels.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

Effects of apolipoprotein E genotype on blood lipid composition and membrane platelet fluidity in Alzheimer's disease.

The blood lipid composition (plasma, platelets and leukocytes), platelet membrane fluidity, apolipoproteins A and B in the plasma of AD patients and control subjects with distinct Apo E genotypes were investigated. No significant differences were found between the Apo E genotype and the cholesterol, phospholipids, triglycerides and Apo B levels in the plasma; cholesterol and phospholipids levels in platelet and leukocyte membranes; and platelet membrane fluidity of AD and control groups. However, the phospholipid levels in the leukocyte membranes of the control subgroup with the genotypes epsilon3/epsilon3 and epsilon3/epsilon4 and the AD subgroups with the genotypes epsilon2/epsilon3 and epsilon3/epsilon3, epsilon3/epsilon4 and epsilon4/epsilon4 were significantly lower than those observed in the control subgroup with the genotype epsilon2/epsilon3. Moreover, the cholesterol and phospholipid levels in the platelet membranes of the AD subgroup with the epsilon2 allele were significantly higher than those in the AD subgroup without the epsilon2 allele and the control subgroups with and without the epsilon2 allele. A strong correlation was found between cholesterol and phospholipids levels in the platelet membranes of the AD and control subgroups without the epsilon2 allele, but the residual cholesterol level in the platelet membranes of the AD subgroup was twice that observed in the control subgroup. Furthermore, the Apo A levels in the plasma of the AD subgroup with the epsilon3 allele were significantly lower than those observed in the AD subgroup without the epsilon3 allele and the control subgroup with the epsilon3 allele. The results are discussed in terms of involvement of lipid metabolism in the etiopathogenesis of AD.     

Effects of adenosine administration on the function and membrane composition of liver mitochondria in carbon tetrachloride-induced cirrhosis.

The effect of chronic carbon tetrachloride (CCl4) administration on liver mitochondria function and the protective action of adenosine on CCl4-induced damage were assessed in rats made cirrhotic by long-term exposure to the hepatotoxin (8 weeks). The CCl4 treatment decreased the ADP-stimulated oxygen consumption, respiratory control, and ADP/O values, mainly for substrates oxidation of site I, in isolated mitochondria. This impaired mitochondrial capacity for substrate oxidation and ATP synthesis was accompanied by an important diminution (approximately 30 mV) of membrane electrical potential. Disturbances of the mitochondrial membrane, induced by CCl4 treatment, were also evidenced as increased mitochondria swelling and altered oscillatory states of mitochondrial volume, both energy-linked processes. The deleterious effects of CCl4 on mitochondrial function were also reflected as a deficient activity of the malate-aspartate shuttle that correlated with abnormal distribution of cholesterol and phospholipids in membranes obtained from submitochondrial particles. Adenosine treatment of CCl4-poisoned rats partially prevented the alterations in mitochondria membrane composition and prevented, almost completely, the impairment of mitochondria function induced by CCl4. Although the nature of the protective action of adenosine on CCl4-induced mitochondria injury remains to be elucidated, such action at this level might play an important role in the partial prevention of liver damage induced by the CCl4.     

Lipid and membrane fluidity abnormalities in platelets and megakaryocytes of the hereditary macrothrombocytopenic Wistar Furth rat.

Biochemical and functional abnormalities of megakaryocytes and platelets were studied in Wistar Furth (WF) rats which have genetically determined macrothrombocytopenia and megakaryocytopenia, and were compared with their counterparts in Sprague-Dawley (SD) rats. Both megakaryocytes and platelets synthesized phospholipids from [14C]acetate. WF and SD megakaryocytes incorporated 0.27 and 0.29 nmol acetate per 10(6) cells, respectively. Phosphatidylcholine (PC) accounted for 64% and 58% of the PL radioactive label in megakaryocytes of SD and WF rats, respectively, (P less than 0.05), while 69% of labeled activity was associated with PC of SD platelets compared to 60% found in PC of WF platelets (P less than 0.01). In WF platelets a significant increase in the levels of lysophosphatidylcholine (6.1% vs. 3.0%) was observed. WF platelets had substantially higher levels of esterified cholesterol, triglycerides, ceramides and a 3-fold increase in the total protein per platelet compared to SD platelets. The fatty acid composition of WF platelet PC showed quantitative abnormalities. Plasma lecithin-cholesterol acyl transferase activity and platelet function monitored by the uptake and release of [14C] serotonin showed nonsignificant variations between SD and WF rats. Compared with the control, platelet membrane fluidity, measured by fluorescence polarization using platelets labeled with 1,6-diphenyl-1,3,5-hexatriene, was significantly decreased in the WF rats.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

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.     

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.     

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.     

Interactions Between Lipoproteins and Platelet Membranes in Obesity

The aim was to investigate low‐density lipoprotein (LDL) composition and Na⁺/K⁺ adenosine triphosphatase (ATPase) and Ca²⁺ ATPase activities and membrane fluidity measured by 1‐(4‐trimethylaminophenyl)‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH) in platelets from obese patients and controls in order to identify, if any, platelet membrane's chemical–physical and/or functional modifications associated with compositional modification of circulating lipoproteins. Moreover, we studied the in vitro effect on both platelet transmembrane cationic transport and fluidity, by incubating LDL from 30 obese subjects with platelets from 30 control subjects. The analysis of the chemical composition of LDL from obese patients showed a significant increase in the percent content of total cholesterol (TC) and triglycerides (TGs) and in the mean levels of lipid hydroperoxides compared to controls' LDL. Platelet Na⁺/K⁺ ATPase and Ca²⁺ ATPase activities showed, respectively, a significant decrease and increase in patients compared to controls; minor significant, respectively, decreases and increases are shown also in control platelets incubated with LDL from obese patients. Anisotropy tested with TMA‐DPH probe was significantly increased both in platelets from obese patients and in control platelets incubated with LDL from obese patients compared to control platelets. This study highlights that obesity induces remarkable modifications both in lipoproteins and platelets. Both platelet hyperfunction and quantitative/qualitative alterations in plasma lipoproteins, as well as an altered interaction between circulating lipoproteins and platelets, might play a relevant role in the increased prevalence of the early atherosclerotic lesions development in obese subjects. The present data point out that obesity might represent a major potentially modifiable risk factor for the onset of numerous complications, in particular cardiovascular ones.     

The effect of changes in the lipid composition of membranes on the functional activity of platelets

The phospholipid and fatty acid composition as well as the effect of platelet lipid composition modifications on the functional parameters of platelets were studied in blood sera from healthy donors and from patients with ischemic heart disease (IHD). It was found that the content of cholesterol and phospholipid hydrolysis products in IHD patients was increased. Reconstitution of the lipid composition of donor platelets by lysophosphatidylcholines, phosphatidic acid, fatty acids and cholesterol led to the increase of the platelet functional activity. It is suggested that the increased adsorption of Ca2+ on platelet surface is due to alterations in the platelet lipid composition in IHD and after modifications.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

The ultrastructure of defective human platelets

Summary Much of our current knowledge about the physiology of hemostasis has come from intensive study of platelets from patients with inherited and acquired bleeding disorders or an increased risk of thrombotic disease. Appreciation of the role of plasma proteins in platelet stickiness, of platelet surface membrane glycoproteins in aggregation, of the substances stored in platelet organelles in cell-cell interaction, vascular injury and atherosclerosis, and of endoperoxides and thromboxanes in platelet intercellular communication have resulted largely from investigations on various types of defective platelets. While the techniques of physiology and biochemistry have generated critical details about abnormal platelets, electron microscopy and ultrastructural cytochemistry have provided an improved morphological framework in which to integrate the new discoveries. The present review has attempted to correlate physiological, biochemical and ultrastructural concepts as they relate to the current understanding of inherited platelet disorders.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

Activation affects access to the platelet receptor for adhesive glycoproteins

Blood platelets have a receptor for macromolecular adhesive glycoproteins, located on a heteroduplex membrane glycoprotein complex (GPIIb/IIIa) that only becomes "exposed" when platelets are activated. Binding of the adhesive glycoproteins, in particular fibrinogen, to the receptor is required for platelet aggregation, which in turn is required to arrest bleeding. A murine monoclonal antibody whose rate of binding to the receptor is affected by platelet activation was both cross-linked and fragmented to assess the effects of changes in molecular size on its rate of binding to unactivated and activated platelets. The results indicate that small molecules can bind more rapidly to the receptors on unactivated platelets than can large molecules and that activation involves a conformational and/or microenvironmental change that permits the large molecules to bind more rapidly.     

Comparison of protein and lipid composition of the human platelet alpha-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and alpha-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to beta 2-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the alpha-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of 125I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000-135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the alpha-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

Autologous cryopreserved platelets and prophylaxis of bleeding in autologous bone marrow transplantation

Autologous platelets were harvested and cryopreserved in eight consecutive patients elected for ablative chemotherapy and autologous bone marrow transplantation (ABMT) for solid malignancy. There was a 19% loss in platelet count after the freeze thaw and wash procedure; with an in vitro functional loss of 40-60%. No correlation could be found for individual platelet transfusions between in vitro functional tests and in vivo recovery. Six consecutive patients received a total of 16 autologous platelet transfusions in the aplastic phase of ABMT. No bleeding was observed during the study period and there was no CMV infection in the recipients. While improvement in freezing and subsequent handling is desirable, autologous cryopreserved platelets can safely be used for the prophylaxis of bleeding during aplasia in patients treated with ABMT.     

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.     

Molecular mechanism underlying impaired platelet responsiveness in liver cirrhosis

We have studied platelet function in 10 patients with severe liver cirrhosis, compared to healthy subjects. Using washed platelets, we have investigated the molecular mechanism underlying the defect in platelet aggregation frequently observed in these patients. We have found that platelets from cirrhotic patients have a reduced responsiveness to thrombin and collagen in terms of aggregation, and receptor-dependent activation of phospholipase C, A2 and cyclooxygenase/thromboxane synthetase. We thus suggest that this impairment in transmembrane signalling is responsible for the defective platelet function observed in cirrhosis.     

Inositol phospholipid turnover in platelets of schizophrenic patients.

Abnormalities in blood cell membrane phospholipid composition and metabolism from schizophrenic patients have been reported by many groups of investigators. Among membrane phospholipids, inositol phospholipids are of special importance as they are involved in transduction system that generates second messengers such as inositol trisphosphate and diacylglycerol. Our studies on platelet inositol phospholipid turnover suggest a significant increase in platelet phosphatidylinositol 4,5-bisphosphate levels, an increased production of inositol trisphosphate in neuroleptic-treated and neuroleptic-free schizophrenic patients platelets and a reduced calcium release by thrombin in neuroleptic-treated schizophrenic patients platelets. The enhanced production of inositol trisphosphate may be due to an increase in its precursor phosphatidylinositol 4,5-bisphosphate with an associated desensitisation of the intracellular inositol trisphosphate receptor by neuroleptics, which may explain the diminished calcium response to thrombin in schizophrenic patients platelets.