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PubMed1989

Activation of human protein C by blood coagulation factor Xa in the presence of anionic phospholipids. Enhancement by sulphated polysaccharides.

The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (20 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca2+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (1:1, w/w) of 75 microM, the apparent Km was 1 microM with a kcat. of 1 min-1. At 25 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-1. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of 2-5 micrograms/ml and 5-10 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca2+ was essential, the dependence being sigmoidal with Hill coefficients ranging from 1.4 to 2.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca2+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like prothrombin fragments F1 or F1+2 could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.

Factor Xa↗

PubMed1989

Characterization of phospholipids accumulated in pulmonary-surfactant compartments of rats intratracheally exposed to silica.

Phospholipids in the lung fractions, i.e. alveolar free cells, extracellular pulmonary surfactant, intracellular pulmonary surfactant (lamellar bodies) and microsomal fractions, of rats were examined 28 days after intratracheal injection of silica (40 mg/kg). Significant accumulations of phospholipids were observed in the extracellular- and intracellular-surfactant fractions of rats exposed to silica. The prominent phospholipid accumulated was phosphatidylcholine (PC), consisting mainly of the dipalmitoyl species. However, a compositional change in acidic phospholipids of surfactant fractions was produced by the silica treatment. The percentage of phosphatidylglycerol (PG) was significantly decreased; in contrast, that of phosphatidylinositol (PI) was increased. Thus the ratio PG/PI in the surfactant fractions was markedly decreased in response to silica. This compositional change in both acidic phospholipids occurred even in the early stages, i.e. before appreciable accumulations of alveolar phospholipids were noticed. The molecular-species profiles of both acidic phospholipids in the surfactant fractions were distinctly different from each other. The dipalmitoyl species accounted for more than 30% of PG and less than 6% of PI, respectively. These species patterns of PG and PI were similar in control and silica-treated rats. These findings suggest two possibilities that (1) PG and PI destined for pulmonary surfactant are synthesized from each specific CDP-diacylglycerol (DG) pool having different molecular species in the lung, or (2) individual enzymes responsible for synthesis of surfactant PG and PI have substrate specificities for molecular species of CDP-DG, thereby producing PG and PI having different molecular species in surfactant compartments.

Animals↗

PubMed1990

The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII.

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

Apoproteins↗

PubMed1995

Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras.

p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.

Amino Acid Sequence↗

PubMed1996

Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis.

Cytosolic phospholipase A2 (cPLA2) is a good candidate for mediating the agonist-stimulated release of arachidonic acid (AA) from membrane phospholipids. This enzyme undergoes a Ca(2+)-dependent translocation from the cytosol to a membrane site in a variety of cell types, and this site has recently been identified as the nuclear envelope in leucocytes. The functional correlate of this finding has not yet been established. The present study was therefore undertaken to determine whether translocation of cPLA2 to the nuclear envelope was associated with localized phospholipid hydrolysis at this site. Rat alveolar epithelial cells, previously shown to contain cPLA2, were prelabelled with [3H]AA and stimulated with the model agonist, ionophore A23187. Ionophore-induced AA release exhibited characteristics typical of a cPLA2-mediated response, in that it was Ca(2+)-dependent, sn-2 AA-selective, and inhibited by arachidonyl trifluoromethyl ketone. As determined by indirect immunofluorescence microscopic analysis as well as subcellular fractionation with immunoblotting, ionophore treatment resulted in a translocation of cPLA2 protein from the cytoplasm to the nuclear envelope. To determine whether the nuclear membrane was indeed the source of released AA, prelabelled cells were incubated in the presence or absence of A23187, after which the phospholipid radioactivity was quantified in nuclear and non-nuclear membrane fractions. [3H]AA was distributed in both nuclear and non-nuclear membrane phospholipids. Following A23187 stimulation, the loss of [3H]AA from nuclear membrane phospholipids accounted for 88.1 +/- 5.8% of the total loss from phospholipids and for 92.9 +/- 2.3% of the total [3H]AA released into the medium. These results demonstrate for the first time that agonist-stimulated translocation of cPLA2 to the nuclear envelope is associated with phospholipid hydrolysis which is preferentially localized to that site.

Animals↗

PubMed1998

The distribution and metabolism of arachidonate-containing phospholipids in cellular nuclei.

The cell nucleus has been identified as a location to which several arachidonic acid-metabolizing enzymes are located in stimulated cells. However, little information exists describing the distribution of arachidonate-containing phospholipids associated with the nucleus or the control of their composition. In this study, nuclei isolated from human monocyte-like THP-1 cells were found to have a distribution of arachidonyl-phospholipids which is markedly different from that of other cellular membranes. THP-1 nuclei which contained 22% of total cellular arachidonate, showed a near equal distribution of arachidonate in 1-acyl-2-arachidonoyl-glycero-3-phosphocholine, 1-acyl-2-arachidonyl-glycero-3-phosphoethanolamine, 1-acyl-2-arachidonoyl-glycero-3-phosphoinositol and 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species. In contrast in non-nuclear membranes, arachidonate was located primarily in 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species which accounted for approximately half of the arachidonate in all non-nuclear phospholipids. Isolated nuclei were incapable of initially acylating arachidonic acid into their phospholipids in the absence of cellular cytosol. However, they were capable of efficiently remodelling existing arachidonate between phospholipid classes and subclasses. Isolated nuclei contained 25-30% of the cellular activity of CoA-independent transacylase, the key enzyme responsible for arachidonate-phospholipid remodelling. This enzyme is also critical in the control of arachidonate availability following cell stimulation. Given that the cellular distribution of arachidonate is such that nuclei are enriched in donor substrates for the CoA-independent transacylase reaction, that non-nuclear membranes are enriched in acceptor substrates and that nuclei have the enzymatic machinery to remodel arachidonate efficiently, these results suggest that CoA-independent transacylase may be responsible for the remodelling of arachidonate not only between different phospholipid species within the same organelles but also between different sub-cellular compartments.

Acyltransferases↗

PubMed1998

Energy requirements for two aspects of phospholipid metabolism in mammalian brain.

Previous estimates have placed the energy requirements of total phospholipid metabolism in mammalian brain at 2% or less of total ATP consumption. This low estimate was consistent with the very long half-lives (up to days) reported for fatty acids esterified within phospholipids. However, using an approach featuring analysis of brain acyl-CoA, which takes into account dilution of the precursor acyl-CoA pool by recycling of fatty acids, we reported that half-lives of fatty acids in phospholipids are some 100 times shorter (min-h) than previously thought. Based on these new estimates of short half-lives, palmitic acid and arachidonic acid were used as prototype fatty acids to calculate energy consumption by fatty acid recycling at the sn-1 and sn-2 positions of brain phospholipids. We calculated that the energy requirements for reacylation of fatty acids into lysophospholipids are 5% of net brain ATP consumption. We also calculated ATP requirements for maintaining asymmetry of the aminophospholipids, phosphatidylserine and phosphatidylethanolamine across brain membrane bilayers. This asymmetry is maintained by a translocase at a stoichiometry of 1 mol of ATP per mol of phospholipid transferred in either direction across the membrane. The energy cost of maintaining membrane bilayer asymmetry of aminophospholipids at steady-state was calculated to be 8% of total ATP consumed. Taken together, deacylation-reacylation and maintenance of membrane asymmetry of phosphatidylserine and phosphatidylethanolamine require about 13% of ATP consumed by brain as a whole. This is a lower limit for energy consumption by processes involving phospholipids, as other processes, including phosphorylation of polyphosphoinositides and de novo phospholipid biosynthesis, were not considered.

Acyl Coenzyme A↗

PubMed2002

Inhibitory effects of acidic phospholipids on the binding of origin-recognition complex to origin DNA.

Origin-recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, shares certain biochemical characteristics with DnaA, the initiator of chromosomal DNA replication in prokaryotes. These similarities include origin-specific DNA binding, ATP binding and ATPase activity. DnaA interacts with acidic phospholipids, such as cardiolipin, and its activity is regulated by these phospholipids. In this study, we examined whether Saccharomyces cerevisiae ORC also interacts with phospholipids. Among the various phospholipids tested, ORC was found to bind specifically to cardiolipin. This binding was inhibited by excess concentrations of salts but unaffected by ATP, adenosine 5'-[gamma-thio]triphosphate or the origin DNA. Cardiolipin weakly inhibited the ATP-binding activity of ORC, whereas it strongly inhibited ORC binding to origin DNA. Acidic phospholipids other than cardiolipin (phosphatidylglycerol and phosphatidylinositol) weakly inhibited ORC binding to origin DNA. Furthermore, total phospholipids extracted from yeast nuclear membranes inhibited ORC binding to origin DNA. We consider that phospholipids may modulate initiation of DNA replication in eukaryotes in a similar manner to that found in prokaryotes.

Adenosine Triphosphatases↗

PubMed1996

Gastric mucosal phospholipids in dogs with familial stomatocytosis-hypertrophic gastritis.

In dogs, hypertrophic gastritis, which resembles Ménétrier's disease in man, has been demonstrated to be part of a hereditary syndrome called familial stomatocytosis-hypertrophic gastritis. In addition to hypertrophic gastritis, affected dogs exhibit abnormal blood phospholipid composition. Phospholipids may play a role in maintaining gastric mucosal integrity, and this may be compromised in gastritis. The question arises whether the differences in blood phospholipids may result from a disorder that might also be revealed in the composition of gastric mucosal phospholipids. We analysed the phospholipid composition of gastric mucosa from four dogs with familial stomatocytosis-hypertrophic gastritis. The general phospholipid composition and the molecular composition of phosphatidylcholine from mucosal tissue in the corpus of the stomach where hypertrophic gastritis was evident were not different from that of the antrum, where the tissue was normal. These results do not corroborate a relation between the gastric mucosal phospholipid composition and hypertrophic gastritis.

Animals↗

PubMed1999

Role of bile salt hydrophobicity in distribution of phospholipid species to carriers in supersaturated model bile solutions.

BACKGROUND: Phospholipid species modulate cholesterol-holding capacity and, therefore, regulate bile metastability. METHODS: In this study, we investigated the effect of bile salt hydrophobicity on the distribution of phospholipids among lipid particles in supersaturated model bile solutions (total lipid concentration, 9 g/dL; taurocholate/phospholipid ratio 3.0, cholesterol saturation index 1.3), by using gel permeation chromatography. RESULTS: With an increase of bile salt hydrophobicity in the elution buffer, the uptake of cholesterol and phospholipids into bile salt micelles was increased, associated with an increased cholesterol/phospholipid molar ratio of the vesicles. In contrast, there was an inverse correlation between the hydrophobicity of the phospholipid species in the vesicles and that of bile salts in the elution buffer, suggesting that hydrophobic bile salts induced preferential uptake of hydrophobic phospholipids into bile salt micelles, while less hydrophobic phospholipids, with a relatively low cholesterol-holding capacity, remained in the vesicles. CONCLUSIONS: These data indicate that bile salt hydrophobicity regulates vesicular cholesterol metastability by modulating the hydrophobicity of phospholipids in vesicles, as well as the lipid distribution among various biliary lipid particles.

Bile↗

PubMed1996

Physiopathological significance of catalytic phospholipids in the generation of thrombin.

Thrombin generation is the culminating event of the coagulation cascade. It is initiated after the expression of tissue factor by endothelial cells and monocytes exposed to thrombogenic stimuli. Anionic phospholipids, chiefly phosphatidylserine, are necessary for the optimal activity of tissue factor and completion of the clotting process. They display a catalytic potential by allowing the formation of the characteristic enzyme complexes at the membrane surface. Platelets are viewed as the main source of procoagulant phospholipid referred to as platelet factor 3. The plasma membrane of resting cells presents an asymmetrical distribution of phospholipids, aminophospholipids being sequestered in the inner leaflet. Procoagulant phospholipids become available at the outer surface after cell stimulation. The collapse of the membrane asymmetry is thought to promote a phospholipid scrambling accompanied by the shedding of microparticles. The plasma membranes of such vesicles bear irreversibly externalized procoagulant phosphatidylserine and contain glycoproteins that testify to their tissue origin. Hence, microparticles could disseminate a dual procoagulant and adhesive potential. Thrombin autoamplification is exerted through feedback activation loops involving either coagulation factors or platelets. This article details the mechanisms by which procoagulant phospholipids promote the generation of an excess of thrombin. A new pharmacological approach of thrombosis is presented, based on the control of the exposure of procoagulant phospholipids and membrane microparticle shedding.

Animals↗

PubMed1981

Rat basophilic leukemia cell lines defective in phospholipid methyltransferase enzymes, Ca2+ influx, and histamine release: reconstitution by hybridization.

Variants of the rat basophilic leukemia (RBL) cell line were isolated and screened for phospholipid methyltransferase I and II activities, enzymes that convert phosphatidylethanolamine to phosphatidylcholine. Two variants were found that had decreased phospholipid methyltransferase enzyme levels and were unable to cause an influx of Ca2+ or release histamine in an IgE-mediated reaction. However, these cells were able to release histamine through an ionophore-induced reaction, indicating that the releasing mechanism distal to the Ca2+ channel was intact. One cell line, 1C1.B1, had low specific activity for phospholipid methyltransferase I. A second variant, 2H3.B6, had reduced phospholipid methyltransferase II activity. Although both variants were unable to incorporate label from [methyl-3H]methionine or [3H]serine into phosphatidylcholine, they were able to incorporate [methyl-3H]choline and myo-[2-3H(N)]inositol into phospholipids. Fusion of the two cell lines and isolation on selective media resulted in the growth of eight independent hybrids. All eight had an increased number of chromosomes and normal phospholipid methyltransferase activities. Stimulation of the hybrids with IgE resulted in CA2+ influx and histamine release. These results indicate that phospholipid methylation precedes and is necessary for Ca2+ influx, and they further support the hypothesis that methylation is a necessary early step in the IgE-mediated histamine release reaction in RBL cells.

Animals↗

PubMed1990

Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H).

Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to greater than 95% purity by sequential phospholipid affinity and ion-exchange chromatography. However, these highly purified aPL antibodies do not bind to the CL antigen when assayed by a modified CL ELISA in which the blocking agent does not contain bovine serum, nor do they bind to phospholipid affinity columns. Binding to the phospholipid antigen will only occur if normal human plasma, human serum, or bovine serum is present, suggesting that the binding of aPL antibodies to CL requires the presence of a plasma/serum cofactor. Using sequential phospholipid affinity, gel-filtration, and ion-exchange chromatography, we have purified this cofactor to homogeneity and shown that the binding of aPL antibodies to CL requires the presence of this cofactor in a dose-dependent manner. N-terminal region sequence analysis of the molecule has identified the cofactor as beta 2-glycoprotein I (beta 2GPI) (apolipoprotein H), a plasma protein known to bind to anionic phospholipids. These findings indicate that the presence of beta 2GPI is an absolute requirement for antibody-phospholipid interaction, suggesting that bound beta 2GPI forms the antigen to which aPL antibodies are directed. Recent evidence indicates that beta 2GPI exerts multiple inhibitory effects on the coagulation pathway and platelet aggregation. Interference with the function of beta 2GPI by aPL antibodies could explain the thrombotic diathesis seen in association with these antibodies.

Amino Acid Sequence↗

PubMed2000

Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2.

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.

Amino Acid Sequence↗

PubMed2003

On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids.

The activation of purified and phospholipid-depleted plasma membrane Ca2+-ATPase by phospholipids and ATP was studied. Enzyme activity increased with [ATP] along biphasic curves representing the sum of two Michaelis-Menten equations. Acidic phospholipids (phosphatidylinositol (PI) and phosphatidylserine (PS)) increased Vmax without affecting apparent affinities of the ATP sites. In the presence of 20 microm ATP, phosphorylation of the enzyme preincubated with Ca2+ (CaE1) was very fast (kapp congruent with 400 s-1). vo of phosphorylation of CaE1 increased with [ATP] along a Michaelis-Menten curve (Km of 15 microm) and was phospholipid-independent. Without Ca2+ preincubation (E1 + E2), vo of phosphorylation was also phospholipid-independent, but was slower and increased with [ATP] along biphasic curves. The high affinity component reflected rapid phosphorylation of CaE1, the low affinity component the E2 --> E1 shift, which accelerated to a rate higher than that of the ATPase activity when ATP was bound to the regulatory site. Dephosphorylation of EP did not occur without ATP. Dephosphorylation increased along a biphasic curve with increasing [ATP], showing that ATP accelerated dephosphorylation independently of phospholipid. PI, but not phosphatidylethanolamine (PE), accelerated dephosphorylation even in the absence of ATP. kapp for dephosphorylation was 57 s-1 at 0 microM ATP; that rate was further increased by ATP. Steady-state [EP] x kapp for dephosphorylation varied with [ATP], and matched the Ca2+-ATPase activity measured under the same conditions. Apparently, the catalytic cycle is rate-limited by dephosphorylation. Acidic phospholipids stimulate Ca2+-ATPase activity by accelerating dephosphorylation, while ATP accelerates both dephosphorylation and the conformational change from E2 to E1, further stimulating the ATPase activity.

Adenosine Triphosphate↗

PubMed2003

Importance of protein S and phospholipid for activated protein C-mediated cleavages in factor Va.

The procoagulant function of activated factor V (FVa) is inhibited by activated protein C (APC) through proteolytic cleavages at Arg306, Arg506, and Arg679. The effect of APC is potentiated by negatively charged phospholipid membranes and the APC cofactor protein S. Protein S has been reported to selectively stimulate cleavage at Arg306, an effect hypothesized to be related to reorientation of the active site of APC closer to the phospholipid membrane. To investigate the importance of protein S and phospholipid in the APC-mediated cleavages of individual sites, recombinant FV variants FV(R306Q/R679Q) and FV(R506Q/R679Q) (can be cleaved only at Arg506 and Arg306, respectively) were created. The cleavage rate was determined for each cleavage site in the presence of varied protein S concentrations and phospholipid compositions. In contrast to results on record, we found that protein S stimulated both APC cleavages in a phospholipid composition-dependent manner. Thus, on vesicles containing both phosphatidylserine and phosphatidylethanolamine, protein S increased the rate of Arg306 cleavage 27-fold and that of Arg506 cleavage 5-fold. Half-maximal stimulation was obtained at approximately 30 nm protein S for both cleavages. In conclusion, we demonstrate that APC-mediated cleavages at both Arg306 and Arg506 in FVa are stimulated by protein S in a phospholipid composition-dependent manner. These results provide new insights into the mechanism of APC cofactor activity of protein S and the importance of phospholipid composition.

Animals↗

PubMedDate not supplied

Influence of early drainage of intraperitoneal phospholipids on efficacy of adhesion prevention.

Postoperative peritoneal adhesions impose a long-term risk of morbidity and mortality. Adjunctive means are needed to prevent these complications. In previous studies we could demonstrate the efficacy and safety of intraperitoneally applied phospholipids with regard to adhesion prevention and wound healing, respectively. The assumption is that phospholipids rapidly adhere to the peritoneal surface and to the mesothelial lesions. This study was designed to evaluate the influence of early drainage of the administered fluid volume on the control of adhesion formation. Forty chinchilla rabbits underwent median laparotomy and standardized abrasion of circumscript areas of the ventral abdominal wall, the cecum, and the ileum. The animals randomly received either 75 mg/kg body weight of phospholipids in a volume of 5.0 mL/kg body weight (n = 20) or the same volume of Ringer's lactate solution (n = 20) prior to closing the laparotomy wounds. In 50% of the rabbits with either medication, 80% of the volume was recovered after 30 min before final closure of the abdominal wall ("drainage"). In the remaining animals the intraabdominal fluid load was not evacuated ("no drainage"). At day 10 after surgery all rabbits were sacrificed for evaluation of adhesion areas by computer-aided planimetry and histopathologic examination. The mean areas of adhesion in both Ringer's lactate groups were significantly larger than in the comparable phospholipid groups (p < .05). In the Ringer's lactate groups, adhesions averaged 341.7 (318.6) mm2 without and 263.3 (275.5) mm2 with drainage. In the phospholipid groups the respective mean areas reached only 24.6 (36.7) mm2 without drainage and 27.0 (49.7) mm2 following evacuation of the fluid 30 min after administration (median, mean in parentheses). These results prove the efficacy of phospholipids after a limited contact period of 30 min. The frequent use of drains in abdominal surgery will not impair the beneficial effect of phospholipids on prevention of adhesions.

Animals↗

PubMed1971

Phospholipid metabolism in intact and modified erythrocyte membranes.

Erythrocyte membranes incorporated labeled phosphate from gamma-adenosine triphosphate (AT(32)P) into phosphatidic acid and the polyphosphoinositides. Inositol-(3)H and palmitate-(14)C were also incorporated into the phospholipids but alpha-glycerophosphate-(32)P was not. The incorporation of gamma-AT(32)P into phospholipids was increased when the erythrocyte ghosts were incubated in hypotonic media which lysed the cells. Lysis had little or no effect on the incorporation of inositol-(3)H and palmitate-(14)C into the phospholipids. If erythrocyte membranes were prepared in 1 mM ethylenediaminetetraacetate (EDTA), instead of 1 mM MgCl(2), then the tonicity of the incubating medium did not influence the incorporation of gamma-AT(32)P into the phospholipids. Erythrocyte ghosts, prepared by lysis in water, EDTA, or 1 mM calcium, lead, mercury, zinc, or cadmium, failed to reconstitute when placed in isotonic medium, inasmuch as they did not retain potassium against a chemical gradient. Ghosts prepared by lysis in 1 mM magnesium, barium, or strontium could be reconstituted. Ghosts which failed to reconstitute incorporated more labeled phosphate from gamma-AT(32)P into the phospholipids than did intact or reconstituted ghosts. The larger incorporation of labeled phosphate by leaky ghosts was not due to a greater entrance of gamma-AT(32)P into those cells. Primaquine phosphate and digitonin, at concentrations which are known to cause cells to form smaller vesicles or to lyse cells by removing cholesterol, did not increase the incorporation of labeled phosphate into the phospholipids. It is suggested that the increased metabolism of phospholipids may be involved in a membrane repair mechanism.

Adenosine Triphosphate↗