Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A₂ (sPLA₂), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA₂ can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA₂ enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.     

Acid dissociation of cyclooctaamylose

Acid dissociation constants of aqueous cyclooctaamylose (8-Cy) have been determined at 15–45°C by pH potentiometry. Standard enthalpies and entropies of dissociation are derived from the temperature dependences of these pK_a's. These results are compared to corresponding measurements of aqueous cyclohexaamylose and cycloheptaamylose, and the observed trends are interpreted in terms of complexation of cycloamylose with hydroxide ion. ¹³C-nmr spectral measurements are reported for 8-Cy in 99.8% D₂O solution, and assignments of the observed lines are made with the help of deuterium-induced differential isotopic shift experiments.     

Kinetic and inhibition studies of phospholipase A2 with short-chain substrates and inhibitors

The action of the phospholipases A2 (PLA2s) from Naja naja naja, Naja naja atra, and Crotalus atrox venoms as well as the enzyme from porcine pancreas on a number of short-chain, water-soluble substrates was studied. The inhibition of these enzymes by short-chain phosphonate- and thiophosphonate-containing phospholipid analogues was also examined. The kinetic patterns observed for the action of the venom PLA2s on substrates containing phosphocholine head groups all deviated from a classical Michaelis-Menten-type behavior. With a substrate containing an anionic head group, the kinetic pattern observed was more normal. In contrast, Michaelis-Menten-type behavior was observed for the action of the porcine pancreatic PLA2 acting on all of the substrates studied. A short-chain phospholipid analogue in which the enzyme-susceptible ester was replaced with a phosphonate group was found to be a tight-binding inhibitor of the venom PLA2s with IC50 values that were some 10(4)-10(5)-fold lower than the concentration of substrate used in the assay. The degree of inhibition was found to depend dramatically on the stereochemical arrangement of substituents in the inhibitor which strongly suggests that the inhibitors are binding directly to the active site of the PLA2s. By comparison, the phosphonate analogue functioned as a poor inhibitor of the porcine pancreatic PLA2. Direct inhibitor binding studies indicated that the short-chain phosphonate inhibitor bound weakly to the venom enzymes in the absence of the short-chain substrates. Several other unusual features of the inhibition were also observed. The data are interpreted in terms of a model in which the enzyme and substrate form a lipid-protein aggregate at substrate concentrations below the critical micelle concentration (cmc). Possible reasons for the selective binding of the inhibitor to the enzyme-substrate microaggregate are discussed.     

Expression of phospholipases A2 in primary human lung macrophages: Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA₂) and secreted phospholipases A₂ (sPLA₂) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA₂s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA₂ (iPLA₂). Two structurally-divergent inhibitors of group IV cPLA₂ completely block arachidonic acid release by macrophages in response to non-physiological (Ca²⁺ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA₂s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA₂s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA₂ inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA₂-alpha, iPLA₂ and several sPLA₂s. Cytosolic PLA₂-alpha is the major enzyme responsible for lipid mediator production in human macrophages.