Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide

BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood

A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are performed. LC-ESI/MS, LC-ESI-MS/MS and High Performance Thin Layer Chromatography (HPTLC) analysis of different lipoprotein fractions collected from pooled plasma revealed the presence of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyeline (SM) only on lipoproteins and phosphatidylcholine (PC), Lyso-PC on both lipoproteins and plasma lipoprotein free fraction (PLFF). Cardiolipin, phosphatidylglycerol (PG) and Phosphatidylserine (PS) were observed neither in the lipoprotein fractions nor in PLFF. All three approaches led to the same results regarding phospholipids occurrence in plasma lipoproteins and PLFF. A high abundancy of PE and SM was observed in VLDL and LDL fractions respectively. This study provides for the first time the knowledge about the phospholipid composition of all defined plasma lipoproteins

Determining the Lipid-Binding Specificity of SMP Domains: An ERMES Subunit as a Case Study.

Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins

Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles

A wide range of methods exist for the on-plate detection of lipids resolved by thin layer chromatography. Fluorescence generally offers improvements in sensitivity over methods that use colorimetric or simple densitometric detection. In this paper, we report that a classic cupric sulfate charring protocol produces a fluorescent signal that sensitively and quantitatively detects a wide range of phospholipids, neutral lipids, and sterols after automated, multi-development high performance thin layer chromatography. The measured lower limits of detection and quantification, respectively, were, on average, 80 and 210 pmol for phospholipids and 43 fmol and 8.7 pmol for sterols. The simple, inexpensive, and highly sensitive approach described here was used to quantitatively analyze the lipid and sterol composition of sea urchin cortical vesicles, a stage-specific model system used to study the mechanism of regulated membrane fusion