Gail Thurman

Analysis of the priming activity of lipids generated during routine storage of platelet concentrates

BACKGROUND: Compounds generated during the routine storage of platelet concentrates may have deleterious effects on the transfusion recipient.

Stored blood components contain agents that prime the neutrophil NADPH oxidase through the platelet-activating-factor receptor

Neutrophils (PMNs) initiate production of toxic oxygen metabolites through stimulation of an NADPH oxidase resulting in the reduction of oxygen to superoxide anion (O2−). The activity of this enzyme system may be primed by a variety of compounds. This laboratory investigated the possibility that stored blood components may contain agents which prime the PMN oxidase. At the time of outdate, packed red blood cells, whole blood, and platelet concentrates contained a priming agent which enhanced the PMN NADPH oxidase activity in response to a soluble stimulus, formyl‐Met‐Leu‐Phe, by 2.1‐to 2.8‐fold. The priming activity was almost completely inhibited by WEB 2170, a specific platelet activating factor antagonist. Fresh plasma or fresh‐frozen plasma did not exhibit priming activity. These data suggest that platelet‐activating factor‐like compounds are generated during the storage of cellular blood components. The presence of these agents in stored blood may suggest a role for specific componds which prime PMNs and possibly mediate other effects which result in severe complications of transfusion therapy.

Compounds biologically similar to platelet activating factor are present in stored blood components

Agents which prime the neutrophil NADPH oxidase develop during routine storae of whole blood and packed red blood cells. This plasma priming activity can be inhibited by bepafant (WEB 2170), a specific platelet activating factor (PAF) receptor antagonist. Quantitation of the priming agent(s), by a commercially available radioimmunoassay for PAF, reproducibly demonstrated high levels of PAF activity. However, analysis of these plasma samples from stored blood components by gas chromatography/mass spectroscopy did not reveal any 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. We conclude that the polyclonal antibody to PAF used in these studies may have recognized different epitopes of a family of heterogeneous, biologically active lipids that manifest their effects through the PFA receptor.

Increased activity of the respiratory burst in cord blood neutrophils: Kinetics of the NADPH oxidase enzyme system in subcellular fractions

ABSTRACT: Previous studies with neutrophils from newborn infants compared to neutrophils from healthy adults have documented increased respiratory burst activity including enhanced superoxide anion (O2-) production, nitroblue tetrazoleum dye reduction, and hexose monophosphate shunt activity. To investigate the biochemical basis for these observations, we examined oxidative metabolism in membrane-rich fractions of neutrophils. Neutrophils from cord blood of vaginally delivered term infants or healthy adults were disrupted by nitrogen cavitation and subcellular fractions collected on discontinuous sucrose density gradients. Subcellular fractions of newborn neutrophils separated in a fashion identical with samples from healthy adults. Activity of alkaline phosphatase, a plasma membrane marker, was increased 4- to 5-fold in disrupted cells free from nuclei (postnuclear supernatant) as well as plasma membrane fractions from newborn samples compared to those from healthy adults. Content of lactoferrin, a specific granule marker, was decreased in postnuclear supernatants but equivalent in specific granule fractions of newborn cells compared to those from adults. No differences were noted in myeloperoxidase content of postnuclear supernatants or any other subcellular fraction. Plasma membrane fractions from phorbol myristate acetate-stimulated cord blood neutrophils made significantly more O2-than samples from adults (newborn 32.9 ± 8.1 nmol O2- min/mg protein mean ± SEM, n = 3 versus adult 10.8 ± 4.2, n = 3; P<0.05). Plasma membrane-rich fractions were also collected by the technique of differential centrifugation and kinetic parameters of the NADPH-dependent oxidase enzyme(s) were measured for vaginally delivered newborn and adult samples. The Kmapp for NADPH for newborn fractions was significantly increased compared with adult samples (newborn 66 ± 10 μM, n = 6 versus adult 30 ± 6, n = 6; P<0.025) but not to the extent that would be associated with abnormal cell function. In contrast, Vmax of newborn membrane-rich fractions was 1.7 times that of adult samples (newborn 30.6 ± 27 nmol O2-/ min/mg protein, n = 6 versus adult 18.0 ± 4.0, n = 6; P<0.025). Plasma membrane-rich fractions from term infants delivered by cesarean section without labor had a Kmapp of 67 ± 20 μM, n = 5 which was different from adult samples (P<0.05) and a Vmax for O2-production of 14.8 ± 4.5 which was less than that measured from samples from vaginally delivered infants (P< 0.01). The Kmapp reimply a qualitative difference in the fetal oxidase enzyme system. In addition, the increased O2-production and Vmax demonstrated in samples from vaginally delivered infants as compared to infants delivered by cesarean section or healthy adults suggests an effect of parturition on the fetal neutrophil which is similar to the effect of certain compounds such as lipopolysaccharide. The increased catalytic activity of the NADPH oxidase enzyme system in neutrophils from newborns may reflect “priming” during parturition.

Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.

Functional characteristics of neutrophils collected and stored after administration of G–CSF

BACKGROUND: Granulocyte transfusion may be used in neutropenic patients with severe bacterial or fungal infections that are unresponsive to antibiotic therapy. However, the inability to store granulocyte concentrates limits their clinical usefulness.

Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca2+ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105kDa protein (Rel-1) requires Ca2+-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10(-9) to 10(-6)M), becoming maximal after 30s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca2+ but chelation negated the effects, including the cytosolic Ca2+ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca2+ decreased the fMLP-mediated Ca2+ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca2+. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca2+-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.

Phox activity of differentiated PLB-985 cells is enhanced, in an agonist specific manner, by the PLA2 activity of Prdx6-PLA2

Peroxiredoxin 6‐phospholipase A2 (Prdx6‐PLA2) is a bi‐functional enzyme with peroxi‐redoxin (Prdx) and phospholipase A2 (PLA2) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB‐985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA‐resistant Prdx6‐PLA2 into the knockdown cells by stable transfection with a Prdx6‐PLA2 expression plasmid restored the fMLF response, as did reintroduction of Prdx6‐PLA2 mutated in the Prdx active site; reintroduction of PLA2 active site mutants, however, failed to restore the response. Thus, the PLA2 activity of Prdx6‐PLA2 in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA2 isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.

Lack of antibody formation to platelet neoantigens after transfusion of riboflavin and ultraviolet light-treated platelet concentrates.

BACKGROUND: Pathogen inactivation technologies provide a potential solution to donor screening and blood testing strategies reducing the risk of transfusion‐transmitted infectious diseases. The Mirasol pathogen reduction technology (PRT) system (CaridianBCT) uses riboflavin and UV light to introduce modifications in nucleic acids, reducing the infectious pathogen load in blood components. This study evaluated serum of patients who received PRT‐treated platelet (PLT) concentrates over a time period of 28 days for the appearance of antibodies to neoantigens on PLTs.

BLOOD COMPONENTS: Lack of antibody formation to platelet neoantigens after transfusion of riboflavin and ultraviolet light–treated platelet concentrates

BACKGROUND: Pathogen inactivation technologies provide a potential solution to donor screening and blood testing strategies reducing the risk of transfusion‐transmitted infectious diseases. The Mirasol pathogen reduction technology (PRT) system (CaridianBCT) uses riboflavin and UV light to introduce modifications in nucleic acids, reducing the infectious pathogen load in blood components. This study evaluated serum of patients who received PRT‐treated platelet (PLT) concentrates over a time period of 28 days for the appearance of antibodies to neoantigens on PLTs.