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G L Dale

Publications and source records attributed to G L Dale.

At least 37 records · Page 2Linked to original sources
PubMed1995

Demonstration that thiazole-orange-positive platelets in the dog are less than 24 hours old.

Approximately 6% of dog platelets are positive for staining with thiazole orange, a dye frequently used to stain ribonucleic acid. In this report, thiazole-orange positivity is shown to mark platelets that are less than 24 hours old. Dog platelets were derivatized in vivo with N-hydroxysuccinimido biotin such that greater than 95% of all platelets were biotinylated. Newly synthesized, nonbiotinylated platelets were then monitored by flow cytometry for their ability to bind thiazole orange. After biotinylation, the percentage of biotin-negative, thiazole-orange-positive platelets increased gradually from 0.72% at 30 minutes to 5.44% at 24 hours. These data indicate that thiazole-orange staining does label newly synthesized platelets.

Animals↗
PubMed1994

Fluorescein derivatization of fibrinogen for flow cytometric analysis of fibrinogen binding to platelets.

Dog and human fibrinogen were derivatized with N-hydroxysuccinimido-fluorescein and utilized for flow cytometric estimation of fibrinogen binding to activated platelets. Fluorescein-fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was saturable, dependent on agonist activation, and inhibited by unlabeled fibrinogen. In addition, EDTA and barbourin, a KGD-containing peptide, were found to inhibit the binding of fluorescein-fibrinogen. Fluorescein-fibrinogen bound to dog platelets with an apparent affinity of 0.31 microM after stimulation with either adenosine-5'-diphosphate (ADP) or plateletactivating factor. The labeled fibrinogen was also used to study the fibrinogen binding capacity of aged, biotinylated platelets. Aged platelets were indistinguishable from young platelets with regard to fibrinogen binding in response to ADP. These studies document that direct derivatization of fibrinogen with fluorescein generates a useful probe for analyzing fibrinogen binding to platelets with flow cytometry.

Adenosine Diphosphate↗
PubMed1994

Alteration of platelet function in dogs mediated by interleukin-6.

To determine if interleukin-6 (IL-6) administration influences platelet function, platelet activation was analyzed sequentially in IL-6-treated (80 micrograms/kg/d) and control dogs. Platelet activation was determined in whole blood by flow cytometry by quantitating the binding of a monoclonal antibody to platelet surface P-selectin after stimulation with graded doses of thrombin. Administration of IL-6 resulted in a twofold decrease in the thrombin concentration required for induction of half-maximal P-selectin expression (ED50) compared with control animals. The ED50 returned to normal after cessation of IL-6 administration. As measured by P-selectin expression, enhanced responsiveness to the strong agonist platelet activating factor (PAF) was also observed in the IL-6-treated dogs. IL-6 had no effect on the susceptibility of platelets to thrombin activation when incubated with anticoagulated dog blood. The data show that, in addition to augmenting the platelet count in normal dogs, IL-6 enhances the sensitivity of platelets to activation in response to thrombin and PAF.

Amino Acid Sequence↗
PubMed1994

Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression.

After the intravenous infusion of N-hydroxysuccinimido biotin into dogs, 80.6% +/- 9.7% (n = 5) of platelets were covalently labeled with biotin. The in vivo survival of the biotinylated platelets was monitored by flow cytometry and was normal as compared with previous reports for dog platelets. The ability of the biotinylated platelets to be activated was analyzed by measuring the expression of cell-surface P-selectin after incubation with graded concentrations of thrombin. When P-selectin expression was examined 3 hours after labeling, biotinylated platelets were indistinguishable from the nonlabeled population of platelets, indicating that biotinylation did not adversely affect the cells. On consecutive days after biotinylation, the thrombin dose-response curves for biotinylated and nonbiotinylated platelets were repeated, and as the biotinylated-platelets aged, they became less responsive to thrombin. On days 3, 4, and 5, the thrombin EC50 for the aged, biotinylated platelets as compared with the total population of platelets was 136%, 150%, and 178%, respectively. Increasing age clearly impairs the reactivity of platelets towards thrombin as quantitated by the expression of cell-surface P-selectin.

Animals↗
PubMed1994

Dog platelets accumulate intracellular fibrinogen as they age.

The alpha granules of circulating platelets are dynamic structures that acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of either receptor-mediated endocytosis or fluid-phase pinocytosis. Despite many detailed studies on the function and content of alpha-granules, little is known of the impact of platelet age on these organelles. In this report, we describe the use of platelet biotinylation to identify and isolate aged platelets for the analysis of alpha-granule contents. When aged platelets were permeabilized and examined by flow cytometry utilizing fluorescently labeled antibodies, two exogenously acquired proteins, fibrinogen and immunoglobulin G, were found to increase significantly with platelet age. The levels of intracellular fibrinogen were found to be elevated relative to control, 114 +/- 2% and 119 +/- 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increased similarly. Levels of two endogenously synthesized proteins, thrombospondin and P-selectin, were not elevated in aged platelets. Confirmation of the flow cytometric data was obtained by isolating aged, biotinylated platelets by fluorescence-activated cell sorting and quantitating the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 +/- 23% of the level for the entire platelet population. These data demonstrate that age-dependent changes in exogenously acquired alpha-granule proteins do occur and that the uptake mechanism for these proteins is active throughout the platelet life span.

Animals↗
PubMed1994

Antibodies against biotinylated proteins are present in normal human serum.

Antibodies against biotinylated proteins have been identified in 10% of individuals tested (6/60). These antibodies bind readily to biotinylated proteins (50% inhibitory concentration = 0.59 mumol/L) but only modestly to free biotin or biocytin. It is unlikely that any clinical consequences occur as a result of these antibodies, because the affinity for free biotin is too low (50% inhibitory concentration = 0.51 mmol/L) to affect the normal level of free plasma biotin, 0.5 nmol/L. The pathogenesis of this antibiotin immune response is unclear. Repeat testing of several individuals 5 months apart indicated that the antibiotin response was stable. In addition, 51 of the individuals tested for antibiotin antibodies were also examined for antiavidin antibodies. Whereas five were positive for antiavidin, only one individual was positive for both antibiotin and antiavidin antibodies. The presence of an antibiotin antibody is unlikely to affect the in vivo use of biotinylated proteins or cells in human subjects because its affinity for biotinylated proteins is modest and the level of biotinylation for in vivo studies is intentionally low. However, these antibodies may affect clinical or laboratory assays based on the biotin-avidin system where an antibiotin antibody may either positively or negatively affect the specific assay.

Antibodies↗
PubMed1993

Biotinylated platelets: a new approach to the measurement of platelet life span.

The in vivo life span of dog platelets was determined by derivatizing whole blood with N-hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 +/- 1.1 d as determined by curve-fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either 111Indium-oxine or 51Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.

Animals↗
PubMed1992

Rheologic properties of senescent erythrocytes: loss of surface area and volume with red blood cell age.

The rheologic properties of senescent erythrocytes have been examined using two models of red blood cell (RBC) aging. In the rabbit, aged erythrocytes were isolated after biotinylation, in vivo aging, and subsequent recovery on an avidin support. Aged RBCs from the mouse were obtained using the Ganzoni hypertransfusion model that suppresses erythropoiesis for prolonged periods of time allowing preexisting cells to age in vivo. In both cases, the aged erythrocytes were found by ektacytometry to have decreased deformability due to diminished surface area and cellular dehydration. The aged rabbit erythrocytes were further characterized by micropipette methods that documented an average surface area decrease of 10.5% and a volume decrease of 8.4% for the cells that were 50 days old. Because both the surface area and volume decreased with cell age, there was little change in surface-to-volume ratio (sphericity) during aging. The aged cells were found to have normal membrane elasticity. In addition, human RBCs were fractionated over Stractan density gradients and the most dense cells were found to have rheologic properties similar to those reported for the aged RBCs from rabbits and mice, although the absolute magnitude of the changes in surface area and volume were considerably greater for the human cells. Thus, stringent density fractionation protocols that result in isolation of the most dense 1% of cells can produce a population of human cells with rheologic properties similar to senescent cells obtained in other species. The data indicate that progressive loss of cell area and cell dehydration are characteristic features of cell aging.

Animals↗
PubMed1991

Quantitation of immunoglobulin associated with senescent erythrocytes from the rabbit.

The biochemical processes that determine the lifespan of mammalian erythrocytes are unknown; one prominent theory suggests that antibody binding to the senescent red blood cell identifies it for removal from the circulation. To address this question, we have used a newly developed procedure for the isolation of aged erythrocytes that involves the biotinylation of rabbit red blood cells, in vivo aging of these cells, and the eventual in vitro recovery of the aged red blood cells by their affinity for an avidin support. Erythrocytes isolated with this method were found to have near-normal levels of cell-associated Ig throughout their 60-day lifespan. These data suggest that IgG accumulation is not part of the normal senescence process for erythrocytes in rabbits.

Animals↗
PubMed1991

Characterization of senescent red cells from the rabbit.

The above data continue to demonstrate the metabolic well being of the aged red cell as it is isolated from rabbits. The abundance of ATP, the absence of surface-bound IgG and a variety of other observations at this time lead to the tentative conclusion that the senescent red cell is amazingly healthy. Many investigators have predicted that the red cell is removed from the circulation as a metabolically exhausted effete cell. There is currently no evidence to support this other than a decrease in deformability of the cells with time, but it is not clear that this decline in deformability is sufficient to keep the cell from circulating. In either case, many of the previously proposed causes of cellular removal are clearly incorrect for the rabbit, and it is now time to focus on new directions for observing either cellular impairment or perhaps the presence of a cellular clock which is independent of the cell's metabolic state. Another point which should be addressed is the reliability of the biotinylation model in rabbits as it relates to red cells in other species. So far several observations in aged red cells isolated with valid models have been reproduced across species boundaries including the rise in ATP, the fall in AMP deaminase activity, the shift in the 4.1a to 4.1b protein ratio, the stability of a number of glycolytic enzymes, and the instability of pyrimidine 5'-nucleotidase activity. To this point, the rabbit has been a reliable model of red cell aging and one with implications for other species.

AMP Deaminase↗
PubMed1990

Density fractionation of erythrocytes by Percoll/hypaque results in only a slight enrichment for aged cells.

Density fractionation has served for many years as a standard procedure for the isolation of aged erythrocyte populations; however, a quantitative evaluation of the technique has not been available. This report describes such an analysis. Rabbits were infused intravenously with N-hydroxysuccinimido biotin to biotinylate greater than 90% of all circulating erythrocytes; the enumeration of these biotinylated cells was possible by monitoring their ability to bind avidin-coated microspheres. The biotinylated erythrocytes were shown to have a normal in vivo survival. As a result of the normal senescence process, only aged red cells would have membrane-bound biotin 50 days after biotinylation. At this time, the rabbit red cells were fractionated over Percoll/hypaque to determine the ability of density fractionation to enrich for the aged, biotinylated cells. Density fractionation resulted in an average enrichment for aged cells of only 2-3-fold above that present in a random population.

Animals↗
PubMed1990

In vivo aging of red cell enzymes: study of biotinylated red blood cells in rabbits.

It is generally recognized that the activities of some of the red cell enzymes decline as the cell ages. However, there is still a controversy regarding the rate at which this aging occurs. In the present study we applied newly developed technology for the specific isolation of maturing reticulocytes/erythrocytes for a more comprehensive study of in vivo aging of red cell enzymes in rabbits. Anemia was induced by repeated phlebotomy, and reticulocyte-rich erythrocytes were labeled with N-hydroxy succinimido-biotin and then transfused into a normal rabbit. These biotinylated cells were isolated at various time points by their affinity for an avidin support, and the enzymatic activity of 19 red cell enzymes was measured. We observed a biphasic pattern of decay for the activity of six age-dependent enzymes--aldolase, glutamate-oxaloacetate transaminase, glucose 6-phosphate dehydrogenase, hexokinase, pyrimidine 5'-nucleotidase, and pyruvate kinase.

Animals↗
PubMed1989

Inhibition of autoantibody binding to platelet glycoprotein IIb/IIIa by anti-idiotypic antibodies in intravenous gammaglobulin.

Intravenous immunoglobulin (IVIgG) causes an acute rise in the platelet count in the majority of patients with chronic immune thrombocytopenic purpura (ITP) but the mechanism(s) of action is still unknown. We evaluated the ability of three different IVIgG preparations to inhibit the in vitro binding of autoantibody to platelet glycoprotein (GP) IIb/IIIa. ITP plasma, known to contain anti-GPIIb/IIIa antibodies, was incubated overnight with either IVIgG or bovine serum albumin (BSA) followed by measurement of the autoantibody titer. Binding of autoantibody from eight ITP patients was inhibited by IVIgG in proportion to the IVIgG concentration. Using 3.2% IVIgG, compatible with therapeutic concentrations expected in vivo, mean inhibition of autoantibody binding ranged from 20.2% to 41.3%. No inhibition by IVIgG of alloantibody binding to the same or different molecules was detected (five patients with anti-GPIIb/IIIa and two with anti-HLA alloantibodies). F(ab')2 fragments of IVIgG also inhibited the binding of both plasma autoantibodies and purified anti-GPIIb/IIIA autoantibodies prepared by elution from antigen affinity columns. A portion of the anti-idiotypic antibodies could be adsorbed from IVIgG using insolubilized, purified anti-GPIIb/IIIa autoantibody. These results show that IVIgG preparations from normal donors contain anti-idiotypic antibodies directed against idiotypes located on GPIIb/IIIa autoantibodies but do not have anti-idiotypes to platelet alloantibodies against the same or different molecules. The importance of these anti-idiotypic antibodies in the therapeutic response to IVIgG remains to be established.

Antibodies, Anti-Idiotypic↗
PubMed1989

Time-dependent loss of adenosine 5'-monophosphate deaminase activity may explain elevated adenosine 5'-triphosphate levels in senescent erythrocytes.

Senescent erythrocytes from rabbits were previously shown to have elevated levels of adenine nucleotides. The present study documents that aged red blood cells have a normal synthetic capacity for adenine nucleotides, as indicated by normal levels of adenosine kinase. However, senescent erythrocytes do have decreased levels of adenosine 5'-monophosphate deaminase, the critical enzyme involved in degrading adenine nucleotides. These circumstances of a normal synthetic capacity in the presence of decreased catabolic ability were observed previously in a human genetic deficiency of adenosine 5'-monophosphate deaminase; the red blood cells in these patients accumulate adenosine 5'-triphosphate as do senescent erythrocytes in rabbits.

AMP Deaminase↗
PubMed1989

Radioisotopic assay for erythrocyte adenosine 5'-monophosphate deaminase.

Adenosine-5'-monophosphate deaminase is a critical enzyme in the regulation of adenine nucleotide levels in the erythrocyte. The routine examination of this enzyme in crude hemolysates is difficult with the commonly used assay which monitors ammonia generated by the deamination reaction. This report details a radioisotopic assay for AMP deaminase which allows separation of the [14C]inosine 5'-monophosphate product from the [14C]adenosine 5'-monophosphate substrate by ion-exchange chromatography at pH 2.2. The radioisotopic assay is linear with respect to time and enzyme concentration over a considerable range and thereby significantly simplifies the monitoring of crude or dilute enzyme preparations.

AMP Deaminase↗
PubMed1989

Membrane proteins in senescent erythrocytes.

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.

Animals↗