PubMed Health⌕ Search

Biomedical subjects

G L Dale

Publications and source records attributed to G L Dale.

At least 19 recordsLinked to original sources
PubMed1998

Rapid production of quasi-stable antibody-phycoerythrin conjugates for use in flow cytometry.

A simple system is described for producing quasi-stable complexes between biotinylated antibodies and fluorochrome-labeled streptavidin. Optimal complexes were produced by mixing biotinylated antibody and phycoerythrin-streptavidin together at a molar ratio of 1:1 for 2 min, followed by the addition of soluble biotin to halt oligomerization. These complexes, which are stable for at least 4 h, exhibit fluorescence with cell binding which approximates that observed with standard two-step labeling techniques. Equally important, multiple biotinylated antibodies complexed with different streptavidin-fluorochromes can be used simultaneously.

Animals↗
PubMed1998

Is there a correlation between raised erythropoietin and thrombotic events in sickle-cell anaemia?

Children with sickle-cell anaemia are predisposed to thrombotic strokes, the aetiology of which is unclear. We propose that erythropoietin, produced in response to chronic anaemia, is responsible for changes in platelet reactivity with a resulting increase in thromboses. This hypothesis is based on reports of enhanced aggregability of erythropoietin-driven platelets and an increased rate of thrombosis in patients receiving large doses of recombinant erythropoietin. Experiments in animals have shown that erythropoietin stimulates synthesis of platelets, that erythropoietin-driven platelets are hyper-reactive compared with age-matched control platelets, and that erythropoietin is pro-thrombotic. These data suggest that erythropoietin-dependent changes in platelet reactivity may potentiate thrombosis in sickle-cell anaemia, particularly in children who, compared with adults, have markedly higher erythropoietin concentrations and incidence of strokes.

Adult↗
PubMed1998

Flow cytometric analysis of platelet activation by different collagen types present in the vessel wall.

The interaction of platelets with collagens of the vessel wall is a critical event in primary haemostasis. Although numerous studies have examined the ability of various collagen types to support platelet adhesion, little is known concerning the relative ability of different collagens to elicit specific activation markers in platelets. In this report, flow cytometric analysis has been utilized to evaluate the ability of various native collagen types to elicit secondary activation events in human platelets. Collagen types I, III, V and VI induced alpha-granule secretion and up-regulation of cell surface glycoprotein (GP) IIb/IIIa. In contrast, collagen type IV did not elicit these responses in the concentration ranges examined. Dose-response curves for alpha-granule secretion induced by the various collagen types indicated that human type III and human type I collagens were less effective than human type V, human type VI and calf skin type I. In addition, the ability of these various collagens to activate GPIIb/IIIa to its ligand binding conformation was even more heterogenous with only human type VI and calf skin type I readily promoting this transition. These data demonstrate that flow cytometric analysis of collagen-induced platelet activation is feasible and that collagen-mediated alpha-granule secretion and membrane glycoprotein redistribution in human platelets are separate events from activation of GPIIb/IIIa.

Blood Platelets↗
PubMed1997

Platelet kinetics.

Platelet survival can be determined with considerable precision using radioisotopic methodologies; however, complications associated with these techniques still catalyze the search for nonisotopic procedures to estimate platelet survival as well as surrogate markers of platelet life span. Recent reports on markers of platelet age have focused on thiazole orange-staining for enumeration of "reticulated platelets," ie, young platelets with residual mRNA. Numerous clinical studies have observed a correlation between thiazole orange-positive platelets and platelet synthesis, thereby providing a noninvasive means to differentiate between synthetic and destructive mechanisms in thrombocytopenia. Additionally, nonisotopic methods for quantitating platelet survival have recently been developed in animal systems, although they have not yet been transferred to human use. And finally, an ancillary topic related to platelet turnover is the impact of cell age on platelet reactivity. Prevailing paradigms concerning age and reactivity are complicated by recent studies with cytokines, such as interleukin-6, which demonstrate that platelet age is not the sole determinant of platelet reactivity.

Animals↗
PubMed1997

Erythropoietin potentiates thrombus development in a canine arterio-venous shunt model.

Erythropoietin (EPO) has been previously shown to affect platelet as well as red cell production. In addition, recent studies demonstrated that platelets from EPO-treated dogs are hyperreactive towards thrombin when compared to age-matched, control platelets. This report extends these observations by quantitating the thrombogenic potential of EPO in dogs. Dogs with arterio-venous (A-V) shunts received 100 U EPO/kg/day for 6 days, and thrombogenicity was serially monitored by insertion of a thrombotic surface into the A-V shunt. The resulting experimental thrombi were analyzed for platelet and erythrocyte content after formalin-fixation and chymotrypsin digestion, a technique which allows non-isotopic quantitation of cellular components. By day 5 of EPO-administration all animals demonstrated a significant increase in platelet and red cell content of the experimental thrombi; the average increase in platelet number was 2.94 +/- 0.12 fold (mean +/- 1 SE; n = 3; p = 0.006) above baseline while that for red cells was 2.46 +/- 0.18 fold above baseline (p = 0.023). After cessation of EPO, thrombogenicity returned to normal. During EPO-treatment, the percentage of thiazole orange-positive (TO+) platelets increased significantly to 17.2 +/- 1.6% (mean +/- 1 SE; n = 3) on day 5 compared to a pre-treatment level of 8.5 +/- 0.9% (p = 0.029). Although the percentage of TO+ erythrocytes also increased during the short course of EPO administration, the change was not significant. Despite the increases in TO+ cells, total platelet and erythrocyte counts did not change significantly within the time frame of these experiments. Fibrin/fibrinogen content of the experimental thrombi was unaltered with EPO-treatment. These data demonstrate that human EPO is pro-thrombotic in dogs and, in conjunction with earlier studies, suggest that hyperreactive platelets may be responsible for the potentiated thrombogenicity.

Animals↗
PubMed1997

Erythropoietin administration increases production and reactivity of platelets in dogs.

Administration of erythropoietin (EPO) to adult dogs resulted in a dramatic increase in the number of thiazole orange-positive (TO+) platelets, also referred to as reticulated platelets. Pre-treatment level of TO+ platelets was 6.2 +/- 0.5% (mean +/- 1 SE: n = 5); following day 5 of treatment with 500 U EPO/kg/day, the percentage of TO+ platelets peaked at 16.8 +/- 2.3% (n = 5; p <0.02). After cessation of the hormone, the number of TO+ platelets fell rapidly to below starting levels. Unexpectedly, there was a significant decline in total platelet count during EPO administration despite an increased level of TO+ platelets. To assess platelet reactivity, total platelets and TO+ platelets from EPO-treated dogs were analyzed for thrombin-responsiveness as quantitated by P-selectin expression on the cell surface; reactivity was expressed as a thrombin EC50, the thrombin concentration required to activate 50% of platelets. Both total and TO+ platelets were hyperreactive during EPO treatment when compared either to pre-treatment values or to control animals. Thrombin EC50 values for total and TO+ platelets on day 5 fell to 66.5 +/- 5.4% (mean +/- 1 SE; n = 5; p <0.02) and 62.2 +/- 8.7% (n = 5; p <0.025), respectively, of pre-treatment levels. These data indicate that EPO not only promotes the synthesis of increased numbers of TO+ platelets in the dog but that these newly produced platelets are hyperreactive when compared to TO+ platelets from control animals.

Animals↗
PubMed1996

Relative reactivity of platelets from thrombopoietin- and interleukin-6-treated dogs.

Previous reports have shown that interleukin-6 (IL-6) enhances the responsiveness of platelets to thrombin stimulation and has modest thrombocytopoietic effects in vivo. Thrombopoietin (TPO; mpl ligand) has been shown to have dramatic thrombocytopoietic effect in vivo, but little is known of its capacity to alter platelet function. In this study, a direct comparison of the effects of IL-6 and TPO on platelet function in dogs has been performed, with modest doses of TPO (1 microgram/kg/d) chosen to match or moderately exceed the platelet counts achieved with IL-6 (40 micrograms/kg/d) for 10 days. Platelet responsiveness to thrombin stimulation was assessed in TPO-treated, IL-6-treated, and control dogs by flow cytometric measurement of P-selectin expression. On day 5, the dose of thrombin promoting half maximal stimulation (EC50) of platelets was not significantly changed in TPO-treated dogs, whereas in IL-6-treated dogs the EC50 decreased to 73.1% +/- 6.1% (mean +/- 1 SD; n = 5) of control values (P < 0.01). These experiments were performed on both gel-filtered platelets and washed whole blood, indicating that the observed changes in EC50 were caused by cytokine-mediated alteration of platelets rather than plasma components. Because it has been shown that thiazole orange specifically labels a subpopulation of dog platelets that is less than 24 hours old, the thrombin responsiveness of these young, newly synthesized platelets was determined. The EC50 of thiazole orange-positive platelets from IL-6-treated dogs decreased dramatically by day 5 to 46.5% +/- 13.1% (n = 4) of control values (P < 0.001), whereas TPO-treated dogs did not significantly change. When TPO was directly incubated with platelets ex vivo, no effects on either thrombin-mediated P-selectin expression or adenosine diphosphate-induced fibrinogen binding were observed. These data show that IL-6 alters platelet function, as measured by reactivity to thrombin, whereas TPO does not. This divergence in function is observed even though TPO is equally, or more, effective at promoting platelet production under these experimental conditions.

Adenosine Diphosphate↗
PubMed1996

Quantitation of platelet life span in splenectomized dogs.

Platelet life spans in dogs were measured pre- and post- splenectomy utilizing in vitro whole blood biotinylation. Four splenectomized dogs were found to have significantly lengthened platelet life spans, 193 +/- 7 hours (mean +/- 1 SD;n=4) postsurgery vs. control life spans of 131 +/- 15 hours (n=6; p< 0.01) when analyzed with the multiple-hit model. Additionally, platelets from normal dogs transfused into splenectomized dogs were found to have convex survival curves with extended life spans approximating that of the splenectomized dog. These data indicate that the spleen is a significant determinant of platelet life span in dogs, with survivals increasing approximately 47% upon splenectomy.

Animals↗
PubMed1996

Non-isotopic method for quantitation of platelets and erythrocytes in experimental thrombi.

Experimental animal models of thrombosis have been established in several species to examine factors responsible for thrombotic disorders in man. One technical facet of all thrombosis models is the need to quantitate cell deposition on thrombogenic surfaces, and this is routinely accomplished with radioisotopic labeling of specific components. Data reported here demonstrate that formalin-fixed thrombi can be hydrolyzed with chymotrypsin allowing recovery and quantitation of platelets and erythrocytes incorporated within the clot. Recovery of platelets from in vitro generated, model thrombi averaged 99 +/- 10% (mean +/- 1 SD; n = 7; range 88-116%) of calculated content; recovery of erythrocytes was 94.1 +/- 1.1% (n = 6) as measured by recovery of cellular hemoglobin after chymotrypsin hydrolysis of clots. Chymotrypsin was also shown to release platelets and erythrocytes from string-bound thrombi generated in vivo with an arterio-venous shunt model in beagle dogs. Platelet recovery from these string clots after chymotrypsin hydrolysis was independently verified with a quantitative Western blot assay of platelet antigens. These data demonstrate that experimental thrombi can be hydrolyzed with chymotrypsin, thereby not only eliminating the need for radioisotopes, but also permitting flow cytometric analysis of cells comprising the thrombus.

Animals↗
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↗