scholarly journals A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1974 ◽  
Vol 138 (2) ◽  
pp. 165-169 ◽  
Author(s):  
John L. Gordon ◽  
Alan H. Drummond
Keyword(s):  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Firefly Luciferase ◽  
Human Platelets ◽  
Luciferase Assay ◽  
Fluorescent Product ◽  
Optimum Conditions ◽  
Before And After ◽  
Rat Platelets

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/108 cells in man and 7.8nmol/108 cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.

Mechanism of platelet plug formation and role of adenosine diphosphate

1964 ◽  
Vol 206 (6) ◽  
pp. 1267-1274 ◽  
Author(s):  
Theodore H. Spaet ◽  
Marjorie B. Zucker
Keyword(s):  
Connective Tissue ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Suitable Model ◽  
Plug Formation ◽  
Rat Platelets

Traumatized rat omentum was used to demonstrate the development of "platelet plugs" following agitation in platelet-rich plasma. In the absence of divalent cation there was only platelet adhesion to connective tissue fibers; in the presence of divalent cation masses of platelets formed (cohesion) even in plasma adequately anticoagulated with heparin. Exposure of these platelet masses to thrombin produced greater compactness and stability. Human and rat platelets behaved alike with the traumatized rat omentum; platelets from two patients with von Willebrand's disease gave normal reactions whereas platelets from a patient with thrombasthenia showed adhesion only. Exposure of human platelets to washed connective-tissue fragments or to thrombin elicited clumping accompanied by release of serotonin and of adenine nucleotides (AN) of which about one-third was adenosine diphosphate. Intermediate concentrations of connective tissue and thrombin also caused clumping but no liberation of AN or serotonin. ADP caused intense clumping but failed to liberate serotonin or additional ADP. It is suggested that cohesion reaction is mediated by release of ADP. The traumatized omentum appears to be a suitable model for studying the hemostatic process.

Extraction of Protein-Bound ATP and ADP from Human Platelets in Plasma

1990 ◽  
Vol 63 (02) ◽  
pp. 286-290 ◽  
Author(s):  
Christina Beurling-Harbury ◽  
Pehr B Harbury
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Binding Protein ◽  
Human Platelets ◽  
Luciferase Assay ◽  
First Time ◽  
Plasma Extraction

SummaryActin is the major ATP and ADP binding protein in platelets, 0.9–1.3 nmol/108 cells, 50–70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/108 cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/108 cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.

Newborn Platelet Dysfunction: a Storage Pool and Release Defect

1976 ◽  
Vol 36 (01) ◽  
pp. 200-207 ◽  
Author(s):  
Donald G. Corby ◽  
Thomas F. Zuck
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Newborn Infants ◽  
Specific Radioactivity ◽  
High Dose ◽  
Platelet Dysfunction ◽  
Paired Samples ◽  
Normal Adults

SummaryPer cent aggregation, release and content of adenine nucleotides, and specific radioactivity were evaluated in citrated platelet-rich plasma (PRP) prepared from paired samples of maternal and cord blood. Platelets of newborn infants aggregated normally in response to high dose ADP (20 μM), strong collagen suspensions, and thrombin; however, when compared with PRP from the mothers or from normal adults, per cent aggregation in response to lower concentrations of ADP (2 μM), weak collagen, and part particularly epinephrine was markedly reduced. Nucleotide release after stimulation of the newborns’ PRP with the latter two inducers was also impaired. ATP and ADP content of the newborns’ platelets was also significantly less than that of their mothers or of normal adults, but specific activity was normal. The data suggest that the impairment of ADP release in the platelets of newborn infants is due to decreased sensitivity to external stimuli. Since metabolic ATP is necessary for the platelet release reaction, it is postulated that the platelet dysfunction results from a lack of metabolic ATP.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

scholarly journals Short communication: possible association of newly absorbed serotonin with nonmetabolic, granule-located adenine nucleotides in human blood platelets

Blood ◽  
1975 ◽  
Vol 45 (3) ◽  
pp. 413-416
Author(s):  
H Holmsen ◽  
CA Setkowsky ◽  
HJ Day
Keyword(s):  
Guinea Pig ◽  
Human Blood ◽  
Short Communication ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Human Platelets ◽  
Serotonin Release ◽  
Release Reaction ◽  
Human Blood Platelets

[3H]-adenine-labeled human platelets in plasma were incubated with or without nonradioactive serotonin. Release reaction was then induced by ADP, epinephrine, collagen, or thrombin. Platelets that had been incubated with serotonin released four times as much serotonin as platelets incubated without serotonin. The specific radioactivities of the ATP and ADP released to plasma during release reaction induced with all four inducers were the same in both systems. This shows that when serotonin is taken up by human platelets, it enters the compartment containing nonmetabolic, granula-stored ATP, and not the compartment with metabolic extragranular ATP. These results suggest that the mechanism of serotonin storage in human platelets is similar to that in other species investigated, i.e., rabbit, guinea pig, and pig.

scholarly journals A simple assay for adenylate cyclase in intact cells by affinity elution chromatography

1978 ◽  
Vol 174 (3) ◽  
pp. 699-702
Author(s):  
A K Sinha ◽  
R W Colman
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Intact Cells ◽  
Atp Pool ◽  
Dependent Protein ◽  
Elution Chromatography ◽  
Cellulose Column

A method using the principle of affinity elution chromatography is described for the assay of adenylate cyclase in intact human platelets. By incubating platelet-rich plasma in the presence of radioactively labelled adenine, the ATP pool of the cells was prelabelled. Formation of labelled cyclic AMP from ATP was determined by extracting the platelets with HC1O4. After removal of the latter as KC1O4, the extract containing cyclic AMP and other adenine nucleotides was adsorbed in a NN-diethyl-N-2-hydroxypropylamino (QAE)-cellulose column. The column was washed, and subsequently cyclic AMP was specifically eluted with a cyclic AMP-dependent protein kinase and the radioactivity of the eluate was determined.

Immune Reactions of Human Blood Platelets

1968 ◽  
Vol 20 (03/04) ◽  
pp. 336-344 ◽  
Author(s):  
Ch Mueller-Eckhardt ◽  
E. F Lüscher
Keyword(s):  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Fibrin Clot ◽  
Human Platelets ◽  
The Other ◽  
Buffer Solution ◽  
Plasma Fractions ◽  
Mediator Substances ◽  
Human Blood Platelets

SummaryThe effect on human platelets of 2 endotoxin preparations (one had known Shwartzman-activity in rabbits, the other was not tested for its biological activity) was investigated in vitro. The following results were found:1. Endotoxin has no effect on washed human platelets suspended in isotonic, plasmafree buffer solution.2. Aggregation or release of adenine nucleotides from human platelets by endotoxin does also not occur if the platelets are suspended in coagulable, complement active, pooled human plasma or plasma fractions.3. Platelets pretreated with α-chymotrypsin do not show aggregation or release of nucleotides by endotoxin.4. The ability of human platelets to retract a fibrin clot is not disturbed by endotoxin. This excludes a functional platelet injury by endotoxin not detectable by aggregation or nucleotide release.5. There is no evidence for the assumption that the effect of endotoxin on platelets is transmitted by yet hypothetical, platelet-damaging mediator substances from leukocytes.6. These results suggest that an immunological injury of platelets by endotoxin comparable with the effect of immune complexes or aggregated gammaglobulin is highly improbable.

Incorporation of Labelled Nucleotides and Aggregation of Human Blood Platelets

1966 ◽  
Vol 15 (01/02) ◽  
pp. 052-068 ◽  
Author(s):  
E. W Salzman ◽  
D. A Chambers ◽  
Lena L. Neri
Keyword(s):  
Platelet Aggregation ◽  
Methyl Ester ◽  
Human Blood ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Temperature Dependent ◽  
Sequence Of Events ◽  
Plasma Factor ◽  
Human Blood Platelets

SummaryAddition of ADP-8-C14 to platelet-rich plasma results in platelet radioactivity through a series of temperature-dependent reactions involving stepwise dephosphorylation of the nucleotide in plasma and then incorporation of the adenosine residue by the platelet, where it participates as a precursor in synthesis of platelet adenine nucleotides. This sequence of events, which requires a heat-labile nondialysable plasma factor, is blocked by 0.01 M KCN, by EDTA, by arginine methyl ester, by adenosine, and by AMP.ADP-induced platelet aggregation does not require the reactions outlined above and proceeds without ADP - breakdown and without binding of the ADP- molecule to the platelet. A possible mechanism of this phenomenon is suggested.

Short communication: possible association of newly absorbed serotonin with nonmetabolic, granule-located adenine nucleotides in human blood platelets

Blood ◽  
1975 ◽  
Vol 45 (3) ◽  
pp. 413-416 ◽  
Author(s):  
H Holmsen ◽  
CA Setkowsky ◽  
HJ Day
Keyword(s):  
Guinea Pig ◽  
Human Blood ◽  
Short Communication ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Human Platelets ◽  
Serotonin Release ◽  
Release Reaction ◽  
Human Blood Platelets

Abstract [3H]-adenine-labeled human platelets in plasma were incubated with or without nonradioactive serotonin. Release reaction was then induced by ADP, epinephrine, collagen, or thrombin. Platelets that had been incubated with serotonin released four times as much serotonin as platelets incubated without serotonin. The specific radioactivities of the ATP and ADP released to plasma during release reaction induced with all four inducers were the same in both systems. This shows that when serotonin is taken up by human platelets, it enters the compartment containing nonmetabolic, granula-stored ATP, and not the compartment with metabolic extragranular ATP. These results suggest that the mechanism of serotonin storage in human platelets is similar to that in other species investigated, i.e., rabbit, guinea pig, and pig.

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