Holm Holmsen

Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma

Abstract A method for ATP-ADP determination in plasma by ethanol extraction and firefly luminescence has been modified for platelets. EDTA had to be included in the ethanol for proper inactivation of platelet adenylate kinase. Presence of EDTA inhibited conversion of ADP to ATP (prior to luminescence measurement), presumably through a drop in pH caused by the interaction between H 2 EDTA 2− and the Mg 2+ of the pyruvate-kinase system employed. Introduction of Tris maleate buffer gave 100% ADP-ATP conversion. Optimal amounts of platelet ATP (99%) and ADP (80–90%) were solubilized by 50% EDTA ethanol; less EDTA ethanol failed to inactivate ATP-ADP degrading platelet enzymes whereas increasing EDTA ethanol concentrations gave decreasing solubilization of the nucleotides. ATP and ADP could be extracted from the EDTA ethanol-insoluble material by 0.6 M HClO 4 . ADP and ATP added to such EDTA-ethanol and HClO 4 extracts of both platelet-rich plasma and suspensions of washed platelets gave luminescence responses per mole nucleotide equaling that of ATP-ADP standards. Optimal conditions for nucleotide determination are described as well as the beneficial use of the DuPont luminescence biometer.

Microdetermination of adenosine diphosphate and adenosine triphosphate in plasma with the firefly luciferase system

A simple device for “dark” injection of firefly lantern extract into solutions is described. Connected to the light-detecting part of a Farrand spectrofluorometer this device permitted estimation of the intensity of the initial light flash of the luciferin-luciferase reaction. This was found to be proportional to the concentration of ATP and of ADP, after conversion to ATP with the pyruvate-kinase system, up to 2 μM. Both nucleotides could be determined in plasma after extraction with an equal volume of 96% ethanol, followed by dilution of the extract with 3 vol of an active (ADP + ATP) and an inactive (ATP) solution of pyruvate kinase and phosphoenolpyruvate. The presence of ethanol and inorganic salts depressed the light emission, and their concentration had to be carefully controlled during the procedure. AMP did not affect determination of ATP or ADP, neither did ADP below 1.6 μM in plasma affect determination of ATP, but ATP caused inaccuracy in the determination of ADP when present in concentrations higher than 5 times the ADP concentration. As little as 0.02 μM ADP or ATP in plasma could be estimated with an accuracy of ±6%.

Hereditary Defect in the Platelet Release Reaction Caused by a Deficiency in the Storage Pool of Platelet Adenine Nucleotides

Summary A large family has recently been described in which impaired platelet aggregation was attributed to defective release of the ADP present, although in decreased amounts, in their platelets (Weiss et al, 1969). It was postulated that these patients might lack the storage, or non‐metabolic, pool of ADP which is selectively released from specialized granules during the platelet release reaction and studies on three affected members of this family were undertaken to test this hypothesis. Citrated platelet‐rich plasma (PRP) was incubated with [8‐14C]adenine for 2 hr at 37°C and the specific activity of platelet nucleotides was determined. The release reaction was then induced by shaking the PRP with collagen fibres. The labelling patterns in the patients platelets were distinctly abnormal. The specific activities of their platelet ADP following incubation with [14C]adenine were 4–6 times those of similarly treated normal platelets and were similar to those obtained in the normal platelets after they had been depleted of their non‐metabolic (unlabelled) ADP by treatment with collagen. The findings are consistent with the hypothesis that the patients platelets contain a normal amount of metabolically active ADP, but are deficient in the storage pool. Similar conclusions were reached for ATP. Incubation of the patients platelets with collagen was accompanied by a normal disappearance of radioactive ATP and accumulation of radioactive IMP and hypoxanthine. Thus the abnormality in the platelet release reaction in these patients appears to be the result of a diminished storage pool of nucleotides rather than a block in the pathway which may provide the energy for this reaction.

Adenine nucleotide metabolism of blood platelets VI. Subcellular localization of nucleotide pools with different functions in the platelet release reaction

Abstract 1. 1. Homogenates of washed human platelets have been fractionated on sucrose gradients, and the amounts of ATP, ADP and serotonin in the fractions have been determined. 2. 2. The bulk (90–95%) of nucleotides was recovered in the soluble fraction, whereas the majority of particle-bound nucleotides was present in a high sucrose density fraction with a ATP ADP ratio of 0.6-1.1. Particle-bound serotonin also had its highest concentration in these particles with an ATP serotonin ratio of 5–7. Incubation of platelet-rich plasma with radioactive adenosine, adenine or orthophosphate gave labeled ATP and ADP in the soluble fraction, membranes and mitochondria, whereas the nucleotides in the high sucrose density particles appeared nonradioactive. 3. 3. When labeled platelets were exposed to collagen, nonradioactive ATP and ADP were released in an ATP ADP ratio equal to that of the granule-bound nucleotides. The amounts of ATP and ADP lost from the cells suggested direct release. During release highly radioactive ATP disappeared and could be recovered intracellularly as IMP. 4. 4. Thrombin released maximally 60% of platelet ATP+ADP in an ATP ADP ratio of 0.78. About 20% ATP disappeared without being released. 5. 5. The results can be explained by assuming 3 pools of ATP+ADP in platelets: One pool (60% of total ATP+ADP, ATP ADP = 0.6−1.1 ) is stored in special granules, does not participate in metabolism and is directly extruded during the release reaction. A second pool consists presumably of ATP only (10% of total ATP+ADP), and is consumed intracellularly during release and converted to IMP. A third pool (30% of total ATP+ADP) present in cytoplasm, mitochondria and membranes is unchanged during release and is in equilibrium with the second pool.

Behaviour of endogenous and newly absorbed serotonin in the platelet release reaction

Abstract The release of endogenous and newly absorbed radioactive serotonin (5-HT) from human platelets has been studied. Prior to release induction the platelets were allowed to absorb an amount of 5-HT being equal to or lower than that of endogenous 5-HT. Thrombin released from washed platelets 5-HT of the same specific radioactivity as 5-HT retained and present in control platelets. Thus, in this system the endogenous and absorbed 5-HT behaved identically. With platelet-rich plasma release was induced in an aggregeometer by ADP, adrenaline, collagen and thrombin as well as by recalcification. The specific activity of released 5-HT was the same or slightly higher than that of 5-HT in control platelets, indicating that newly absorbed 5-HT behaves like endogenous 5-HT, except for a slight tendency to be more easily released. Absorption of 5-HT caused a small reduction of the platelets release capacity. It is concluded that under the conditions used, measurement of release of newly absorbed 5-HT is a reasonably good measure of the release of endogenous 5-HT.

An evaluation of the arachidonate pathway of platelets from companion and food-producing animals, mink, and man.

Abstract The arachidonate pathway of human, feline, canine, equine, mink, porcine, and bovine platelets was evaluated by determining the formation of arachidonate-induced malondialdehyde (MDA), thrombin-induced MDA and thrombin-induced thromboxane (Tx) B 2 . In addition, arachidonate-induced platelet aggregation responses were monitored. Arachidonate activated platelets from every animal species evaluated and induced formation of TxB 2 and MDA. There were, however, considerable species differences in the importance of the pathway in mediating the basic platelet reaction. Platelets from mink, pigs, and cows did not aggregate to arachidonate (0.5 mM) and in response to thrombin produced less than 0.5 nmoles of MDA/3 × 10 8 platelets and less than 10 nmoles of TxB 2 /10 11 platelets. Human platelets had a well-developed arachidonate pathway, as they formed more than 1.0 nmoles of MDA/3 × 10 8 platelets and more than 50 nmoles of TxB 2 /10 11 platelets in response to thrombin and irreversibly aggregated in response to arachidonate. Feline platelets exhibited considerable intraspecies variation in the arachidonate pathway. Canine platelets generally formed more than 1.0 nmole of MDA/3 × 10 8 platelets in response to thrombin; yet, platelets from some dogs did not irreversibly aggregate in response to arachidonate. Equine platelets aggregated in response to arachidonate but the aggregation was reversible and they formed between 1.0 and 0.5 nmoles of MDA/3 × 10 8 platelets when incubated with thrombin.

Determination of acid hydrolases in human platelets

Abstract Most acid hydrolases in platelets are determined by methods developed for other tissues. Therefore, we have established the optimal conditions for the measurements of 13 acid glycosidases, aryl phosphatase, aryl sulfatase, and β-glycerophosphatase in crude and dialyzed lysates of human platelets. The substrates used were the glycosides, phosphates and sulfates of p-nitrophenol and 4-methylumbelliferone, phenolphthalein-β-glucuronide, p-nitrocatechol sulfate and β-glycerophosphate. The pH optimum for each enzyme activity did not depend on the type of substrate, whereas Km and V could vary as much as 100-fold. For most of the acid hydrolases, V was higher than the enzyme level determined in the usual way, with the highest substrate concentration obtainable, because this concentration was close to Km. The possible existence of low molecular weight enzyme inhibitors in the lysates was tested by comparing Km at two different lysate concentrations and by comparing the enzyme level in crude lysate with that obtained after dialysis. Only inhibitors for α- and β-galactosidase and aryl sulfatase were found. α-Glucosidase had two pH optima, one at pH 4.1 and the other at 6.0. p-Nitrocatechol sulfatase had a nonlinear time course, similar to the enzyme present in liver. Where possible, Km and V were determined in both citrate-phosphate and acetate buffers, and differences were found for β-fucosidase, α-galactosidase, β-glucosidase, β-N-acetylglucosaminidase, and α-mannosidase. The sensitivity and accuracy of the fluorophor-based assays with 4-methylumbelliferone-containing substrates were markedly better than the chromophore-based assays. In contrast, the determination of β-glycerophosphatase was particularly inaccurate due to formation of large amounts of phosphate from endogenous sources in the lysates. The following acid hydrolases activities were found in human platelet lysates (listed in descending order of total levels): Aryl phosphatase, β-N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, α-mannosidase, aryl sulfatase (p-nitrocatechol substrate), α-arabinosidase, β-N-acetylgalactosaminidase, α-galactosidase, α-fucosidase, β-fucosidase, β-glucosidase, and α-glucosidase (pH 4.1). β-Cellubiosidase and α-xylosidase were not present in the lysates.

Adenine nucleotide metabolism of blood platelets: I. Adenosine kinase and nucleotide formation from exogenous adenosine and AMP

Abstract 1. 1. The metabolism of [14C10]adenosine and [14C10]AMP added to| platelet-rich plasma, washed platelets in saline and platelet extracts was investigated. 2. 2. The metabolites were extracted with ethanol, identified by radioautography and their radioactivity measured after two-dimensional paper chromatography. 3. 3. Approx. 30 % of the [14C10]adenosine was transported into the platelets and appeared mainly as radioactive ADP and ATP whereas the remainder was converted in plasma via inosine to hypoxanthine and two unidentified compounds. 4. 4. AMP was not transported across the platelet membrane, but first had to be dephosphorylated to adenosine in plasma. 5. 5. Platelet lysates in presence of ATP and Mg2+ (or Mn2+) converted [14C10]-adenosine to radioactive AMP, ADP and ATP. The reaction was optimal at pH 5.6 and it was shown that radioactive AMP was the immediate product, but its radio-activity was converted to ATP and ADP within a few seconds by platelet adenylate kinase. The lysates did not incorporate either [8-14C]adenine in the presence of ribose 1-phosphate or 32Pi into adenine nucleotides, suggesting that the adenosine-AMP conversion was catalyzed by adenosine kinase. 6. 6. Platelet lysates contained adenosine deaminase, 5′-AMP-deaminase as well as purine nucleoside phosphorylase.

[17] Measurement of secretion of serotonin

Publisher Summary The principle of the measurement of secretion of serotonin states that the radiolabeled serotonin that has been taken up equilibrates rapidly and totally with the endogenous serotonin and, therefore, behaves functionally as endogenous serotonin. This allows the quantitation of serotonin by a simple measurement of the total radioactivity. However, this ability also poses difficulties for secretion experiments because the secreted serotonin will rapidly re-enter the same platelets that secreted it. In the procedures outlined in this chapter, imipramine is added to a platelet suspension before secretion is induced. Alternatively, serotonin forms a fluorophore with O-phthalaldehyde (OPT), which can be easily detected down to 22 pmol under optimal fluorimetric conditions. The content of serotonin varies in platelets from different species, from 300 μ mol/10 11 human platelets to 6,930 μ mol/10 11 rabbit platelets. The OPT method described in the chapter is, therefore, suitable for human platelet suspensions of 1–3 × 10 8 cells/mL.

Adenine nucleotide metabolism of blood platelets: II. Uptake of adenosine and inhibition of ADP-induced platelet aggregation

Abstract 1. 1. Inhibition of ADP-induced aggregation of human platelets in plasma by AMP, adenosine monoacetate and adenosine monopropionate has been studied. These compounds produced inhibition only after hydrolysis to adenosine in plasma. 2. 2. The concentration of adenosine in plasma and the amount phosphorylated in the platelets during inhibition of aggregation was estimated by use of [ 14 C 10 ]adenosine and [ 14 C 10 ]AMP. Inhibition correlated well with the rate of phosphorylation, but poorly with the adenosine concentration. 3. 3. Inhibition of platelet aggregation remained present after rapid clearance of the adenosine from plasma on addition of adenosine deaminase. 4. 4. Rising concentrations of adenosine did not induce 100 % inhibition of aggregation. Both inhibition and the rate of phosphorylation were saturated at the same adenosine concentration. 5. 5. d -2-Deoxyglucose and antimycin together strongly inhibited ADP-induced platelet aggregation, reduced adenosine phosphorylation by 50 % and conversion of [8- 14 C]adenine to nucleotides by 80 %. 6. 6. These results suggest that adenosine is transported across the membrane and perhaps phosphorylated before inducing inhibition. Inhibition might be caused by competition for energy required for both platelet aggregation and the adenosine transport—phosphorylation process.