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D C Mills

Publications and source records attributed to D C Mills.

53 records · Page 3Linked to original sources
PubMed1971

The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3':5'-cyclic monophosphate in platelets.

1. The involvement of intracellular 3':5'-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E(1), isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate (14)C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3':5'-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3':5'-cyclic AMP phosphodiesterase from platelets. At concentrations of 3':5'-cyclic AMP greater than 50mum the most active inhibitor was dipyridamole; at 3':5'-cyclic AMP concentrations less than 19mum, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50mum), the effectiveness of prostaglandin E(1) as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E(1) of the incorporation of radioactivity into 3':5'-cyclic AMP. 4. Compound RA233 (50mum) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70-100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3':5'-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100-500mum) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3':5'-cyclic AMP. Partial positive correlations were found between the radioactivity of 3':5'-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E(1) and drugs that inhibit platelet 3':5'-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3':5'-cyclic AMP.

Adenine Nucleotides↗
PubMed1968

The release of nucleotides, 5-hydroxytryptamine and enzymes from human blood platelets during aggregation.

1. Adenosine diphosphate (ADP) and adrenaline caused the aggregation of human platelets suspended in plasma containing citrate anticoagulant and stirred at 37 degrees C. The aggregation occurred in two phases and the second phase was associated with the appearance in the plasma of up to 30% of the ATP and 55% of the ADP present in the platelets. The concentration of ADP appearing in the plasma was up to 7 times the concentration added.2. Radioactivity was released by ADP and by adrenaline from platelets labelled with radioactive 5-hydroxytryptamine; this release was closely correlated with the second phase of aggregation and with the release of nucleotides.3. Acid phosphatase, beta-glucuronidase and adenylate kinase were released to a small extent during second phase aggregation by ADP or adrenaline; thrombin and collagen particles caused significantly greater release of beta-glucuronidase than of either acid phosphatase or of adenylate kinase.4. Morphological changes indicating degranulation of the platelets were observed during the second phase of aggregation produced by adrenaline and by ADP.5. The second phase of aggregation, degranulation of platelets, and the release of nucleotides, of labelled 5-hydroxytryptamine and of enzymes, were all inhibited by concentrations of amitriptyline which did not inhibit aggregation.

Acid Phosphatase↗
PubMed1967

The adenylate kinase of human plasma, erythrocytes and platelets in relation to the degradation of adenosine diphosphate in plasma.

1. Adenylate kinase (EC 2.7.4.3) has been shown to be present in human plasma obtained by conventional means and the adenylate-kinase activities of plasma and of lysed and intact human platelets and erythrocytes have been measured at 37 degrees by sensitive spectrophotometric methods. 2. The activities found in plasma ranged from 2.7 to 22.9mumoles of ADP formed/min./l. and in lysed platelets and lysed erythrocytes mean values of 0.79 and 12.0mumoles of ADP formed/min./10(9) cells respectively were found. Intact platelets and erythrocytes showed little or no activity. 3. The apparent K(m) of plasma adenylate kinase for ADP was found to be 1.4-1.6mm. 4. The adenylate-kinase activity of plasma was correlated with the free haemoglobin present and the larger part of the activity could be accounted for by haemolysis occurring either during the withdrawal of the blood or in vivo. 5. Aggregation of platelets by ADP, collagen fibres or thrombin released up to 16% of the platelet adenylate kinase into the suspending medium. 6. Measurement of the rate of breakdown of 1.6mum-ADP in plasma gave values of about 0.1mmu-mole/min./ml. This was not increased by addition of sufficient erythrocyte lysate to increase the activity of plasma adenylate kinase five to ten times. 7. It was concluded that the activity of adenylate kinase found in plasma, even after aggregation of the platelets, is insufficient to account for the rate of breakdown of low concentrations of ADP usually observed, and that another enzyme is responsible for this process.

Adenine Nucleotides↗
PubMed1967

Effects of adrenaline on human blood platelets.

1. Adrenaline at concentrations too low to cause aggregation of human platelets potentiates the aggregation by adenosine diphosphate. Noradrenaline has the same effect but is less active than adrenaline; isopropylnoradrenaline is inactive or inhibitory.2. The potentiation of adenosine diphosphate by catecholamines is blocked by the adrenergic alpha-receptor antagonists phentolamine and dihydroergotamine but not by 2-halogenoethylamines or by adrenergic beta-receptor antagonists.3. Both the first and second phases of adenosine diphosphate aggregation are potentiated by catecholamines but the second phase more than the first.4. The release from the platelets of adenine nucleotides which is associated with the second phase of aggregation is also increased by adrenaline.

Adenine Nucleotides↗
PubMed1966

Detection and determination of adenosine diphosphate and related substances in plasma.

1. A method is described for detecting and determining the products of metabolism of ADP added to plasma at initial concentrations of about 1mum-ADP. 2. ATP, ADP, AMP, adenosine, inosine and hypoxanthine were detected in human platelet-rich plasma after incubation with ADP and in the presence of either heparin or heparin-citrate. 3. The products of incubation of ADP with human platelet-poor plasma in the presence of heparin were the same as with platelet-rich plasma, except that, when the initial concentration of ADP was 1.5mum, little or no ATP was detected. 4. The ATP detected in platelet-rich plasma when 1.5mum-ADP was initially incubated was present in the platelets and not in the plasma. 5. The time for 50% decay of ADP in either platelet-rich or platelet-poor plasma in the presence of heparin was about 20min. when the initial concentration of ADP was 200mum, but was 6-9min. when the initial ADP concentration was 1.5-2.5mum. The corresponding values in the presence of heparin-citrate were about 45min. and about 9-12min. respectively. 6. Hypoxanthine accumulated to a greater extent in platelet-rich than in platelet-poor plasma after the addition of ADP. 7. After incubation for 15-20min. of either platelet-rich plasma or suspensions of washed platelets in saline with adenosine at an initial concentration of about 3-4mum, ATP, ADP and AMP were detected in the platelets. Similar incubations of washed platelets with inosine also showed the formation of these substances, but to a much less extent. 8. After the addition of adenosine to suspensions of washed platelets in saline, inosine and hypoxanthine were detected in the incubation mixture. After the addition of inosine, hypoxanthine was detected. 9. When ADP at an initial concentration of 1.5mum was added to platelet-rich plasma containing adenosine deaminase, no adenosine was detected in the incubation mixture. There was no difference in the rate of decay of ADP in the presence or absence of the deaminase, but ATP formation was decreased in its presence.

Adenine Nucleotides↗
PubMedDate not supplied

Characterization of P2 purinergic receptors on human erythro leukemia cells.

We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.

Adenosine Diphosphate↗