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At least 19 recordsLinked to original sources
PubMed1977

Hydrolysis of synthetic pyrophosphoric esters by an isoenzyme of apyrase from Solanum tuberosum.

A highly purified isoenzyme of apyrase obtained from potatoes (Solanum tuberosum var. Pimpernel) exhibits a low specificity for the organic moiety of synthetic pyro- and triphosphates. Methyl di- and tri-phosphates were hydrolysed at higher rates than ADP and ATP, but their Km values were also higher. Steric hindrance at the carbon atom linked to the pyrophosphate chain decreases both binding and maximum rate, whereas length or polarity of the organic chain do not have systematic effects. t-Butyl diphosphate, inorganic pyrophosphate, adenosine 5'-[alpha,beta-methylene]triphosphate and adenosine 5'-[beta,gamma-methylene]triphosphate are competitive inhibitors of the hydrolysis of ATP and ADP.

Adenosine Triphosphatases↗
PubMed1978

Role of platelets in aspirin-sensitive bronchoconstriction in the guinea-pig; interactions with salicylic acid.

1 The bronchoconstriction caused in the guinea-pig by arachidonic acid (AA), bradykinin, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) was correlated with effects on platelets. ATP and ADP produced a brief thrombocytopenia and AA a more prolonged one. Bradykinin had no effect on platelets.2 Aspirin inhibited bronchoconstriction and thrombocytopenia produced by AA and part of the bronchoconstriction produced by ATP, but had no effect against ADP. Thrombocytopenia produced by ADP and ATP was not affected by aspirin or indomethacin.3 Platelet depletion by antiserum prevented bronchoconstriction in response to ADP and to ATP, but not in response to bradykinin or to AA, showing that platelets are not involved in aspirin-sensitive bronchoconstriction. Infusions of ADP reduced bronchoconstriction and thrombocytopenia in response to ADP itself and to ATP, but not to AA. Bronchoconstriction by ADP or ATP involves an action on platelets. Only that due to ATP is partially dependent on the activity of prostaglandin synthetase.4 ATP induced aggregation in vitro in guinea-pig platelet-rich plasma (PRP). Rabbit PRP responded only when ATP was first incubated with guinea-pig plasma. The aggregating compound formed was probably ADP, since it was destroyed by apyrase. Its formation was not inhibited by aspirin or indomethacin, indicating that aspirin inhibits ATP-induced bronchoconstriction by a different mechanism.5 The aggregating effect of ATP on guinea-pig platelets was inhibited by concentrations of apyrase that block ADP-induced aggregation, and potentiated by lower concentrations of apyrase.6 Adenosine 5'-tetraphosphate did not aggregate platelets in vivo or in vitro. In vitro aggregation occurred when apyrase was added, suggesting transformation into ADP. Adenosine 5'-tetraphosphate and apyrase inhibited aggregation due to ADP, but failed to affect that due to AA. This suggests that aggregation involving products of prostaglandin synthesis does not require ADP.7 Salicylic acid did not interfere with bronchoconstriction or aggregation due to AA, but prevented inhibition by aspirin when the weight ratio, salicylic acid:aspirin was 4:1. Salicyclic acid may be useful in studies of potential inhibitors of thromboxane A2 synthesis and of thromboxane A2-dependent processes in vivo and in vitro.

Adenosine Diphosphate↗
PubMed1975

ADP, thrombin, and Bothrops atrox thrombinlike enzyme in platelet-dependent fibrin retraction.

Clots formed upon the addition of thrombin to human platelet-rich plasma (PRP) retracted readily but the clotting enzyme from Bothrops atrox venom did not cause retraction in PRP unless ADP, collagen, epinephrine, or low concentrations of thrombin (0.1 U) were added. The latter type of retraction was inhibited by apyrase and creatine phosphate kinase in the presence of creatine phosphate, but that induced with higher concentration of thrombin (2 U) was not. In a system composed of washed human platelets and purified fibrinogen, Bothrops marajoensis (BM) thrombinlike enzyme (highly purified preparations of viper venom) did not cause clot retraction. Addition of ADP to the platelet-fibrinogen mixture prior to BM enzyme resulted in stimulation of clot retraction that could be dissociated from the release of platelet constituents. Addition of low concentrations of thrombin (0.1 U/ml) caused retraction associated with a considerable release of adenine nucleotides that was inhibited by potato apyrase. Electron micrographs showed platelet-fibrin aggregates in all types of retracted clots. Nonretracted clots formed in the presence of potato apyrase contained discoidal platelets that were not in close association with fibrin. It has been postulated that platelet-dependent fibrin clot retraction induced by collagen, epinephrine, and low concentration of thrombin is mediated by ADP. High concentrations of thormbin may possibly promote clot retraction independently of ADP.

Adenosine Diphosphate↗
PubMed1977

ADP-induced refractory state of platelets in vitro. II. Functional and ultra studies on gel filtered platelets.

Gelfiltered platelets (GFP) in calcium free Tyrode solution containing albumin, glucose and adenosine deaminase were preincubated with 1 micronM 14C-ADP or 0.15 M NaCl (control) at 37 degrees C. The breakdown of extracellular 14C-ADP was markedly inhibited in this medium. No aggregation took place without fibrinogen, but the platelets underwent a disc to sphere transformation with development of refactoriness towards ADP. Presence of 2 mM CaCl2 in the incubation medium did not prevent refractoriness as reported earlier with washed rabbit platelets. When the ADP degrading enzyme, apyrase, was added at 30 min of incubation a partial recovery of the aggregability was observed. Electron microscopic studies showed that the partial restoration of the aggregation response, due to ADP degradation by apyrase, was accompanied by a return of discoidal morphology of the platelets. The ultrastructural studies showed further that spherical form with large number of pseudopods is not by itself a necessary or sufficient indication of platelets in a refractory state. However, the results indicated that spherical platelets are more vulnerable to external factors. It was concluded that refractoriness was mainly caused by a direct effect on the platelets by ADP itself, but the studies also suggested that deteriorating, irreversible, intracellular changes may take place when platelets are in spherical shape. An artificial medium, mechanical stress, incubation at 37 degrees C are factors that probably speed up these changes.

Adenine↗
PubMed1978

In vitro interaction between cultured cells and human blood platelets.

The effects of washed human cultured cells (tumour cells and Chang liver cells) on human blood platelets in heparinized plasma were studied. Platelet aggregation was induced by suspensions of the tumour cells. Ultrastructural examination showed that the platelets, especially in the central regions of the aggregates, were tightly packed and the alpha-granules were mostly present. In the periphery of the aggregates the platelets appeared swollen and devoid of organelles, and fibrin strands were seen. The platelet aggregation was not completely abolished by incubation with apyrase. The washing fluids from the tumour cells also induced platelet aggregation, but the aggregation could be abolished by incubation with apyrase. When three different lines of the Chang cells were used, suspensions of two lines of these cells induced platelet aggregation, but the third line did not. Presence of ADP could be demonstrated in the washing fluids from the cultured cells, except for the one line of Chang cells which did not induce platelet aggregation. The experiments indicated that the platelet aggregation induced by the various types of cells was mediated via ADP, but with a possible additional effect of coagulation activity.

Adenosine Diphosphate↗
PubMed1975

In vitro evaluation of inhibitors of platelet release and aggregation.

In order to determine which drugs would be most effective as inhibitors of platelet release and aggregation, in vitro release reactions and platelet aggregometry were used to evaluate aspirin, dipyridamole, sulfinpyrazone, flurbiprofen, low molecular weight dextran (dextran 40), prostaglandin E1 (PGE1), apyrase, and adenosine. Adenosine diphosphate-induced aggregation was most effectively inhibited by PGF1, sulfinpyrazone, and dipyridamole. The latter had to be used in large doses. Collagen and epinephrine-induced release and aggregation were inhibited by the same drugs as well as by aspirin and apyrase. Antihynocyte globulin (ATG)-induced release and aggregation could only be partially blocked by these agents. In vitro studies suggest that sulfinpyrazone is one of the most effective of platelet inhibitors currently available for clinical testing.

Adenosine↗
PubMed1977

Metabolism and function of human platelets washed by albumin density gradient separation.

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium-free Tyrode's solution. The mean recovery of platelets after two separations relative to platelet-rich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [14C]5-hydroxytryptamine (5HT) in response to adenosine diphosphate (adp) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP-induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP-induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [14C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE1 or PGD2. Platelet coagulant activities were not made available and heparin-neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37degreesC compared with 25degreesC did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.

Adenine Nucleotides↗
PubMed1975

Inhibition of platelet adherence to damaged surface of rabbit aorta.

A method has been developed for quantitative measurement of adherence of rabbit platelets to the damaged intimal surface of everted segments of rabbit thoracic aorta. Platelets were labeled with 51Cr, washed, and resuspended in Tyrode solution containing 0.35 per cent albumin and apyrase. This suspending medium contains physiologic concentrations of calcium and magnesium; apyrase degrades any ADP lost from the platelets or from the damaged wall. Everted aorta segments were rotated in the platelet suspensions. Neither platelet aggregation nor lysis occurred and the platelets adhered to the subendothelium either as individual platelets or as a single layer. Damage caused by scraping the everted segments with a scalpel blade increased adherence 50-fold. Acetylsalicylic acid (ASA) in vitro, or administered orally to the rabbits from which platelet suspensions were prepared, significantly reduced the number of platelets adherent to the damaged aorta wall. ASA affected only the platelets, and did not affect the damaged wall. Platelet adherence to the damaged wall was also reduced by the use of 4 per cent albumin in the suspending medium, or by the addition of citrate. Adherence of platelets resuspended in citrated plasma was low and further inhibition by ASA was not demonstrable. ASA may affect two aspects of thrombus formation: platelet adherence to subendothelial structures and the platelet release reaction induced by collagen (and possibly by other subendothelial structures). These studies show that ASA has a marked effect on adherence of platelets to subendothelium under conditions in which aggregation and thrombus formation are prevented.

Albumins↗
PubMed1976

Reactions of polylysine with human platelets in plasma and in suspensions of washed platelets.

The effects of polylysine on human platelets have been examined in citrated platelet-rich plasma (PRP) and in suspensions of washed platelets in various media. In PRP, polylysine caused aggregation after a lag phase. Heparin inhibited this completely. At certain concentrations of polylysine, two phases of aggregation occurred, the second being associated with release of 14C-serotonin from prelabelled platelets; this phase was inhibitable with prostaglandin E1, acetylsalicylic acid, sulphinpyrazone, adenosine, apyrase, or creatine phosphate/creatine phosphokinase. Polylysine-induced release also occurred in PRP with EDTA or hirudin as anticoagulant. In suspensions of washed platelets in Tyrode solution containing 0.35% or 4% albumin, or 1% gelatin, polylysine caused immediate platelet-to-platelet adherence and very little release of 14C-serotonin or platelet lysis. Heparin inhibited aggregation, but acetylsalicylic acid, prostaglandin E1, adenosine, apyrase, creatine phosphate/creatine phosphokinase or EDTA did not. In a modified Tyrode-albumin medium containing 1 mM magnesium but no calcium, polylysine-induced aggregation was associated with the release of 14C-serotonin which could be inhibited by acetylsalicylic acid or indomethacin; this is similar to the effect of ADP in this medium. In Tyrode solution without albumin or gelatin, polylysine-induced platelet aggregation was associated with release of a large percentage of 14C-serotonin, together with as much as 18% lysis; indomethacin inhibited this release reaction.

Albumins↗
PubMed1977

The control of ionized calcium in squid axons.

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 mumol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 mumol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 mumol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.

Adenosine Triphosphate↗
PubMed1977

Intracellular calcium buffering capacity in isolated squid axons.

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 mumol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/muM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 muM is taken up by this system.

Adenosine Triphosphate↗
PubMed1975

Dissociation of clot retraction from platelet granule fusion and degranulation: an ultrastructural study of Reptilase-human platelet-rich plasma clots.

Human platelet-rich plasma clots, formed with Reptilase, do not retract. Electron microscopy of such a clot reveals platelets of normal shape and ultrastructural features surrounded by fibrin. When ADP is added before Reptilase, the clot strongly retracts. A sequential ultrastructural study of this process shows that, before visible fibrin formation, the platelets form small clusters and show shape change, granule centralization and pseudopod formation. Upon immobilization of the platelets by fibrin, the pseudopods develop into large cytoplasmatic protrusions, which allow cell contact. During the retraction process, the platelet granules remain intact; some granule fusion only becomes apparent in fully retracted clots. When Thrombofax is added to platelet-rich plasma before Reptilase, the clots formed also retract. With this aggregation inducer, granule fusion occurs earlier and in more platelets. Platelet pseudopod enlargement and fibrin concentration around the platelet mass are similar to that in ADP-treated samples. Inhibitors of granule fusion and secretion (suprofen, indomethacin) do not modify ADP-Reptilase nor Thrombofax-Reptilase clot retraction but reduce the incidence of granule fusion in Thrombofax-Reptilase clots. Retraction of Thrombofax-Reptilase clots is unaffected by concentrations of apyrase which completely block ADP-Reptilase clot retraction. Prostaglandin E1, papaverine and amitryptiline inhibit both ADP-and Thrombofax-Reptilase clot retraction, platelet pseudopod formation and cell-to-cell adhesion. These findings suggest that interaction of fibrin with the mass of fused granules (granulomere) or platelet secretion are not responsible for clot retraction. Rather, contraction around adhesion sites formed by cytoplasmic protrusions from adjacent platelets would seem to be involved.

Adenosine Diphosphate↗
PubMed1976

Factor VIII and human platelet aggregation. II. Characteristics of aggregation of human platelets by bovine factor VIII.

Bovine factor VIII aggregates human platelets either in a strong single wave at high concentration (10 mug/ml platelet suspension) or in two waves at low concentration (0.2-I mug/ml). The strong single wave of aggregation is not associated with release of [14C]serotonin or beta-glucuronidase; the high concentration does not induce retraction of reptilase-clotted platelet-rich plasma. Wtih the low concentration, relase of [14C]serotonin is observed just prior to the onset of the second wave of aggregation; release of beta-glucuronidase does not occur at any moment. The low concentration of bovine factor VIII induces moderate retraction of reptilase-clotted platelet-clotted platelet-rich plasma, which is inhibited by acetylsalicylic acid, indomethacin and apyrase, indicating that it is a consequence of release of platelet adenosine-5'-diphosphate. It has previously been suggested tht carbohydrate groups are involved in the human platelet-bovine factor VIII interaction, since galactose oxidase and periodate oxidation abolish the platelet aggregating activity of bovine factor VIII. The present study shows that these oxidizing substances also induce a degradation of bovine factor VIII, so that the exact role of carbohydrate groups in the aggregation process remains to be established.

Animals↗
PubMed1976

Reptilase clot retraction induced by electrical stimulation.

Retraction of platelet rich plasma clotted by reptilase is induced by electrical stimulation. Optimal retraction is obtained by stimuli, applied for more than 4 min, with the following characteristics: intensity = 150 volts, duration = 50 msec each, frequency = 10/sec. Electrically induced reptilase clot retraction is shown to be inhibited by EDTA, EGTA, methyl-xanthines, PGE1, acetylsalicylic acid, indomethacin, but not by apyrase or by phosphoenolpyruvate-pyruvate kinase and MgCl2. The results indicate that electrical stimulation induces retraction of PRP clotted by reptilase by triggering off an increased availability of Ca2+ in the intracellular space.

Apyrase↗
PubMed1975

Detection of bacteriuria by luciferase assay of adenosine triphosphate.

A selective method for distinguishing bacterial and nonbacterial adenosine triphosphate (ATP) in clinical bacteriological specimens was studied. The method involved incubation of samples with the detergent Triton X-100 and the ATP-hydrolyzing enzyme apyrase. The incubation selectively destroyed ATP in suspensions of various human cells while not affecting the ATP content in microbial cells. ATP remaining in the sample after incubation was extracted in boiling buffer and assayed by the firefly luciferase assay. Application of the method to 469 clinical urine specimens showed that the ATP level after treatment with Triton/apyrase was correlated to bacterial counts and that the sensitivity of the assay was sufficient for the detection of 10(5) bacteria/ml. The ATP levels per bacterial cell remaining in the urine specimen after treatment with Triton/apyrase were close to values observed in laboratory-grown cultures. The specificity and sensitivity of the luciferase assay for the detection of urinary bacteria and its possible use as a bacteriuria screening method are discussed.

Adenosine Triphosphate↗
PubMed1978

Superoxide-independent platelet response to xanthine oxidase.

Xanthine oxidase (1--5 microgram/ml) from cow's milk induces shape change, aggregation, and the release reaction of human washed platelets. Xanthine oxidase plus xanthine produce superoxide radicals, which reduce nitro blue tetrazolium. Superoxide dismutase, allopurinol, or ommission of xanthine inhibits the reduction of nitro blue tetrazolium but has no influence on the platelet response to xanthine oxidase. In contrast, small amounts of plasma or apyrase from potatoes abolish the effect on platelets, but not the enzyme activity of xanthine oxidase. Comparison of two xanthine oxidase preparations shows that higher specific enzyme activity corresponds to a lesser effect on platelets. The results suggest that platelet and enzyme activities reside in different components of xanthine oxidase preparations.

Apyrase↗
PubMed1976

Aggregation of platelets and inert particles induced by thrombin.

Thrombin-induced platelet aggregation and release were investigated in washed platelet suspensions and in suspensions of inert particles in order to evaluate the role of fibrinogen-fibrin transformation in aggregometer tracings. Thrombin (0.25-2.0 U/ml) produced two waves of light transmission increase (LTI) in both platelet and inert particle suspensions containing fibrinogen, and concomittantly aggregates were observed under phase microscopy. Without fibrinogen, thrombin induced rapid release of platelet ADP but failed to cause second wave of LTI. The kinetics of LTI in platelet and inert particle systems were related to both thrombin and fibrinogen concentrations. A rapid second wave of LTI could be produced by direct interaction of thrombin-treated platelets or inert particles with polymerizing fibrin, and was inhibited by sodium sulfite and low pH of 5.1 which prevent fibrin monomer polymerization. No fibrin strands were noted in platelet aggregates fixed at the completion of the second wave of LTI. Apyrase and PGE1 inhibited the rate of first but not that of second wave LTI. The results suggest that the release of platelet ADP induced by thrombin primarily affects the first phase aggregation, and the second phase may result from interaction of thrombin-exposed platelets and polymerizing fibrin. Thus, the blood coagulation mechanism may be directly involved in platelet aggregation.

Adenosine Diphosphate↗