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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↗
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↗
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↗
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↗
PubMed1979

Aggregation of gel-filtered guinea-pig platelets by lipoproteins.

Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) were isolated from serum of hypercholesterolemic guinea-pigs, and the effect of these lipoproteins on guinea-pig platelets was studied. VLDL (greater than 100 microgram/ml) and LDL (greater than 400 microgram/ml) were found to cause aggregation of gel-filtered platelets (GFP), although the extent of GFP aggregation by LDL was smaller than that by VLDL. In platelet-rich plasma, however, lipoproteins could not induce platelet aggregation. VLDL and LDL even at the low concentrations at which lipoproteins alone could not induce aggregation potentiated ADP-induced aggregation of GFP. VLDL-induced aggregation of GFP was inhibited by apyrase (0.2--1.0 mg/ml) in a concentration-related manner. Prostaglandin E1, dipyridamole, potassium cyanide and ethylenediaminetetraacetic acid inhibited VLDL- and ADP-induced aggregation of GFP in the almost same degree. Inhibitions of VLDL-induced GFP aggregation by acetylsalicylic acid and albumin were slightly stronger than that of ADP-induced aggregation. These findings suggest that lipoproteins modulate platelets so that endogenous ADP can be released from platelets.

Adenosine↗
PubMed1979

The action mechanism of the purified platelet aggregation principle of Trimeresurus mucrosquamatus venom.

The minimal concentration of the platelet aggregation principle (Platelet Aggregoserpentin, PAS) necessary to induce platelet aggregation was 10 ng/ml, about one-hundredth of that of the crude venom. PAS induced the release of platelet factors 3 and 4 from platelets, but the released platelet factor 3 was easily inactivated by the anti-phospholipid effect of PAS. Pretreatment of platelets with neuraminidase potentiated PAS-induced platelet aggregation. PAS-induced platelet aggregation was independent on released ADP; it could occur in the ADP-removing systems, such as apyrase or a combination of phosphoenolpyruvate and pyruvate kinase. However, PAS-induced platelet aggregation could be inhibited by adenine nucleotides and adenosine. PAS-induced platelet aggregation was inhibited by some anti-inflammatory agents, antimalarial drugs, local anesthetics, antihistamine and smooth muscle relaxants. After deaggregation of PAS-treated platelets, thrombin and sodium arachidonate could further induce platelet aggregation, but ADP and second dose of PAS could not. It is concluded that PAS-induced platelet aggregation is due to prostaglandin synthesis. Recent literatures on the mechanism of platelet aggregation were surveyed and the actions of PAS were discussed.

Animals↗
PubMed1979

Collagen-induced platelet aggregation:--evidence against the essential role of platelet adenosine diphosphate.

The hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 microM ADP, were not inhibited by 500 microM adenosine, a concentration that greatly reduced the effect of 300 microM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

Adenosine↗
PubMed1979

Inhibition of platelet thrombus formation by chlorpromazine acting to diminish haemolysis.

There is increasing evidence that the sudden, unpredicatable event initiating myocardial infarction is fissuring of an atherosclerotic plaque. The resulting haemorrhage into the arterial wall produces obstructive platelet thrombi, just as arterial haemorrhages elsewhere produce haemostatic platelet plugs. It has been suggested that such platelet aggregation depends on ADP originating in red cells which are subjected to excessive haemodynamic stress at the site of haemorrhage. The release of ADP from red cells has been demonstrated in vitro in equivalent condtions of shear stress; and other mechansims, such as activation by collagen, cannot account for the rapidity with which the platelets react. One of us (G.V.R.B.) has suggested that drugs capable of counteracting haemolysis might diminish the activating effect of erythrocytes on platelets and so inhibit their aggregation as thrombi. Thus, chlorpromazine, added to human blood at concentrations which diminish haemoylsis but do not directly affect platelet aggregation, prolonged the 'bleeding time' from small holes in artificial vessels where extravasation is terminated, as in living arterioles, by aggregated platelets. The bleeding time was also prolonged by apyrase, consistent with the conclusion that the chlorpromazine acted through decreasing plasma ADP. We show here that this occurs through the anti-haemolytic action of chlorpromazine.

Adenosine Diphosphate↗
PubMed1975

Factors responsible for ADP-induced release reaction of human platelets.

Extensive aggregation of human platelets can be induced by ADP without secondaryaggregation or release of granule contents. This occurs with washed platelets in Tyrode solution containing 0.35% albumin, human fibrinogen, and apyrase, and in platelet-rich, heparin- or hirudin-plasma. Conditions that caused release during ADP-inducedaggregation were-citrate as the anticoagulant in platelet-rich plasma; addition of citrate (11-15 mM) to a suspension of washed platelets, or to hirudin-plasma or heparin-plasma; suspension of platelets in a medium containing magnesium but no calcium;and the presence of trace amounts of thrombin or aggregated gamma globulin in the platelet suspensions. Acetylsalicylic acid, phenylbutazone, or sulfinpyrazone inhibited secondary aggregation and release in all these circumstances. Heparin or hirudin inhibited ADP-INDUCED SECONDARY AGGREGATION AND RELEASE PROMOTED BY TRACES OF THROMBIN. Although fibrinogen is required for ADP-induced primary aggregation, it does not support secondary aggregation and release, provided that it has no clot-promoting activity. The main agent responsible for ADP-induced secondary aggregation and release in human, citrated, platelet-rich plasma appears to be sodium citrate. Suspending washed human platelets in a medium without calcium mimics the effect of citrate.

Adenosine Diphosphate↗
PubMed1978

Adenosinetriphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells.

In Paramecium cells a synchronized discharge of trichocysts (which involves only the final exocytosis steps of membrane fusion, content discharge and membrane resealing) was achieved with ATPase-blockers, Ca2+-ionophores, lipid solvents (including lysolecithin), polyethyleneglycol, anaesthetics (Dibucain) and cationic detergents (cetyltrimethylammonium bromide (CTMAB) and cetylpyridinium chloride (CPC). Only Dibucain--and to some extent cationic detergents--can trigger exocytosis independently of extracellular Ca2+, possibly by mobilizing intracellular Ca2+. The internal free [Ca2+] necessary for exocytosis can be estimated to be greater than 10(-6) to 10(-4) M. Membrane-free trichocyst contents were isolated by density gradient centrifugation; they are converted from the contracted to the expanded state by Dibucain, CTMAB and CPC, and also by exogenous ATPase (Apyrase). Thus, it is possible to de-couple the discharge (stretching) process from membrane-related phenomena. Since only the latter are inhibited by low temperature (0 degrees C), membrane lipids probably have to be in a fluid state for exocytosis to occur. At least 2 steps appear to be involved: when membrane fusion is initiated, an independent matrix-bound system is activated for the synchronized stretching process. The energy requirement for one discharge event is estimated to be about 14 X 10(6) ATP molecules.

Adenosine Triphosphate↗
PubMed1975

Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg2+, or K+.

We have adapted the gel filtration technique for separation of human platelets from the plasma constituents to permit use of an eluant containing no added Ca2+, Mg2+, and K+, and hence allow direct determination of the intracellular concentrations of these ions in the isolated platelets. The eluant employed is modified Ca2+-free Tyrode's buffer which contains Sr2+ (0.2 mM) as a substitute for Mg2+ and lacks added K+. The functional metabolic, and morphological properties of these isolated platelets have been determined ael-filtered platelets (GFP) to low concentrations of ADP and adrenaline was qualitatively similar to that of platelet-rich plasma (PRP). However, a slower response was observed for the GFP. This rate difference was partially or completely reversed by addition of apyrase to the medium. Analysis of the total adenine nucleotide content and the pattern of 14C incorporation into the metabolic adenine nucleotide pool indicated that isolation in this medium caused to significant change in the ATP and ADP contents or in the adenylate energy change in comparison with the PRP. However, a significant increase in the extent of hypoxanthine production from ATP was noted in GFP isolated in media lacking Mg2+. Inclusion of Mg2+ in the elution media prevented this increased hypoxanthine production. The intracellular concentrations of Ca2+, Mg2+, and K+ of the GFP as determined by atomic absorption analysis were in good agreement with the values obtained for platelets separated from plasma by high-speed centrifugation. Platelet Ca2+ and Mg2+ levels remained stable despite the lack of significant extracellular levels of these ions. However platelet K+ fell to about 30 per cent of its initial value after incubation of 90 minutes at 23 degrees C. and a coincident increase was observed in extracellular K+ concentration. This procedure for platelet isolation may be of particular value for studies on the role of Ca2+, Mg2+, and K+ in platelet physiology and metabolic processes.

Adenine↗
PubMed1977

Properties of washed human platelets.

We have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentration of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.

Arachidonic Acids↗
PubMed1978

Endotoxin-induced changes in human platelet membranes: morphologic evidence.

Interaction between human platelets and bacterial endotoxin was studied in vitro with transmission and scanning electron microscopy. Washed human platelets, whose aggregation was blocked with apyrase, were incubated in a plasma-free medium containing crude endotoxin that had previously been complexed with copper. Thirty minutes of incubation resulted in adherence of endotoxin particles to the platelet surface, breaks in the platelet plasma membrane with apparent attempts at repair, pseudoped formation, and centralization of platelet organelles. Copper appeared to potentiate these phenomena, since neither Cu2+ at low concentrations nor endotoxin alone altered the morphology of the platelet membrane. This platelet-endotoxin interaction may be an intermediary step in the detoxification and clearance of endotoxin from the plasma.

Blood Platelets↗
PubMed1977

Involvement of mediators in the interaction of platelets and carrageenan.

Carrageenan or thrombin-induced aggregation of plasma-free rabbit platelets was inhibited by calcium and magnesium chelating agents, by N-ethylmaleimide and by drugs that increase the intra-cellular cyclic AMP content. Inhibitors of prostaglandin (PG) synthetase were only partially active, and had to be present in the platelet suspension to inhibit aggregation. Inhibition of PG synthetase, as evaluated by bioassay and by AA-induced platelet aggregation, was not reduced when inhibitors were washed from platelets. The phospholipase A2 inhibitors bromophenacyl bromide and mepacrine, the chymotrypsin inhibitor tosylphenylalaninechloromethylketone, catalase and dithiothreitol also inhibited aggregation, whereas inhibitors of trypsin failed to do so. Incubation of rabbit platelet-rich plasma with carrageenan was followed by generation of PG-like and of rabbit aorta contracting activities. Generation of these activities was inhibited by drugs effective against aggregation, and also by non-steroidal anti-inflammatory drugs. Aggregation of rabbit platelets by carrageenan and by thrombin does not appear to be dependent upon activation of PG synthetase, although PG-like substances are formed during aggregation.

Animals↗