Toxicity assessment of 16 inorganic environmental pollutants by six bioassays

ATP and ADP are simultaneously released from activated platelets in equimolar concentrations. Micromolar concentrations of ATP inhibit platelet aggregation by both competitive and non-competitive mechanisms. The current studies addressed the question of how platelets respond to agonists in the presence of nanomolar and micromolar concentrations of ATP and ADP alone or in combination. This is a significant issue since the concentration of ATP +/- ADP may vary widely within a microenvironment depending upon the source and cause for the release of the nucleotides. ATP (1-10 nM) was found to significantly enhance the thromboxane A2 analog, U44619-, collagen- and thrombin-induced platelet aggregations. Conversely, ATP at 1-100 microM inhibited these same reactions. ADP, in general, behaved exactly opposite to ATP. When equal amounts of ATP and ADP were added together the ADP response appeared to predominate. The observed ATP-induced response was not due to a hydrolytic product as evidenced by an unaltered response to ATP in the presence of adenosine deaminase or the ATP generating system, creatine phosphate plus creatine phosphokinase. Adenosine (1-10 nM), like ADP, inhibited agonist-induced platelet aggregation. The stimulation of agonist-induced platelet aggregation by 1-10 nM extracellular ATP appears to depend upon the phosphorylation of platelet membrane ecto proteins. The ATP analog, beta gamma-methylene ATP, that is incapable of serving as a phosphate donor for protein kinases, inhibited rather than stimulated agonist-induced platelet aggregation. The dual response of platelets to low and high concentrations of extracellular ATP +/- ADP may play a physiological role in hemostasis and thrombosis under normal and pathological conditions.     

Effects of some antineoplastic drugs (vincristine, doxorubicin and epirubicin) on human platelet aggregation

The effects of some antineoplastic drugs (vincristine, doxorubicin and epirubicin) on collagen- and ADP-induced human platelet aggregation are investigated. Platelet rich plasma (PRP) and platelet poor plasma (PPP) from healthy male and female donors were used. The PRP was adjusted with analogous PPP to 300,000 platelets/microliters. Platelet aggregation was studied according to Born's turbidimetric technique using an Aggrecorder II PA 3220 with collagen at a concentration of 10 micrograms/ml and ADP at a concentration of 30 microM. Vincristine, doxorubicin and epirubicin significantly (p < 0.01) inhibited collagen- and ADP-induced platelet aggregation. The vincristine induced inhibition was higher than that induced by doxorubicin or epirubicin. The effects of doxorubicin and epirubicin were more intense on ADP-induced platelet aggregation than on the collagen induced one. Moreover, the doxorubicin inhibition of ADP-induced platelet aggregation was greater than the epirubicin one. In conclusion, our study shows that vincristine, doxorubicin and epirubicin inhibit human platelet aggregation. The present results may improve the therapeutic use of these drugs since it has been clearly shown that drugs with antiplatelet activity could block metastases.     

Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.     

The effect of halofenate or halofenate free acid on human, rat and guinea pig platelet aggregation

Halofenate free acid (HFA), the major metabolite of the hypolipemic agent halofenate, blocked the secondary phase of human platelet aggregation induced by ADP, epinephrine, or thrombin; higher concentrations of clofibrate free acid (CFA) were required to produce similar inhibitory effects on platelet aggregation. HFA and CFA inhibited collagen-induced aggregation of human, rat, or guinea pig platelets. Halofenate orally administered to rats caused inhibition of collagen-induced aggregation when plasma levels of HFA exceeded 300 mug/ml, a clinically achievable human plasma concentration. The platelet inhibitory effects of clofibrate administration were less than those observed with halofenate administration.     

Platelet sialic acid and platelet survival after aggregation by ADP

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP-induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.     

Modulatory effect of 8-iso-PGE2 on platelets

1. 8-Iso-PGE2 induced either reversible or irreversible aggregation of platelets in human platelet-rich plasma (PRP) or in the suspension of washed platelets (WP). The values of EC50 for irreversible aggregation in PRP and WP were 4 and 2 microM, respectively. 2. In rabbit PRP, 8-iso-PGE2 (0.1-100 microM) itself did not induce or induced only reversible aggregation. 3. 8-Iso-PGE2 (0.1-20 microM) potentiated adenosine diphosphate-(ADP) induced platelet aggregation in both human and rabbit. The same effect also was found for adrenaline-induced platelet aggregation in rabbit. 4. The lower concentrations (0.2-0.5 microM) of 8-iso-PGE2 decreased, and higher concentrations (1-2 microM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE2 (0.02-200 microM) had only a decreasing effect on PAF-induced aggregation. 5. The results suggest that low concentrations of 8-iso-PGE2 can amplify or weaken platelet aggregation induced by various aggregatory agents.     

A potent 5-hydroxytryptamine receptor (5-HT2A) antagonist, DV-7028, delays arterial thrombosis development in rats

In our study, we demonstrated that DV-7028: (3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6, 7,8,9-tetrahydro-2H-pyrido [1,2,-a]-1,3,5-triazine-2, 4(3H)-dione maleate)--a selective 5-HT2A receptor antagonist, inhibited thrombus formation in the arterial thrombosis model and was completely ineffective in the prevention of venous thrombosis in the rat. In washed platelets prelabelled with 3H-serotonin, DV-7028 inhibited, in a dose-dependent manner, the collagen-induced secretion of serotonin. However, the uptake of serotonin into platelets was not affected by this substance. Administration of DV-7028 also inhibited platelet aggregation in the whole blood and platelet-rich plasma (PRP) induced by collagen, and diminished serotonin-induced aggregation of rat platelets in the presence of a sensitizing but nonaggregating amount of ADP, whereas it did not modify aggregation in PRP when induced by ADP. DV-7028 caused a concentration-dependent, almost parallel shift to the right of the concentration-response to serotonin for its pressor effect in the rat perfused tail artery. The present data demonstrate that DV-7028 exhibits 5-HT2A receptor antagonistic properties in the rat cardiovascular system, exhibits antithrombotic effect in the model of arterial but not venous thrombosis in rats. These results constitute further evidence of the possible importance of serotonin as a mediator of platelet thrombosis in arteries. Moreover, they can provide a useful tool for the prevention of various thrombotic diseases.     

Effect of xanthinol nicotinate treatment on platelet aggregation

Treatment of 24 male patients with 3 g/day of xanthinol nicotinate did not change the in vitro measurements of ADP-induced platelet aggregation but produced a marked inhibition of collagen-induced platelet aggregation. This effect may be connected with the drug-induced depression of the ATP level in platelet-rich plasma. Changes in the platelets in the patients' blood or in the lipid composition and the concentration of uric acid in their serum were ruled out as reasons for the decrease of the collagen-induced aggregation. The activity of the three serum enzymes y-GT, GOT, and GPT and the concentration of the blood sugar did not change.     

Instrumental non invasive diagnosis of arterial circulation disorders of the extremities

1 In citrated platelet-rich plasma, freshly prepared from rabbit blood, the velocity of platelet aggregation was within limits proportional to the log of the concentration of added adenosine diphosphate (ADP). 2 Addition of either adenosine triphosphate (ATP) or its beta,y-methylene analogue inhibited aggregation similarly except that the analogue was about half as potent as ATP. beta,y-Methylene ATP also reversed the optical effects associated with the shape change of platelets very similarly to ATP itself. 3 As beta,y-methylene ATP is not a substrate for nucleoside diphosphokinase, these observations do not support the proposition that inhibition of aggregation by added ATP is due to its utilization by the nucleoside diphosphokinase of platelets.     

Inhibitory effects of Acanthopanax gracilistylus saponins on human platelet aggregation and platelet factor 4 liberation in vitro

AIM: To study the effects of Acanthopanax gracilistylus var pubescens Li saponins (AGVPS) on human platelet aggregation and platelet factor 4 (PF4) liberation in vitro. METHODS: Human platelet aggregations induced by ADP, adrenaline, and collagen were measured turbidimetrically. The aggregation curve was recorded on a platelet aggregometer and the maximal aggregation rate (ARmax), effective deaggregation rate in 5 min (DR5 min) and lag time (LT) were autocalculated by the built-in microcomputer; PF4 liberation from human platelets stimulated by ADP and collagen was determined by recording the heparin thrombin clotting time (HTCT). Thrombosis was tested by weighing the wet and dry thrombi formed in a siliconized revolving ring. RESULTS: AGVPS inhibited in vitro the ARmax with IC50 of 1.33 (95% confidence limits: 1.09-1.63, ADP-induced), 1.66 (1.54-1.79, adrenaline-induced), and 4.2 g.L-1 (0.6-29, collagen-induced). The DR5 min (on ADP-induced aggregation) and LT (collagen-induced) were also increased as well. Meanwhile, AGVPS 0.63-2.50 g.L-1 prolonged HTCT on ADP- and collagen-stimulated PF4 liberation. At 0.34-1.39 g.L-1, AGVPS reduced the wet and dry weight of thrombi formed in vitro. CONCLUSION: AGVPS inhibits human platelet aggregation, liberation, and thrombosis in vitro, suggesting its possible antithrombotic action in man.     

Characterization of platelet hypofunctions in rats under SART stress (repeated cold stress)

Platelet hypoaggregability has been reported in rats exposed to a chronic form of environmental stress induced by long-lasting fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress. This study examines functional characteristics of platelets from stressed rats in more detail. Exposure to stress reduced aggregation and ATP release in platelets stimulated with collagen, as determined using platelet-rich plasma (PRP). The resting levels of ATP but not ADP in platelets from stressed rats were lower than those from unstressed ones. Collagen-induced release and resting level of serotonin also decreased in platelets from stressed rats. In contrast, stress failed to cause hypoaggregability of washed platelets. Circulating platelet aggregates were detected in stressed rats. From these data, SART stress appears to cause intravascular activation of platelets in spite of in vitro hypofunctions. Alteration in plasma milieu may be associated with stress-induced platelet hypofunctions in PRP.     

The antiplatelet activity of ancrod on administration to rabbits

Ancrod, a thrombin-like enzyme purified from the venom of Calloselasma rhodostoma, was administered to rabbits intravenously, and blood samples were obtained at 1, 3, 6, 10, and 24 hours after infusion. Ancrod caused a rapid and sustained defibrinogenation within the first 6 hours, with production of fibrinogen degradation products (FDPs) peaking at 1 hour and declining to background level at 6 hours. No significant changes in platelet count, white cell count, or hematocrit was observed. Citrated PRP prepared 1, 3, and 6 hours after ancrod infusion showed diminished aggregation, adenosine triphosphate (ATP) release, and thromboxane B2 formation on the addition of collagen. Although platelet suspension prepared from defibrinogenated platelet-rich plasma (PRP) at 3 hours showed no significant change in aggregation and ATP-releasing activity, the latent period of platelet aggregation was prolonged. When the remaining platelet-poor plasma obtained from defibrinogenated PRP at 3 hours was used to suspend the normal washed platelets prepared from PRP before ancrod infusion, the platelets showed a similar defect in aggregation and release action. Addition of fibrinogen (200 micrograms/ml to 2 mg/ml) to the above preparation partially restored aggregation but not capacity for secretion and thromboxane formation. When normal washed platelets were suspended with the defibrinogenated plasma, prepared by mixing ancrod with normal plasma in vitro and removing the formed fibrin, the platelet suspension showed impaired platelet aggregability, and the aggregability could be restored to the normal level by the addition of exogenous fibrinogen to this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 microM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 microM) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 microM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 microM). A3P5P (1 mM) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 +/- 50 to 611 +/- 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 +/- 68 sites per platelet and further decreased to 55 +/- 12 sites in the presence of A3P5P (1 mM). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Enzymatic removal of ADP from plasma: unaltered platelet adhesion but reduced aggregation on subendothelium and collagen fibrils

Subendothelium of rabbit aorta and fibrillar collagen were exposed to citrated human or rabbit blood which was circulated through a perfusion chamber under flow conditions similar to those found in arteries. The resulting platelet adhesion and subsequent formation of platelet microthrombi on the exposed surfaces were measured in 0.8 mum thich sections by a morphometric technique using light microscopy. Removal of plasma ADP by the substrate-enzyme combination CP-CPK (creatine phosphate-creatine phosphokinase; 3 mM and 90 U/ml blood) did not affect the initial attachment and spreading of platelets on subendothelium, whereas platelet thrombus formation was strongly inhibited. On free collagen fibrils CP-CPK was much less inhibitory on platelet thrombus formation but platelet adhesion again was not affected. It is concluded that platelet aggregation induced by thrombogenic surfaces in the presence of arterial blood flow is at least partially governed by ADP released from adhering platelets. Platelet adhesion to the examined surfaces, however, does not seem to be mediated by plasma ADP.     

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel

This study aimed to determine the binding characteristics of [3H]alpha,beta-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo. Binding of [3H]alpha,beta-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [3H]alpha,beta-Me-ATP bound to one population of specific binding sites with high affinity (KD = 23.6 +/- 1.6 nM; Bmax = 690 +/- 24 fmole/10[8]cells) (n=3). Unlabelled alpha,beta-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [3H]alpha,beta-Me-ATP with IC50 values of 19.0 +/- 6.6, 103 +/- 20 and 1120 +/- 80 nM respectively (n=3). Other nucleotide analogues such as ATP, ATP-gammaS, UTP and GTP also antagonized [3H]alpha,beta-Me-ATP binding. When administered orally (10mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [3H]alpha,beta-Me-ATP to rat platelets ex vivo. In vitro, alpha,beta-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.     

Inhibitory mechanisms of naloxone on human platelets

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.     

Stability of metallothionein isoforms by capillary zone electrophoresis

Advanced glyco-oxidation end products (AGEs) generate oxygen free radicals that potentiate the development of atherosclerosis. Thus, AGEs may potentiate the aggregation of human platelets through oxidative stress. AGE-bovine serum albumin (BSA) and AGE-poly-L-lysine were evaluated for aggregation of human platelets. Superoxide in platelet-rich plasma (PRP) was measured using lucigenin-derived chemiluminescence. The platelet aggregation induced by ADP or U46619 was potentiated by preincubation with AGE-BSA, by 40% and by 59%, P < .05, respectively, vs BSA. Aggregation was increased by AGEs in a dose-dependent manner. The production of superoxide was significantly greater in PRP incubated with AGE-BSA vs BSA. The other Maillard reaction products, such as Amadori-, pentosidine-, and carboxymethyl lysine (CML)-BSA had no effect. Superoxide dismutase or indomethacin abolished the enhancing effect of AGEs on the platelet aggregation. AGEs potentiate platelet aggregation possibly with superoxide anions and prostanoids. AGE-induced potentiation of platelet aggregation may be involved in the development of atherosclerosis.     

ADP-induced platelet activation

Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.     

Platelet activation by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate is mediated by its binding to a putative ADP receptor, aggregin

Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.     

Different roles of endothelium-derived substances on inhibiting platelet aggregation in whole blood

1. The inhibitory effects of adenosine, nitroprusside (a nitric oxide donor) and prostacyclin on collagen-induced rabbit platelet aggregation were studied under two different conditions: in whole blood with an impedance method and in platelet-rich plasma (PRP) with a turbidimetric method. 2. All substances tested were less potent in whole blood than in PRP, and the differences in IC50 value between whole blood and PRP were not of the same order of magnitude; adenosine (669-fold), nitroprusside (54-fold) and prostacyclin (2-fold). 3. These results imply that (a) some other, as yet unknown, factors in blood modulate the platelet aggregation; (b) adenosine and nitric oxide act close to the endothelium, and (c) prostacyclin acts as a relatively long lasting circulating hormone.