5′-nucleotidase activity is altered by hypo- and hyperthyroidism in platelets from adult rats

Thyroid disorders are associated to a number of vascular diseases that involve processes such as platelet aggregation and vascular tone control. Since, these processes can be also affected by ATP, ADP and adenosine levels, we investigate the hydrolysis of these nucleotides in platelets from hyperthyroid, hypothyroid, and hypothyroid with hormonal replacement rats. Hyperthyroidism was induced by daily injections of L-thyroxine (T4) 25?μg/100?g body weight for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) for 14 days. In the hormonal replacement group, hypothyroid rats were injected with T4 (5?μg/100?g body weight, i.p.) for 5 days. The AMP hydrolysis by platelets was increased 49% in hyperthyroid rats and decreased 50% in response to hypothyroidism, while the ATP and ADP hydrolysis was not altered in both groups. Besides, the T4 replacement significantly reversed the inhibition of the AMP hydrolysis observed in hypothyroid rats. Our findings indicate that the thyroid disorders affect the 5′-nucleotidase activity and consequently can alter the adenosine levels in a reversible manner in platelet fraction. Since, adenosine is able to inhibit platelet aggregation and acts as a potent vasodilator, these results can contribute to a better comprehension of the vascular events described in thyroid disorders.     

Ecto-hydrolysis of adenine nucleotides in rat blood platelets are altered by ovariectomy

There is evidence that estrogen is associated with a reduction on cardiovascular disease risk through inhibition of platelet aggregation and action on vascular function. The process of haemostasis can also be affected by adenine nucleotides and adenosine. Consequently, regulation of enzymes that hydrolyze these nucleotides in the bloodstream is essential in the modulation of the processes of platelet aggregation, vasodilatation and coronary flow. Ecto-ATP diphosphohydrolase and ecto-5′-nucleotidase from platelets are enzymes related to nucleotide hydrolysis. In the present study, we examined the effect of ovariectomy (OVX) and estradiol replacement therapy (ER) on the activity of the enzymes that degrade adenine nucleotides in platelets of female rats. The OVX group significantly decrease the hydrolysis of ATP, ADP and AMP by 42, 52 and 29.3%, respectively, when compared to a control group. ER did not reverse the inhibition of nucleotide hydrolysis observed in OVX rats. Our findings indicate that hormonal deprivation affects ATP, ADP and AMP hydrolysis by platelets and consequently the level of these nucleotides and adenosine in the circulation. Since, ADP is the most important platelet agonist and recruiting agent present in the microenvironment of the thrombus, our findings was contribute to a better comprehension of the cardiovascular complications described in alterations of sexual hormonal status.     

Ecto-hydrolysis of adenine nucleotides in rat blood platelets are altered by ovariectomy

There is evidence that estrogen is associated with a reduction on cardiovascular disease risk through inhibition of platelet aggregation and action on vascular function. The process of haemostasis can also be affected by adenine nucleotides and adenosine. Consequently, regulation of enzymes that hydrolyze these nucleotides in the bloodstream is essential in the modulation of the processes of platelet aggregation, vasodilatation and coronary flow. Ecto-ATP diphosphohydrolase and ecto-5'-nucleotidase from platelets are enzymes related to nucleotide hydrolysis. In the present study, we examined the effect of ovariectomy (OVX) and estradiol replacement therapy (ER) on the activity of the enzymes that degrade adenine nucleotides in platelets of female rats. The OVX group significantly decrease the hydrolysis of ATP, ADP and AMP by 42, 52 and 29.3%, respectively, when compared to a control group. ER did not reverse the inhibition of nucleotide hydrolysis observed in OVX rats. Our findings indicate that hormonal deprivation affects ATP, ADP and AMP hydrolysis by platelets and consequently the level of these nucleotides and adenosine in the circulation. Since, ADP is the most important platelet agonist and recruiting agent present in the microenvironment of the thrombus, our findings was contribute to a better comprehension of the cardiovascular complications described in alterations of sexual hormonal status.     

Ectonucleotidase activities are altered in serum and platelets of L-NAME-treated rats

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of Nomega-Nitro-L-arginine methyl ester hydrochloride(L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects;thus, ADP is the most important platelet agonist and recruiting ag ent, while adenosine, an end product of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5'-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonucleotidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%,respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation.     

NTPDase and 5'-nucleotidase activities in rats with alloxan-induced diabetes

Diabetes is associated with a hypercoagulable state. In this study, we investigated the potential effects of alloxan-induced diabetes on the activities of the enzymes NTPDase (E.C. 3.6.1.5, apyrase, ATP diphosphohydrolase, ecto/CD39) and 5'-nucleotidase (E.C. 3.1.3.5, CD73) that can control the levels of ADP and adenosine, two substances that regulates platelet aggregation. In the alloxan-treated rats, NTPDase activity was significantly increased by 88 and 35% with ATP as substrate and by 156 and 58% with ADP as substrate in platelets and synaptosomes, respectively (P< 0.05). AMP hydrolysis was increased by 142% (platelets) and 70% (synaptosomes) in diabetic rats compared to control. These results demonstrate that alloxan-induced diabetes interferes with ATP, ADP, and AMP hydrolysis in platelets and synaptosomes. Taken together, these results may indicate that in diabetic rats both NTPDase and 5'-nuleotidase from the central nervous system (CNS) and platelets respond similarly with increased activity. Thus, we speculate that platelets could be used as a potential peripheral marker of central alterations in NTPDase and 5'-nucleotidase activities in diabetes.     

High-intensity intermittent exercise increases adenosine hydrolysis in platelets and lymphocytes and promotes platelet aggregation in futsal athletes

Acute bouts of high-intensity intermittent exercise (HIIE) or sports are associated with changes in lymphocytes and platelet functions and we hypothesized that the purinergic system is involved with these alterations. We investigated the activity of ectonucleotidases in platelets and lymphocytes as well as the platelet aggregation of futsal players in response to an acute protocol of HIIE. Thus, 19 male semi-professional futsal players were submitted to 40 min of HIIE on a treadmill. Blood samples were collected three-time points: before exercise, immediately after, and 30 min after the end of the session. Platelet-rich plasma (PRP) and lymphocytes were isolated. ATP, ADP, AMP, and adenosine hydrolysis, NTPDase1 (CD39) expression as well as platelet aggregation were measured. Our results showed HIIE induced a decrease in ATP and ADP hydrolysis in platelets, an increase in adenosine hydrolysis and an increase in platelet aggregation immediately after exercise. After 30 min of recovery, enzymatic activity and platelet aggregation returned to baseline levels. In lymphocytes, adenosine hydrolysis was augmented immediately after exercise and remained increased even after 30 min of recovery. In conclusion, acute HIIE triggers a transient proaggregant status that is reverted after a 30 min of recovery. The effects of HIIE in lymphocytes remained after 30 min of recovery, indicating a pro-inflammatory response. This work elucidated some of the mechanisms by which purinergic system regulates lymphocytes and platelets activities related to HIIE, suggesting that the type of exercise may influence an increase in platelet aggregation even in trained individuals.     

Modulation of platelet function by extracellular adenosine triphosphate

A potential physiologic role of extracellular adenosine triphosphate (ATP) on platelet function is proposed in this report. It is widely accepted that ATP competitively inhibits adenosine diphosphate (ADP)- induced platelet aggregation. Our observations of platelet aggregation with the agonists, collagen, epinephrine, and ADP in the presence of 180 mumol/L ATP could support this competitive nature of ATP. However, the disaggregation of maximally aggregated platelets induced by ATP, theophylline, or ATP plus theophylline indicates that additional mechanisms of ATP action may be present. Extracellular gamma-32P-ATP (7 pmol) labels surface-membrane proteins in intact platelets as demonstrated by several criteria. The reaction is Ca++-dependent. Stimulation by calcium occurs in the physiologic range of 1 to 5 mmol/L. Significant levels of phosphorylation occur within one minute with near maximal levels reached by five minutes. Platelet cyclic AMP (cAMP) levels were elevated in a dose-dependent fashion in cells incubated for four minutes with increasing amounts of extracellular ATP (18 to 540 nmol). The addition of ATP plus theophylline resulted in a synergistic stimulation of cAMP levels. ATP was not being hydrolyzed to adenosine by plasma nucleotidases, as demonstrated by the lack of effect of ten U of adenosine deaminase. The phosphorylation of surface proteins by extracellular ATP released from activated platelets may modulate platelet responsiveness to agonists at distances removed from the site of vascular injury. Phosphorylation may also play a role in signal transduction to regulate the levels of intracellular cAMP, which further inhibits platelet activation.     

ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galphaq

Platelets from Galphaq knockout mice are unable to aggregate in response to physiological agonists like adenosine 5'-diphosphate (ADP), thromboxane A(2), thrombin, or collagen, although shape change still occurs in response to all of these agonists except ADP. ADP-induced platelet aggregation results from simultaneous activation of the purinergic P2Y(1) receptor coupled to calcium mobilization and shape change and of a distinct P2 receptor, P2cyc, coupled through Gi to adenylyl cyclase inhibition, which is responsible for completion and amplification of the response. P2cyc could be the molecular target of the antithrombotic drug clopidogrel and the adenosine triphosphate (ATP) analogs AR-C69931MX, AR-C67085, and AR-C66096. The aim of the present study was to determine whether externally added ADP could still act through the Gi pathway in Galphaq-deficient mouse platelets and thereby amplify the residual responses to agonists such as thrombin or collagen. It was found that (1) ADP and adrenaline still inhibited cyclic AMP accumulation in Galphaq-deficient platelets; (2) both agonists restored collagen- but not thrombin-induced aggregation in these platelets; (3) the effects of ADP were selectively inhibited in vitro by the ATP analog AR-C69931MX and ex vivo by clopidogrel and hence were apparently mediated by the P2cyc receptor; and (4) high concentrations of ADP (100 micromol/L) induced aggregation without shape change in Galphaq-deficient platelets through activation of P2cyc. Since adrenaline was not able to induce platelet aggregation even at high concentrations, we conclude that the effects of ADP mediated by P2cyc are not restricted to the inhibition of adenylyl cyclase through Gi(2).     

Abnormal platelet aggregation in severe combined immunodeficiency disease with adenosine deaminase deficiency

The platelets from a boy with adenosine deaminase deficiency-severe combined immunodeficiency disease (ADA-SCID) showed markedly subnormal aggregation in response to collagen and ADP. In contrast with normal platelets, ATP, AMP and adenosine had little effect in inhibiting ADP-induced aggregation of ADA-SCID platelets. These observations suggest that altered adenosine metabolism exists in ADA-SCID platelets.     

Platelet aggregation and adenosine diphosphate/adenosine triphosphate receptors: a historical perspective

The work of many investigators since adenosine diphosphate (ADP) was recognized as a platelet aggregating agent in 1961 has led to an appreciation of the important part that ADP plays in hemostasis and thrombosis. Recently, interest has focused on the platelet receptors for ADP and adenosine triphosphate (ATP). Platelets are unique because they have two P2Y receptors that must act in concert to achieve a normal aggregation response. The P2Y (1) receptor is responsible for mobilizing internal calcium, platelet shape change, and weak aggregation. The P2Y (12) receptor inhibits adenylyl cyclase, but the concentration of cyclic AMP is reduced only if it has been raised from its low basal levels by stimulation of adenylyl cyclase by an aggregation inhibitor such as adenosine or prostaglandin I (2). The abnormal bleeding of the rare patients whose platelets lack P2Y (12) and the beneficial clinical effects of ticlopidine and clopidogrel that block this receptor indicate that P2Y (12), in addition to inhibiting adenylyl cyclase, may have an as yet unidentified role that is needed for its cooperative aggregation effect with P2Y (1). ATP stimulates a rapid influx of calcium into platelets through the P2X (1) receptor, and it may synergize with ADP when these two nucleotides are released from platelets at a site of vessel injury.     

Contribution of ecto-5'-nucleotidase to the inhibition of platelet aggregation by human endothelial cells

We studied the role of adenosine (Ado), which is generated from adenine nucleotides via the activity of ecto-5'-nucleotidase (ecto-5'-NT), in the inhibition of platelet aggregation by endothelial cells (ECs). The enzymatic activity of nucleotidases on human umbilical vein endothelial cells (HUVECs) was examined with regard to (1) the inhibition of adenosine diphosphate (ADP)-induced platelet aggregation and (2) the liberation of inorganic phosphate from adenine nucleotides. Adenosine 5'-monophosphate (AMP) preincubated with HUVECs significantly inhibited ADP-induced platelet aggregation. This was completely blocked by the treatment of HUVECs with a specific inhibitor of ecto-5'-NT, 5'-[alphabeta-methylene] diphosphate (APCP), or by the addition of an A(2a) receptor antagonist. Neither nitric oxide nor prostacyclin was involved in this inhibitory activity, suggesting that Ado generated in the incubation medium by the activity of 5'-NT on HUVECs inhibited platelet aggregation. When ADP was incubated on HUVECs, it lost most of its agonistic activity for platelets. Pretreatment of HUVECs with APCP at a concentration that abolished ecto-5'-NT activity partially restored ADP-induced platelet aggregation. Ecto-5'-NT contributes to EC function by inhibiting platelet aggregation in cooperation with ATP diphosphohydrolase, which degrades ADP to AMP.     

The influence of the thyroid state on energy turnover during tetanic stimulation in the fast-twitch (mixed type) muscle of rats

The effects of thyroid hormones on energy metabolism and force development during tetanic stimulation of fast-twitch skeletal muscle (mixed type) were studied. Hypothyroid, euthyroid, and hyperthyroid (ten days of daily administration of 15 micrograms T3/100 gm body weight) rats showed the same tension-time integral (force x time) under tetanic stimulation. The ATP turnover rate was significantly lower in the hypothyroid group than in the euthyroid and hyperthyroid animals. The ratio difference in the energy/force ratio in the Tx group cannot be ascribed to differences in internal work. Lactate production was diminished in the hypothyroid group; this was probably not the result of a block in glycogenolysis but rather was a reflection of more economic energy expenditure during contraction. After tetanic stimulation, the phosphorylation potential ([ATP]/[ADP]f[Pi]) was decreased most strongly in the hyperthyroid group, mainly because of the higher accumulation of Pi compared with the other groups, which suggests that during the recovery phase the stimulation of oxidative phosphorylation is greater in this group.     

Bioluminescent assay of adenine nucleotides: rapid analysis of ATP and ADP in red cells and platelets using the LKB luminometer

Summary The assessment of both red cell and platelet function requires assay of adenine nucleotides. We describe the use of the new LKB luminometer to measure adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by bioluminescence in both normal and abnormal red cells and platelets. ATP was measured a in a lysate of red cells suspended in buffer, pH 7.4, b in ethanol extracts of platelets from platelet-rich plasma (PRP) and c in ethanol extracts of supernatant platelet-poor plasma (PPP) following platelet aggregation by collagen. ADP was measured as ATP after phosphorylation by pyruvate kinase (PK) with phosphoenolpyruvate (PEP). Duplicate assays showed a variance of < 3%. Red cell lysates and ethanol extracts of PRP and PPP stored at ?40°C were stable for 4 weeks. Duplicate assays of ATP and ADP in eight samples plus standards could be performed in 2 h. Normal values were: red cells (μM/ml red cells) (n = 20), ATP 1.15 ± 0.17 (1 SD), ADP 0.22 ± 0.07, ATP/ADP (mean ratio) 5.76:1, platelets (nM/10⁹ platelets) (n = 20), ATP 53.3 ± 7.6, ADP 27.8 ± 5.8, ATP/ADP (mean ratio: 1.96:1). Abnormalities of red cell ATP and/or ADP could be demonstrated in chronic renal failure, hereditary glycolytic enzyme deficiencies and other haemolytic states. In myeloproliferative disorders defective platelet aggregation associated with storage pool deficiency and/or impaired release of ADP and ATP could be shown. We conclude that this is a reliable, rapid and economical technique for measuring red cell and platelet adenine nucleotides.     

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2x10(3) leukocytes/microl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1-10 microM) and ATP (10-100 microM) was markedly reduced, but to ATP (1000 microM) was enhanced (all p<0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t1/2=4 min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4 min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.     

Cigarette smoke inhibits adenine nucleotide hydrolysis by human platelets

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis and thrombotic events. In athero-thrombotic diseases, the extracellular adenine nucleotides play an important role by triggering a range of effects such as the recruitment and activation of platelets, endothelial cell activation and vasoconstriction. NTPDase, a plasma membrane-bound enzyme, is the most relevant enzyme involved in the hydrolysis of extracellular tri- and di-phosphate nucleotides to adenosine monophosphate, which is further degraded by 5′ectonucleotidase to the anti-thrombotic and anti-inflammatory mediator adenosine. Thus, the preserved activity of these enzymes, regulating the extracellular concentrations of nucleotides, is critical in thromboregulatory functions. In the present in vitro study, performed on human platelets suspended in undiluted or diluted aqueous cigarette smoke extract (aCSE), we demonstrated that undiluted and 1 : 2 diluted aCSE is able to significantly reduce ADP hydrolysis (?24% and 12%, respectively) by intact human platelets. ATP degradation was also reduced (?31%) by undiluted aCSE. Conversely, aCSE did not alter platelet AMP hydrolysis. Results obtained by using N-acetylcysteine, a thiol-containing antioxidant, suggest that stable oxidants present in aCSE are responsible for the platelet NTPDase inhibition induced by aCSE. The decreased adenine nucleotide degradation could play a significant role in the extensive platelet activation and vascular inflammation observed in chronic smokers.     

Cigarette smoke inhibits adenine nucleotide hydrolysis by human platelets

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis and thrombotic events. In athero-thrombotic diseases, the extracellular adenine nucleotides play an important role by triggering a range of effects such as the recruitment and activation of platelets, endothelial cell activation and vasoconstriction. NTPDase, a plasma membrane-bound enzyme, is the most relevant enzyme involved in the hydrolysis of extracellular tri- and di-phosphate nucleotides to adenosine monophosphate, which is further degraded by 5'ectonucleotidase to the anti-thrombotic and anti-inflammatory mediator adenosine. Thus, the preserved activity of these enzymes, regulating the extracellular concentrations of nucleotides, is critical in thromboregulatory functions. In the present in vitro study, performed on human platelets suspended in undiluted or diluted aqueous cigarette smoke extract (aCSE), we demonstrated that undiluted and 1 : 2 diluted aCSE is able to significantly reduce ADP hydrolysis (-24% and 12%, respectively) by intact human platelets. ATP degradation was also reduced (-31%) by undiluted aCSE. Conversely, aCSE did not alter platelet AMP hydrolysis. Results obtained by using N-acetylcysteine, a thiol-containing antioxidant, suggest that stable oxidants present in aCSE are responsible for the platelet NTPDase inhibition induced by aCSE. The decreased adenine nucleotide degradation could play a significant role in the extensive platelet activation and vascular inflammation observed in chronic smokers.     

The Effect of Pyrimidine Compounds on the Potentiation of Adenosine Inhibition of Aggregation, on Adenosine Phosphorylation and Phosphodiesterase Activity of Blood Platelets

S ummary . Three pyrimidine compounds (dipyridamole, RA233 and VK 744) produced variable degrees of direct inhibition as well as potentiation of adenosineinduced inhibition of ADP aggregation of blood platelets. RA233 and VK744 were more potent direct inhibitors of platelet aggregation, whereas RA233 was a more powerful potentiator of adenosine–induced inhibition. A relation between the effect of these compounds on platelet aggregation and on adenosine phosphorylation and phosphodiesterase activity of platelets was looked for in an attempt to elucidate possible modes of action.
RA233 and dipyridamole were powerful inhibitors of [¹⁴C]adenosine phosphorylation by platelets and VK744 had no effect. There was no correlation with inhibition of platelet aggregation. Adenosine-induced inhibition of platelet aggregation in the presence of dipyridamole could be maintained for at least 10 min after the rapid clearance of the adenosine with exogenous deaminase. The presence of the pyrimidine so modified the inhibition response that there was no correlation between the concentration of adenosine in the system and the degree or rate of recovery from the inhibition.
Dipyridamole, VK744 and RA233 produced 45%, 45% and 55% inhibition of phosphodiesterase activity as measured by the rate of breakdown of [³H]cyclic AMP to AMP in platelet lysates. There was possible correlation between the effect of the three pyrimidine compounds on platelet phosphodiesterase activity and their potentiatory action on adenosine-induced inhibition of platelet aggregation. These findings suggested that adenosine might induce inhibition of platelet aggregation through the adenyl cyclase, cyclic AMP, phosphodiesterase system.     

Thyroid-catecholamine interactions in isolated rat brown adipocytes

The effects of hyperthyroidism and hypothyroidism on the metabolism of adipocytes isolated from rat brown adipose tissue were as follows: The yield of brown adipocytes was 65% less in the hypothyroid as compared to the control rats. No change in cell recovery was observed in the hyperthyroid group as compared to controls but there was a 65% increase in cell volume. The stimulation of respiration by isoproterenol and forskolin was markedly greater in cells from hyperthyroid as compared to euthyroid rats. In the adipocytes from hypothyroid rats, respiration and lipolysis were reduced but there was no defect in stimulation of adenylate cyclase by forskolin or isoproterenol as compared to euthyroid controls. The effects of different thyroid states on respiration did not correlate with changes in adenosine 3':5'-cyclic phosphate (cyclic AMP) or lipolysis. Phenylephrine in the presence of alprenolol or octanoate were as potent stimulators of respiration as 10 mumol/L isoproterenol in adipocytes from hypothyroid rats. In cells from hyperthyroid rats phenylephrine was twice, octanoate three times, and isoproterenol 16 times more effective in stimulating respiration than in cells from hypothyroid rats. These data indicate that thyroid status regulates beta-catecholamine and forskolin stimulation of respiration in brown adipocytes.     

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2?×?10³?leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10?µM) and ATP (10–100?µM) was markedly reduced, but to ATP (1000?µM) was enhanced (all p?<?0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t_1/2?=?4?min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4?min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.