Decreased sensitivity of platelets to prostacyclin in patients with diabetes mellitus

In order to ascertain the platelet sensitivity to prostacyclin (PGI2) in patients with diabetes mellitus, we determined the percentage inhibition of platelet aggregation and platelet ATP secretion following PGI2 addition in an in vitro system. The percentage inhibition of platelet aggregation caused by PGI2 in final concentration of 1.25, 2.5, or 5.0 ng/ml was significantly lower in diabetics than in healthy controls. That of platelet ATP secretion by 1.25 or 2.5 ng/ml of PGI2 was also significantly lower in diabetics. These data suggested that in patients with diabetes mellitus, the decreased sensitivity of platelets to PGI2 will bring about hypercoagulability and may become one of the risk factors of diabetic microangiopathy in cooperation with lowered vascular PGI2 generation.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

The effects of short-term training on platelet functions and total antioxidant capacity in rats

The purpose of the present study was to investigate the effect of short-term endurance training on plasma total antioxidant status (TAS) and on in vitro platelet aggregation and ATP release. Blood samples were collected from the abdominal aorta of rats following short-term treadmill exercise (25 m/min, 0 % grade, 30 min) for three consecutive days, as well as in non-exercised control group. Platelet aggregation and platelet ATP release were evaluated by impedance and bioluminescence techniques, respectively. Plasma TAS was measured spectrophotometrically. Plasma TAS was higher and ADP-induced platelet ATP release was lower in the short-term training group with respect to the control group (p<0.001). Significant negative correlation (r = -0.56, p<0.05) was found between plasma TAS and ADP-induced platelet ATP release. Neither ADP- and collagen-induced maximum aggregation rate nor collagen-induced platelet ATP release were significantly different between the groups. According to these results, short-term training caused an alteration in platelet functions limited to the secretion response, which may be related to the oxidant/antioxidant balance changes favoring the antioxidants. The improved plasma total antioxidant capacity was possibly sufficient to prevent exercise-induced oxidative damage, and the adaptive response of platelets might be associated with enhanced antioxidant status.     

Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5-hydroxytryptamine in rats

Sarpogrelate, a specific 5-HT_2A receptor antagonist is reported to produce a number of beneficial cardiovascular effects in diabetes mellitus. In the present investigation we have studied the effects of sarpogrelate on 5-HT receptors in heart and platelets in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg) and sarpogrelate (1 mg/kg, i.p.) was administered daily for 6 weeks. Injection of STZ produced significant loss of body weight, polyphagia, polydypsia, hyperglycemia, hypoinsulinemia, hypertension and bradycardia. Treatment with sarpogrelate significantly lowered fasting glucose levels with corresponding increase in insulin levels. It also significantly prevented STZ-induced polydypsia, hyperphagia, hypertension, and bradycardia but not the loss of body weight. 5-HT produced dose-dependent positive inotropic effect that was found to be decreased significantly in STZ-diabetic rats. Hearts obtained from sarpogrelate treated diabetic rats did not show any decrease in responsiveness to 5-HT. Relative platelet aggregation per se was found to be higher in STZ-diabetic rats as compared to control and this was significantly prevented by sarpogrelate treatment. 5-HT produced a dose-dependent increase in platelet aggregation in non-diabetic and sarpogrelate treated diabetic rats. However, 5-HT failed to produce any increase in platelet aggregation in untreated diabetic rats. Our data suggest that STZ-induced diabetes may produce down-regulation of cardiac 5-HT_2A receptors and increased platelet aggregation. Treatment with sarpogrelate seems to prevent STZ-induced down-regulation of 5-HT receptors and increase in platelet activity in diabetic rats.     

Antithrombotic effect of onion in streptozotocin-induced diabetic rat

In the present study, we investigated whether onion has antithrombotic effect in streptozotocin (STZ)-induced diabetic rats. In diabetic rats, serum thromboxane B(2) (TXB(2)) level was elevated compared to that in normal, and this elevation in diabetes was significantly inhibited by treatment with onion (0.5 g/ml/kg/day, i.p.) for 4 weeks. In normal rats, the serum TXB(2) level remained unaltered after the treatment with onion. To investigate in vitro effect of onion, we examined its effect on TXB(2) formation, platelet aggregation and arachidonic acid (AA)-release in platelets from diabetic and normal rats. Onion showed a significant inhibitory effect on collagen- or AA-induced TXB(2) formation with greater potency in diabetic platelets than in normal. Similarly, more potent inhibitory effects of onion in diabetes were observed in collagen- or AA-induced platelet aggregation and collagen-induced AA release response. In conclusion, these results suggest that onion can produce more beneficial antithrombotic effect in diabetes.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platelets

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.     

Interaction between ATP and catecholamines in stimulation of platelet aggregation

Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1-5 microM) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 microM of adenosine 3'-phosphate 5'-phosphosulfate, a selective P(2)Y(1) receptor antagonist. ATP alone did not cause release of intracellular Ca(2+), but produced a significant Ca(2+) response in the presence of norepinephrine. In contrast, the P(2)X(1) receptor agonists P(1),P(6)-diadenosine-5' hexophosphate and alpha,beta-methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 microM. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress.     

Inhibition of oxidative-stress-induced platelet aggregation by androgen at physiological levels via its receptor is associated with the reduction of thromboxane A2 release from platelets

BACKGROUND: Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis. The purposes of our study are to assess the effect of androgen at physiological concentration via its receptor on oxidative-stress-induced platelet aggregation and to further elucidate the possible mechanism. METHODS AND RESULTS: Plasma dihydrotestosterone (DHT) was determined by ELISA using a commercially available kit. Platelet aggregometer was used to measure platelet aggregation. The contents of thromboxane B(2) (TXB(2)) were assayed with radio-immunoassay. Our results showed that addition of DHT (2 nM) significantly inhibited platelet aggregation induced by hydrogen peroxide (H(2)O(2)) (10 mM, 25 mM) in PRP diluted with Tyrode's buffer. Moreover, H(2)O(2)-induced platelet aggregation decreased in sham-operated rats. However, H(2)O(2)-induced platelet aggregation significantly increased in castrated rats. Replacement of DHT inhibited H(2)O(2)-induced platelet aggregation in castrated rats. After PRP was pretreated with flutamide, H(2)O(2)-induced platelet aggregation increased in castrated rats again. Presence of DHT (2 nM) obviously inhibited H(2)O(2)-induced thromboxane A(2) (TXA(2)) release in castrated rats. Pretreatment of DHT and flutamide increased H(2)O(2)-stimulated TXA(2) release from platelet in castrated rats again. Castration caused a significant reduction in plasma testosterone and DHT levels, whereas DHT replaced at a dose of 0.25 mg/rat restored the circulating DHT to physiological levels, without being altered by treatment with flutamide. The plasma TXB(2) increased in castrated rats as compared with that in sham-operated rats. Replacement with DHT reduced plasma TXB(2) contents in castrated rats. However, flutamide supplementation increased plasma contents of TXB(2) in castrated rats again. CONCLUSION: Androgen at physiological doses via its receptor inhibits oxidative-stress-induced platelet aggregation, which is associated with the reduction of TXA(2) release from platelets.     

Moderate Red Wine and Grape Juice Consumption Modulates the Hydrolysis of the Adenine Nucleotides and Decreases Platelet Aggregation in Streptozotocin-Induced Diabetic Rats

This study investigated the ex vivo effects of the moderate red wine (RW) and grape juice (GJ) consumption, and the in vitro effects of the resveratrol, caffeic acid, gallic acid, quercetin, and rutin on NTPDase (nucleoside triphosphate diphosphohydrolase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), 5′-nucleotidase, and adenosine deaminase (ADA) activities in platelets and platelet aggregation from streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 10): control/saline, control/GJ, control/RW, diabetic/saline, diabetic/GJ, and diabetic/RW. RW and GJ were administered for 45 days; after this period, the blood was collected for experimental determinations. Results showed that NTPDase, E-NPP, 5′-nucleotidase, and ADA activities as well as platelet aggregation were increased in the diabetic/saline group compared to the control/saline group. Treatment with RW and GJ increased ectonucleotidases activities and prevented the increase in the ADA activity in the diabetic/GJ and diabetic/RW groups. Platelet aggregation was also decreased by the treatment with RW and GJ in the diabetic/GJ and diabetic/RW groups. In the in vitro tests, resveratrol, caffeic acid, and gallic acid increased ATP, ADP, and AMP hydrolysis, while quercetin and rutin decreased the hydrolysis of these nucleotides in platelets of diabetic rats. The ADA activity and platelet aggregation were reduced in platelets of diabetic rats in the presence of all polyphenols tested in vitro. These findings suggest that RW, GJ, and all polyphenols tested were able to modulate the ectoenzymes activities. Moreover, a decrease in the platelet aggregation was observed and it could contribute to the prevention of platelet abnormality, and consequently vascular complications in diabetic state.     

Experimental investigation of the rheological activation of blood platelets

In order to define various aspects of platelet rheological activation, samples of whole blood and platelet-rich plasma (PRP) from the same donors were subjected for 5 min to shear rates increasing from 10 to 10000 sec-1 (shear stresses from 10(-2) to 30 Pa approximatively) in a Couette type viscometer. The following parameters were measured: erythrocyte hemolysis; lactic dehydrogenase activity; plasma B-Thromboglobulin (B-TG); adenine nucleotides, and platelet photometric aggregation. The experimental results reveal that: In whole blood, hemolysis only reached at maximum 2% of the total hemolysis. Plasma LDH activity increased regularly beyond 500 sec-1, in close correlation with B-TG plasma concentration. In contrast, ADP and ATP levels remained stable up to 1000 sec-1 then increased slowly. In PRP, the LDH, ADP and ATP levels remain practically stable up to shear rates around 5000 sec-1. In contrast, B-TG appeared to be released in plasma at shear rate values of 3000 sec-1 and its progression is only correlated with the other parameters, when the platelet lysis occurred. Finally, a rapid and complete inhibition of platelet aggregation to ADP was observed from 5000 sec-1.     

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.     

Evidence for the dependence of arterial haemostasis on ADP

The possible involvement of adenosine diphosphate (ADP) in haemostatic platelet aggregation was investigated by determining the duration of primary haemorrhage as standardized bleeding times from punctures of small mesenteric arteries in anaesthetized rats. The bleeding times were highly significantly increased by infusing into the mesenteric arterial blood flowing towards the punctures either the nucleotide-dephosphorylating enzyme apyrase or the ADP-receptor antagonists ATP, adenosine 5'-(beta,gamma-methylene)triphosphonate (AMP-PCP) or 2-methylthioadenosine 5'-(beta,gamma-methylene)triphosphonate (2-MeS-AMP-PCP). The increases in bleeding times could not be accounted for by local vasodilator effects of the agents. It is concluded that the presence of ADP through local release and/or formation at sites of vascular injury contributes significantly to haemostasis, presumably by accelerating platelet aggregation.     

Effect of chronic intramuscular administration of low molecular weight heparin-collagen complex on hemostatic indices and development of alloxan-induced diabetes

Repeated intramuscular administration of low molecular weight heparin-collagen complex proved to increase fibrinolytic activity and to decrease platelet aggregation in the blood of rats (11 months) with depressed anticoagulant system. Administration of diabetogenic alloxan dose induced no diabetes mellitus in such animals.     

Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats.

This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was produced in rats by occluding of the right middle cerebral artery, together with the right common carotid artery. rCBF was monitored by H2 clearance method with a tissue blood-flow meter. Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. (i.v.) improved rCBF in rats after ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the platelet aggregation induced by collagen, ADP, and AA with IC50 values of 0.197, 2.22 and 3.29 x 10(3) mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the ischemic hemisphere and inhibit platelet aggregation in rats.     

一种快速、灵敏的血小板释放功能检测方法及其在抗血小板药物研究中的应用

本文报道了一种快速、灵敏的血小板释放功能检测方法:利用荧光素-荧光素酶在有ATP、Mg~(2+)、O_2存在时产生的生物发光素测定血小板ATP的释放量,以反映血小板的释放功能;研究了ADP、AA、胶原、凝血酶等四种诱导剂对血小板释放功能的作用,发现ADP的诱导释放能力较其他三者为弱;观察在不同剂量ADP和AA的诱导下,血小板聚集强度和释放能力之间的关系,研究了血小板数等因素对ATP释放功能测定的影响。应用该方法研究了Aspirin及活血化淤药物川芎嗪,毛冬青甲素对血小板释放功能的影响,发现Aspirin对AA诱导的释放反应有强烈的抑制作用。在以ADP诱导的释放反应中,川芎嗪的抑制作用较毛冬青甲素更为强烈。     

Morphologic and hematologic characteristics of storage pool deficiency in beige rats (Chédiak-Higashi syndrome of rats)

Characterization of beige rats as having a platelet storage pool deficiency (SPD) was undertaken. Platelets from beige rats, an animal model of Chédiak-Higashi syndrome (CHS), completely lacked the ability to aggregate when stimulated with high dosages of collagen (50 micrograms/ml), and lacked secondary aggregation induced by adenosine diphosphate (ADP). Concentrations of ADP, ATP, and serotonin in the platelets of beige rats were significantly lower than those of control rats. However, platelet count remained within normal values. Electron microscopy revealed that platelets had fewer dense granules, whereas other organelles had normal structure. This morphologic and functional evidence confirms that platelets of beige rats have the typical characteristics of SPD.     

Effects of 2-tetradecylglycidic acid on rat platelet energy metabolism and aggregation.

We investigated the role of energy supplied by long-chain fatty acid oxidation in rat platelet function. Inhibition of the mitochondrial uptake of long-chain fatty acids was achieved by treating rats with 2-tetradecylglycidic acid (TDGA), a potent inhibitor of the overt form of carnitine palmitoyltransferase (CPT-I). The maximum aggregation rate (MAR), CPT-I activity, lactate production, oxygen consumption and adenine nucleotide content of isolated rat platelets were then studied in vitro. 4 h after the in vivo administration of TDGA, the CPT-I activity in saponin-permeabilized platelets was nearly completely inhibited along with a significant reduction in the MAR induced by ADP, thrombin and ionophore A23187. The ATP level, adenylate energy charge (ATP + 1/2 ADP)/(ATP + ADP + AMP) and ATP/ADP ratio in the platelet cytoplasmic pool were also reduced. Platelets from TDGA-treated rats showed lower oxygen consumption rates in both the basal respiratory and oxygen burst states. These results indicate that mitochondrial long-chain fatty acid oxidation coupled to oxidative phosphorylation is an important energy source in rat platelets and is probably involved in the maintenance of platelet function. Enhanced in vitro lactate production in platelets from TDGA-treated rats may have resulted from a compensatory increase in glycolysis which only partly compensated for impaired long-chain fatty acid oxidation.     

E-NTPase/E-NTPDase: a potential regulatory role in E-kinase/PKA-mediated CD36 activation

CD36 is a platelet surface receptor protein that plays a major role in platelet aggregation and accumulation that is mediated by parasitic attachment. The CD36 receptor is constitutively phosphorylated by E-kinase/PKA, resulting in increased affinity for collagen, but preventing spontaneous platelet aggregation. Dephosphorylation of CD36 by protein phosphatase 2A (PP2A) leads to increased affinity for thrombospondin at a different rate than that of collagen-mediated platelet aggregation. Depletion of the E-kinase/PKA substrate [ATP](0)by E-NTPase-mediated hydrolysis, in conjunction with inhibition of PP2A by okadaic acid, could prove to be a valuable tool in inhibiting CD36 activation, thus preventing platelet aggregation and thrombus formation.     

Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-(14)C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3':5'-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3':5'-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3':5'-cyclic AMP occurred by 10-30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3':5'-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3':5'-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3':5'-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3':5'-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3':5'-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.