Blood platelet function in patients with obliterative arteriosclerosis of the lower limbs

The specific markers of platelet activation, e.g. platelet aggregation induced with ADP, AA and PAF as well as the levels of Beta-TG, TXB2, 6-keto-PGF1 alpha and cyclic AMP in the patients suffering from obliterative arteriosclerosis of the lower limbs were measured. It was found that these patients revealed hyperfunction of blood platelets expressed in increased sensitivity of platelets to ADP and PAF, increased levels of Beta-TG and TXB2 as well as decreased levels of 6-keto-PGF1 alpha and cyclic AMP. Obtained results support the concept that atherosclerosis consists of a wide-spread functional alteration of various types of cells.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase.

The events involved in platelet shape change, aggregation, the release reaction and contraction are thought to be mediated by the availability of Ca2+. Increased cytoplasmic calcium, released from intracellular stores, triggers platelet activity, and increased concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits platelet alterations. We have studied the hypothesis that cyclic AMP may regulate the level of platelet cytoplasmic calcium by stimulating calcium removal by a membrane system. Such a hypothesis would be consistent with the reversibility of most manifestations of platelet activation. Human platelets were sonicated and unlysed platelets, mitochondria and granules were removed by centrifugation at 19 000 X g. Electron microscopy shows that the sediment, after centrifugation of the supernatant at 40 000 X g consists to a large extent of membrane vesicles. Such preparations actively concentrate calcium, as measured by the uptake of 45Ca, and also have the maximal calcium-stimulated ATPase activity. Optimal calcium uptake requires ATP and oxalate, and release of calcium from loaded vesicles was stimulated by the calcium ionophore A23187 and inhibited by LaCl3. These data indicate that calcium was being actively concentrated within membrane vesicles. After washing of such preparations in the absence of ATP, their capacity to take up Ca2+ is reduced to an initial value of 2.8 nmol/mg protein per min. In the presence of 2 - 10(6) M cyclic AMP to which was added a protein kinase preparation from human platelets, up to a 3-fold increase of this rate of uptake was observed. These results suggest that in platelets, as in muscle, cyclic AMP is a regulatory factor in the control of cytoplasmic calcium. Although the cyclic nucleotide may have still other functions, it appears likely that the well-known inhibition of many platelet activities by high intracellular cyclic AMP concentrations is directly linked to the stimulation of the removal of Ca2+ from the cytoplasm.     

Cyclic AMP in locust fat body: Correlation with octopamine and adipokinetic hormones during flight

The effects of flight upon the level of cyclic AMP in the fat body of Locusta migratoria have been examined. Flight induced two phases of cyclic AMP elevation; the first during the initial 10 min of flight, the second between 20–30 min of flight. Neck-ligated locusts have increased levels of cyclic AMP after 10 min of flight, indicating that the adipokinetic hormones are not necessary for this elevation. Injection of 6 mg trehalose, a procedure known to delay the release of adipokinetic hormones, prevented the increases seen at 10 and 30 min of flight. Injection of synthetic adipokinetic hormone I increased the levels of cyclic AMP within 5 min, and these were maintained for up to 15 min. The roles of octopamine and the adipokinetic hormones in increasing fat body cyclic AMP, and thereby regulating haemolymph lipid, during flight are discussed.     

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [³²P]cyclic AMP (N₃ cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5′-AMP. Binding was unaffected by 5′-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Cyclic AMP induces synthesis of prostaglandin E1 in platelets

Although platelets are known to synthesize small amounts of prostaglandin E1 the control of the formation of this prostanoid has not been investigated. Incubation of human platelet-rich plasma with various compounds which are known to increase cyclic AMP concentration in platelets and inhibit platelet aggregation also increased intracellular prostaglandin E1 synthesis. The prostaglandin E1 was isolated by high pressure liquid chromatography and definitively identified by negative and positive ionization mass spectroscopy. The amounts of prostaglandin E1 formed were proportional to the concentration of cyclic AMP in platelets. Prostacyclin (10 nM) which is the most potent stimulator of cyclic AMP formation increased intracellular cyclic AMP by 4.6 fold and prostaglandin E1 level by 3 fold over the basal levels. Addition of theophylline, a cyclic AMP phosphodiesterase inhibitor, together with prostacyclin increased cyclic AMP concentration 8.7-fold and prostaglandin E1 level 12-fold compared to basal concentrations. Dibutyryl cyclic AMP (2 mM) and 8-bromo cyclic AMP (0.1 mM) increased prostaglandin E1 levels by 3 fold and 2 fold over the basal level, respectively. Prostaglandin D2 (3 microM) when added to platelet-rich plasma increased the cyclic nucleotide levels by 2 fold concomitant with 2 fold increase in prostaglandin E1 concentration. In contrast prostaglandin E2 or prostaglandin F2 alpha which had no effect on cyclic AMP level did not affect the prostaglandin E1 synthesis. Addition of 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, to platelet-rich plasma inhibited both the increase of intracellular prostaglandin E1 and cyclic AMP levels induced by prostacyclin.     

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

Impaired cAMP metabolism associated with abnormal function of thrombopathic canine platelets

The effects of forskolin and 1-Methyl-3-isobutyl-xanthine on thrombopathic platelet function and cyclic AMP accumulation were examined. The concentration of forskolin required to inhibit gamma-thrombin- induced platelet aggregation and secretion by 50% was significantly lower for thrombopathic than for normal platelets. This inhibition was accompanied by a marked elevation of cyclic AMP. 1-Methyl-3-isobutyl-xanthine, alone or in combination with forskolin, augmented both the cyclic AMP accumulation and the inhibition of platelet function. These results demonstrate that cyclic AMP metabolism is abnormal in thrombopathic platelets and imply that cyclic AMP-phosphodiesterase activity is impaired.     

The influence of azathioprine on the behaviour of blood platelets in vitro.

The influence of azathioprine on human platelets was studied in vitro. Azathioprine in a final concentration of 10(-4) M caused significant inhibition of spontaneous sedimentation and aggregation of platelets, but did not effect platelet adhesiveness or resistance to freezing and thawing. The influence of azathioprine is probably due to thioimidazole, this last being a potent stimulating agent of phosphodiesterase activity, involved in the metabolism of cyclic AMP to 5' AMP. These observations indicate that the widely used platelet function assays could be influenced by drugs. The current therapy should therefore be taken into account when the results of the tests are evaluated for clinical purposes.     

Modulation of peripheral blood mononuclear cell cyclic adenosine monophosphate levels by human very low density lipoprotein

Normal human plasma very low density lipoprotein (VLDL) can inhibit mitogen-induced lymphocyte DNA synthesis. Since early events in lymphocyte activation (e.g., cyclic nucleotide metabolism) are thought to influence the magnitude of later events (e.g., [³H]thymidine uptake) we designed the current studies to compare the effects of VLDL on these two cellular processes. Two separate effects of VLDL on peripheral blood mononuclear cell (PBM) cyclic adenosine monophosphate (AMP) metabolism were observed at VLDL concentrations which inhibit phytohemagglutinin (PHA)-induced [³H]thymidine uptake. VLDL suppressed the early, transient increase in PBM cyclic AMP which occurs within minutes of the addition of mitogen. VLDL exposure also stimulated a delayed (greater than 24 hr) and spontaneous increase in PBM cyclic AMP levels which corresponded temporally with progressive cellular refractoriness to mitogen stimulation. If mitogen-induced lymphoproliferation is influenced by early changes in cyclic nucleotide metabolism, as claimed by some investigators, then perhaps the ability of VLDL to modulate intracellular cyclic AMP levels may explain some of the antiproliferative properties of this bioregulatory lipoprotein.     

Platelet aggregation. I. Regulation by cyclic AMP and prostaglandin E1

Platelet aggregation plays a major role in thrombogenesis. This study was undertaken to examine the inhibition of platelet aggregation induced by adenosine diphosphate. It is known that cyclic AMP (adenosine monophosphate) and its dibutyryl derivative inhibit platelet aggregation. This study showed that prostaglandin E1 (PGE1) also inhibits platelet aggregation and stimulates cyclic AMP synthesis by stimulation of adenyl cyclose. Caffeine, on the other hand, inhibits platelet phosphodiesterase, and increases cyclic AMP levels. PGA1 and PGF1 alpha can also inhibit platelet aggregation but only at very high concentrations.     

Interaction of cyclic nucleotides and ecdysterone in breaking the pupal diapause of the bertha armyworm, Mamestra configurata WLK.

Cyclic GMP and cyclic AMP have distinct and opposite effects upon the action of ecdysterone in diapausing pupae of the Bertha armyworm, $\text{Mamestra}$$\text{configurata}$. Cyclic GMP enhanced the effectiveness of suboptimal doses of ecdysterone in breaking diapause; the amount of cyclic GMP required to lower the ED50 of ecdysterone by half was 80 μg/g. Dibutyryl cyclic GMP had no apparent effect on the action of ecdysterone over a wide dose range (0.07 – 70 μg/g). On the other hand, cyclic AMP and dibutyryl cyclic AMP effectively blocked the diapause-breaking action of ecdysterone when administered simultaneously with the steroid hormone. The amount of cyclic AMP required to reduce the incidence of diapause termination from 100% to 50% was 60 μg/g; for dibutyryl cyclic AMP the amount required was only 14 μg/g. No cyclic nucleotide tested in the study could by itself break the pupal diapause of $\text{M.}$$\text{configurata}$. The concept that cyclic GMP and cyclic AMP provide at least different if not opposing regulatory influences in certain insect systems is discussed briefly in the light of these observations.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La3+ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B2 by washed platelets exposed to collagen or thrombin. In addition, La3+ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B2 was not affected. La3+ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E1, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La3+, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane. La3+ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E1 and to F-, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La3+ could be reversed by the addition of Ca2+ up to 10 mM. The stimulation of cyclic AMP production by La3+ may largely explain the inhibitory effect of La3+ upon platelet secretion and thromboxane B2 production. These results also suggest that Ca2+ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

Metabolic control mechanisms in mammalian systems. Involvement of adenosine 3′:5′-cyclic monophosphate in androgen action

1. The ability of exogenously administered cyclic AMP (adenosine 3':5'-monophosphate) to exert andromimetic action on certain carbohydrate-metabolizing enzymes was investigated in the rat prostate gland and seminal vesicles. 2. Cyclic AMP, when injected concurrently with theophylline, produced marked increases in hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, and two hexose monophosphate-shunt enzymes, as well as alpha-glycerophosphate dehydrogenase activity in accessory sexual tissues of castrated rats. The 6-N,2'-O-dibutyryl analogue of cyclic AMP caused increases of enzyme activity that were greater than those induced by the parent compound. 3. Time-course studies demonstrated that, whereas significant increases in the activities of most enzymes occurred within 4h after the injection of cyclic AMP, maximal increases were attained at 16-24h. 4. Increase in the activity of the various prostatic and vesicular enzymes was dependent on the dose of cyclic AMP; in most instances, 2.5mg of the cyclic nucleotide/rat was sufficient to elicit a statistically significant response. 5. Administration of cyclic AMP and theophylline also produced stimulation of enzyme activities in secondary sexual tissues of immature rats. 6. Cyclic AMP and theophylline did not affect significantly any of the enzymes studied in hepatic tissue. 7. Stimulation of various carbohydrate-metabolizing enzymes in the prostate gland and seminal vesicles by cyclic AMP was independent of adrenal function. 8. Concurrent treatment with actinomycin or cycloheximide prevented the cyclic AMP- and theophylline-induced increases in enzyme activities in both castrated and adrenalectomized-castrated animals. 9. Administration of a single dose of testosterone propionate (5.0mg/100g) to castrated rats caused a significant increase in cyclic AMP concentration in both accessory sexual tissues. 10. In addition, treatment with theophylline potentiated the effects of a submaximal dose of testosterone (1.0mg/100g) on all those prostatic and seminal-vesicular enzymes that are increased by exogenous cyclic AMP. 11. The evidence indicates that cyclic AMP may be involved in triggering the known metabolic actions of androgens on secondary sexual tissues of the rat.     

Inhibitory mechanism of cis-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca2+ mobilization, cyclic AMP levels and membrane fluidity

The in vitro inhibitory effects of cis-polyunsaturated fatty acids, linolenic (18:2 delta 9,12), alpha-linoleic (18:3 delta 9,12,15) and eicosatrienoic (20:3 delta 11,14,17) acid, on bovine platelet aggregation and their inhibitory mechanism were investigated. These fatty acids inhibited platelet aggregation induced by ADP and thrombin to similar extent. Fluorescence analyses with fura-2-loaded platelets showed that, in the concentration ranges that inhibited aggregation, they also inhibited agonist-induced increase in cytoplasmic Ca2+. According to radioimmunoassay study, addition of these fatty acids increased cyclic AMP contents in the presence of theophylline corresponded with their inhibitory effects on aggregation. These fatty acids induced a 1.6-1.8-fold increase over basal concentration of cyclic AMP in the concentration ranges that fully inhibited aggregation. On the other hand, saturated fatty acid, stearic acid, affected neither aggregation nor cyclic AMP levels. As reported previously [1985) Biochim. Biophys. Acta 818, 391), these unsaturated fatty acids induced increase in membrane fluidity in the same concentration range. These results suggest that inhibition of platelet aggregation by cis-polyunsaturated fatty acids is due to the increase in cyclic AMP levels. This increase seems to be due to stimulation of adenylate cyclase which is mediated by membrane perturbation.     

Direct evidence for the modulation of human platelet cytosolic free Ca2+ by intracellular cyclic AMP produced with a photoactivatable derivative

We have previously reported that intraplatelet "cyclic AMP jumps" produced with newly synthesized photoactivatable cyclic AMP analogue, inhibited washed rat platelet aggregation and serotonin release as induced by thrombin. Using the same approach on human platelets, thrombin-induced platelet aggregation was dose-dependently inhibited only when a flash was delivered. The mechanism of action of intraplatelet cyclic AMP as resulting from photolysis could be by controlling the level of cytosolic Ca2+. In order to test this hypothesis, the same protocol was used on human platelets preloaded with the internal Ca2+ fluorescent indicator, Quin 2, we found that the extent and the rate of the rise of the cytosolic Ca2+ induced by thrombin were dramatically decreased, in the presence of the photoactivatable cyclic AMP, only following photoirradiation. In addition, the flashes were produced, in the presence of photoactivatable cyclic AMP, after the thrombin-induced rise of internal Ca2+ had reached its peak. In these conditions, photoirradiation caused a rapid fall in fluorescence. These experiments provide the first direct evidence that intracellular cyclic AMP is involved in the control of platelet cytosolic Ca2+ by inhibition of its mobilization and by stimulation of its sequestration.     

Platelet cyclic 3':5'-nucleotide phosphodiesterase released by thrombin and calcium ionophore.

Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La³⁺ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B₂ by washed platelets exposed to collagen or thrombin. In addition, La³⁺ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B₂ was not affected.La³⁺ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E₁, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La³⁺, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane.La³⁺ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E₁ and to F^?, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La³⁺ could be reversed by the addition of Ca²⁺ up to 10 mM. The stimulation of cyclic AMP production by La³⁺ may largely explain the inhibitory effect of La³⁺ upon platelet secretion and thromboxane B₂ production. These results also suggest that Ca²⁺ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.