Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

Cyclic nucleotide phosphodiesterase 3 signaling complexes

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.     

The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function.

The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP- and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP-deficient mice had normal agonist-induced contraction, and normal cAMP- and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP- and cGMP-mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP- and cGMP-dependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.     

Cross-Regulation of Intracellular cGMP and cAMP in Cultured Human Corpus Cavernosum Smooth Muscle Cells

The goal of this study was to assess the potential cross-regulation of cyclic nucleotides in human corpus cavernosum (HCC). Incubation of primary cultures of HCC smooth muscle cells with either the NO donor sodium nitroprusside (SNP, 10 μM) or the phosphodiesterase type 5 (PDE 5) inhibitor sildenafil (50 nM) produced little or no changes in the intracellular cGMP levels. Incubation with both SNP and sildenafil produced marked increases in cGMP. Interestingly, incubation of cells with 10 μM of forskolin or PGE₁ produced significant enhancement of cGMP accumulation. These increases were not further enhanced by the addition of SNP and sildenafil. Kinetic analyses of cGMP hydrolysis by PDE 5 showed that high concentrations of cAMP reversibly inhibited the enzyme with a K_i of 258 ± 54 μM. The increase in cGMP levels in response to cAMP generating agents is not due to assay artifact since cAMP did not cross-react with cGMP antibody. Our data suggest that cAMP up-regulates intracellular levels of cGMP, in part, by inhibition of PDE 5. We also noted that cGMP down-regulates cAMP synthesis via a mechanism requiring G-protein coupling of adenylyl cyclase. These observations may have important implications in the utility of pharmacotherapeutic agents targeting cyclic nucleotide metabolism for the treatment of erectile dysfunction.     

Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil

Trapidil (N,N-diethyl-5-methyl[l,2,4]triazolo[l,5-α]pyrimidine-7-amine) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (K_i = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6β-PGI_i). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.     

Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation

Cyclic nucleotides have been previously shown to modulate cell death processes in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP as well as cGMP analogs suppressed tumor necrosis factor alpha (TNFalpha) plus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cultured primary hepatocytes. Furthermore, forskolin, which increases intracellular cAMP levels, also effectively suppressed TNFalpha+ActD-induced apoptosis. Activation of multiple caspases was suppressed in cells exposed to TNFalpha+ActD in the presence of cAMP or cGMP analogs. TNFalpha+ActD-induced cytochrome c release from mitochondria was also inhibited by cAMP or cGMP, reinforcing our conclusion that cyclic nucleotides interfere with the early signaling events of TNFalpha-mediated apoptosis. We evaluated the possibility that cAMP and cGMP inhibit apoptosis by activating the serine/threonine kinase Akt, which is known to promote cell survival. Both cAMP- and cGMP-elevating agents led to marked increases in Akt activation that was inhibited by the phosphatidylinositol 3'-kinase inhibitors, LY294002 and wortmannin. However, complete inhibition of cyclic nucleotide-induced Akt activation had little effect on cyclic nucleotide-mediated cell survival, indicating the existence of other survival pathways. Interestingly, the specific inhibitor of protein kinase A (PKA), KT5720, blocked cGMP-mediated protection but only partially prevented the anti-apoptotic effect of cAMP, indicating that both PKA-dependent and -independent mechanisms are involved in cAMP-mediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides delay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms.     

Metabolic aspects of the secretion of stored compounds from blood platelets. IV. Effects of ionophore X537A on washed platelets.

1. X537A at concentrations below 10 muM can liberate platelet serotonin from washed human platelets without inducing the platelet release reaction. Up to 100% of serotonin preabsorbed by the platelets can be liberated before initiation of the release reaction. 2. Concentrations of X537A above 10muM initiate the platelet release reaction, with a maximum release of adenine nucleotides and platelet factor 4 antigen comparable to that obtained with 1.25 units thrombin/ml. 3. The changes in ATP metabolism at the concentration necessary for X537A-induced release are more profound than those in platelets exposed to concentrations of thrombin or A23187 giving the same degree of release, and approach those seen with high concentrations of A23187. At concentrations where serotonin is liberated but no adenine nucleotide or platelet factor 4 antigen is released, short time incubation causes no change in the level of metabolic ATP.     

Effect of sildenafil on renin secretion in human subjects

Sildenafil is a potent and selective inhibitor of the cyclic GMP-specific phosphodiesterase (PDE5) that is very effective in the treatment of male impotence. It inhibits breakdown of cyclic guanosine monophosphate (cGMP) formed in penile smooth muscle cells in response to stimulation by nitric oxide resulting in muscle relaxation. PDE5 is widely distributed in the body, being present in the vasculature, platelets, and kidneys. In the kidney, PDE5 is involved in the regulation of sodium excretion and renin secretion. The aim of the present investigation was to investigate the effect of sildenafil, in doses used clinically, on renin secretion in human subjects. The studies were performed in two groups of healthy normotensive subjects: one in which sodium intake was unrestricted, and one in which sodium intake was restricted to 600 mg/day. Blood pressure and heart rate were monitored throughout the study, and blood samples for the measurement of plasma cGMP and cAMP concentrations and plasma renin activity (PRA) were collected. After control measurements, the subjects ingested a capsule containing sildenafil or placebo. Cardiovascular measurements and blood sampling continued for the next 120 min. Sildenafil had only minor cardiovascular effects. Diastolic pressure tended to be lower and heart rate was generally higher after sildenafil than after placebo, but the differences were small. Sildenafil caused a prompt and sustained increase in plasma cGMP concentration and a more gradual increase in plasma cAMP concentration. After the subjects received placebo, there was a progressive decrease in PRA during the 2-hr observation period, presumably reflecting the circadian rhythm in renin secretion. In contrast, PRA failed to decrease after the subjects received sildenafil, thus indicating that sildenafil exerts a stimulatory action on renin secretion. This action on renin secretion may help explain why sildenafil only has minor effect on blood pressure despite the widespread distribution of PDE5 in vascular tissues.     

Characterization of the Isoenzymes of cyclic nucleotide phosphodiesterase in human platelets and the effects of E4021

In extracts of human platelets, three isoenzymes of cyclic nucleotide phosphodiesterase (PDE), namely, PDE2, PDE3, and PDE5, were identified; activities of PDE1 and PDE4 were not detected. In human platelets, the cGMP-hydrolytic activity was about six times higher than the cAMP-hydrolytic activity, and PDE5 and PDE3 are the major phosphodiesterase isoenzymes that hydrolyze cGMP and CAMP, respectively. PDE5 exhibited organ-specific expression in humans, and platelets were among the tissues richest in PDE5. A novel inhibitor of PDE5, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl] piperidine-4-carboxylate sesquihydrate (E4021), was a potent and highly selective inhibitor of human platelet PDE5. However, E4021 (up to 10 μM) did not inhibit 9,11-epithio-11,12-methano-thromboxane A₂-induced platelet aggregation, in vitro. E4021 plus SIN-1 (3-morpholino-sydnonimine), at concentrations that had little effect individually, inhibited aggregation. These results suggest the unique distribution of phosphodiesterase isoenzymes in human platelets and the PDE5 inhibitors might be useful as a new class of antiplatelet drugs.     

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

Role of phosphodiesterase type 3A and 3B in regulating platelet and cardiac function using subtype-selective knockout mice

Phosphodiesterase type 3 (PDE3) is an important regulator of cAMP-mediated responses within the cardiovascular system. PDE3 exists as two subtypes: PDE3A and PDE3B, with distinct cellular and subcellular locations. Due to the lack of subtype-specific pharmacological tools, the definitive role of each subtype in regulating cardiovascular function has not been determined. In this study, we investigated platelet and cardiac function, using PDE3A and PDE3B gene knockout (KO) mice. Platelet-rich-plasma was prepared from the blood of KO and age-matched wild-type (WT) mice. PGE1 (1 microg/mL) almost completely inhibited aggregation of platelets from WT, PDE3A KO and PDE3B KO mice. In platelets from WT mice, cilostamide (100 microM), a selective PDE3 inhibitor, blocked collagen- and ADP-induced aggregation. In contrast, cilostamide had no effect on aggregation of platelets from PDE3A KO mice. In PDE3B KO mice, inhibition of collagen- and ADP-induced platelet aggregation was similar to that in WT mice. The resting intra-platelet cAMP concentration in platelets from PDE3A KO mice was twice that in the WT platelets. After PGE1 (0.1 microg/mL) stimulation, intra-cellular cAMP concentration was increased significantly more in platelets from PDE3A KO mice compared to WT mice. In vivo, PDE3A KO mice were protected against collagen/epinephrine-induced pulmonary thrombosis and death, while no such protection was observed in PDE3B KO mice. The heart rate of PDE3A KO mice was significantly higher, compared with age-matched WT mice, while that of PDE3B KO mice was similar to WT. There was no difference in cardiac contractility between PDE3A or PDE3B KO mice. Heart rate and contractility were increased in a similar dose-dependent fashion by isoproterenol in both types of KO mice. Cilostamide increased heart rate and contractility in WT and PDE3B KO but not in PDE3A KO mice. Compared to WT and PDE3B KO mice, cyclic AMP-PDE activity in membrane fractions prepared from the hearts of PDE3A KO mice was lower and not inhibited by cilostamide. The data suggest that PDE3A is the main subtype of PDE3 expressed in platelets and cardiac ventricular myocytes, and is responsible for the functional changes caused by PDE3 inhibition.     

cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties]

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.     

Platelet aggregation. IV. Platelet phosphodiesterase and its inhibition by vasodilators

Platelet cyclic AMP phosphodiesterase (PDE) has been partially purified, its properties studied and inhibition by certain vasodilators observed. Quazodine, dipyridamole, papaverine, Paveril^® and other agents inhibit platelet adenosine uptake, potentiate both the inhibition of aggregation and PGE₁ stimulated cyclic AMP synthesis and inhibit PDE. The mechanism of action of vasodilators as inhibitors of aggregation is discussed.     

Effect of bed rest on the adenine nucleotides concentration in human blood platelets.

The effect of immobilization in bed on metabolism and function of human blood platelet was studied. Blood platelets taken from patients with bone fractures after long term bed rest (14 days and 28 days) demonstrated significantly reduced concentration of total adenine nucleotides (after 28 days reduction about 30%). This decrease of total platelet adenine nucleotides after immobilization in bed is probably caused by stimulation of platelet secretory process. Thrombin which released from control platelets 58.2% +/- 1.5% of total adenine nucleotides liberated decreased amounts (only 23.1% +/- 3.3% of total) of nucleotides from patient platelets isolated after 28 days of immobilization in bed. Loss of nucleotides from platelets was accompanied by slightly increased extent of platelet aggregation. It is concluded that during bed rest the reactivity of blood platelets (aggregation and release reaction) is stimulated.     

A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle

Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M₂AChR pharmacological profile. Therefore, a novel Ca²⁺/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58?kDa) being the 58?kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [³H]cGMP and [³H]cAMP exhibiting a higher affinity as K_m (μM) for cGMP than cAMP but being close values with V_max cAMP/cGMP ratio of 1.5. The co-factor Mg²⁺ showed similar K_(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC₅₀ of 4.9 and 4.6?μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K_(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M₂AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.     

Stimulatory effects of endotoxin on the platelet secretory process

The effects of the lipopolysaccharides (LPS) of Proteus mirabilis (smooth and rough types), differing significantly in their composition on the release of compounds stored in specific platelet granules, were studied. There are two main types of secretory granules in blood platelets. Dense granules contain adenine nucleotides, and in alpha-granules different proteins are stored. The LPS were found to cause a dose-dependent release of proteins and adenine nucleotides. In the extracellular medium LDH activity was not present. The results presented in this study indicate that LPS from P. mirabilis act directly on blood platelets and induce the platelet secretory process. In comparison with thrombin, a strong platelet agonist, the action of the endotoxins tested was weak.     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.     

Activation of human platelets by N-substituted aminophospholipids

N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylserine (NBD-PS), a fluorescent phospholipid synthesized from phosphatidylserine by reaction with NBD-chloride, caused platelet shape change and aggregation when added at micromolar concentrations to suspensions of washed human platelets in the absence of added fibrinogen. Platelet aggregation by NBD-PS was accompanied by thromboxane synthesis and secretion of contents from dense, alpha-, and lysosomal granules in the absence of appreciable platelet damage. Indomethacin completely inhibited NBD-PS-induced thromboxane synthesis, but platelet aggregation and [14C]serotonin secretion were only slightly inhibited. Neither inhibition of the ADP-dependent pathway with creatine phosphate/creatine kinase plus ATP, alone or in combination with indomethacin, nor maximum elevation of cyclic AMP by treatment with prostaglandin I2 and theophylline completely inhibited NBD-PS-induced platelet aggregation or [14C]serotonin secretion. Platelet effects of NBD-PS were specific in that neither phosphatidylserine nor lyso-NBD-PS were similarly active. The activation of platelets by NBD-PS is not attributable to the NBD moiety exclusively since acylation of the amino group with 5-dimethylaminonaphthalene-1-sulfonyl-chloride yielded a similarly active derivative. Dansylated phosphatidylethanolamine was also active. The findings indicate that NBD-PS and other N-substituted aminophospholipids can activate a central pathway of platelet secretion and aggregation that is independent of released ADP and thromboxane formation and is only partially controlled by platelet cyclic AMP.     

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)