Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets.

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.     

Adenine nucleotide metabolism of blood platelets IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (³H-labelled), which was essentially complete in 30 s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. β-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and α-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O₂ consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9–10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4–5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.     

Characteristics of collagen-induced fibrinogen binding to human platelets

Polymerized type I calf skin collagen induced a time-dependent specific binding of 125I-fibrinogen to washed human platelets. Binding occurred more rapidly in a shaken rather than in an unstirred system. It was linear in the range 0.05-0.3 microM added fibrinogen and was saturated at higher fibrinogen concentrations (more than 0.8 microM). Scatchard analysis showed a single population of binding sites (16530 +/- 5410 per platelet) with a Kd = 0.53 +/- 0.23 microM. Collagen-induced 125I-fibrinogen binding to platelets was completely inhibited by ADP antagonists such as creatine phosphate/creatine phosphokinase and AMP, and partially inhibited by pretreatment of the platelets with aspirin. With both normal and aspirin-treated platelets a close correlation was observed between the amount of 125I-fibrinogen bound and the extent of dense granule secretion. Our results confirm that fibrinogen becomes bound to platelet surface receptors during collagen-induced platelet aggregation and suggest that secreted ADP is an essential cofactor in this process.     

Quantification of energy consumption in platelets during thrombin-induced aggregation and secretion. Tight coupling between platelet responses and the increment in energy consumption.

The involvement of metabolic energy in platelet responses was investigated by measuring the energy consumption during aggregation and secretion from dense, alpha- and acid-hydrolase-containing granules. Gel-filtered human platelets were stimulated with different amounts of thrombin (0.05-5.0 units X ml-1). At various stages during aggregation and secretion the energy consumption was measured from the changes in metabolic ATP and ADP following abrupt arrest of ATP resynthesis. Stimulation with 5 units of thrombin X ml-1 increased the energy consumption from 6.2 +/- 0.9 to 17.8 +/- 0.4 mumol of ATPeq. X min-1 X (10(11) platelets)-1 during the first 15 s. It decreased thereafter and returned to values found in resting cells after about 30 s. With 0.05 unit of thrombin X ml-1, the energy consumption accelerated more slowly and took at least 3 min before it normalized. A strong positive correlation was found between the velocities of the three secretion responses and the concurrent energy consumption (a) at different stages of the responses induced by a given dose of thrombin, and (b) at different secretion velocities initiated by different amounts of thrombin. When, at different stages of the responses, the extent of secretion was compared with the amount of energy that had been consumed, a strong linear correlation was found with the increment in energy consumption but not with the total energy consumption. This correlation was independent of the concentration of thrombin and indicated that complete secretion from dense, alpha- and acid-hydrolase-containing granules was paralleled by an increment of 4.0, 6.5 and 6.7 mumol of ATPeq. X (10(11) platelets)-1, respectively. An energy cost of 0.7 mumol of ATPeq. X (10(11) platelets)-1 was calculated for separate dense-granule secretion, whereas the combined alpha- and acid-hydrolase granule secretion required 5.3 mumol of ATPeq. X (10(11) platelets)-1. There was no correlation between energy consumption and optical aggregation. In contrast, the rate of single platelet disappearance, which is a measure for the early formation of small aggregates, correlated closely with the rate of energy consumption. Compared with secretion, however, the energy requirement of single platelet disappearance was minor, since 2mM-EDTA completely prevented this response but decreased the energy consumption only slightly. An increase of 0.5-1.0 mumol of ATPeq. X (10(11) platelets)-1 was seen before single platelet disappearance and the three secretion responses were initiated, indicating an increase in energy consuming processes that preceded these responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.     

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.     

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.     

Influence of calmodulin antagonist (stelazine) on agonist-induced calcium mobilization and platelet activation

Phenothiazines at high concentrations inhibit platelet aggregation and the secretion of granule contents. In this study we have evaluated the influence of stelazine on platelet function at low concentrations. Stelazine alone had no influence on resting calcium levels in platelets but facilitated agonist-induced elevation of cytosolic calcium. Platelets combined with low concentrations of stelazine (10 microM) and stimulated with subthreshold concentrations of thrombin (0.05 mu/ml) aggregated irreversibly and released significant quantities of ATP. Results of these studies suggest a new role for the calmodulin antagonist stelazine in platelet activation.     

Comparative studies on the energetics of platelet responses induced by different agonists.

The correlation between energy consumption and platelet responses induced by collagen, A23187 and ADP was investigated and compared with the energetics of thrombin-stimulated platelets established in earlier work. Aggregation, measured as single-platelet disappearance, and secretion correlated quantitatively with the increment but not with the total consumption of energy, suggesting that the former reflects the energy cost of these responses. The cost of complete aggregation was 2-3 mumol of ATP equivalents/10(11) platelets with collagen, ADP and thrombin as the stimulus. The cost of complete dense-granule secretion was 0.5-0.8 mumol of ATP equivalents/10(11) platelets with all agonists tested. The cost of combined secretion of alpha-granule and acid hydrolase granule contents was 5-7 mumol of ATP equivalents/10(11) platelets with thrombin and collagen. However, in the presence of A23187 much more energy was consumed during aggregation and secretion. Also ADP triggered more energy consumption during secretion than was seen with the other inducers. The effect of inhibitors of aggregation and secretion was investigated in thrombin-stimulated platelets. Raising the cellular cyclic AMP content sharply decreased the increment in energy consumption as well as aggregation and secretion. The cytoskeleton-disrupting agents cytochalasin B and colchicine left the increment in energy consumption intact, but decreased the basal consumption seen in unstimulated platelets. This was accompanied by normal (cytochalasin B) or diminished (colchicine) aggregation and secretion. Apart from the latter exception, all inhibitors decreased secretion and incremental energy consumption in parallel, thereby preserving the energy-versus-secretion relationship established in earlier work. In contrast, aggregation and energy consumption varied independently, suggesting that the coupling with energy consumption is much weaker for this response.     

Copper deficiency alters protein kinase C mediation of thrombin-induced dense granule secretion from rat platelets

Experiments were conducted to determine if copper deficiency enhances the rate of thrombin-induced dense granule secretion by modifying the major signal transduction pathways of rat platelets. Platelets were obtained from male, weanling Sprague-Dawley rats fed diets containing either deficient ( < 0.5 μg/g diet) or adequate (5.5 μg/g diet) copper for 5 weeks. Following stimulation with thrombin (0.1 U/mL), the rate of dense granule secretion as measured by ATP release was 160% higher in platelets from copper-deficient than from control rats. Inhibition of the rate of thrombin-induced ATP release by (6-aminohexyl)-1-naphthalene-sulfonamide, a calmodulin antagonist was independent of copper status. However, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the rate of ATP release only in platelets from copper-deficient rats. Aspirin had no effect on ATP release from platelets obtained from either copper-deficient or control rats. This suggests that copper deficiency alters the role of protein kinase C in regulating dense granule secretion. Analysis of autoradiographs showing [³²P]-labeled platelet proteins indicated that the phosphorylation of a 40 kDa protein, a known substrate for protein kinase C in platelets, was significantly less following thrombin stimulation in platelets from copper-deficient than from control rats. When protein kinase C was activated by phorbol 12-myristate 13-acetate prior to thrombin stimulation, ATP release was attenuated regardless of copper status. These findings suggest that protein kinase C can still function as a feedback inhibitor of platelet dense granule secretion in copper deficiency, but impaired activation of this enzyme following thrombin stimulation may prevent it from achieving full regulatory capacity.     

Metabolic and functional consequences of introducing inositol 1,4,5-trisphosphate into saponin-permeabilized human platelets.

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.     

Sex-specific changes in platelet aggregation and secretion with sexual maturity in pigs.

Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2-3 mo) and sexually mature (adult; 5-6 mo) male and female pigs (n=8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 microM) and collagen (6 microg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.     

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.     

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.     

Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets

Several platelet agonists, including thrombin, collagen, and thromboxane A(2), cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCdelta was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets. Only agonists that caused platelet dense granule secretion activated PKCdelta. SFLLRN- or AYPGKF-induced dense granule release and PKCdelta phosphorylation occurred at the same respective agonist concentration. Furthermore, AYPGKF and SFLLRN-induced dense granule release was blocked by rottlerin, a PKCdelta selective inhibitor. In contrast, convulxin-induced dense granule secretion was potentiated by rottlerin but was abolished by Go6976, a classical PKC isoform inhibitor. However, SFLLRN-induced dense granule release was unaffected in the presence of Go6976. Finally, rottlerin did not affect SFLLRN-induced platelet aggregation, even in the presence of dimethyl-BAPTA, indicating that PKCdelta has no role in platelet fibrinogen receptor activation. We conclude that PKCdelta and the classical PKC isoforms play a differential role in platelet dense granule release mediated by protease-activated receptors and glycoprotein VI. Furthermore, PKCdelta plays a positive role in protease-activated receptor-mediated dense granule secretion, whereas it functions as a negative regulator downstream of glycoprotein VI signaling.     

A platelet secretion pathway mediated by cGMP-dependent protein kinase

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.     

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.