Influence of nitric oxide on agonist-mediated calcium mobilization in platelets

Recent studies have characterized endothelium-derived relaxing factor as nitric oxide. It appears to exert its effect by elevating intracellular levels of cyclic GMP. In this study we confirm that nitric oxide is a potent inhibitor of agonist-induced irreversible aggregation. At the concentrations tested nitric oxide effectively blocked thrombin-stimulated mobilization of cytosolic-free calcium in Fura 2-loaded platelets. In addition, nitric oxide prevented the inositol 1,4,5-trisphosphate-stimulated calcium rise in cytosolic calcium in saponin-permeabilized Fura 2-loaded platelets. Similar to the action of adenylate cyclase stimulators, nitric oxide facilitated lowering of calcium levels raised by the action of agonists. The specific mechanism by which it exerts its effect on intracellular levels of calcium is not clear.     

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.     

Platelet-activating factor phosphatidate,but not platelet-activating factor,is a powerful calcium ionophore in the human red cell

Platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, PAF) is a potent inducer of shape-change, aggregation and secretion in platelets. PAF causes a rapid increase in intracellular calcium, but has no calcium gating effect in intact lipid bilayers. Human red cells (RBC) did not metabolize either PAF or PAF-phosphatidate (PAF-PA). While PAF (10 μM) was devoid of calcium ionophoretic activity, PAF-PA (1–5 μM) stimulated calcium influx into intact human RBC. In addition, PAF-PA (1–10 μM), but not PAF (10 μM), elicited a series of satellite effects related to the rise of intracellular calcium: 1) increased efflux of intracellular potassium (Gàrdos effect); 2) alkalinization of unbuffered RBC suspensions; 3) stimulation of ATP consumption and production, and enhancement of glycolytic flux with crossover at the glyceraldehyde 3-phosphate dehydrogenase step. These effects exactly duplicate those brought about by the calcium ionophore A23187. The ionophoretic potency of PAF-PA was about half that of A23187. Approximately the same concentrations of PAF-PA as those that stimulate calcium influx into RBC elicit full aggregatory response in human platelets. It is possible that transformation of PAF into PAF-PA by the combined action of phospholipase C and diacylglycerol kinase contributes to the increase of calcium influx in platelets.     

Impaired platelet activation in familial high density lipoprotein deficiency (Tangier disease)

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.     

Lack of evidence for voltage dependent calcium channels on platelets

Intracellular calcium was measured in human platelets using the fluorescent calcium indicator Quin 2. A concentration dependent increase was observed with thrombin. Depolarisation induced by high KCl concentrations did not alter [Ca++]i. The calcium agonist Bay K 8644 did not affect resting levels or thrombin stimulated elevation of intracellular calcium. The calcium antagonists diltiazem, verapamil and PN 200-110 did not inhibit the thrombin stimulated elevation in [Ca++]i. Pretreatment of platelets with adenylate cyclase stimulants reduced the rate and magnitude of the maximal [Ca++]i elevation due to thrombin. In addition, thrombin stimulation of 45Ca++ influx was insensitive to Bay K 8644, verapamil, diltiazem and Pn 200-110. We conclude that functional voltage sensitive calcium channels are not present on human platelets.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Oxidation by hypochlorite converts protective HDL into a potent platelet agonist

High density lipoproteins (HDL) represent a protective factor of central importance that counteracts the development of cardiovascular disease, in part by normalizing platelet (hyper)reactivity. As HDL represent an efficient scavenger of the naturally occurring oxidant hypochlorite, this work was intended to investigate the influence of hypochlorite-oxidized HDL on platelet function. Addition of hypochlorite-oxidized HDL to human platelets results in an immediate and transient raise in intracellular calcium, surface expression of P-selectin and platelet aggregation. The observed effects are dose dependent and can be blocked by an antibody directed against the lipoprotein-binding domain of platelet thrombospondin- and scavenger receptor CD36.     

Augmented platelet calcium uptake in response to serotonin stimulation in patients with major depression measured using Mn2+ influx and 45Ca2+ uptake

There is an augmented platelet intracellular calcium response to serotonin stimulation in major depression. The role that calcium influx has in this process is not known. The objective of this study was to determine platelet calcium influx in response to serotonin by two methods, Mn2+ influx and 45Ca2+ uptake, in order to observe if the uptake response to serotonin was augmented in major depression by comparing the response to normal controls. The use of the two methods of calcium influx showed that serotonin stimulates calcium uptake into platelets. Furthermore, patients with major depression have significantly augmented platelet calcium uptake in response to serotonin. The interesting finding was that calcium uptake into platelets is biphasic, occurring immediately and after five minutes. These results may support the two pool model for calcium oscillations within cells whereby extracellular calcium is needed for intracellular calcium release, and for replenishment of depleted stores once intracellular calcium is released.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

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.     

Synaptotagmin II could confer Ca(2+) sensitivity to phagocytosis in human neutrophils

To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.     

The roles of ATP and calcium in morphological changes and cytotoxicity induced by 1,4-benzoquinone in platelets

To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.     

Epinephrine potentiates activation of human platelets by low density lipoproteins

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.     

Epinephrine potentiates activation of human platelets by low density lipoproteins.

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.     

19F-NMR study of the effect of lead on intracellular free calcium in human platelets

Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrations of lead in the environment (approximately 1 microM) on the intracellular free Ca2+ concentration in any human tissue. We have investigated the effect of lead on the intracellular free Ca2+ concentration of human blood platelets using 19F-NMR and a fluorinated intracellular Ca2+ indicator. We report a basal intracellular free Ca2+ value of 172 +/- 8 nM. Treatment with 1, 5, 10 and 25 microM Pb2+ resulted in average increases in intracellular free Ca2+ of 39%, 91%, 135% and 172%, respectively. The percent increase in intracellular free Ca2+ was linearly and positively correlated with the log of Pb2+ concentration. Using atomic absorption spectroscopy, a significant increase in total calcium of approx. 10 nmol/mg protein was found in 25 microM Pb2+ treated platelets. This indicates that influx of external Ca2+ contributes to the observed increase in free Ca2+. The results provide an explanation for the previously reported effects of lead on platelet function, and suggest a mechanism for low level lead-induced hypertension.     

The Influence of Epinephrine on Washed Human Platelets: Shape Change and Protein Phosphorylation

Abstract

Protein phosphorylation is an important regulator of the properties or functions of many proteins and is associated with the platelet activation response to a number of chemically and functionally different agents such as thrombin, plateletactivating factor, serotonin and collagen. The physiological responses of platelets to these agents are similar, and the common intracellular messenger for activation is an elevated concentration of calcium. Platelets possess alpha-2-receptors, and treatment with epinephrine produces an elevation in platelet cytosolic free calcium concentrations. Methods are described for studying hormone sensitive shape change and protein phosphorylation in washed human platelets. Epinephrine induces platelet shape change, and this process is independent of extracellular calcium. Treatment of [³²P]-orthophosphate-labelled platelets with epinephrine produces an increase in ³²P-incorporation into two platelet proteins with molecular weights of 47000 and 20000. This phosphorylation response is both dose and time dependent. Extracellular calcium is not absolutely essential for epinephrine-induced phosphorylation, but does enhance the maximum levels of ³²P-incorporation. Epinephrine sensitive phosphorylation is completely inhibited following pretreatment with verapamil or nitrendipine. Shape change in response to epinephrine occurs in the absence of enhanced protein phosphorylation. The data suggest that epinephrine mobilizes intracellular calcium, and induces platelet shape change and phosphorylation responses characteristic of platelet activation.     

Modification of human platelet response to sodium arachidonate by membrane modulation

Previous studies have shown that the majority of mongrel dogs have platelets that do not aggregate when stirred with sodium arachidonate, but respond normally to other aggregating agents. Here we have created a model in which human platelets mimic the unusual behavior of canine cells. Brief exposure of human platelets to low concentrations of prostacyclin will convert them to a physiologic state resembling that of canine platelets without causing elevation of intracellular levels of cAMP. Studies on the conversion of arachidonic acid showed that prostacyclin did not cause any inhibition of thromboxane B₂ generation. Exposure of prostacyclin treated platelets to epinephrine restored their sensitivity to arachidonate without causing any detectable changes in levels of cAMP. The selective inhibition achieved by PGI₂ could be mimicked by the endoperoxide analog, U44069, a thromboxane inhibitor, U-51605, and an endoperoxide/thromboxane receptor blocker, 13-APA. Inhibition induced by these compounds was also reversed by epinephrine. A calcium channel blocker, verapamil, and an alpha blocker, dihydroergocryptine, effectively blocked the corrective influence of epinephrine on prostacyclin-treated platelets. Results of these studies show that catecholamines and prostaglandin receptors share close sites on the membrane and exhibit a degree of cooperativity in calcium modulation. Dog platelets that do not respond to products of AA metabolism and the human platelets made to behave like canine platelets may have a defect in calcium mobilization and this defect is corrected by adrenalin through an alpha receptor modulation.     

A Novel,Rapid Method to Quantify Intraplatelet Calcium Dynamics by Ratiometric Flow Cytometry

Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar⁺-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.     

Ethanol stimulates shape change in human platelets by activation of phosphoinositide-specific phospholipase C

Administration of ethanol to human platelets resulted in a rapid shape change which was maximal within 30 s. Ethanol did not cause aggregation or secretion of ATP at any time and inhibited aggregation induced by collagen. In platelets that were loaded with the intracellular calcium indicator fura2, ethanol induced a rapid mobilization of calcium from internal, thrombin-sensitive pools. Cytosolic calcium increased to a maximum within 5 s and decreased slowly over the ensuing 5 min to near basal levels. The mobilization of calcium by ethanol coincided with the rapid formation of phosphatidic acid and a decrease in the level of phosphatidylinositol 4,5-bisphosphate, as measured in 32P-labeled platelets. In platelets labeled with myo-[2-3H]inositol, ethanol caused a 20-30% increase in the levels of inositol (1,4,5)-trisphosphate and inositol bisphosphate within 10 s. Ethanol also induced the transient phosphorylation of myosin light chain (20 kDa) and a 40 kDa protein, a known substrate for protein kinase C. The results indicate that ethanol activates phosphoinositide-specific phospholipase C in human platelets. The subsequent mobilization of intracellular calcium and activation of protein kinase C can account for the shape change induced by ethanol.     

Measurement of Receptor Induced Changes in Intracellular Free Calcium in Human Platelets

Abstract

The intracellular free calcium concentration plays a key role as second messenger in the regulation of cellular reactions. Post-receptor events can be investigated by measurement of free calcium concentration in cells. Our experience in measuring the intracellular free calcium concentration in platelets with the use of the fluorescent indicator quin-2-tetraacetoxymethyl- ester is described. Possible pitfalls in the preparation procedures of the platelets are discussed as well as critical steps and the limitations of the quin-2-method. The methodological approach is demonstrated by the presentation of an investigation with the adenylate cyclase inhibitors adenosine-5′-diphosphate and epinephrine as well as their interrelationship. The potentiation effect of epinephrine to adenosine 5′-diphosphate on platelet function such as aggregation is accompanied by a potentiation of the effect of these two platelet activators in elevating the intracellular free calcium concentration. Adenosine 5′-diphosphate elevates intra-platelet free calcium alone, whereas epinephrine acting through stimulation of the alpha₂-receptor needs another permissive factor to immediately elevate the intra-platelet free calcium.