Influence of calcium antagonists on thrombin-induced calcium mobilization and platelet-vessel wall interactions.

Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Measurement of ionized calcium in blood platelets with a new generation calcium indicator

Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.     

Block of L-type calcium channels by charged dihydropyridines. Sensitivity to side of application and calcium.

We have studied block of L-type calcium channels by intracellular and extracellular application of the ionized dihydropyridine derivatives amlodipine and SDZ 207-180. We find that extracellular application of either drug causes voltage-dependent block of calcium channels. However, neither drug is effective when applied intracellularly. The insensitivity of calcium channels to intracellular drug is not due to the low concentrations of cytosolic calcium, because voltage-dependent block by ionized amlodipine, SDZ 207-180, and the neutral drug nisoldipine persists under conditions in which Ca0 is buffered by EGTA. In fact, the time course of the development of block by the ionized but not neutral drug molecules studied, is slower in the presence than in the absence of calcium. Our results indicate that the DHP binding site of the L-type calcium channel is close to the extracellular surface of the cell membrane and that ionized DHP molecules may interact with the receptor in a manner that is uniquely affected by calcium.     

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.     

Advantages of calcium green-1 over other fluorescent dyes in measuring cytosolic calcium in platelets.

Fluorescent indicators are widely used in the measurements of cytosolic calcium in many cell types for many purposes because they are relatively easy to use. Notwithstanding, they have some defects to prevent accurate measurements under certain conditions, such as significant dye leakage and UV-quenching effect. Menadione, a representative quinone derivative with antiaggregating effect, is also UV-absorbent. To investigate whether menadione can affect the change of cytosolic calcium in platelets by agonist, we measured the change of cytosolic calcium level using calcium green-1. Since this dye has not been used previously in platelets, we determined that the optimal loading of calcium green-1 to platelets was achieved using 3 microM dye incubated for 60 min at 37 degrees C. Our study compared the use of calcium green-1 with fura-2 and fluo-3 (two widely used dyes) in measurements of cytosolic calcium. Fura-2 is UV-excited, so when menadione was treated in fura-2-loaded cells, it had a quenching effect. Fluo-3, the other visible fluorescent indicator, leaked from platelets very rapidly and required the use of anion channel blockers which are known to affect physiological response of platelets. Our study demonstrated that changes in cytosolic calcium levels can be accurately measured without these problems by using calcium green-1. We therefore were able to demonstrate that menadione inhibited calcium increase by thrombin in a dose-dependent manner similar to menadione's antiaggregating effect in platelets.     

Activation of human platelets by ADP causes a rapid rise in cytosolic free calcium without hydrolysis of phosphatidylinositol-4,5-bisphosphate

Phosphatidylinositol-4,5-bisphosphate decreased 40% within 10 seconds after the addition of thrombin to platelets. This thrombin-induced loss was accompanied by a corresponding increase of inositol phosphates. In contrast, within the first 60 seconds after exposure of platelets to ADP there was no detectable change in the amounts of phosphatidylinositol-4,5-bisphosphate or inositol phosphates. Both thrombin and ADP, however, caused a very rapid rise of cytosolic free calcium, as measured by Quin-2. The magnitude of this rise of calcium was similar for the two agonists. These results suggest that in platelets, agonist stimulation may lead to increased cytosolic free calcium independently of phosphatidylinositol-4,5-bisphosphate degradation.     

Alterations in calcium homeostasis as biological marker for mild Alzheimer's disease?

The calcium hypothesis of neurodegenerative disorders such as Alzheimer's disease (AD) suggests that altered cytosolic Ca(2+) levels ( Ca(2+) (i)) and/or disturbances in Ca2+ homeostasis concern cellular mechanisms underlying neuronal pathology. To search for a diagnostic marker of Alzheimer's disease, we measured cytosolic calcium concentrations in platelets of AD patients, age-matched control subjects (AMC), and vascular dementia (VD) patients. The ( Ca(2+) (i)) was determined using long wavelength indicator Fluo-3AM in 21 mild AD patients, 17 AMC, and 23 patients with VD. The basal values of [Ca(2+)](i) were significantly lower in AD compared to AMC. After the addition of 1 mM calcium, the [Ca(2+)](i) markedly increased in platelets of AD compared to AMC and VD. Measurement of calcium homeostasis could provide a very sensitive, but less specific biological marker of AD. These results support the hypothesis that influencing calcium homeostasis may provide a therapeutic strategy in dementia.     

Response of human platelets exposed to arachidonate upon stimulation with other agonists: possible role of Ca2+i movement

Arachidonate (25 microM) induced only 20% maximal platelet aggregation but 80% maximal cytoplasmic ionized calcium movement (as measured by the photoprotein aequorin). Preincubation with 25 microM arachidonate prevented platelet aggregation and cytoplasmic ionized calcium movement induced by threshold concentrations of the thromboxane A2 analogue U-46619, but did not prevent platelet aggregation induced by threshold concentrations of thrombin, despite a marked reduction of cytoplasmic ionized calcium movement. These findings suggest that thrombin may employ pools of cytoplasmic ionized calcium and/or mechanisms to mobilize it different from those utilized by arachidonate and its metabolites.     

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2.

The recently synthesized calcium indicator quin -2 was incorporated into synaptosomes from guinea-pig cerebral cortex following uptake and internal hydrolysis of quin -2 tetra-acetoxymethyl ester. Incubation in physiological media containing 1 mM- or 2 mM-CaCl2 led to equilibrium cytosolic ionized calcium concentrations of 85 +/- 10 nM and 205 +/- 5 nM respectively (mean +/- S.E.M. from eight and eighteen preparations respectively). Cytosolic Ca2+ was elevated following increases in external Ca2+ concentration, plasma membrane depolarization, mitochondrial inhibition, calcium ionophore addition or replacement of external sodium by lithium. Preliminary experiments were performed to assess changes in cytosolic Ca2+ accompanying the release of the neurotransmitter acetylcholine.     

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.     

Calcium fluxes into and out of cytosol in human platelets: analysis of experimental data

The purpose of this research was to analyse experimental data concerning cytosolic calcium concentration in view of the mechanisms involved in calcium fluxes in human platelets. The parameters of model curves are related to the properties of the entities responsible for control or maintenance of cytosolic calcium concentration. It has been shown that: (a) biphasicity of increase in cytosolic calcium concentration caused by inhibition of SERCAs either by TBHQ and TG or by TG alone is related to fast and slow discharge of acidic calcium stores and DTS; (b) biphasicity of decline in cytosolic calcium concentration after its rise caused by stimulation of platelets by the agonists is related to non-synchronous extrusion of calcium by PMCA and NCX; (c) NCX is active only in calcium containing medium: calcium ion(s) are necessary to be bound to the site(s) located on the medium-facing side of the (macro)molecule; (d) PMCA is likely to be activated either by binding calcium ion(s) to the site(s) located on its cytosol-facing side or by unbinding identical ion(s) from the site(s) on its medium-facing side.     

Reduction of the metallochromic indicators arsenazo III and antipyrylazo III to their free radical metabolites by cytoplasmic enzymes

At a concentration much lower than that usually employed for measuring cytosolic ionized Ca²⁺ concentrations, arsenazo III underwent a one-electron reduction by rat liver cytosolic fraction or a hypoxanthinexanthine oxidase system to produce an azo anion radical metabolite. NADH, NADPH, N¹-methylnicotinamide, hypoxanthine, and xanthine, in that order, could serve as a source of reducing equivalents for the production of this free radical by the cytosolic fraction. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated O₂ consumption were enhanced by calcium and magnesium. Antipyrylazo III was ineffective in increasing O₂ consumption by rat liver cytosolic fraction and gave a much weaker ESR signal of an azo anion radical with both the liver cytosolic fraction, in the presence of NADH, and the hypoxanthine-xanthine oxidase system.     

A method of estimating the amount of calcium bound to the metallochromic indicator arsenazo III

As a metallochromic indicator for ionized calcium, arsenazo III is approximately 50 times more sensitive than murexide. However, because of the high binding constant for calcium, the following problems may occur: (a) a considerable amount of calcium is bound to arsenazo III, thereby causing an error in estimating the concentration of ionized calcium; (b) the amount of bound calcium varies with the concentrations of calcium, arsenazo III, magnesium ion and monovalent cations; (c) the amount also varies with pH, (d) the relationship between the absorbance change and the concentration of ionized calcium is nonlinear; and (e) the binding constant of arsenazo III for calcium cannot be determined by the conventional double reciprocal plot. A new experimental and theoretical method is presented which copes with these problems.     

A method of estimating the amount of calcium bound to the metallochromic indicator arsenazo III.

As a metallochromic indicator for ionized calcium, arsenazo III is approximately 50 times more sensitive than murexide. However, because of the high binding constant for calcium, the following problems may occur: (a) a considerable amount of calcium is bound to arsenazo III, thereby causing an error in estimating the concentration of ionized calcium; (b) the amount of bound calcium varies with the concentrations of calcium;, arsenazo III, magnesium ion and monovalent cations; (c) the amount also varies with pH, (d) the relationship between the absorbance change and the concentration of ionized calcium is nonlinear; and (e) the binding constant of arsenazo III for calcium cannot be determined by the conventional double reciprocal plot. A new experimental and theoretical method is presented which copes with these problems.     

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.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

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.