激光扫描共聚焦显微镜术检测单个血小板钙波动方法的探讨

目的 探讨血小板贴壁的简易方法,同时,观察单个血小板激活前后细胞内钙波动及形态变化的规律。方法 采用多种粘附剂固定血小板,利用钙荧光探针（Fluo-3/AM)（终浓度10－20μmol)进行染色,在激光扫描共聚焦显同镜检测下加入二磷酸腺苷,观察和分析血小板内Ca^2 浓度及形态变化。结果 多聚赖氨酸促进血小板贴壁固定的效果最佳;单个血小板活后细胞内Ca^2 浓度为激活前的128％,形态圆形或椭形转为不规则形,有空泡,突起形成。结论 多聚赖氨酸是血小板贴壁的理想粘附剂,本方法能简易,快捷地监测血小板激活过程中胞浆内钙离子动态变化及形态改变,有助于血小板功能的深入研究。     

Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers.

To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist-induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+)-dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.     

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.     

The rapid desensitization of receptors for platelet derived growth factor, bradykinin and ATP: studies on individual cells using quantitative digital video fluorescence microscopy

The rise in free cytosolic Ca2+ of individual response to growth factors was studied in serum starved cultures of 3T3 fibroblasts. Quantitative digital video fluorescence microscopy revealed that with platelet derived growth factor (PDGF) there was a lag period between stimulation and Ca2+ response, with considerable cell-to-cell variation, whereas ATP, bradykinin and fetal calf serum induced an immediate, synchronous response. A coverslip with attached cells was mounted on a small flow chamber, allowing complete change of medium in 2 sec. Using this technique, homologous desensitization to a second addition of agonist 2 min after removal of the first addition was found for all agonists. Unusual heterologous desensitization was observed in that PDGF desensitized the cells to the other agonists, yet the reverse did not occur.     

The interaction of Tb3+ with the human platelet surface

The interaction of the lanthanide Tb3+ with washed, human platelets was examined. When bound to the platelet surface, the fluorescence of this Ca2+ analog was increased approximately 200-fold, most likely by a F?rster mechanism involving platelet surface protein aromatic residues. The binding of Tb3+ to the unactivated platelet was specific and saturable with an apparent approximate Kd of 195 microM. Both Ca2+ and La3+ effectively displaced Tb3+ from platelet surface sites, but neither cation did so completely. Plasmin treatment of the platelet surface reduced Tb3+ fluorescence by 68% at saturation without significantly affecting the approximate apparent Kd. Activating washed, aspirinated platelets with ADP induced a 78% increase in Tb3+ fluorescence at saturation. Tb3+ competed effectively and completely for platelet surface-bound 45Ca2+ with an approximate IC50 of 10 microM. These data indicate the potential utility of this fluorescent lanthanide in characterizing Ca2+-binding sites on the human platelet.     

Calpain I activation is not correlated with aggregation in human platelets.

Calpain-catalysed hydrolysis of platelet substrates such as cytoskeletal and calmodulin-binding proteins, and of protein kinase C, is assumed to contribute to platelet aggregation. We have measured calpain I activation by immunoblotting, and [Ca2+]i (cytoplasmic Ca2+ concn.) by fura-2 fluorescence, in parallel with measurement of aggregation, in stirred human platelets treated at different [Ca2+]ext (extend Ca2+ concns.) with A23187, leupeptin, phorbol ester and thrombin. Hydrolysis of actin-binding protein, and [3H]5-hydroxytryptamine release, were also measured in some cases. A rise in [Ca2+]i, platelet aggregation and calpain activation often occurred together. With some combinations of agonists and [Ca2+]ext, however, this correlation was clearly not maintained. It was shown: (a) that activation of calpain and its hydrolysis of platelet substrates were not strictly necessary conditions for platelet secretion and aggregation; (b) conversely, that calpain activation could occur without aggregation.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry.

We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.     

Evaluation of platelet function by flow cytometry

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.     

Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.     

Digitonin-aided loading of Fluo-3 into embryogenic plant cells

This paper describes a method to load embryogenic plant cells with Fluo-3 in its cell impermeant form with the aid of digitonin. Attempts to load cells with Fluo-3/AM were all unsuccessful. Presumably the indicator is cleaved outside the cells and cannot penetrate in its acidic form. At a low pH, Fluo-3 enters the plant cells but normal Ca2+ homeostasis seems to be disturbed. Successful loading of Fluo-3 was achieved by adding 0.1% digitonin during incubation with the Ca(2+)-indicator. A bright fluorescence was observed in the epidermal layer of heart and torpedo shaped somatic embryos of carrot with confocal scanning laser microscopy. Vacuoles were always without fluorescence which indicates that the dye, after loading, remains in the cytosol and does not leak out. The fluorescence intensity was sensitive to treatments with A23187 and EGTA. We conclude that Fluo-3 can be effectively loaded, with the aid of digitonin, into plant embryogenic cells in liquid culture. Therefore, we expect this technique to be very useful for the study of changes in cytosolic free Ca2+ levels during plant growth and development.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

Role of glycosaminoglycans in calcium metabolism of human platelets

Changes in intracellular Ca2+, [Ca2+]i, were measured in control and chondroitin ABC lyase-pretreated platelets. [Ca2+]i was measured with the fluorescent calcium probe Quin2. Chondroitin ABC lyase removed chondroitin 4-sulfate from the platelet surface without inducing shape change or release of serotonin. Compared to similarly prepared controls, enzyme treated platelets showed an increase of [Ca2+]i in response to stimulation by various agonists at high (1 mM) extracellular Ca2+ concentration. At low Ca2+ in the medium (1 mM EGTA), such platelets responded to agonists with a decreased rise in [Ca2+]i compared to the controls. These studies indicate that selective removal of glycosaminoglycans may sensitize platelets to the action of platelet aggregating agents. In addition, glycosaminoglycans may have a calcium storage function.     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

槲皮素硫酸酯对猪血小板胞浆游离钙浓度和蛋白激酶C的影响

槲皮素（ｑｕｅｒｃｅｔｉｎ,Ｑｕｅ）,是一种天然的黄酮类化合物,具有多种生物活性［１］,但是Ｑｕｅ水溶性差,口服时胃肠难以吸收［２］．因此,为进一步开发和利用Ｑｕｅ,人工合成水溶性Ｑｕｅ——槲桷皮素硫酸酯（ｓｏｄｉｕｍｑｕｅｒｃｅｔｉｎｓｕｌｆａｔｅ...     

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.     

Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization.

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.     

Irregular spiking in free calcium concentration in single, human platelets. Regulation by modulation of the inositol trisphosphate receptors.

Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP3)-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+]i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca2+]i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 microm) and U73122 (10 microm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP3-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3-dependent Ca2+-induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3. In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.     

Intracellular calcium fluxes in human platelets

Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 microM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane-associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.