Calcium Entry In Human Platelets Research Articles

A key role for dense granule secretion in potentiation of the Ca 2+ signal arising from store-operated calcium entry in human platelets

Recent work has demonstrated a role for Na +/Ca 2+ exchange in potentiation of the Ca 2+ entry elicited through the human platelet store-operated channel by controlling a Mn 2+-impermeable Ca 2+ entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na +/Ca 2+ exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn 2+-impermeable Ca 2+ entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca 2+ signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca 2+ signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca 2+stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca 2+entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca 2+-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca 2+content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca 2+in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 μM TBHQ, individually or simultaneously, accelerated Ca 2+ store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca 2+ influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca 2+ extrusion by PMCA or the Na +/Ca 2+ exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca2+ entry (SOCE) regulated by two independent Ca2+ stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP3 receptor (IP3RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca2+ entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca2+-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca2+ stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca2+ stores and the DTS, the major releasable Ca2+ store in platelets. These findings were confirmed by using Sr2+ as a surrogate for Ca2+ entry. Colchicine attenuated the coupling between IP3RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP3RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca2+. These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca2+ stores.

A role for the intracellular protease calpain in the activation of store-operated calcium entry in human platelets

Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca 2+ entry activated in human platelets by thapsigargin-evoked Ca 2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC- l-leucyl- l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca 2+ store depletion even in the absence of a rise in cytosolic [Ca 2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca 2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca 2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca 2+ entry pathways.

Carbon monoxide inhibits capacitative calcium entry in human platelets

The cytosolic calcium concentration in human platelets is elevated by several agonists via receptor-operated mechanisms involving both Ca 2+ release from intracellular stores and Ca 2+ entry. In order to get a mechanistic insight in the effect of carbon monoxide (CO)-containing solutions, this work examines the changes in [Ca 2+] i induced by 100 μM adenosine 5′diphosphate (ADP), 0.1 IU/ ml thrombin, 0.5 μM thapsigargin or 0.5 μM ionomycin in human platelets. In a saline solution bubbled with CO, the increase of [Ca 2+] i produced by thrombin was 72±4% of the response evoked in the control solution (CO-free) and the response elicited by ADP was 64±8% of the control. When a mixture of 5% CO/95% N 2 was used, the responses were 70±7% of control for thrombin and 79±6% of control for ADP. The mobilization of stored calcium produced by thrombin in a calcium-free solution and the increase of [Ca 2+] i produced by subsequent introduction of 1 mM extracellular calcium were both reduced in the presence of CO (82±6% and 78±5% of control, respectively). Similar reductions in the presence of CO were found when platelets were stimulated by ADP (62±8% and 60±8% for mobilization in calcium-free media and calcium entry, respectively). Although the change in [Ca 2+] i induced by ionomycin in the presence of extracellular calcium was almost the same in the absence or presence of CO (97±5% of control), the entry induced by depletion of reservoirs with the ionophore undergoes a significant reduction in a solution bubbled with CO (84±5% of control). In agreement with the concept that CO has a direct inhibitory effect on capacitative calcium entry, a reduction to 47±6% of control was obtained when sarco/endoplasmic reticulum ATPase was blocked by thapsigargin. Diverse mechanisms could be responsible for the effect of CO on calcium entry. On the one hand, a decrease in the calcium release from intracellular stores or an increase in the rate of its back-sequestration could occur, being the reduction of capacitative calcium entry an indirect consequence of a diminished emptying of reservoirs. On the other hand, CO could have a direct inhibitory effect on the pathway that produces the calcium entry. The decrease in the Ca 2+ signal in the presence of CO evoked by receptor-independent emptying of reservoirs indicates that a direct effect of CO on capacitative calcium entry participates in the antiaggregatory properties of CO. The proposal that CO inhibits directly store-operated calcium influx widens the potential mechanisms by which heme oxygenase regulates cell functions.

Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets

Redox regulation is important for the modulation of cytosolic Ca 2+ concentration. Hence, we have investigated the effect of H 2O 2 on store-mediated Ca 2+ entry (SMCE). In fura-2-loaded human platelets treatment with H 2O 2 resulted in a concentration-dependent increase in Ca 2+ release from intracellular stores, while the effect on Ca 2+ entry was biphasic. In addition, 1 mM H 2O 2 reduced SMCE induced by agonists. The inhibitory effect of 1 mM H 2O 2 was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10 μM H 2O 2 and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1 mM H 2O 2 shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H 2O 2-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1 mM H 2O 2 significantly increased store depletion-induced p60 src activation. We conclude that H 2O 2 exerted a biphasic effect on SMCE. The inhibitory role of high H 2O 2 concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways.

49 Effects of PH on capacitative calcium entry in human platelets

49 Effects of PH on capacitative calcium entry in human platelets

Role of the ERK Pathway in the Activation of Store-mediated Calcium Entry in Human Platelets

Extracellular signal-regulated kinases (ERKs), are common participants in a broad variety of signal transduction pathways. Several studies have demonstrated the presence of ERKs in human platelets and their activation by the physiological agonist thrombin. Here we report the involvement of the ERK cascade in store-mediated Ca(2+) entry in human platelets. Treatment of dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-loaded platelets with thapsigargin to deplete the intracellular Ca(2+) stores resulted in a time- and concentration-dependent activation of ERK1 and ERK2. Incubation with either U0126 or PD 184352, specific inhibitors of mitogen-activated protein kinase kinase (MEK), prevented thapsigargin-induced ERK activation. Furthermore, U0126 and PD 184352 reduced Ca(2+) entry stimulated by thapsigargin or thrombin, in a concentration-dependent manner. The role of ERK in store-mediated Ca(2+) entry was found to be independent of phosphatidylinositol 3- and 4-kinases, the tyrosine kinase pathway, and actin polymerization but sensitive to treatment with inhibitors of Ras, suggesting that the ERK pathway might be a downstream effector of Ras in mediating store-mediated Ca(2+) entry in human platelets. In addition, we have found that store depletion stimulated ERK activation does not require PKC activity. This study demonstrates for the first time a novel mechanism for regulation of store-mediated Ca(2+) entry in human platelets involving the ERK cascade.

Phosphoinositides Are Required for Store-mediated Calcium Entry in Human Platelets

We have recently observed that small GTP-binding proteins are important for mediation of store-mediated Ca(2+) entry in human platelets through the reorganization of the actin cytoskeleton. Because it has been shown in platelets and other cells that small GTP-binding proteins regulate the activity of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, whose products, phosphoinositides, play a key role in the reorganization of the actin cytoskeleton, we have investigated the role of these lipid kinases in store-mediated Ca(2+) entry. Treatment of platelets with LY294002, an inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinases, resulted in a concentration-dependent inhibition of Ca(2+) entry stimulated by thapsigargin or the physiological agonist, thrombin. In addition, wortmannin, another inhibitor of these kinases, which is structurally unrelated to LY294002, significantly reduced store-mediated Ca(2+) entry. The inhibitory effect of LY294002 was not mediated either by blockage of Ca(2+) channels or by modification of membrane potential. LY294002 inhibited actin polymerization stimulated by thrombin or thapsigargin. These results indicate that both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase are required for activation of store-mediated Ca(2+) entry in human platelets and that the mechanism could involve the reorganization of the actin cytoskeleton.

A Role for the Actin Cytoskeleton in the Initiation and Maintenance of Store-mediated Calcium Entry in Human Platelets: EVIDENCE FOR CONFORMATIONAL COUPLING

The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.

Two pathways for store-mediated calcium entry in human platelets

In human platelets and other non-excitable cell types depletion of the intracellular calcium stores promotes calcium entry across the plasma membrane. Although the mechanism of this store-mediated calcium entry remains uncertain, it has been suggested that a tyrosine phosphorylation step could be involved. In support of this hypothesis various tyrosine kinase inhibitors have been shown to reduce store-mediated calcium entry in platelets, although this inhibition is never complete. Here we investigate the properties of store-mediated calcium entry in human platelets during the time course of its activation. Our data suggest that at least two pathways may contribute to store-mediated calcium entry in these cells. An early component, activated soon after the initiation of Ca 2+ store depletion, is insensitive to trivalent cations, SKF 96365 and tyrosine kinase inhibitors. This is followed by a second component which is inhibited by La 3+ , SKF 96365 and by tyrosine kinase inhibitors. These results suggest a role for tyrosine kinases in generating only the later stages of store-mediated calcium entry in platelets and may explain the incomplete inhibition of this pathway by inhibitors of tyrosine kinases.