Cisplatin induces platelet apoptosis through the ERK signaling pathway

Cisplatin (cis-diamminedichloroplatinum II) is one of the most widely used anti-tumor agents. However, cisplatin-based chemotherapy is usually accompanied by adverse side effects such as thrombocytopenia, and the mechanism remains unclear. Here we show that cisplatin induced several platelet apoptotic events including up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X_L, mitochondrial translocation of Bax, mitochondrial inner transmembrane potential depolarization, caspase-3 activation and phosphatidylserine (PS) exposure. Cisplatin dose-dependently induced activation of extracellular signal-regulated protein kinase (ERK) in platelets. Caspase-3 inhibitor z-DEVD-fmk dramatically inhibited cisplatin-induced caspase-3 activation and PS exposure without affecting ERK activation. Blockade of the ERK pathway significantly prevented platelet apoptosis. Furthermore, levels of reactive oxygen species (ROS) and Ca^2 + were significantly elevated by cisplatin, and scavenging of ROS and Ca^2 + obviously inhibited platelet apoptosis induced by cisplatin. In addition, cisplatin did not induce platelet activation, whereas it obviously impaired platelet functions. These data indicate that cisplatin induces platelet apoptosis through the ERK signaling pathway, which might contribute to cisplatin-related haematological toxicity.     

Altered platelet calsequestrin abundance,Na/Ca exchange and Ca signaling responses with the progression of diabetes mellitus

Introduction

Downregulation of calsequestrin (CSQ), a major Ca^2 + storage protein, may contribute significantly to the hyperactivity of internal Ca^2 + ([Ca^2 +]_i) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca^2 + signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca^2 + influx in diabetic platelets.

Materials and methods

Type 1 diabetes was induced by streptozotocin in rats. Platelet [Ca^2 +]_i and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting.

Results

During the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca^2 +]_i and ADP-induced Ca^2 + release were progressively altered in diabetic platelets, while the elevated Ca^2 + influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca^2 + channel blocker, almost completely abolished ADP-induced Ca^2 + influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na⁺/Ca^2 + exchange induced much slower Ca^2 + extrusion and more Ca^2 + influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca^2 +-ATPase inhibition, ionomycin caused greater Ca^2 + mobilization and Ca^2 + influx in diabetic platelets than in normal platelets.

Conclusions

These data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca^2 + homeostasis, insufficient Na⁺/Ca^2 + exchange also contributes, at least in part, to the hyperactive Ca^2 + response to stimulation in diabetic platelets.     

Calmodulin activity regulates group I metabotropic glutamate receptor‐mediated signal transduction and synaptic depression

Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR 5 (mGluR1/5), are coupled to Gq and modulate activity‐dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation‐dependent long‐term depression (LTD). Although it has been established that intracellular Ca²⁺ and the Gq‐regulated signaling molecules are required for mGluR1/5 LTD, whether and how Ca²⁺ regulates Gq signaling and upregulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of the Ca²⁺ sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor N‐[4‐aminobutyl]‐5‐chloro‐2‐naphthalenesulfonamide hydrochloride (W13) suppressed the mGluR1/5‐stimulated activation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and p70‐S6 kinase 1 (S6K1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist‐induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca²⁺/CaM‐dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Furthermore, disruption of the CaM–CaMK–ERK1/2 signaling cascade suppressed the mGluR1/5‐stimulated upregulation of Arc expression. Altogether, our data suggest CaM as a new Gq signaling component for coupling Ca²⁺ and protein upregulation and regulating mGluR1/5‐mediated synaptic modification. © 2016 Wiley Periodicals, Inc.     

Investigation of the Relocation of Cytosolic Phospholipase A and Annexin V in Activated Platelets

Cytosolic phospholipase A₂ is a Ca²⁺-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A₂. Annexin V is a Ca²⁺-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A₂. Here, we have studied the association of cytosolic phospholipase A₂ and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A₂ and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A₂ to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A₂ or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A₂ remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A₂ with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A₂ can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A₂ copurify annexin V and cytosolic phospholipase A₂ from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A₂ and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A₂ and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A₂ into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

The anti-aggregation effects of ondansetron on platelets involve IP3 signaling and MAP kinase pathway, but not 5-HT3-dependent pathway

Ondansetron is a 5-HT3 receptor antagonist with potent antiemetic, analgesic, and antiphlogistic effects. Literature concerning 5-HT3 antagonists on platelets is limited. In this report we examined the pharmacological effects of ondansetron on human washed platelets. Platelet aggregation induced by thrombin (0.1 U/mL), collagen (2 μg/mL), arachidonic acid (0.5 mM), ADP (10 μM), or U46619 (2 μM) was observed. The effects of ondansetron on platelet aggregation and ATP release were investigated at different concentrations. Cytosolic Ca²⁺ influx concentration, TXB2, IP3, and the levels of cAMP and cGMP were monitored, and flow cytometric analysis and immunoblotting were performed to investigate downstream signaling components. Our results showed that ondansetron, in a concentration-dependent manner, inhibited agonist-induced platelet aggregation. At 75 μM, ondansetron significantly attenuated intracellular Ca²⁺ mobilization, thromboxane B2 formation, and ATP release by human washed platelets activated by thrombin, collagen, or U46619, whereas it only partially attenuated arachidonic acid-driven platelet activation. Administration of ondansetron resulted in attenuated IP3 production in the washed platelets stimulated by thrombin, as determined by reduced IP1 levels, as well as diminished p38 and ERK2 phosphorylation in response to thrombin. No effect of ondansetron on the levels of either cAMP or cGMP in washed platelets was observed. Furthermore, ondansetron-mediated inhibition of platelet aggregation was not impacted by SR 57227A, the 5-HT3 agonist. Thus, rather than involving the 5-HT3-dependent pathway, the negative effect of ondansetron on platelet aggregation is instead manifested through the attenuation of agonist-induced IP3 production and MAPK (p38 and ERK2) phosphorylation that results in suppressed intracellular Ca²⁺ mobilization, TXB2 formation, and ATP release.     

Effects of peroxisome proliferator-activated receptor γ in simvastatin antiplatelet activity: Influences on cAMP and mitogen-activated protein kinases

Statins are widely used as hypolipidemic drugs, and have beneficial effects in reducing cardiovascular events. In addition, recent studies on the pleiotropic effects of statins (i.e., simvastatin) reveal that these drugs have many additional anti-atherogenic effects, including antiplatelet activity. The mechanisms may be partly related to activation of peroxisome proliferator-activated receptors (PPARs), which are present in human platelets, and whose activation inhibits platelet aggregation. However, the details of the signaling pathway by which simvastatin inhibits platelet activation via PPARs have not yet been completely established. The aim of this study was to examine the mechanisms by which the PPAR-mediated pathways contribute to the antiplatelet activity of simvastatin. Simvastatin (3-50 μM) induced PPARα and PPARγ activation in a dose-dependent manner in washed platelets. Additionally, simvastatin inhibited collagen-induced platelet aggregation, expression of CD62 and PAC-1, and Ca^2 + mobilization. These effects of simvastatin on platelet responses were strongly reduced by adding a selective PPARγ antagonist (GW9662), but not PPARα antagonist (GW6471). Moreover, in the presence of GW9662, simvastatin-mediated increase of cyclic adenosine monophosphate (cAMP) production, vasodilator-stimulated phosphoprotein (VASP) Ser¹⁵⁷ phosphorylation and inhibition of Akt phosphorylation were markedly reversed. Furthermore, simvastatin was found to inhibit phosphorylation of mitogen-activated protein kinases (MAPKs, i.e., p38 MAPK, ERK) by increasing the association between PPARγ and the components of MAPKs after platelet activation. Taken together, the present results confirm that simvastatin inhibition of platelet activation is mediated by PPARγ-dependent processes, which involves mediating MAPKs signaling, increase of cAMP formation and VASP Ser¹⁵⁷ phosphorylation, inhibition of Akt phosphorylation and intracellular Ca^2 + mobilization.     

Blockade of glycoprotein IIb/IIIa by crotavirin, a member of disintegrins, prevents platelet from activation and aggregation by Staphylococcus aureus bacteria

Interaction with circulating platelets is considered an important virulent mechanism for Staphylococcus aureus (S. aureus) bacteria to induce endocarditis, a severe infectious disease with high incidence of systemic thrombosis. It therefore represents an important target for pharmacological intervention. In this study, we found that the clinical isolate S. aureus 30326 induced activation and aggregation of washed human platelets in a fibrinogen-dependent manner and this platelet reactivity was abrogated by crotavirin, a snake venom-derived glycoprotein (GP) IIb/IIIa antagonist, indicating that crotavirin is able to protect platelets from activation and aggregation by S. aureus 30326. When tested at a concentration that prevented the platelet reactivity of S. aureus 30326, crotavirin also interfered with the binding of bacteria to washed human platelets supplemented with fibrinogen. The fibrinogen-binding activity of S. aureus has been shown to be essential for S. aureus to trigger platelet activation and aggregation. Crotavirin failed to affect the fibrinogen binding of S. aureus 30326 and neither did it bind to this microbe, suggesting that the inhibitory action of crotavirin on the S. aureus 30326-platelet interaction resulted from the occupation of platelet GPIIb/IIIa. Taken together, these results demonstrate an important role for GPIIb/IIIa in mediating the interaction of platelets with S. aureus in the presence of fibrinogen and platelet GPIIb/IIIa thus appears to be a new target for the intervention of S. aureus-platelet interaction.     

Effect of chronic sleep restriction and aging on calcium signaling and apoptosis in the hippocampus of young and aged animals

Aging leads to progressive deterioration of physiological function and diminished responses to environmental stress. Organic and functional alterations are frequently observed in elderly subjects. Although chronic sleep loss is observed during senescence, little is known about the impact of insufficient sleep on cellular function in aging neurons. Disruption of neuronal calcium (Ca^2 +) signaling is related to impaired neuronal function and cell death. It has been hypothesized that sleep deprivation may compromise neuronal stability and induce cell death in young neurons; however, it is necessary to evaluate the impact of aging on this process. Therefore, the aim of this study was to evaluate the effects of chronic sleep restriction (CSR) on Ca^2 + signaling and cell death in the hippocampus of young and aged animals. We found that glutamate and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced a greater elevation in cytosolic Ca^2 + ([Ca^2 +]_c) in hippocampal slices from aged rats subjected to CSR compared to age-matched controls. Interestingly, aged-matched controls showed a reduced Ca^2 + response to glutamate and FCCP, relative to both CSR and control young animals. Apoptotic nuclei were observed in aged rats from both treatment groups; however, the profile of apoptotic nuclei in aged CSR rats was highly variable. Bax and Bcl-2 protein expression did not change with aging in the CSR groups. Our study indicates that aging promotes changes in Ca^2 + signaling, which may also be affected by CSR. These age-dependent changes in Ca^2 + signaling may increase cellular vulnerability during CSR and contribute to Ca^2 + signaling dysregulation, which may ultimately induce cell death.     

I(4), a new synthetic sulfonylurea compound, inhibits the action of TXA(2)in vivo and in vitro on platelets and aorta vascular smooth muscle

Introduction

1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl]phenylsulfonyl]-3-(trans-4-methylcy-clohexyl)urea(I₄, CAS865483-06-3); a totally synthetic new sulfonylurea compound, combining the hypoglycemic active structure of Glimepiride (CAS 93479-97-1) and anti-TXA₂ receptor (TP) active structure of BM-531(CAS 284464-46-6), was designed and synthesized. Its effects on TXA₂ synthesis and TP have not been reported yet.

Aim

To study the inhibitory effects of I₄ and its mechanisms of action on TXA₂ and TP.

Methods

Platelet aggregation studies were performed on human platelet, rat whole blood platelet and rabbit platelet, platelets aggregation was induced by TP agonist U-46619(stable analog of TXA₂, CAS 56985-40-1). Plasma TXB₂ and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were used as markers to determine the effect of I₄ on thromboxane synthesis. Fluo-3-AM was used to measure the cytosolic Ca^2 + concentrations ([Ca^2 +]_i) in rabbit platelet. Aorta rings with and without endothelium were prepared and aorta contraction was induced by U-46619. A model of type 2 diabetes mellitus was established by intraperitoneal injection of low dose of streptozocin to rats fed a high-calorie diet. Both normal rats and type 2 diabetic rats were used to assay the inhibitory effect of I₄ on platelet aggregation induced by U-46619.

Results

I₄ exhibited a higher inhibitory potency than Glimepiride on U-46619 induced platelet aggregation in vitro and in vivo. I₄ increased the ratio of plasma PGI₂/TXA₂ and decreased [Ca^2 +]_i release from platelet internal stores. In addition, I₄ presented a vasorelaxant activity on isolated rat aorta contraction induced by U-46619.Oral administration of I₄ (1 ~ 10 mg/kg) markedly and dose-dependently inhibited platelet aggregation in both normal rats and type 2 diabetic rats.

Conclusion

I₄ significantly inhibited platelet aggregation induced by U-46619 in vitro and in vivo, and rat aorta contraction. It probably acts by partly blocking TXA₂ action, decreasing the platelet intracellular Ca^2 +, and increasing the PGI₂/TXA₂ ratio.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

MC-002 exhibits positive effects against platelets aggregation and endothelial dysfunction through thromboxane A2 inhibition

Introduction

Thromboxane A₂ (TXA₂) induces platelet aggregation and vasoconstriction, and agents that inhibit TXA₂ production or interaction with receptors may exert potential application in stroke therapy.

Aim

To illustrate the platelet aggregation antagonistic and endothelial protective effect of (E) - 3 - (3 - methoxy - 4 - ((3, 5, 6 - trimethylpyrazin - 2 - yl) methoxy) phenyl) sodium acrylate (MC-002) through TXA₂ inhibition and underline mechanisms.

Materials and methods

Platelets aggregation and thoracic aorta ring contraction of rabbits were induced by U46619. Human umbilical vein endothelial cells (HUVECs) were further applied to explore the protective effect of MC-002 on endothelium when exposed to tumor necrosis factor - α (TNF-α). MTT method was used to assess cell damage, and ELISA analysis was exerted to estimate nitrogen monoxide (NO), endothelin-1 (ET-1), thromboxane B₂ (TXB₂) and 6-keto-prostaglandin F1α (6-keto-PGF1α) releasing. Fluorescence spectrophotometry was conducted to determine intracellular calcium concentration ([Ca^2 +]_i), and western blotting method was applied to evaluate the protein expressions of intracellular adhesion molecule-1 (ICAM-1), P-selectin and nuclear factor-kappa B (NF-κB).

Results and conclusions

TXA₂ analog U46619 mediated obvious platelet aggregation and vasoconstriction. MC-002 inhibited platelet aggregation through administration in vivo and incubation with platelet in vitro, and relaxed aorta ring in endothelium dependent manner. MC-002 alleviated cell damage, [Ca^2 +]_i overload, ET-1 overexcretion and TXB₂ activation, but improved NO availability reduction in HUVECs treated with TNF-α. Furthermore, MC-002 downregulated ICAM-1, P-selectin and NF-κB overexpression induced by TNF-α. In conclusion, MC-002 exerted antiplatelet aggregation effect through TXA₂ inhibition and relieved inflammatory injury of endothelial cells through NF-κB signal pathway.     

Possible involvement of calmodulin-dependent kinases in Ca-dependent enhancement of [H]5-hydroxytryptamine uptake in rat cortex

Effects of Ca²⁺ on [³H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca²⁺ in Krebs-Ringer medium and the uptake at 0.3–5 mM Ca²⁺ was 2.4–2.7 times greater than that observed in the absence of Ca²⁺. The maximal increase at the concentration of 1 mM Ca²⁺ was achieved after 2 min preincubation. Ca²⁺-dependent enhancement of the [³H]5-HT uptake reflected an increase in V_max of the uptake process. However, K_d and B_max values for [³H]paroxetine were not significantly changed in the presence of 1 mM Ca²⁺ compared with Ca²⁺-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca²⁺-free medium after preincubation with 1 mM Ca²⁺. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca²⁺-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-

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-tyrosyl]-4-phenylpiperazine (KN-62, inhibitor of Ca²⁺/calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca²⁺-channel blockers suppressed enhancement of the uptake. These results indicate that Ca²⁺-dependent enhancement of [³H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake.     

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation

The effect of prostaglandin E₁ (PGE₁) on platelets is mediated through the PGE₁ receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE₁ and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 gm. The time course of the extension length (Dp, in μg) of the platelets in response to aspiration with a negative pressure (ΔP) of 5 cm H₂ O (ΔP × Rp = 0.15 dynes/cm) was analyzed. PGE₁ treatment (0.1 μM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE₁ -treated) / Dp/Rp (control), was significantly reduced to 0.90 ± 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE₁ -treated platelets had a higher [cAMP]_i than controls. Platelets treated with PGE₁ or dbcAMP showed a reduced [Ca²⁺]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE₁ and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]_i and low [Ca²⁺]_i after PGE₁ treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.     

In vitro and ex vivo autoradiography of the NK-1 antagonist [³H]-LY686017 in Guinea pig brain

NK-1 receptor antagonists have shown potential for the clinical treatment of chemotherapy-induced nausea and vomiting, depression and alcoholism. In a recent study, we disclosed the potential for the NK-1 antagonist, LY686017, to treat alcoholism in a clinical population. To assess whether this compound could be utilized as a platform for a brain imaging ligand, we evaluated the binding of [³H]-LY686017 to sections of guinea pig in vitro. In these studies, [³H]-LY686017 bound with a distribution and pharmacology consistent with the NK-1 receptor. Using sections through the region of the caudate nucleus, we obtained a K_d of 0.34 nM and a B_max of 31.37 fmoles/mg tissue. Based on its high potency and low nonspecific binding in vitro, we initiated studies to evaluate the radioligand as a tool to measure in vivo receptor occupancy. In initial studies, 25 microCi of [³H]-LY686017 was administered via an indwelling jugular catheter and accumulation of radioactivity in the caudate (NK-1 containing tissue) and cerebellum (low NK-1 expression) were assessed. The ratios of caudate to cerebellum radioactivity were optimal 2 h after radioligand administration so this time point was used for subsequent studies. To assess the pharmacological specificity of the radioactivity accumulation, we administered various doses of Aprepitant, a potent NK-1 antagonists 1 h prior to intravenous administration of [³H]-LY686017. Aprepitant produced a dose-dependent reduction in radioactivity in the caudate with an approximate 70% reduction at 10 mg/kg. To image NK-1 receptors, 100 microCi of [³H]-LY686017 was administered and the brains sectioned for autoradiography. In these studies, a characteristic distribution on NK-1 receptors was observed. Based on these results, LY686017 should serve as a suitable chemical platform for future imaging ligand development.     

Apoptosis induced by Aβ25–35 peptide is Ca2+‐IP3 signaling‐dependent in murine astrocytes

Although the accumulation of the neurotoxic peptide β‐amyloid (Aβ) in the central nervous system is a hallmark of Alzheimer's disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca²⁺ dysregulation, induced by a neurotoxic fragment (Aβ25–35), caused apoptosis in a concentration‐dependent manner, leading to cytoplasmic Ca²⁺ mobilization from extra‐ and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP₃ receptor activation. This mechanism was related to Aβ‐mediated apoptosis by the intrinsic pathway because the expression of pro‐apoptotic Bax was accompanied by its translocation in cells transfected with GFP‐Bax. Aβ‐mediated apoptosis was reduced by BAPTA‐AM, a fast Ca²⁺ chelator, indicating that an increase in intracellular Ca²⁺ was involved in cell death. Interestingly, the Bax translocation was dependent on Ca²⁺ mobilization from IP₃ receptors because pre‐incubation with xestospongin C, a selective IP₃ receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca²⁺ homeostasis induces ER depletion of Ca²⁺ stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments.     

Mitochondrial division inhibitor 1 disrupts oligodendrocyte Ca2+ homeostasis and mitochondrial function

Mitochondrial fission mediated by cytosolic dynamin related protein 1 (Drp1) is essential for mitochondrial quality control but may contribute to apoptosis as well. Blockade of Drp1 with mitochondrial division inhibitor 1 (mdivi-1) provides neuroprotection in several models of neurodegeneration and cerebral ischemia and has emerged as a promising therapeutic drug. In oligodendrocytes, overactivation of AMPA-type ionotropic glutamate receptors (AMPARs) induces intracellular Ca²⁺ overload and excitotoxic death that contributes to demyelinating diseases. Mitochondria are key to Ca²⁺ homeostasis, however it is unclear how it is disrupted during oligodendroglial excitotoxicity. In the current study, we have analyzed mitochondrial dynamics during AMPAR activation and the effects of mdivi-1 on excitotoxicity in optic nerve-derived oligodendrocytes. Sublethal AMPAR activation triggered Drp1-dependent mitochondrial fission, whereas toxic AMPAR activation produced Drp1-independent mitochondrial swelling. Accordingly, mdivi-1 efficiently inhibited Drp1-mediated mitochondrial fission and did not prevent oligodendrocyte excitotoxicity. Unexpectedly, mdivi-1 also induced mitochondrial depolarization, ER Ca²⁺ depletion and modulation of AMPA-induced Ca²⁺ signaling. These off-target effects of mdivi-1 sensitized oligodendrocytes to excitotoxicity and ER stress and eventually produced oxidative stress and apoptosis. Interestingly, in cultured astrocytes mdivi-1 induced nondetrimental mitochondrial depolarization and oxidative stress that did not cause toxicity or sensitization to apoptotic stimuli. In summary, our results provide evidence of Drp1-mediated mitochondrial fission during activation of ionotropic glutamate receptors in oligodendrocytes, and uncover a deleterious and Drp1-independent effect of mdivi-1 on mitochondrial and ER function in these cells. These off-target effects of mdivi-1 limit its therapeutic potential and should be taken into account in clinical studies.     

Elevated platelet angiostatin and circulating endothelial microfragments in idiopathic pulmonary arterial hypertension: A preliminary study

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by pulmonary arteriolar narrowing and degeneration associated with in situ thrombosis. We hypothesized that microvascular endothelial injury and apoptosis may be an initiating mechanism in IPAH. Endothelial apoptosis generates endothelial microfragments (EMF), which can activate platelets. Platelets release both VEGF and angiostatin, which depending the balance can inhibit or induce endothelial apoptosis, respectively.

Materials and Methods

We measured EMFs from blood of IPAH patients as index of endothelial cell apoptosis/injury and levels of pro- and anti- EC apoptotic factors found in platelets. EMFs and platelets in blood samples from control subjects and patients with IPAH were measured using a 4-color flow cytometry protocol, and platelet levels of VEGF and angiostatin were determined by ELISAs and immunoblotting.

Results

Compared to controls, IPAH patients exhibited higher numbers of circulating EMFs and more activated/apoptotic platelets. IPAH patients also exhibited higher levels of platelet angiostatin; however, no significant difference was detected in platelet VEGF levels between the two groups.

Conclusions

These results are consistent with an increase in EC dysfunction in patients with IPAH, possibly contributing to the progression of IPAH and its associated thrombosis.     

The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca signaling pathways

Introduction

Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated.

Methods

The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry.

Results

PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca^2 + mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca^2 + chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it.

Conclusion

The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca^2 + signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.