Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective

The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and Results

We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84^−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84^−/− mice. In vivo, Cd84^−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion

These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Platelet Activation Determines Angiopoietin-1 and VEGF Levels in Malaria: Implications for Their Use as Biomarkers

Introduction

The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, in vivo platelet activation and inadvertent platelet activation during blood processing. We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7.

Methods

Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls.

Results

Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls.

Conclusion

In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.     

Spatial Distribution of Factor Xa,Thrombin, and Fibrin(ogen) on Thrombi at Venous Shear

Background

The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.

Methodology/Principal Findings

Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca²⁺ signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl₃. Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).

Conclusions/Significance

FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).     

The P2Y12 Antagonists, 2MeSAMP and Cangrelor,Inhibit Platelet Activation through P2Y12/Gi-Dependent Mechanism

Background

ADP is an important physiological agonist that induces integrin activation and platelet aggregation through its receptors P2Y₁ (Gα_q-coupled) and P2Y₁₂ (Gα_i-coupled). P2Y₁₂ plays a critical role in platelet activation and thrombosis. Adenosine-based P2Y₁₂ antagonists, 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2MeSAMP) and Cangrelor (AR-C69931MX) have been widely used to demonstrate the role of P2Y₁₂ in platelet function. Cangrelor is being evaluated in clinical trials of thrombotic diseases. However, a recent study reported that both 2MeSAMP and Cangrelor raise intra-platelet cAMP levels and inhibit platelet aggregation through a P2Y₁₂-independent mechanism.

Methodology/Principal Findings

The present work, using P2Y₁₂ deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and Cangrelor inhibit platelet activation and thrombosis. 2MeSAMP and Cangrelor inhibited aggregation and ATP release of wild-type but not P2Y₁₂ deficient platelets. 2MeSAMP and Cangrelor neither raised intracellular cAMP concentrations nor induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in washed human or mouse platelets. Furthermore, unlike the activators (PGI₂ and forskolin) of the cAMP pathway, 2MeSAMP and Cangrelor failed to inhibit Ca²⁺ mobilization, Akt phosphorylation, and Rap1b activation in P2Y₁₂ deficient platelets. Importantly, while injection of Cangrelor inhibited thrombus formation in a FeCl₃-induced thrombosis model in wild-type mice, it failed to affect thrombus formation in P2Y₁₂ deficient mice.

Conclusions

These data together demonstrate that 2MeSAMP and Cangrelor inhibit platelet function through the P2Y₁₂-dependent mechanism both in vitro and in vivo.     

Platelet Content of Nitric Oxide Synthase 3 Phosphorylated At Serine1177 Is Associated with the Functional Response of Platelets to Aspirin

Objective

To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO).

Patients/Methods

Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100).

Results

ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position −786 (T⁻⁷⁸⁶→C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)¹¹⁷⁷, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser¹¹⁷⁷ phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser¹¹⁷⁷. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser¹¹⁷⁷. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets.

Conclusions

Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser¹¹⁷⁷.     

Aggravation of myocardial dysfunction by injurious mechanical ventilation in LPS-induced pneumonia in rats

Background

Mechanical ventilation (MV) may cause ventilator-induced lung injury (VILI) and may thereby contribute to fatal multiple organ failure. We tested the hypothesis that injurious MV of lipopolysaccharide (LPS) pre-injured lungs induces myocardial inflammation and further dysfunction ex vivo, through calcium (Ca²⁺)-dependent mechanism.

Materials and methods

N = 35 male anesthetized and paralyzed male Wistar rats were randomized to intratracheal instillation of 2 mg/kg LPS or nothing and subsequent MV with lung-protective settings (low tidal volume (V_t) of 6 mL/kg and 5 cmH₂O positive end-expiratory pressure (PEEP)) or injurious ventilation (high V_t of 19 mL/kg and 1 cmH₂O PEEP) for 4 hours. Myocardial function ex vivo was evaluated in a Langendorff setup and Ca²⁺ exposure. Key mediators were determined in lung and heart at the mRNA level.

Results

Instillation of LPS and high V_t MV impaired gas exchange and, particularly when combined, increased pulmonary wet/dry ratio; heat shock protein (HSP)70 mRNA expression also increased by the interaction between LPS and high V_t MV. For the heart, C-X-C motif ligand (CXCL)1 and Toll-like receptor (TLR)2 mRNA expression increased, and ventricular (LV) systolic pressure, LV developed pressure, LV +dP/dt_max and contractile responses to increasing Ca²⁺ exposure ex vivo decreased by LPS. High V_t ventilation aggravated the effects of LPS on myocardial inflammation and dysfunction but not on Ca²⁺ responses.

Conclusions

Injurious MV by high V_t aggravates the effects of intratracheal instillation of LPS on myocardial dysfunction, possibly through enhancing myocardial inflammation via pulmonary release of HSP70 stimulating cardiac TLR2, not involving Ca²⁺ handling and sensitivity.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and Aims

Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested.

Methods

Human washed platelet aggregation and adhesion assays, as well as flow cytometry for α_IIbβ₃ integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte).

Results

BAY 60-2770 (0.001–10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca²⁺ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca²⁺ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced α_IIbβ₃ activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770.

Conclusion

The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca²⁺ levels and α_IIbβ₃ activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.     

Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

Background

It has recently been suggested that RhoA plays an important role in the enhancement of the Ca²⁺ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca²⁺ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.

Methods

Ovalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.

Results

Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K⁺-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca²⁺ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca²⁺ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca²⁺ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.

Conclusion

These findings suggest that the augmentation of Ca²⁺ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.     

Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

Background

Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet.

Results

Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca²⁺ uptake. Ca²⁺-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca²⁺ release from the endoplasmic reticulum via both Ins₃P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca²⁺uptake.

Conclusions

Our data indicate that TG2 might act as a Ca²⁺ sensor to amplify endoplasmic reticulum-derived Ca²⁺ signals to enhance mitochondria Ca²⁺ uptake. Since enhanced mitochondrial Ca²⁺ levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca²⁺ signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca²⁺ homeostasis.     

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

Background

Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca²⁺ concentration, which is a fundamental prerequisite for any Ca²⁺-based signalling system, is accomplished by complex mechanisms including Ca²⁺ binding proteins acting as Ca²⁺ buffers. In this work we investigated the occurrence of Ca²⁺ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results

A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca²⁺-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca²⁺-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca²⁺ binding ability of the M. loti protein was demonstrated by ⁴⁵Ca²⁺-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca²⁺ adducts.

Conclusions

The present data indicate that ferredoxin II is a major Ca²⁺ binding protein in M. loti that may participate in Ca²⁺ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca²⁺ regulatory systems.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.     

Platelets Induce Apoptosis during Sepsis in a Contact-Dependent Manner That Is Inhibited by GPIIb/IIIa Blockade

Purpose

End-organ apoptosis is well-described in progressive sepsis and Multiple Organ Dysfunction Syndrome (MODS), especially where platelets accumulate (e.g. spleen and lung). We previously reported an acute sepsis-induced cytotoxic platelet phenotype expressing serine protease granzyme B. We now aim to define the site(s) of and mechanism(s) by which platelet granzyme B induces end-organ apoptosis in sepsis.

Methods

End-organ apoptosis in murine sepsis (i.e. polymicrobial peritonitis) was analyzed by immunohistochemistry. Platelet cytotoxicity was measured by flow cytometry following 90 minute ex vivo co-incubation with healthy murine splenocytes. Sepsis progression was measured via validated preclinical murine sepsis score.

Measurements and Main Results

There was evident apoptosis in spleen, lung, and kidney sections from septic wild type mice. In contrast, there was a lack of TUNEL staining in spleens and lungs from septic granzyme B null mice and these mice survived longer following induction of sepsis than wild type mice. In co-incubation experiments, physical separation of septic platelets from splenocytes by a semi-permeable membrane reduced splenocyte apoptosis to a rate indistinguishable from negative controls. Chemical separation by the platelet GPIIb/IIIa receptor inhibitor eptifibatide decreased apoptosis by 66.6±10.6% (p = 0.008). Mice treated with eptifibatide in vivo survived longer following induction of sepsis than vehicle control mice.

Conclusions

In sepsis, platelet granzyme B-mediated apoptosis occurs in spleen and lung, and absence of granzyme B slows sepsis progression. This process proceeds in a contact-dependent manner that is inhibited ex vivo and in vivo by the platelet GPIIb/IIIa receptor inhibitor eptifibatide. The GPIIb/IIIa inhibitors and other classes of anti-platelet drugs may be protective in sepsis.     

Aerobic Interval Training Partly Reverse Contractile Dysfunction and Impaired Ca2+ Handling in Atrial Myocytes from Rats with Post Infarction Heart Failure

Background

There is limited knowledge about atrial myocyte Ca²⁺ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca²⁺ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.

Methods and results

Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca²⁺ amplitude, decreased SR Ca²⁺ content, and slower Ca²⁺ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca²⁺ ATPase activity, increased Na⁺/Ca²⁺-exchanger activity and increased diastolic Ca²⁺ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca²⁺ handling in HF.

Conclusion

Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca²⁺ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells

Background

Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca²⁺ homeostasis and tTG activity.

Methods/Principal Findings

We studied Ca²⁺ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31–43 and 57–68 rapidly mobilized Ca²⁺ from intracellular stores. Specifically, peptide 31–43 mobilized Ca²⁺ from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57–68 mobilized Ca²⁺ only from mitochondria. We also found that gliadin peptide-induced Ca²⁺ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31–43, but not peptide 57–68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31–43, but not of peptide 57–68, induces the expression of both genes.

Conclusions

By inducing Ca²⁺ mobilization from the ER, peptide 31–43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31–43 and 57–68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin–tTG crosslinking in enterocytes and specialized antigen-presenting cells.     

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity

Background and Aims

Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca²⁺ and Cl⁻ as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl⁻ and Ca²⁺, but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides.

Methods

Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT–PCR and time-resolved chlorophyll fluorescence were used.

Key Results

Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein.

Conclusions

In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.Key words: Photosystem II, PsbQ, PsbP, halophytes, Salicornia veneta     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

Leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide

Background

Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS).

Methodology/Principal Findings

Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens.

Conclusions/Significance

Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.