CaV ß controls the endocytic turnover of CaV 1.2 L-type calcium channel.

Membrane depolarization activates the multisubunit CaV 1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV 1.2-mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV 1.2α1 pore-forming and auxiliary CaV ß subunits and analyzed their trafficking dynamics from single-particle-tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest-mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaV ß to CaV 1.2α1 suggesting that high-speed transport occurs in association with CaV ß. Selective tracking of CaV 1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaV ß-independent retrograde transport. Retrograde speeds of CaV 1.2α1 are compatible with myosin VI-mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV 1.2α1 was covalently linked to CaV ß. Thus, CaV ß promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L-type calcium channels.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Direct interaction of CaVß with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes.

Expression of the ß-subunit (CaVß) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVß binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVß encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVß and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVß2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVß2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVß mediated L-type up-regulation, and CaVß-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVß that is directly involved in vesicular trafficking. We propose a model in which CaVß promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane.

The N-terminal domain tethers the voltage-gated calcium channel ß2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions.

The ß-subunit associates with the α1 pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four ß-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, ß2a and ß2e, associate with the plasma membrane in the absence of the α1-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of ß2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which ß2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in ß2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic ß1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type ß2e, but not with a ß2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the ß2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the ß-subunit family that also sustains slowed inactivation.

Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

BACKGROUND: An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. METHODS: With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. RESULTS: The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. CONCLUSIONS: We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Atorvastatin reduces the proadhesive and prothrombotic endothelial cell phenotype induced by cocaine and plasma from cocaine consumers in vitro.

OBJECTIVE: Cocaine consumption is a risk factor for vascular ischemic complications. Although endothelial dysfunction and accelerated atherosclerosis have been observed in cocaine consumers, the mechanisms underlying their pathogenesis are not fully understood. This study aimed at identifying the effects of atorvastatin in relation to a proadhesive and prothrombotic phenotype induced by cocaine and plasma from chronic cocaine users on endothelial cells. APPROACH AND RESULTS: Human umbilical vein endothelial cells were exposed to either cocaine or platelet-free plasma (PFP) from chronic cocaine consumers in the presence or absence of 10 µmol/L of atorvastatin. Atorvastatin significantly reduced the enhanced platelet adhesion that was induced by cocaine and PFP from chronic cocaine consumers, as well as the release of the von Willebrand factor. Atorvastatin also avoided striking alterations on cell monolayer structure triggered by both stimuli and enhanced NO reduction because of cocaine stimulation through disrupting interactions between endothelial nitric oxide synthase (eNOS) and caveolin-1, thus increasing eNOS bioavailability. Cocaine-increased tissue factor-dependent procoagulant activity and reactive oxygen species generation were not counteracted by atorvastatin. Although monocyte chemoattractant protein-1 levels were not significantly higher than controls either under cocaine or PFP stimulation, atorvastatin completely avoided monocyte chemoattractant protein-1 release in both conditions. Platelets stimulated with cocaine or PFP did not express P-selectin, glycoprotein IIb/IIIa, or CD40L and failed to adhere to resting human umbilical vein endothelial cell. CONCLUSIONS: Cocaine and patient plasma equally induced a proadhesive and prothrombotic phenotype in endothelial cells, except for von Willebrand Factor release, which was only induced by PFP from chronic cocaine consumers. Atorvastatin improved endothelial cell function by reducing cocaine-induced and PFP from chronic cocaine consumer-induced effects on platelet adhesion, cell architecture, and NO production.

An orally available Cav2.2 calcium channel inhibitor for the treatment of neuropathic pain.

BACKGROUND AND PURPOSE: Neuropathic pain affects up to 10% of the global population and is caused by an injury or a disease affecting the somatosensory, peripheral, or central nervous system. NP is characterized by chronic, severe and opioid-resistant properties. Therefore, its clinical management remains very challenging. The N-type voltage-gated calcium channel, Cav2.2, is a validated target for therapeutic intervention in chronic and neuropathic pain. The conotoxin ziconotide (Prialt®) is an FDA-approved drug that blocks Cav2.2 channel but needs to be administered intrathecally. Thus, although being principally efficient, the required application route is very much in disfavour. EXPERIMENTAL APPROACH AND KEY RESULTS: Here, we describe an orally available drug candidate, RD2, which competes with ziconotide binding to Cav2.2 at nanomolar concentrations and inhibits Cav2.2 almost completely reversible. Other voltage-gated calcium channel subtypes, like Cav1.2 and Cav3.2, were affected by RD2 only at concentrations higher than 10 µM. Data from sciatic inflammatory neuritis rat model demonstrated the in vivo proof of concept, as low-dose RD2 (5 mg·kg-1) administered orally alleviated neuropathic pain compared with vehicle controls. High-dose RD2 (50 mg·kg-1) was necessary to reduce pain sensation in acute thermal response assessed by the tail flick test. CONCLUSIONS AND IMPLICATIONS: Taken together, these results demonstrate that RD2 has antiallodynic properties. RD2 is orally available, which is the most convenient application form for patients and caregivers. The surprising and novel result from standard receptor screens opens the room for further optimization into new promising drug candidates, which address an unmet medical need.

A short polybasic segment between the two conserved domains of the ß2a-subunit modulates the rate of inactivation of R-type calcium channel.

Besides opening and closing, high voltage-activated calcium channels transit to a nonconducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of a deleterious rise in calcium concentration. R-type high voltage-activated channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular ß-subunit isoform, ß(2a), inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N terminus anchors ß(2a) to the plasma membrane. The current view is that membrane-anchored ß(2a) immobilizes the channel inactivation machinery and confers slow inactivation phenotype. ß-Subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here, we identified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient ß(2a) to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slow inactivation-conferring phenotype. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the ß(2a)-subunit that might involve electrostatic interactions with an unknown target on the channel's inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the ß(2a)-subunit.

Homodimerization of the Src homology 3 domain of the calcium channel ß-subunit drives dynamin-dependent endocytosis.

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory ß-subunit (Ca(V)ß), a membrane associated guanylate kinase containing one guanylate kinase (ß-GK) and one Src homology 3 (ß-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although ß-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, ß-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that ß-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the ß-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)ß form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)ß with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that ß-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)ß. We propose that a change in the oligomeric state of Ca(V)ß is the functional switch between channel activator and channel internalizer.

The guanylate kinase domain of the beta-subunit of voltage-gated calcium channels suffices to modulate gating.

Inactivation of voltage-gated calcium channels is crucial for the spatiotemporal coordination of calcium signals and prevention of toxic calcium buildup. Only one member of the highly conserved family of calcium channel beta-subunits--Ca(V)beta--inhibits inactivation. This unique property has been attributed to short variable regions of the protein; however, here we report that this inhibition actually is conferred by a conserved guanylate kinase (GK) domain and, moreover, that this domain alone recapitulates Ca(V)beta-mediated modulation of channel activation. We expressed and refolded the GK domain of Ca(V)beta(2a), the unique variant that inhibits inactivation, and of Ca(V)beta(1b), an isoform that facilitates it. The refolded domains of both Ca(V)beta variants were found to inhibit inactivation of Ca(V)2.3 channels expressed in Xenopus laevis oocytes. These findings suggest that the GK domain endows calcium channels with a brake restraining voltage-dependent inactivation, and thus facilitation of inactivation by full-length Ca(V)beta requires additional structural determinants to antagonize the GK effect. We found that Ca(V)beta can switch the inactivation phenotype conferred to Ca(V)2.3 from slow to fast after posttranslational modifications during channel biogenesis. Our findings provide a framework within which to understand the modulation of inactivation and a new functional map of Ca(V)beta in which the GK domain regulates channel gating and the other conserved domain (Src homology 3) may couple calcium channels to other signaling pathways.

A Tripartite Interaction Among the Calcium Channel α1- and ß-Subunits and F-Actin Increases the Readily Releasable Pool of Vesicles and Its Recovery After Depletion.

Neurotransmitter release is initiated by the influx of Ca2+ via voltage-gated calcium channels. The accessory ß-subunit (CaVß) of these channels shapes synaptic transmission by associating with the pore-forming subunit (CaVα1) and up-regulating presynaptic calcium currents. Besides CaVα1, CaVß interacts with several partners including actin filaments (F-actin). These filaments are known to associate with synaptic vesicles (SVs) at the presynaptic terminals and support their translocation within different pools, but the role of CaVß/F-actin association on synaptic transmission has not yet been explored. We here study how CaVß4, the major calcium channel ß isoform in mamalian brain, modifies synaptic transmission in concert with F-actin in cultured hippocampal neurons. We analyzed the effect of exogenous CaVß4 before and after pharmacological disruption of the actin cytoskeleton and dissected calcium channel-dependent and -independent functions by comparing the effects of the wild-type subunit with the one bearing a double mutation that impairs binding to CaVα1. We found that exogenously expressed wild-type CaVß4 enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. CaVß4 increases the size of the readily releasable pool (RRP) of SVs at resting conditions and accelerates their recovery after depletion. The enhanced neurotransmitter release induced by CaVß4 is abolished upon disruption of the actin cytoskeleton. The CaVα1 association-deficient CaVß4 mutant associates with actin filaments, but neither alters postsynaptic responses nor the time course of the RRP recovery. Furthermore, this mutant protein preserves the ability to increase the RRP size. These results indicate that the interplay between CaVß4 and F-actin also support the recruitment of SVs to the RRP in a CaVα1-independent manner. Our studies show an emerging role of CaVß in determining SV maturation toward the priming state and its replenishment after release. We envision that this subunit plays a role in coupling exocytosis to endocytosis during the vesicle cycle.

High Incidence of Sleep-Related Breathing Disorders in Children with Down Syndrome Referred to a High-Altitude Sleep Laboratory.

Aims: The aim of the study was to assess the incidence of sleep-related breathing disorders (SRBD) in children with Down Syndrome (DS) living at high altitude. Methods: A retrospective descriptive study was conducted on 53 children with DS who underwent polysomnography (PSG) at San Ignacio University Hospital (2640 m/8660 ft above sea level) from 2009 to 2016. Data were extracted from official PSG reports and analyzed using measures of central tendency and dispersion, frequency calculation, ranges, and confidence intervals. Associations were examined using t-test, chi-square test, and analysis of variance test. Results: Obstructive sleep apnea (OSA) was present in 90.5% of children. Central sleep apnea was evident in 11.3%. Periodic breathing was seen in 15.1% of patients. Snoring was able to predict OSA with a sensitivity of 61.7%, a specificity of 100%, and negative predictive value of 25%. Conclusion: Children with DS who live at high altitude have a high incidence of SRBD. Our findings show a higher incidence of SRBD than previously reported in the population with DS. Furthermore, snoring was not sensitive enough to predict OSA. This high risk of SRBD may increase the risk of other comorbid conditions seen in the population with DS. Our results support the need for routine PSG screening independent of symptoms such as snoring status.

Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains.

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

Rapid Turnover of the Cardiac L-Type CaV1.2 Channel by Endocytic Recycling Regulates Its Cell Surface Availability.

Calcium entry through CaV1.2 L-type calcium channels regulates cardiac contractility. Here, we study the impact of exocytic and post-endocytic trafficking on cell surface channel abundance in cardiomyocytes. Single-molecule localization and confocal microscopy reveal an intracellular CaV1.2 pool tightly associated with microtubules from the perinuclear region to the cell periphery, and with actin filaments at the cell cortex. Channels newly inserted into the plasma membrane become internalized with an average time constant of 7.5 min and are sorted out to the Rab11a-recycling compartment. CaV1.2 recycling suffices for maintaining stable L-type current amplitudes over 20 hr independent of de novo channel transport along microtubules. Disruption of the actin cytoskeleton re-routes CaV1.2 from recycling toward lysosomal degradation. We identify endocytic recycling as essential for the homeostatic regulation of voltage-dependent calcium influx into cardiomyocytes. This mechanism provides the basis for a dynamic adjustment of the channel's surface availability and thus, of heart's contraction.

Multiplicity of protein interactions and functions of the voltage-gated calcium channel beta-subunit.

For a long time the auxiliary beta-subunit of voltage-gated calcium channels was thought to be engaged exclusively in the regulation of calcium channel function, including gating, intracellular trafficking, assembly and membrane expression. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins (MAGUK) that comprises a series of protein interaction motifs. Two such domains, a Src homology 3 and a guanylate kinase domain are present in the beta-subunit. Recently, it was shown that this subunit interacts with a variety of proteins and regulates diverse cellular processes ranging from gene expression to hormone secretion and endocytosis. In light of these new findings, the beta-subunit deserves to be promoted to the category of multifunctional regulatory protein. Some of these new functions support a tighter regulation of calcium influx through voltage-gated calcium channels and others apparently serve channel unrelated processes. Here we discuss a variety of protein-protein interactions held by the beta-subunit of voltage-gated calcium channels and their functional consequences. Certainly the identification of additional binding partners and effector pathways will help to understand how the different beta-subunit-mediated processes are interwoven.

The S4---S5 linker - gearbox of TRP channel gating.

Transient receptor potential (TRP) channels are cation channels which participate in a wide variety of physiological processes in organisms ranging from fungi to humans. They fulfill roles in body homeostasis, are sensors for noxious chemicals and temperature in the mammalian somatosensory system and are activated by light stimulated phospholipase C activity in Drosophila or by hypertonicity in yeast. The transmembrane topology of TRP channels is similar to that of voltage-gated cation channels. TRP proteins assemble as tetramers with each subunit containing six transmembrane helices (S1-S6) and intracellular N- and C-termini. Here we focus on the emerging functions of the cytosolic S4-S5 linker on TRP channel gating. Most of this knowledge comes from pathogenic mutations within the S4-S5 linker that alter TRP channel activities. This knowledge has stimulated forward genetic approaches to identify additional residues around this region which are essential for channel gating and is supported, in part, by recent structures obtained for TRPV1, TRPV2, TRPV6, TRPA1, and TRPP2.

Platelet tissue factor activity and membrane cholesterol are increased in hypercholesterolemia and normalized by rosuvastatin, but not by atorvastatin.

BACKGROUND AND AIMS: High plasma LDL-cholesterol (LDL-C) and platelet responses have major pathogenic roles in atherothrombosis. Thus, statins and anti-platelet drugs constitute mainstays in cardiovascular prevention/treatment. However, the role of platelet tissue factor-dependent procoagulant activity (TF-PCA) has remained unexplored in hypercholesterolemia. We aimed to study platelet TF-PCA and its relationship with membrane cholesterol in vitro and in 45 hypercholesterolemic patients (HC-patients) (LDL-C >3.37 mmol/L, 130 mg/dL) and 37 control subjects (LDL-C <3.37 mmol/L). The effect of 1-month administration of 80 mg/day atorvastatin (n = 21) and 20 mg/day rosuvastatin (n = 24) was compared. METHODS: Platelet TF-PCA was induced by GPIbα activation with VWF-ristocetin. RESULTS: Cholesterol-enriched platelets in vitro had augmented aggregation/secretion and platelet FXa generation (1.65-fold increase, p = 0.01). HC-patients had 1.5-, 2.3- and 2.5-fold increases in platelet cholesterol, TF protein and activity, respectively; their platelets had neither hyper-aggregation nor endogenous thrombin generation (ETP). Rosuvastatin, but not atorvastatin, normalized platelet cholesterol, TF protein and FXa generation. It also increased slightly the plasma HDL-C levels, which correlated negatively with TF-PCA. CONCLUSIONS: Platelets from HC-patients were not hyper-responsive to low concentrations of classical agonists and had normal PRP-ETP, before and after statin administration. However, washed platelets from HC-patients had increased membrane cholesterol, TF protein and TF-PCA. The platelet TF-dependent PCA was specifically expressed after VWF-induced GPIbα activation. Rosuvastatin, but not atorvastatin treatment, normalized the membrane cholesterol, TF protein and TF-PCA in HC-patients, possibly unveiling a new pleiotropic effect of rosuvastatin. Modulation of platelet TF-PCA may become a novel target to prevent/treat atherothrombosis without increasing bleeding risks.

Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Association of Cardiovascular Disease and Sleep Apnea at Different Altitudes.

Otero, Liliana, Patricia Hidalgo, Rafael González, and Carlos A. Morillo. Association of cardiovascular disease and sleep apnea at different altitudes. High Alt Med Biol. 17:336-341, 2016.-We evaluated the prevalence of sleep apnea (SA) in patients with cardiovascular disease (CVD) at different altitudes. A total of 398 subjects with coronary artery disease (CAD), 144 subjects with atrial fibrillation (AF), and 292 controls (without CVD) were recruited in three cities at sea level, moderate altitude, and high altitude. All participants underwent polysomnography. Multinomial logistic regression, X2, and Hosmer and Lemeshow tests were used to determine interactions among CVD, SA, and altitude. Men and women with CVD at high altitude had a higher risk for SA than men and women living at lower altitudes. The highest risk of SA was observed in men with AF and men with CAD living at high altitude. Obstructive SA (OSA) prevalence was significantly increased in CVD subjects living at high altitude (OR: 5.52; p < 0.0001). Central SA (CSA) was more frequent in subjects with CVD than control group (OR: 2.44; p < 0.021). OSA was the most frequent type of SA in subjects with CVD and overweight subjects, and in control individuals with obesity or being overweight. Significant differences in the prevalence of SA associated with altitude and gender were noted in subjects with CAD and AF.

[Phenotypic and genotypic diagnosis of bone and miliary tuberculosis in an HIV+ patient in Bogotá, Colombia]. / Diagnóstico genotípico y fenotípico de tuberculosis ósea y miliar en un paciente positivo para HIV en Bogotá, Colombia.

Tuberculosis is the single most frequent cause of death by an infectious agent worldwide. Diagnosis of extra-pulmonary tuberculosis is not always possible through conventional methods, due to the long time required for cultures and the paucibacillary nature of samples; hence the need of rapid molecular methods. HIV infection increases the risk of tuberculosis, and HIV/tuberculosis coinfection is associated with higher mortality. We describe the case of a 56-year old mestizo male patient suspected of having tuberculosis who consulted the San Ignacio Hospital in Bogotá with a two-month history of a painful ulcerated lesion over the distal third area of the right forearm and in whom HIV coinfection was confirmed. Bone and pulmonary histological examination evidenced multiple granulomas, giant cells and fibrosis. Cultures and IS6110-PCR from lung and bone tissues were positive for Mycobacterium tuberculosis complex. Mycobacterium tuberculosis isolates were sensitive to first line drugs.