Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

Background

An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced.

Results

A known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N ⁶-chain elongated functionalized congener ADAC (N ⁶-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A₃ AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (K_{i app} = 2.4 nM) and functional assays (EC₅₀ = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A₁ AR subtype, but there was no selectivity for the A₃ AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A₃ AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A₃ AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A₃ receptor but did not bind cells that did not express the receptor.

Conclusion

This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A₃ AR dimers.     

Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres

Background

Dendrimers are highly branched synthetic macromolecules with a globular shape. They have been successfully used for generation of nanospheres at mild conditions via biomimetic silicification. Encapsulation of enzyme molecules within these nanospheres during their synthesis is a promising method for rapid and efficient entrapment of several enzymes. However, encapsulation of proteolytic enzymes has been rarely done via biomimetic silicification. As well, the operational stability of encapsulated enzyme has not been systematically reported.

Methods

A proteolytic enzyme, either α-Chymotrypsin or a fungal protease from Aspergilus Oryzea was encapsulated along with iron oxide nanoparticles within particles yielded via biomimetic silicification of different generations of polyamidoamine (PAMAM) dendrimers. Stability of encapsulated enzyme was compared to that of free enzyme during storage at room temperature. As well, their thermal and ultrasonic stabilities were measured. Scanning electron microscopy, transmission electron microscopy and optical microscopy were used to investigate the morphology of nanospheres.

Results

Determination of encapsulation efficiency revealed that ∼ 85% of fungal protease with concentration 1.4 mg mL^− 1 stock solution was immobilized within particles yielded by generation 0. Based on microscopic images the generated particles interconnected with each other and had spherical morphologies independent of generation. Kinetic analysis of encapsulated fungal protease demonstrated that Mechaelis-Menten constant (K_m) slightly increased.

Conclusion

PAMAM dendrimer generation 0 could be effectively used for rapid encapsulation of a fungal protease from Aspegilus Oryzae.

General significance

Encapsulation significantly enhances the thermal and ultrasonic stabilities of enzymes, suggesting a range of diverse applications for them.     

Theoretical study on aluminum carbide endohedral fullerene-Al4C@C80

The possibility of a new endohedral fullerene with a trapped aluminum carbide cluster, Al₄C @C₈₀-I _h , was theoretical investigated. The geometries and electronic properties of it were investigated using density functional theory methods. The Al₄C unit formally transfers six electrons to the C₈₀ cage which induces stabilization of Al₄C@C₈₀. A favorable binding energy, relatively large HOMO-LUMO gap, electron affinities and ionization potentials suggested the Al₄C@C₈₀ is rather stable. The analysis of vertical ionization potential and vertical electron affinity indicate Al₄C@C₈₀ is a good electron acceptor.

Modulation of pro-inflammatory activation of monocytes and dendritic cells by aza-bis-phosphonate dendrimer as an experimental therapeutic agent

Introduction

Our objective was to assess the capacity of dendrimer aza-bis-phosphonate (ABP) to modulate phenotype of monocytes (Mo) and monocytes derived dendritic cells (MoDC) activated in response to toll-like receptor 4 (TLR4) and interferon γ (IFN- γ) stimulation.

Methods

Mo (n = 12) and MoDC (n = 11) from peripheral blood of healthy donors were prepared. Cells were preincubated or not for 1 hour with dendrimer ABP, then incubated with lipopolysaccharide (LPS; as a TLR4 ligand) and (IFN-γ) for 38 hours. Secretion of tumor necrosis factor α (TNFα), interleukin (IL) -1, IL-6, IL-12, IL-10 and IL-23 in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Bead Array. Differentiation and subsequent maturation of MoDC from nine donors in the presence of LPS were analyzed by flow cytometry using CD80, CD86, CD83 and CD1a surface expression as markers.

Results

Mo and MoDC were orientated to a pro-inflammatory state. In activated Mo, TNFα, IL-1β and IL-23 levels were significantly lower after prior incubation with dendrimer ABP. In activated MoDC, dendrimer ABP promoted IL-10 secretion while decreasing dramatically the level of IL-12. TNFα and IL-6 secretion were significantly lower in the presence of dendrimer ABP. LPS driven maturation of MoDC was impaired by dendrimer ABP treatment, as attested by the significantly lower expression of CD80 and CD86.

Conclusion

Our data indicate that dendrimer ABP possesses immunomodulatory properties on human Mo and MoDC, in TLR4 + IFN-γ stimulation model, by inducing M2 alternative activation of Mo and promoting tolerogenic MoDC.     

Interactions of calcium with the external surfaces of fullerenes and endofullerenes doped with radioactive sodium iodide

We report first-principles calculations carried out to analyze the adsorption of calcium on the outer surface of the fullerene C₆₀, yielding [C₆₀?+?mCa]. Geometric optimization (GO) and molecular dynamics (MD) simulation were performed using the plane-wave pseudopotential method within the framework of density functional theory (DFT) and time-dependent DFT (TD-DFT) to investigate the configurations, the associated energies in the ground state, and the stabilities of fullerenes and endofullerenes doped with radioactive sodium iodide when they interact with calcium atoms on the outer fullerene surface (i.e., [nNa¹³¹I@C₆₀?+?mCa]). The reason for investigating these calcium-functionalized (endo)fullerene systems was to gauge their potential stability when used as vectors to deliver radioiodine to cancerous tissue in the human body. In the simulations, we found that the geometric limit on the number of calcium atoms that can be physisorbed on the outer surface of an empty fullerene while maintaining its structural stability is 28 calcium atoms, which also takes into account the proportional expansion of the fullerene as the number of absorbed calcium atoms increases. However, the stability of a fullerene system during calcium adsorption also strongly depends on whether any atoms or molecules are being encapsulated by the fullerene, as these encapsulated atoms/molecules can also interact with the fullerene and influence its stability. A Mulliken electronegativity analysis revealed that, when atoms inside and/or outside the fullerene donate charge (electrons) to the fullerene, the fullerene expands. The excess charge on the carbon atoms of the fullerene weakens some of the carbon–carbon bonds, potentially causing them to break, in which case the fullerene loses its ability to encapsulate molecules and releases them.

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Graphical Abstract DFT simulation of a endo fullerene doped with radioactive sodium iodide interacting with 28 calcium atoms in a geometric arrangement

     

Ni-hemin metal–organic framework with highly efficient peroxidase catalytic activity: toward colorimetric cancer cell detection and targeted therapeutics

Background

Given the great benefits of artificial enzymes, a simple approach is proposed via assembling of Ni²⁺ with hemin for synthesis of Ni-hemin metal–organic-frameworks (Ni-hemin MOFs) mimic enzyme. The formation of the Ni-hemin MOFs was verified by scanning electron microscopy, Transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Energy-dispersive X-ray spectroscopy and UV–vis absorption spectroscopy. This novel nanocomposite exhibited surprising peroxidase like activity monitored by catalytic oxidation of a typical peroxidase substrate, 3,3,5,5′-tetramethylbenzidine, in the presence of H₂O₂. By using folic acid conjugated MOF nanocomposite as a recognition element, we develop a colorimetric assay for the direct detection of cancer cells.

Results

The proposed sensor presented high sensitivity and selectivity for the detection of human breast cancer cells (MCF-7) and Human Caucasian gastric adenocarcinoma. By measuring UV–vis absorbance response, a wide detection range from 50 to 10⁵ cells/mL with a detection limit as low as 10 cells/mLwas reached for MCF-7 cells. We further discuss therapeutics efficiency of Ni-hemin MOFs in the presence of H₂O₂ and ascorbic acid. Peroxidase-mimic Ni-hemin MOFs as reactive oxygen species which could damage MCF-7 cancer cells, however for normal cells (human embryonic kidney HEK 293 cells) killing effect was negligible.

Conclusions

Based on these behaviors, the developed method offers a fast, easy and cheap assay for the interest in future diagnostic and treatment application.

     

Investigation of a MMP-2 Activity-Dependent Anchoring Probe for Nuclear Imaging of Cancer

Purpose

Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments.

Methods

We designed and synthesized MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀, which consist of 4 independent moieties: RI unit (¹¹¹In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates.

Results

MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀ were cleaved by MMP-2 in a concentration-dependent manner. MDAP₃₀₀₀ pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP₃₀₀₀ exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP₃₀₀₀ exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP₃₀₀₀ existed in tumor xenografts in vivo.

Conclusions

The results indicate the possible usefulness of our MDAP strategy for tumor imaging.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

Background  

Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.     

Immobilization of β-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications

Background

β-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction.

Methodology/Principal Findings

Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (E_a) remains unchanged whereas optimum pH and K_m were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene.

Significance

Immobilized β-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.     

Diversity in forms of C4 in the genus Cleome (Cleomaceae)

Background and Aims

Cleomaceae is one of 19 angiosperm families in which C₄ photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C₄ in genus Cleome.

Methods

Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC).

Key Results

Three species with C₄-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C₄ photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C₄ cycle (compared with the C₃–C₄ intermediate C. paradoxa and the C₃ species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells.

Conclusions

NAD-malic enzyme-type C₄ photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C₃–C₄ intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.     

The ultrastructural basis for the identification of cell types in the pancreatic islets I. Guinea pig

Summary The pancreatic islets of the guinea pig have been studied by light and electron microscopy. The B granules in glutaraldehyde-fixed tissue often are cup-shaped with an indentation visible on one side of the granule. Phosphotungstic acid hematoxylin (PTAH) positive cells have been characterized by electron microscopy as three subtypes based on the size of the secretory granules. A_a cells are the most common and have secretory granules around 200 m in diameter. A_b cells have large secretory granules around 300 m in diameter and are relatively infrequent. A_c cells are the least common and have small (160 m) granules. Characteristic D cells are identifiable by electron microscopy and, on the basis of the subsequent study (Munger, Caramia, and Lacy, 1965), are identified as the silver positive cells observed by light microscopy.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases.-The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donohue.     

Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

Larval fish of Antarctica have very narrow rings on their otoliths (<1 m) that may not be resolved with light microscopy. In this study, age data from the otoliths of larval Nototheniidae (Gobionotothen gibberifrons and Lepidonotothen larseni), determined using light and scanning electron microscopy, are compared. Rings 0.4 m wide were observed on otoliths viewed under electron microscopy; however, light microscopy could only resolve rings 0.5 m wide. Scanning electron microscopy is more time consuming and costly than light microscopy but has greater resolving power and is recommended to validate ring counts made using light microscopy in otolith studies with Antarctic larval fish.     

A ring-like nucleoid is not necessary for radioresistance in the <Emphasis Type="Italic">Deinococcaceae</Emphasis>

Background  

Transmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures.     

Fluorescence analysis of the action of soluble derivatives of fullerene C<Subscript>60</Subscript> on amyloid fibrils of the brain peptide Aβ(1–42)

It has been shown by fluorescence analysis in vitro that the water-soluble sodium salt of the polycarboxylic derivative of fullerene C₆₀, fullerenol, and complexes of fullerene C₆₀ with polyvinylpyrrolidone (mol. wt. 25000 and 10000) destroy amyloid fibrils of the brain peptide Aβ(1–42) and prevent their formation. The results of fluorescence analysis confirmed the data obtained earlier by high-resolution electron microscopy. Fluorescence analysis and electron microscopy are complementary methods for the selection of effective antiamyloid drugs.     

Oxidation of glucose by syntrophic association between <Emphasis Type="Italic">Geobacter</Emphasis> and hydrogenotrophic methanogens in microbial fuel cell

Objective

To investigate a syntrophic interaction between Geobacter sulfurreducens and hydrogenotrophic methanogens in sludge-inoculated microbial fuel cell (MFC) systems running on glucose with an improved electron recovery at the anode.

Results

The presence of archaea in MFC reduces Coulombic efficiency (CE) due to their electron scavenging capability but, here, we demonstrate that a syntrophic interaction can occur between G. sulfurreducens and hydrogenotrophic methanogens via interspecies H₂ transfer with improvement in CE and power density. The addition of the methanogenesis inhibitor, 2-bromoethanesulfonate (BES), resulted in the reduction in power density from 5.29 to 2 W/m³, and then gradually increased to the peak value of 5.5 W/m³ when BES addition was stopped.

Conclusion

Reduction of H₂ partial pressure by archaea is an efficient approach in improving power output in a glucose-fed MFC system using Geobacter sp. as an inoculum.

     

Treatment of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> with 2-bromopalmitate alters morphology,endocytosis, differentiation and infectivity

Background

The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease.

Results

The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC₅₀/48 h value for culture epimastigotes was estimated as 130 μM. The IC₅₀/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC₅₀/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity.

Conclusion

Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.

     

Foliar CO2photoassimilation and chloroplast linear electron transport rates in nitrogen-sufficient and nitrogen-limited soybean plants

Leaflets of soybean plants which are moderately inorganic nitrogen (N)-limited exhibit either no difference in the rate of net photosynthesis or as much as a 15–23% lower net photosynthesis rate per unit area than leaflets of N-sufficient plants [Robinson JM (1996) Photosynth Res 50: 133–148; Robinson JM (1997a) Int J Plant Sci 158: 32–43]. However, mature leaflets of N-limited soybean plants have a higher CO₂photoassimilation rate per unit chlorophyll than leaflets of N-sufficient soybean plants at both moderate light intensity (500 µmol m^-2s^-1) and saturating light intensity (1200 µmol m^-2s^-1) [Robinson JM (1996) Photosynth Res 50: 133–148]. This study was undertaken to determine whether chloroplast thylakoids isolated from the leaflets of nitrogen-limited soybean plants displayed similar or higher linear electron transport rates (H₂O ferredoxin NADP) per unit chlorophyll than thylakoids isolated from leaflets of N-sufficient plants. Chlorophyll concentration in reaction mixtures containing chloroplast thylakoids prepared from leaflets of N-limited plants was manipulated so that it was similar to the chlorophyll concentration in reaction mixtures of thylakoids prepared from leaflets of N-sufficient plants. Measurements of ferredoxin dependent, NADP dependent, O₂photo-evolution in thylakoid isolates were carried out in saturating light (1500 µmol m^-2s^-1) and with (an uncoupler) in the chloroplast reaction mixtures. Chloroplast thylakoids isolated from N-limited soybean plant leaflets routinely had a 1.5 to 1.7 times higher rate of uncoupled, whole chain electron transport per unit chlorophyll in saturating light than did chloroplast thylakoids isolated from leaflets of N-sufficient plants. The results suggest that the photosystems and photosynthetic electron transport chain components are more active per unit Chl in leaflet chloroplast thylakoids of N-limited soybean plants than in thylakoids of N-sufficient plants.     

Polyhedrocytes in blood clots of type 2 diabetic patients with high cardiovascular risk: association with glycemia,oxidative stress and platelet activation

Background

Little is known about factors that affect the composition of contracted blood clots in specific diseases. We investigated the content of polyhedral erythrocytes (polyhedrocytes) formed in blood clots and its determinants in type 2 diabetes (T2D) patients.

Methods

In 97 patients with long-standing T2D [median HbA_1c, 6.4% (interquartile range 5.9–7.8)], we measured in vitro the composition of blood clots, including a clot area covered by polyhedrocytes using scanning electron microscopy and the erythrocyte compression index (ECI), defined as a ratio of the mean polyhedrocyte area to the mean native erythrocyte area. Moreover, plasma fibrin clot permeability (K_s), clot lysis time (CLT), thrombin generation, oxidative stress [total protein carbonyl (total PC), total antioxidant capacity and thiobarbituric acid reactive substances (TBARS)], and platelet activation markers were determined. The impact of glucose concentration on polyhedrocytes formation was assessed in vitro.

Results

Polyhedrocytes content in contracted clots was positively correlated with glucose (r?=?0.24, p?=?0.028), glycated hemoglobin (r?=?0.40, p?=?0.024), total cholesterol (r?=?0.22, p?=?0.044), TBARS (r?=?0.60, p?=?0.0027), P-selectin (r?=?0.54, p?=?0.0078) and platelet factor-4, PF4 (r?=?0.59, p?=?0.0032), but not with thrombin generation, platelet count, K_s or CLT. Patients who formed more polyhedrocytes (≥?10th percentile) (n?=?83, 85.6%) had higher glucose (+?15.7%, p?=?0.018), fibrinogen (+?16.6%, p?=?0.004), lower red blood cell distribution width (RDW, ??8.8%, p?=?0.034), reduced plasma clot density (??21.8% K_s, p?=?0.011) and impaired fibrinolysis (+?6.5% CLT, p?=?0.037) when compared to patients with lesser amount of polyhedrocytes (<?10th percentile). ECI and the content of polyhedrocytes were strongly associated with total PC (r?=?0.79, p?=?0.036 and r?=?0.67, p?=?0.0004, respectively). In vitro an increase of glucose concentration by 10 mmol/L was associated with 94% higher polyhedrocytes content (p?=?0.033) when compared to the baseline (7.1 mM). After adjustment for age, sex and fibrinogen, multiple regression analysis showed that RDW was the only independent predictor of polyhedrocytes content in T2D (OR?=?0.61, 95% CI 0.39–0.92).

Conclusions

Poor glycemic control, together with enhanced platelet activation and oxidative stress, increase the content of polyhedrocytes in blood clots generated in T2D patients.