Structural analysis of platelet fragments and extracellular vesicles produced by apheresis platelets during storage.

ABSTRACT: Platelets (PLTs) for transfusion can be stored for up to 7 days at room temperature (RT). The quality of apheresis PLTs decreases over storage time, which affects PLT hemostatic functions. Here, we characterized the membranous particles produced by PLT storage lesion (PSLPs), including degranulated PLTs, PLT ghosts, membrane fragments, and extracellular membrane vesicles (PEVs). The PSLPs generated in apheresis platelet units were analyzed on days 1, 3, 5, and 7 of RT storage. A differential centrifugation and a sucrose density gradient were used to separate PSLP populations. PSLPs were characterized using scanning and transmission electron microscopy (EM), flow cytometry (FC), and nanoparticle tracking analysis (NTA). PSLPs have different morphologies and a broad size distribution; FC and NTA showed that the concentration of small and large PSLPs increases with storage time. The density gradient separated 3 PSLP populations: (1) degranulated PLTs, PLT ghosts, and large PLT fragments; (2) PEVs originated from PLT activation and organelles released by necrotic PLTs; and (3) PEV ghosts. Most PSLPs expressed phosphatidyl serine and induced thrombin generation in the plasma. PSLPs contained extracellular mitochondria and some had the autophagosome marker LC3. PSLPs encompass degranulated PLTs, PLT ghosts, large PLT fragments, large and dense PEVs, and low-density PEV ghosts. The activation-related PSLPs are released, particularly during early stage of storage (days 1-3), and the release of apoptosis- and necrosis-related PSLPs prevails after that. No elevation of LC3- and TOM20-positive PSLPs indicates that the increase of extracellular mitochondria during later-stage storage is not associated with PLT mitophagy.

Analysis of the mechanism of damage produced by thiazole orange photoinactivation in apheresis platelets.

BACKGROUND: Pathogen Reduction Technologies (PRTs) are broad spectrum nucleic acid replication-blocking antimicrobial treatments designed to mitigate risk of infection from blood product transfusions. Thiazole Orange (TO), a photosensitizing nucleic acid dye, was previously shown to photoinactivate several types of bacterial and viral pathogens in RBC suspensions without adverse effects on function. In this report we extended TO treatment to platelet concentrates (PCs) to see whether it is compatible with in vitro platelet functions also, and thus, could serve as a candidate technology for further evaluation. MATERIAL AND METHODS: PCs were treated with TO, and an effective treatment dose for inactivation of Staphylococci was identified. Platelet function and physiology were then evaluated by various assays in vitro. RESULTS: Phototreatment of PCs yielded significant reduction (≥4-log) in Staphylococci at TO concentrations ≥20 µM. However, treatment with TO reduced aggregation response to collagen over time, and platelets became unresponsive by 24 hours post-treatment (from >80% at 1 h to 0% at 24 h). TO treatment also significantly increased CD62P expression (<1% CD62P+ for untreated and >50% for TO treated at 1 h) and induced apoptosis in platelets (<1% Annexin V+ for untreated and >50% for TO treated at 1 h) and damaged mitochondrial DNA. A mitochondria-targeted antioxidant and reactive oxygen species (ROS) scavenger Mito-Tempo mitigated these adverse effects. DISCUSSION: The results demonstrate that TO compromises mitochondria and perturbs internal signaling that activates platelets and triggers apoptosis. This study illustrates that protecting platelet mitochondria and its functions should be a fundamental consideration in selecting a PRT for transfusion units containing platelets, such as PCs.

Essential Role of Neutrophils in the Protective Immune Response Induced by a Live Attenuated Leishmania Vaccine.

No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene-deleted L. donovani (LdCen-/- ) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen-/- with neutrophils, we immunized mice intradermally in the ear pinna with LdCen-/- Compared with LdWT infection, LdCen-/- parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6-18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen-/- -immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nß subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen-/- -immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen-/- parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen-/- -immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen-/- -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen-/- parasite vaccine.

Cell membrane disintegration and extracellular vesicle release in a model of different size and charge PAMAM dendrimers effects on cultured endothelial cells.

Different nanomaterials are under development for various biomedical applications in which nanoparticles contact blood and vasculature. Therefore, investigating the interactions between nanomaterials and vascular endothelial cells (ECs) is of great importance. Here, we show the effects of polyamidoamine (PAMAM) dendrimers of two different sizes, generation 2 (G2; approximately 3 nm diameter) and generation 7 (G7; 9 nm), with neutral (OH-terminated), anionic (COOH-terminated), and cationic (NH2-terminated) surface modifications on cultured human umbilical vein ECs (HUVECs). We found that only cationic dendrimers (5-100 µg/mL G7-NH2 and 100 µg/mL G2-NH2) and not anionic or neutral dendrimers were cytotoxic to HUVECs. In addition, cationic dendrimers at low concentrations (5 µg/mL) markedly increased the HUVEC surface expression of the proinflammatory activation marker ICAM-1 and phosphatidylserine (PS). Both G2-NH2 and G7-NH2 dendrimers caused g1 arrest, but only G7-NH2 dendrimers induced significant HUVEC apoptosis. G7-NH2 interacted strongly with HUVEC plasma membranes and mitochondrial membranes, and phospholipid vesicles containing G7-NH2 formed, which resulted in extensive plasma membrane blebbing and disintegration. Furthermore, flow cytometric analysis showed that G7-NH2-treated HUVECs released large numbers of extracellular vesicles (EVs) positive for CD105 and PS. A notable population of EVs positive for the mitochondrial marker TOM20 but negative for the autophagosome marker LC3 was found. In summary, large cationic PAMAM dendrimers (G7-NH2) showed both proinflammatory and proapoptotic effects in ECs; at high dendrimer concentrations, these effects were accompanied by necrotic cytotoxicity. G7-NH2 caused plasma and mitochondrial membrane disintegration and the release of EVs, including EVs of mitochondrial origin that were not associated with mitophagy.

Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent ß-globin posttranslational modifications, including the irreversible oxidation of ßCys93 and the ubiquitination of ßLys96 and ßLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, ßCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

An effective "three-in-one" screening assay for testing drug and nanoparticle toxicity in human endothelial cells.

Evaluating nanoparticle (NP) toxicity in human cell systems is a fundamental requirement for future NP biomedical applications. In this study, we have designed a screening assay for assessing different types of cell death induced by NPs in human umbilical vein endothelial cell (HUVEC) culture. This assay consists of WST-8, LDH and Hoechst 33342 staining, all performed in one well, which enables an evaluation of cell viability, necrosis and apoptosis, respectively, in the same cell sample. The 96-well format and automated processing of fluorescent images enhances the assay rapidity and reproducibility. After testing the assay functionality with agents that induced different types of cell death, we investigated the endothelial toxicity of superparamagnetic iron oxide nanoparticles (SPIONs, 8 nm), silica nanoparticles (SiNPs, 7-14 nm) and carboxylated multiwall carbon nanotubes (CNTCOOHs, 60 nm). Our results indicated that all the tested NP types induced decreases in cell viability after 24 hours at a concentration of 100 µg/ml. SPIONs caused the lowest toxicity in HUVECs. By contrast, SiNPs induced pronounced necrosis and apoptosis. A time course experiment showed the gradual toxic effect of all the tested NPs. CNTCOOHs inhibited tetrazolium derivatives at 100 µg/ml, causing false negative results from the WST-8 and LDH assay. In summary, our data demonstrate that the presented "three-in-one" screening assay is capable of evaluating NP toxicity effectively and reliably. Due to its simultaneous utilization of two different methods to assess cell viability, this assay is also capable of revealing, if NPs interfere with tetrazolium salts.

Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20+LC3+ PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

The effects of nanomaterials on blood coagulation in hemostasis and thrombosis.

The blood coagulation balance in the organism is achieved by the interaction of the blood platelets (PLTs) with the plasma coagulation system (PCS) and the vascular endothelial cells. In healthy organism, these systems prevent thrombosis and, in events of vascular damage, enable blood clotting to stop bleeding. The dysregulation of hemostasis may cause serious thrombotic and/or hemorrhagic pathologies. Numerous engineered nanomaterials are being investigated for biomedical purposes and are unavoidably exposed to the blood. Also, nanomaterials may access vascular system after occupational, environmental, or other types of exposure. Thus, it is essential to evaluate the effects of engineered nanomaterials on hemostasis. This review focuses on investigations of nanomaterial interactions with the blood components involved in blood coagulation: the PCS and PLTs. Particular emphases include the pathophysiology of effects of nanomaterials on the PCS, including the kallikrein-kinin system, and on PLTs. Methods for investigating these interactions are briefly described, and a review of the most important studies on the interactions of nanomaterials with plasma coagulation and platelets is provided. WIREs Nanomed Nanobiotechnol 2017, 9:e1448. doi: 10.1002/wnan.1448 For further resources related to this article, please visit the WIREs website.

Image-guided thermal therapy with a dual-contrast magnetic nanoparticle formulation: A feasibility study.

PURPOSE/OBJECTIVE: The aim of this study was to develop and investigate the properties of a magnetic iron oxide nanoparticle-ethiodised oil formulation for image-guided thermal therapy of liver cancer. MATERIALS AND METHODS: The formulation comprises bionised nano-ferrite (BNF) nanoparticles suspended in ethiodised oil, emulsified with polysorbate 20 (BNF-lip). Nanoparticle size was measured via photon correlation spectroscopy and transmission electron microscopy. In vivo thermal therapy capability was tested in two groups of male Foxn1(nu) mice bearing subcutaneous HepG2 xenograft tumours. Group I (n = 12) was used to screen conditions for group II (n = 48). In group II, mice received one of BNF-lip (n = 18), BNF alone (n = 16), or PBS (n = 14), followed by alternating magnetic field (AMF) hyperthermia, with either varied duration (15 or 20 min) or amplitude (0, 16, 20, or 24 kA/m). Image-guided fluoroscopic intra-arterial injection of BNF-lip was tested in New Zealand white rabbits (n = 10), bearing liver VX2 tumours. The animals were subsequently imaged with CT and 3 T MRI, up to 7 days post-injection. The tumours were histopathologically evaluated for distribution of BNF-lip. RESULTS: The BNF showed larger aggregate diameters when suspended in BNF-lip, compared to clear solution. The BNF-lip formulation produced maximum tumour temperatures with AMF >20 kA/m and showed positive X-ray visibility and substantial shortening of T1 and T2 relaxation time, with sustained intratumoural retention up to 7 days post-injection. On pathology, intratumoural BNF-lip distribution correlated well with CT imaging of intratumoural BNF-lip distribution. CONCLUSION: The BNF-lip formulation has favourable thermal and dual imaging capabilities for image-guided thermal therapy of liver cancer, suggesting further exploration for clinical applications.

Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets.

BACKGROUND: Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs. METHODS: CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test. RESULTS: SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing-thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a(+) PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent. CONCLUSIONS: Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing-thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

Subvisible Particle Content, Formulation, and Dose of an Erythropoietin Peptide Mimetic Product Are Associated With Severe Adverse Postmarketing Events.

Peginesatide (Omontys(®); Affymax, Inc., Cupertino, CA) was voluntarily withdrawn from the market less than a year after the product launch. Although clinical trials had demonstrated the drug to be safe and efficacious, 49 cases of anaphylaxis, including 7 fatalities, were reported not long after market introduction. Commercialization was initiated with a multiuse vial presentation, which differs in formulation from the single-use vial presentation used in phase 3 studies. Standard physical and chemical testing did not indicate any deviation from product specifications in either formulation. However, an analysis of subvisible particulates using nanoparticle tracking analysis and flow imaging revealed a significantly higher concentration of subvisible particles in the multiuse vial presentation linked to the hypersensitivity cases. Although it is unknown whether the elevated particulate content is causally related to these serious adverse events, this report illustrates the utility of characterizing subvisible particulates not captured by conventional light obscuration.

First demonstration of transmissible spongiform encephalopathy-associated prion protein (PrPTSE) in extracellular vesicles from plasma of mice infected with mouse-adapted variant Creutzfeldt-Jakob disease by in vitro amplification.

The development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced red blood cells from asymptomatic donors who subsequently developed the disease has confirmed existing concerns about the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. In addition, the presence of disease-associated misfolded prion protein (PrP(TSE)), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are still unknown. Various studies have identified cellular prion protein (PrP(C)) among the protein cargo in human blood-circulating extracellular vesicles released from endothelial cells and platelets, and exosomes isolated from the conditioned media of TSE-infected cells have caused the disease when injected into experimental mice. In this study, we demonstrate the detection of PrP(TSE) in extracellular vesicles isolated from plasma samples collected during the preclinical and clinical phases of the disease from mice infected with mouse-adapted vCJD and confirm the presence of the exosomal marker Hsp70 in these preparations.

The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets.

Carbon nanotubes (CNT) are one of the most promising nanomaterials for use in medicine. The blood biocompatibility of CNT is a critical safety issue. In the bloodstream, proteins bind to CNT through non-covalent interactions to form a protein corona, thereby largely defining the biological properties of the CNT. Here, we characterize the interactions of carboxylated-multiwalled carbon nanotubes (CNTCOOH) with common human proteins and investigate the effect of the different protein coronas on the interaction of CNTCOOH with human blood platelets (PLT). Molecular modeling and different photophysical techniques were employed to characterize the binding of albumin (HSA), fibrinogen (FBG), γ-globulins (IgG) and histone H1 (H1) on CNTCOOH. We found that the identity of protein forming the corona greatly affects the outcome of CNTCOOH's interaction with blood PLT. Bare CNTCOOH-induced PLT aggregation and the release of platelet membrane microparticles (PMP). HSA corona attenuated the PLT aggregating activity of CNTCOOH, while FBG caused the agglomeration of CNTCOOH nanomaterial, thereby diminishing the effect of CNTCOOH on PLT. In contrast, the IgG corona caused PLT fragmentation, and the H1 corona induced a strong PLT aggregation, thus potentiating the release of PMP.

Toxicity of carboxylated carbon nanotubes in endothelial cells is attenuated by stimulation of the autophagic flux with the release of nanomaterial in autophagic vesicles.

Carbon nanotubes (CNTs) exhibit a number of unique properties that make them attractive for various nanomedicine applications including their intravascular use. Therefore, the vascular toxicity of CNTs is a critical safety concern and methods of CNTs toxicity modulation are of great interest. Here, we report that carboxylated multiwalled carbon nanotubes (MWCNTs) induce a decrease in viability of cultured human umbilical vein endothelial cells (HUVECs) associated with the profound accumulation of autophagosomes. This autophagosome accumulation was mTOR kinase independent and was caused by blockade of the autophagic flux rather than by activation of autophagy. Stimulation of the autophagic flux with 1nmol/L bafilomycin A1 attenuated the cytotoxicity of carboxylated MWCNTs in HUVECs and was associated with the extracellular release of the nanomaterial in autophagic microvesicles. Thus, pharmacological stimulation of the autophagic flux may represent a new method of cytoprotection against toxic effects of nanomaterials. FROM THE CLINICAL EDITOR: This study investigates the mechanisms of toxicity of multiwalled carbon nanutubes on human endothelial cells, concluding that pharmacological stimulation of autophagic flux may represent a new method of cytoprotection against the toxic effects of these nanomaterials.