Jan Simak

Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria

We analysed endothelial cell membrane microparticles (ECMP) in the peripheral blood of patients with paroxysmal nocturnal haemoglobinuria (PNH) (n = 9), aplastic anaemia (AA) (n = 10), sickle cell disease (SCD) (n = 8), and healthy donors (HD) (n = 11). There was no clinically manifested thrombosis in the PNH or AA group, except one cured thrombophlebitis (PNH), while all SCD patients had a history of vaso‐occlusive crises. We used three‐colour flow cytometry with blood cell‐specific antibodies and antibodies to endothelial antigens CD105 and CD144. Phosphatidylserine‐positive microparticles were detected using the annexin V‐binding (AVB) assay. The population of CD105+AVB+ ECMP was significantly (P < 0·05) higher in SCD (median: 0·568 × 109/l; 25–75th percentile range: 0·351–0·976 × 109/l) and PNH (0·401 × 109/l ; 0·19–0·441 × 109/l) patients when compared with AA (0·122 × 109/l; 0·061–0·172 × 109/l) or HD (0·180 × 109/l; 0·137–0·217 × 109/l) group. Even more pronounced differences were observed in ECMP exhibiting a marker of inflammatory stimulation CD54 (CD105+CD54+). Similarly, ECMP that exhibited endothelial specific and proteolysis‐sensitive antigen CD144 were increased in SCD and PNH, but not in AA. Elevated CD54+ ECMP may reflect the inflammatory status of endothelial cells in SCD and PNH, while CD144+ ECMP could indicate continuous endothelial stimulation and/or injury. Analysis of circulating ECMP appears promising to provide useful information on the status of the vascular endothelium in PNH and SCD.

Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin

BackgroundElevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail.ResultsOvernight stimulation of HUVEC with either LPS or TNFα, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 μM camptothecin, documented by 60–70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (~80,000 MP/103 cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3–1 μm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.ConclusionsCamptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.

Carbon nanotubes activate blood platelets by inducing extracellular Ca2+ influx sensitive to calcium entry inhibitors.

To elucidate a mechanism of prothrombotic effects of carbon nanotubes (CNTs), we report here that multiwalled CNTs activate blood platelets by inducing extracellular Ca(2+) influx that could be inhibited by calcium channel blockers SKF 96365 and 2-APB. We also demonstrate platelet aggregating activity of different single-walled and multiwalled CNTs. In addition, we show that CNT-induced platelet activation is associated with a marked release of platelet membrane microparticles positive for the granular secretion markers CD62P and CD63.

Cellular prion protein is expressed on endothelial cells and is released during apoptosis on membrane microparticles found in human plasma.

BACKGROUND: Blood and plasma of animals experimentally infected with transmissible spongiform encephalopathies (TSEs) can transmit TSE infection by transfusion. A conformational isoform of prion protein (PrPsc) is believed to be the TSE‐infectious agent that propagates by converting the cellular prion protein (PrPc) to additional molecules of PrPsc. In orally infected animals, PrPsc accumulates in intestinal endothelial cells. In blood, two thirds of PrPc resides in plasma, but its source is not known.

Flow Cytometric Analysis of Cell Membrane Microparticles

Cell membrane microparticles (MPs) are phospholipid microvesicles shed from the plasma membrane of most eukaryotic cells undergoing activation or apoptosis. The presence of MPs is common in healthy individuals. However, an increase in their release is a controlled event and is considered a hallmark of cellular alteration. Microparticles display cell surface proteins that indicate their cellular origin. In addition, they may also express other markers, e.g., markers of cellular activation. Elevated levels of circulating MPs are associated with various vascular pathologies and their pathogenic potential has been widely documented. MPs have been analyzed in plasma and cell cultures by means of flow cytometry or solid phase assays. Here we present a three-color flow cytometric assay for immunophenotyping of MPs in plasma. This assay has been used to study elevated counts of different phenotypes of circulating endothelial MPs in several hematological and vascular diseases. A modified version of this assay can also be used for MP analysis in blood products and cell cultures.

Meeting report on protein particles and immunogenicity of therapeutic proteins: Filling in the gaps in risk evaluation and mitigation

This meeting was successful in achieving its main goals: (1) summarize currently available information on the origin, detection, quantification and characterization of sub-visible particulates in protein products, available information on their clinical importance, and potential strategies for evaluating and mitigating risk to product quality, and (2) foster communication among academic, industry, and regulatory scientists to define the capabilities of current analytical methods, to promote the development of improved methods, and to stimulate investigations into the impact of large protein aggregates on immunogenicity. There was a general consensus that a considerable amount of interesting scientific information was presented and many stimulating conversations were begun. It is clear that this aspect of protein characterization is in its initial stages. As the development of these new methods progress, it is hoped that they will shed light on the role of protein particulates on product quality, safety, and efficacy. A topic which seemed appropriate for short term follow up was to hold further discussions concerning the development and preparation of one or more standard preparations of protein particulates. This would be generally useful to facilitate comparison of results among different studies, methods, and laboratories, and to foster further development of a common understanding among laboratories and health authorities which is essential to making further progress in this emerging field.

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 CNTCOOHs 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.

Carbon nanotubes activate store-operated calcium entry in human blood platelets.

Carbon nanotubes (CNTs) are known to potentiate arterial thrombosis in animal models, which raises serious safety issues concerning environmental or occupational exposure to CNTs and their use in various biomedical applications. We have shown previously that different CNTs, but not fullerene (nC60), induce the aggregation of human blood platelets. To date, however, a mechanism of potentially thrombogenic CNT-induced platelet activation has not been elucidated. Here we show that pristine multiwalled CNTs (MWCNTs) penetrate platelet plasma membrane without any discernible damage but interact with the dense tubular system (DTS) causing depletion of platelet intracellular Ca(2+) stores. This process is accompanied by the clustering of stromal interaction molecule 1 (STIM1) colocalized with Orai1, indicating the activation of store-operated Ca(2+) entry (SOCE). Our findings reveal the molecular mechanism of CNT-induced platelet activation which is critical in the evaluation of the biocompatibility of carbon nanomaterials with blood.

Adverse effects of fullerenes on endothelial cells: Fullerenol C60(OH)24 induced tissue factor and ICAM-1 membrane expression and apoptosis in vitro

We studied the effects of a C60 water suspension at 4 μg/mL (nC60) and the water soluble fullerenol C60(OH)24 at final concentrations of 1–100 μg/mL on human umbilical vein endothelial cells (HUVECs) in culture. We found that a 24 hr treatment of HUVECs with C60(OH)24 at 100 μg/mL significantly increased cell surface expression of ICAM-1(CD54) (67 ± 4% CD54+ cells vs. 19 ± 2 % CD54+ cells in control; p < 0.001). In addition, this treatment induced the expression of tissue factor (CD142) on HUVECs (54 ± 20% CD142+ cells vs 4 ± 2% CD142+ cells in control; p = 0.008) and increased exposure of phosphatidylserine (PS) (29 ± 2% PS+ cells vs. 12 ± 5% PS+ cells in control; p < 0.001). Analysis of cell cycle and DNA fragmentation (TUNEL) showed that both nC60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21 ± 2% TUNEL+ cells at 100 μg/mL of C60(OH)24 vs. 4 ± 2% TUNEL+ cells in control; p < 0.001). We also demonstrated that both nC60 and C60(OH)24 induced a rapid concentration dependent elevation of intracellular calcium [Ca2+]i. This could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C60(OH)24 had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.

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.