Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers.

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.

Plaque Vulnerability Index Predicts Cardiovascular Events: A Histological Study of an Endarterectomy Cohort.

Background The balance between stabilizing and destabilizing atherosclerotic plaque components is used in experimental studies and in imaging studies to identify rupture prone plaques. However, we lack the evidence that this balance predicts future cardiovascular events. Here we explore whether a calculated histological ratio, referred to as vulnerability index (VI), can predict patients at higher risk to suffer from future cardiovascular events. Methods and Results Carotid plaques and clinical information from 194 patients were studied. Tissue sections were used for histological analysis to calculate the VI (CD68 [cluster of differentiation 68], alpha-actin, Oil red O, Movat pentachrome, and glycophorin A). Postoperative cardiovascular events were identified through the Swedish National Inpatient Health Register (2005-2013). During the follow-up (60 months) 45 postoperative cardiovascular events were registered. Patients with a plaque VI in the fourth quartile compared with the first to third quartiles had significantly higher risk to suffer from a future cardiovascular event (P=0.0002). The VI was an independent predictor and none of the 5 histological variables analyzed separately predicted events. In the 13 patients who underwent bilateral carotid endarterectomy, the VI of the right plaque correlated with the VI of the left plaque and vice versa (r=0.7, P=0.01). Conclusions Our findings demonstrate that subjects with a high plaque VI have an increased risk of future cardiovascular events, independently of symptoms and other known cardiovascular risk factors . This strongly supports that techniques which image such plaques can facilitate risk stratification for subjects in need of more intense treatment.

Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

Coronary high-signal-intensity plaques on T1-weighted magnetic resonance imaging reflect intraplaque hemorrhage.

Coronary high-signal-intensity plaques (HIPs) detected by T1-weighted magnetic resonance imaging are associated with future cardiovascular events. This study aimed to identify pathological findings reflecting HIPs in coronary arteries obtained from autopsy cases. Formalin-fixed hearts were imaged with noncontrast T1-weighted imaging with a 1.5-T magnetic resonance system. We defined HIPs or non-HIPs as a coronary plaque to myocardial signal intensity ratio (PMR) of ≥1.4 or <1.4, respectively. We found HIPs in 4 of 37 (10.8%) hearts and analyzed 7 hearts in detail. The corresponding sections to HIPs (n=11) or non-HIPs (n=25) were histologically and immunohistochemically analyzed. We calculated the T1 relaxation time of human venous blood in vitro. Plaque and necrotic core areas, and the frequency of intraplaque hemorrhage in HIPs were significantly larger/higher than those in non-HIPs. HIPs were immunopositive for CD68 (11/11), glycophorin A (10/11), and fibrin (11/11). Glycophorin-A-, matrix metalloprotease 9 (MMP9)-, and tissue factor-immunopositive areas were larger in HIPs than in non-HIPs. The PMR was positively correlated with glycophorin-A-, fibrin-, MMP9-, and tissue factor-immunopositive areas. Blood coagulation shortened the T1 relaxation time of the blood and plasma, and the T1 relaxation times in coagulated whole blood and erythrocyte-rich blood were significantly shorter than those in plasma. Coronary HIPs may reflect intraplaque hemorrhage and may be a novel marker for plaque instability and thrombogenic potential.

Identification and detection of protein markers to differentiate between forensically relevant body fluids.

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen-antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction.

The Gerbich blood group system: old knowledge, new importance.

Antigens of the Gerbich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), minor sialoglycoproteins of human erythrocytes. GPC and GPD help maintain erythrocyte shape of and contributes to the stability of its membrane. There are six high-prevalence Gerbich antigens: Ge2, Ge3, Ge4, GEPL (GE10), GEAT (GE11), GETI (GE12) and five low-prevalence Gerbich antigens: Wb (GE5), Lsa (GE6), Ana (GE7), Dha (GE8), GEIS (GE9). Some Gerbich antigens (Ge4, Wb, Dha, GEAT) are expressed only on GPC, two (Ge2, Ana) are expressed only on GPD, while others (Ge3, Lsa, GEIS, GEPL, GETI) are expressed on both GPC and GPD. Antibodies recognizing GPC/GPD may arise naturally (so-called "naturally-occurring RBC antibodies") or as the result of alloimmunization, and some of them may be clinically relevant. Gerbich antibodies usually do not cause serious hemolytic transfusion reactions (HTR); autoantibodies of anti-Ge2- or anti-Ge3 specificity can cause autoimmune hemolytic anemia (AIHA).

Value of immunohistochemical expression of podocalyxin in active lupus nephritis / Valor de la expresión inmunohistoquímica de la podocalixina en la nefritis lúpica activa

Podocalyxin is an electronegative sialoglycoprotein that prevents the podocyte foot process from collapsing. The aim of this study was to detect an association between the glomerular immunohistochemical (IHC) expression of podocalyxin and the degree of podocyte effacement detected by electron microscopy, and to evaluate the role of podocalyxin IHC expression as a novel marker for disease activity in lupus nephritis (LN). Methods: Thirty-two renal biopsies of active lupus nephritis patients were studied. Clinical assessment by the systemic lupus activity measure (SLAM-R) score and laboratory data were included [serum creatinine, 24-h urinary protein, antinuclear antibodies (ANA), anti-double-strand DNA antibodies (anti-dsDNA), C3 and C4]. Light (L/M) and electron microscopic (E/M) examination was conducted. Podocyte loss was evaluated by immunohistochemistry with monoclonal anti-podocalyxin antibodies by means of a semiquantitative score that was graded from 0 to 4+ according to the percentage of glomerular involvement. Results: 22 cases (68.8%) with LN class IV, 6 (18.8%) with class III and 4 (12.5%) with class V. The mean age was (25.41±10.13) years. There was a significant negative correlation between IHC podocalyxin score and LN class, and NIH activity parameters such as leukocyte infiltration, endocapillary proliferation, fibrinoid necrosis and cellular crescent and disease activity index but not chronicity index. There was a highly significant negative correlation between IHC podocalyxin and podocyte effacement by E/M (rs=−0.903, P=0.000), and E/M immune deposits (r=−0.53, P=0.001), and a significant association with degree of proteinuria, ANA and SLAM score (P<0.05). Conclusions: Podocyte loss indicated by podocalyxin immunohistochemical expression reflects the degree of activity and severity of LN and the degree of podocyte effacement by E/M (AU)

La podocalixina es una sialoglicoproteína electronegativa que evita el colapso del proceso podocitario. Nuestro objetivo fue detectar una asociación entre la expresión inmunohistoquímica (IHQ) glomerular de la podocalixina y el grado de borramiento podocitario detectado mediante microscopia electrónica, además de evaluar la función de la expresión IHQ de la podocalixina como un nuevo marcador de la actividad de la enfermedad en la nefritis lúpica (NL). Métodos: Se evaluaron 32 biopsias renales de pacientes con NL activa. Se incluyeron la evaluación clínica mediante la puntuación de la determinación de la actividad del lupus sistémico (systemic lupus activity measure, SLAM-R) y datos analíticos (creatinina sérica, proteína en la orina de 24h, anticuerpos antinucleares [AAN], anticuerpos anti-ADN de doble cadena [anti-ADNdc], C3 y C4). Evaluación mediante microscopio de luz (M/L) y microscopio electrónico (M/E). La evaluación de la pérdida podocitaria se realizó mediante inmunohistoquímica con anticuerpos antipodocalixina monoclonales, por medio de una puntuación semicuantitativa que se clasificó de 0 a 4+ en función del porcentaje de afectación glomerular. Resultados: Encontramos 22 (68,8%) casos con clase IV de NL, 6 (18,8%) con clase III y 4 (12,5%) con clase V. La media de edad fue de 25,41±10,13 años. Se observó una asociación negativa significativa entre la puntuación de la podocalixina en la IHQ con la clase de NL y los parámetros de actividad del NIH, como la infiltración leucocitaria, la proliferación endocapilar, la necrosis fibrinoide y los drepanocitos y el índice de actividad de la enfermedad, pero no el índice de cronicidad. Se observó una correlación negativa muy significativa entre la podocalixina en la IHQ y el borramiento podocitario mediante M/E (rs=−0,903; p=0,000), depósitos inmunes mediante M/E (r=−0,53; p=0,001) y una asociación significativa con el grado de proteinuria, AAN y puntuación en el índice SLAM (p<0,05). Conclusiones: La pérdida podocitaria indicada mediante la expresión IQH de la podocalixina refleja el grado de actividad y la intensidad de la NL, así como el grado de borramiento podocitario mediante M/E (AU)

Protease sensitivity and redistribution of CD71 and glycophorin A on K562 cells.

Transmembrane proteins are delivered to plasma membrane from the endoplasmic reticulum and Golgi complex by vesicular transport along with the cytoskeletal network. Disruption of this process likely affects transmembrane protein expression. K562 cells were digested with Streptomyces griseus protease for different periods of time, and then re-cultured with different cytoskeletal and glycosylation inhibitors. Cell viability and surface expression of transferrin receptor (CD71) and glycophorin A (GPA) were analyzed before and after re-culture by flow cytometry. We found that digestion with protease almost completely removed extracellular CD71 and GPA but their expression recovered to the initial levels after re-culture for 8 h and 24 h, respectively. The microtubule depolymerizer colchicine promoted cell surface recovery of CD71 but inhibited that of GPA; the microtubule stabilizer paclitaxel inhibited cell surface recovery of CD71 but promoted that of GPA; the microfilament depolymerizer cytochalasin D had no effect on cell surface recovery of CD71 and GPA; the microfilament stabilizer phalloidin inhibited cell surface recovery of GPA. The glycosylation inhibitor tunicamycin inhibited the recovery of both CD71 and GPA, and BADGP inhibited the recovery of GPA. These studies show differential sensitivities of surface proteins on K562 cells to proteases, and suggest molecular mechanisms of transmembrane protein transport and cycling.

Acetylcholinesterase provides new insights into red blood cell ageing in vivo and in vitro.

BACKGROUND: During its 120 days sojourn in the circulation, the red blood cell (RBC) remodels its membrane. Acetylcholinesterase (AChE) is a glycosylphosphatidylinositol (GPI)-linked enzyme that may serve as a marker for membrane processes occurring this ageing-associated remodelling process. MATERIALS AND METHODS: Expression and enzymatic activity of AChE were determined on RBCs of various ages, as obtained by separation based on volume and density (ageing in vivo), and on RBCs of various times of storage in blood bank conditions (ageing in vitro), as well as on RBC-derived vesicles. RESULTS: During ageing in vivo, the enzymatic activity of AChE decreases, but not the AChE protein concentration. In contrast, neither AChE activity nor concentration show a consistent, significant decrease during ageing in vitro. CD59, another GPI-linked protein that protects against complement-induced removal, also remains constant during storage. The cellular content of the integral membrane protein glycophorin A, however, decreases with storage time in the more dense RBC fractions. The latter are enriched in echinocytes and other misshapen cells during storage. DISCUSSION: Our findings suggest that, during RBC ageing, GPI-linked proteins and integral membrane proteins are differentially sorted. Also, the vesicles that are generated in vitro show a fast and extensive loss of AChE activity, but not of AChE expression. Thus, AChE characteristics may constitute sensitive biomarkers of RBC ageing in vivo, and a source of information on the structural and functional changes that GPI-linked proteins undergo during ageing in vivo and in vitro. This information may help to understand RBC homeostasis and the effects of transfusion, especially in immunologically compromised patients.

Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides.

Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

Do Not Forget the Glycophorin A: An Unusual Case of Myeloid Sarcoma.

Acute erythroid leukemia is rare, with isolated reports on presentation as an extramedullary tumor mass (myeloid sarcoma). We describe a case of pure erythroid leukemia presenting as an orbital mass in a 1-year, 9-month-old girl. This is only the second case described in a child. Tissue biopsy of the tumor mass showed medium-sized cells that were glycophorin A positive and negative with conventional myeloid markers. Flow cytometry, bone marrow aspirate, and trephine confirmed the diagnosis of pure erythroid leukemia.

Immunophenotypic Profiling of Erythroid Progenitor-Derived Extracellular Vesicles in Diamond-Blackfan Anaemia: A New Diagnostic Strategy.

Diamond-Blackfan Anaemia (DBA) is a rare inherited anaemia caused by heterozygous mutations in one of 13 ribosomal protein genes. Erythroid progenitors (BFU-E and CFU-E) in bone marrow (BM) show a proapoptotic phenotype. Suspicion of DBA is reached after exclusion of other forms of BM failure syndromes. To improve DBA diagnosis, which is confirmed by mutation analysis, we tested a new approach based on the study of extracellular vesicles (EVs) isolated from plasma by differential centrifugations and analysed by flow cytometry. We chose CD34, CD71 and CD235a markers to study erythroid EVs. We characterised the EVs immunophentoypic profiles of 13 DBA patients, 22 healthy controls and 16 patients with other haematological diseases. Among the three EVs clusters we found, only the CD34+/CD71low population showed statistically significant differences between DBA patients and controls (p< 0.05). The absence of this cluster is in agreement with the low levels of BFU-E found in DBA patients. The assessment of ROC curves demonstrated the potential diagnostic value of this population. We suggest that this assay may be useful to improve DBA diagnosis as a quicker and less invasive alternative to BM BFU-E culture analysis.

Aqueous-filled polymer microcavity arrays: versatile & stable lipid bilayer platforms offering high lateral mobility to incorporated membrane proteins.

A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled, arrays of spherical cap microcavities formed from microsphere template polydimethoxysilane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepared using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coefficients comparable to free-standing vesicles in solution. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aqueous reservoirs, reconstituted membrane proteins experience an aqueous interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbß3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coefficients.

Trifluoroethanol-containing RP-HPLC mobile phases for the separation of transmembrane peptides human glycophorin-A, integrin alpha-1, and p24: analysis and prevention of potential side reactions due to formic acid.

Reversed-phase high-pressure liquid chromatography analysis and purification of three hydrophobic, aggregation-prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2-trifluoroethanol (TFE). The addition of 10-16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE-containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711: 181-186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM-peptide purification. Characterization of the potential side reactions using MALDI and ESI-LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE-formic acid possibly induces O-formylation of the Ser residue and N-formylation of the N-terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation-prone TM peptides and proteins.

Resveratrol induces human K562 cell apoptosis, erythroid differentiation, and autophagy.

Resveratrol (Res) is a naturally occurring phytoalexin with apoptotic and inducing-glob effects in leukemic cells, but the potential induction of erythroid differentiation in cells is not fully understood. Here, we investigated the effects of Res on human erythro-megakaryoblastic leukemia cell line K562. Among the treated cells, proliferation was inhibited and the occurrence of cell apoptosis and cell death were detected. Erythroid differentiation assay was explored, and we found that Res could increase the expression of glycophorin A (GPA), HBA1, HBB, and γ-globin genes and enforced the expression of GPA, CD71, and Band3 proteins. Res also induced K562 cell autophagy when the concentration of Res was increased up to 50 or 100 µM. Our findings suggested that Res possesses the potency not only inducing apoptosis but also inducing erythroid differentiation and autophagy in K562 cells. These results provide that Res may be a therapeutic candidate for chronic myelogenous leukemia treatment.

A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants.

BACKGROUND: GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. STUDY DESIGN AND METHODS: To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. RESULTS: Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing. CONCLUSION: This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

F-cell levels are altered with erythrocyte density in sickle cell disease.

Lighter cells from density fractionated erythrocytes of sickle cell disease (SCD) patients carry higher amount of externalized phosphatidylserine (PS) and cell surface glycophorins compared to the denser counterparts. Further analysis also revealed that the denser cells contained higher levels of fetal hemoglobin (HbF) compared to the lighter cells, supported by the presence of larger number of F-cells in these populations. In this report, we have found direct evidence on the higher survival of the HbF rich erythrocytes in SCD.

An alternative to the human hemoglobin test in the investigation of bloodstains treated with active oxygen: the human glycophorin A test.

In criminal investigations, there are three stages involved when studying bloodstains: search and orientation, confirmation, and individualization. Confirmatory tests have two aims: to show that the stain contains a human biological fluid and to confirm the type of biological fluid. The need to determine the nature of the evidence is reflected in the latest bibliography, where the possibility of employing mRNA and miRNA markers for this purpose is proposed. While these new proposals are being investigated, the kits for determining human hemoglobin currently provide a simple solution for resolving this issue. With these kits, the possibility of obtaining false positives and false negatives is well known. However, recently, a new problem has been detected. This involves the interference caused by new cleaning products that contain sodium percarbonate (or active oxygen) when determining human hemoglobin. With the aim to resolve this problem, this work studied the ability of the human glycophorin A test to determine human blood in samples that have been treated with active oxygen. Our results show that the human glycophorin A test has a greater resistance to the destructive effect of the new detergents containing active oxygen; consequently, it provides an alternative to be taken into consideration in the confirmatory diagnoses of bloodstains.

Phenolic metabolites of benzene inhibited the erythroid differentiation of K562 cells.

Benzene is a common occupational hazard and a ubiquitous environmental pollutant. Benzene exposure at the levels even below 1ppm still showed hematotoxicity. It is widely accepted that the metabolites of benzene play important roles in the benzene toxicity to the hematopoietic system, but little is known about the effects of benzene metabolites on erythropoiesis. In present study, erythroid progenitor-like K562 cells were used to determine the effects of phenolic metabolites of benzene, including phenol, hydroquinone and 1,2,4-benzenetriol, on the erythroid differentiation. After the treatment with these benzene metabolites at the concentrations with no obvious cytotoxicity, the hemin-induced hemoglobin synthesis in K562 cells decreased in a concentration- and time-dependent manner, and the expression of CD71 and GPA protein on the surface of K562 cells was also inhibited. The reverse transcription-PCR was used to determine the mRNA level of the erythroid related genes in the K562 cells that were treated with benzene metabolites. The hemin-induced expression of globin genes, including α-, ß- and γ-globin genes, and the gene encoding the heme synthesis enzyme porphobilinogen deaminase was inhibited by benzene metabolites. When the K562 cells were pretreated with benzene metabolites, the hemin-induced expression of two transcription factor genes GATA-1 and NF-E2 was distinctly reduced, and the pre-treatment with benzene metabolites promoted the decrease of the mRNA level of transcription factor gene GATA-2 by hemin. These results indicated that benzene metabolites inhibited the hemin-induced erythroid differentiation through affecting the transcription of the erythroid related genes.

Flow cytometric analysis of microparticle phenotype and their role in thrombin generation.

BACKGROUND: Microparticles may be generated from a number of cell types and are known to play a role in haemostasis by a variety of mechanisms. We investigated the role of platelet, red cell, and leucocyte-derived microparticles in the measurement of thrombin generation. METHODS: Four parameters of thrombin generation (the endogenous thrombin potential (ETP), lag time, time to peak, peak height) and microparticle content was determined in 35 plasma samples from normal individuals pre and post filtration to remove microparticles. Immunofluorescent flow cytometry was used to identify and enumerate platelet, leucocyte, monocyte and red cell derived microparticles in plasma samples based on the expression of CD42b, CD45, CD15, and Glycophorin A respectively. Expression of phosphatidylserine and tissue factor by microparticles was determined by Annexin V and anti CD142 binding. The pre and post filtration results were compared. RESULTS: There was a significant decrease in ETP and Peak Height, and an increase in the time to peak post filtration (P < 0.001). A significant decrease in the number of CD42+, CD45+, CD15+, CD142+, and Annexin V+ microparticles was also observed. The change in CD42b+ microparticles correlated highly with the change in Annexin V+ microparticles (r = 0.68). Whilst the change in ETP correlated best with the change in CD15+ microparticles (r = 0.45) and the change in time to peak correlated with the change in Annexin V binding (r = 0.52) (P < 0.01). CONCLUSION: The presence of micropartcles in plasma significantly affects thrombin generation.