BACKGROUNDCisplatin based chemotherapy is a well recognized risk factor for coagulation disorders and thrombosis. The pathophysiological mechanisms by which cisplatin promote thrombosis are not well understood.

METHODSRed blood cells (RBCs) were separated from peripheral blood of patients with breast cancer (n = 10) and healthy adults (n = 6) and treated with cisplatin. Coagulation time of RBCs was assessed by one step recalcification time and the productions of thrombin, intrinsic and extrinsic factor Xa were measured in the presence or absence of various concentrations of lactadherin. Exposed phosphatidylserine was stained with lactadherin and observed by confocal microscopy and flow cytometry.

RESULTSNeither fresh RBCs nor RBCs treated without cisplatin had potent procoagulant activity. Cisplatin treatment increased procoagulant activity of RBCs in a cell number- and concentration-dependent manner. Exposed phosphatidylserine was stained with lactadherin and after cisplatin treatment, strong fluorescence was revealed by confocal microscopy. Lactadherin bound RBCs from patients with breast cancer increased from (1.9 +/- 0.5)% on control RBCs to (68.0 +/- 3.5)% on RBCs treated with 10 micromol/L cisplatin for 24 hours.

CONCLUSIONSCisplatin treatment increases procoagulant activity of RBCs, which have a strong association with exposure of phosphatidylserine. The increased procoagulant activity may contribute to the pathogenesis of thrombophilia during cisplatin based chemotherapy in breast cancer patients.

Antineoplastic Agents ; pharmacology ; Blood Coagulation ; drug effects ; physiology ; Cisplatin ; pharmacology ; Erythrocytes ; drug effects ; physiology ; Humans ; In Vitro Techniques

OBJECTIVETo summarize the results of general survey, primary structure analysis and related functional studies of abnormal hemoglobins (Hbs) found in Hunan Province.

DATA SOURCESInternational Hb journals, Chinese biochemical and biomedical journals and other articles relevant to hematology.

STUDY SELECTIONAll Hb variants found in Hunan and identified by primary structure analysis during 1980 - 1991 were included.

DATA EXTRACTIONData concerning 11 types of Hb variants found in 3 districts and 7 counties in Hunan Province were briefly documented. Their frequencies of occurrence were calculated and their distributions among Han, Yao, Tujia and Dong ethnic groups were listed.

RESULTSThirty-six cases with abnormal Hb were identified out of 7412 individuals screened in Hunan. 11 different types of Hb variants were recognized by primary structure analysis in 19 propositi along with their family members, including 5 alpha-chain variants, 4 beta-chain variants, 1 delta-chain variant and 1 delta-beta chain fusion variant. Oxygen equilibrium characteristics, reaction dynamics, the rate of globin chain synthesis (RGCS), morphology observation by electron microscopy and DNA analysis were all used in the functional studies of hemoglobinopathies.

CONCLUSIONSThe average incidence of abnormal Hbs in Hunan is 0.486%. In Jianghua County, whose inhabitants are mostly of the Yao ethnic group, the incidence is significantly higher (1.09%). Hb Jianghua [beta120(GH3) Lys-->lle] and Hb Shuangfeng (SF) [alpha27(B8) Glu-->Lys] were two new variants first reported in international literature; whereas Hb Lille [alpha74(EF3) Asp-->Ala], HbA(2) Flatbush [delta22(B4) Ala-->Glu] and Hb Lepore-Boston [delta87(F3)-beta116(G18)] were the first three instances to be found in China. Hb SF displayed an oxygen affinity 1.5-fold higher than that of HbA at pH 7.4 and 25 degrees C with its oxygen equilibrium curve shifted to the left. Reticulocytes of Hb SF heterozygote showed unbalanced RGCS, quite similar to that found in beta-thalassemia minor. Erythrocytes of Hb SF heterozygote were changed to spherocytes and began to lyse after incubation with sodium salicylate or sulfadiazine (pH 7.4, 37 degrees C) for 2 - 4 h. These findings explained the sudden attack of hemolytic anemia provoked by two drugs in Hb SF propositus. The genotype of a patient with Hb Q-H disease is identified as -,-/-,alpha(Q) by DNA restriction mapping.

Erythrocytes ; drug effects ; Globins ; biosynthesis ; Hemoglobins, Abnormal ; chemistry ; genetics ; physiology ; Humans ; Mutation

Rh is a very important blood group like ABO blood system in transfusion medicine. It causes severe transfusion reaction and hemolytic disease of the newborn (HDN) if RhD blood group does not match between the donor and the recipient. The population of RhD negative is only about 0.2% - 0.5% in Chinese. Conversion of RhD positive RBCs to RhD negative is very important in clinical transfusion. This study was to try to modify RhD antigen located on the surface of A, B, O and AB red blood cells in order to convert RhD positive to RhD negative by the modification of four kinds of methoxypolyethylene glycol (mPEG) derivatives and to observe the effect of mPEG modification on cell morphology, structure and function. The result demonstrated that modification efficiency of mPEG-BTC (mPEG-benzotriazole carbonate) was better than other three kinds of mPEG derivatives. It could camouflage RhD antigen efficiently when the concentration reached to 1 mmol/L. The result also showed that there were no harmful effects of mPEG modification on cell morphology, osmotic fragility, hemolysis, AchE, cholesterol, ATP, 2,3-DPG and deformability. It is suggested that success in converting RhD positive RBCs to RhD negative was preliminarily achieved.
Erythrocytes ; drug effects ; immunology ; physiology ; Humans ; Polyethylene Glycols ; pharmacology ; Rh-Hr Blood-Group System ; immunology

In order to optimize the preservation of blood, 3 kinds of antioxidant were selected and each of them can be injected directly into vein, then the optimal dose of these antioxidants was chosen using statistical method; ISMC (injectio salvia miltiorrhizae composita), ginaton and the combination of ISMC and ginaton were added into blood as optimal dose, some references as ATP, EI and so on were observed during blood preservation. The results showed that all of the three kinds of antioxidants increased ATP, EI and decreased FHb during blood preservation. It is concluded that both of ISMC and ginaton can effectively optimize the preservation of blood and combination of ISMC and ginaton can produce additive effect.
Adenosine Triphosphate ; metabolism ; Antioxidants ; pharmacology ; Blood Preservation ; Erythrocytes ; drug effects ; physiology ; Humans ; Salvia miltiorrhiza

In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields.
ABO Blood-Group System ; immunology ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes ; drug effects ; Fermentation ; physiology ; Hemagglutination ; drug effects ; Humans ; Pichia ; genetics ; metabolism ; alpha-Galactosidase ; genetics ; metabolism ; pharmacology

The present paper was aimed to explore the effect of Shuxuetong on the membrane viscoelasticity of erythrocyte taken from the acute phase patients suffering from chronic pulmonary heart disease. The membrane viscoelasticity of erythrocyte was taken from the acute phase patients suffering from chronic pulmonary heart disease. The changes of membrane viscoelasticity of erythrocyte after treated with shuxuetong were detected by micropipette aspiration technique. The results showed that the Shuxuetong of certain concentration could cause the decrease of membrane elastic modulus and viscous coefficients in acute phase patients suffering from chronic pulmonary heart disease. The study offers experimental evidences that the comprehensive treatment of pulmonary heart disease should involve the drug or measure to improve the erythrocyte deformability.
Aged ; Blood Viscosity ; drug effects ; Chronic Disease ; Drugs, Chinese Herbal ; therapeutic use ; Elasticity ; drug effects ; Erythrocyte Deformability ; physiology ; Erythrocyte Membrane ; physiology ; Erythrocytes ; physiology ; Female ; Humans ; Male ; Middle Aged ; Phytotherapy ; Pulmonary Heart Disease ; blood ; drug therapy

A modified electrometric method was described and validated for measurement of plasma and erythrocyte cholinesterase activities in 6~18 months old goats. The enzymatic reaction mixture contained 3 ml distilled water, 3 ml barbital-phosphate buffer (pH 8.1), 0.2 ml plasma or erythrocytes and 0.1 ml acetylthiocholine iodide (7.5%) as a substrate. The mixture was incubated at 37 degrees C for 40 minutes. The pH of the reaction mixture was determined by a pH meter before and after the incubation. The initial pH was measured before the substrate addition. The enzyme activity was expressed as deltapH/40 min. The coefficients of variation of the described method in measuring plasma and erythrocyte cholinesterase activities were 4 and 2%, respectively. Preliminary reference values (n = 14) of the mean cholinesterase activity (deltapH/40 min) and 95% confidence interval in the plasma were 0.194 and 0.184~ 0.204, respectively, and those of the erythrocytes were 0.416 and 0.396~0.436, respectively. The pseudocholinesterase activity of the plasma cholinesterase was 63.5% as determined by quinidine sulfate inhibition. The organophosphorus insecticides dichlorvos and diazinon at 0.5~4 micrometer and the carbamate insecticide carbaryl at 5~20 micrometer in the reaction mixture significantly inhibited plasma (13.7~85.5%) and erythrocyte (16.4~71.9%) cholinesterases in vitro in a concentration-dependent manner. The results suggest that the described electrometric method is simple, precise and efficient in measuring blood cholinesterase activity in goats.
Acid-Base Equilibrium/physiology ; Animals ; Carbaryl/pharmacology ; Cholinesterase Inhibitors/pharmacology ; Cholinesterases/*blood/drug effects ; Diazinon/pharmacology ; Dichlorvos/pharmacology ; Enzyme Activation/drug effects/physiology ; Erythrocytes/metabolism ; Goats/*blood ; Plasma/metabolism

This paper was to explore the effect of blood oxygen saturation (SO2) on oxidative damages of erythrocytes under the condition of oxidative stress. Keeping SO2 of cultured erythrocytes in vitro at the states of 0.3, 0.5, 0.7, 0.9 and 0.98, respectively, we induced oxidative stress by tert-buthylhydroperoxide (BHP, 0.15 mmol/L of final concentration). After incubation, antioxidant capacity was assessed by measuring content of reduced glutathin hormone (GSH) in erythrocytes. Methemoglobin (MetHb) content, lipid peroxidation (thiobarbituric acid-reactive substances, TBARS) and denatured globin-chains on the plasma membrane were measured to assess the extent of oxidative damages. The results showed that in the presence of BHP, GSH contents increased from 0.3 to 0.98 groups; MetHb, TBARS and globin-chains levels all dropped with the rise of SO2. In conclusion, antioxidant capacity and oxidative damages of erythrocytes are closely related to SO2, declined SO2 could promote oxidative damages of erythrocytes.
Cells, Cultured ; Erythrocytes ; cytology ; metabolism ; physiology ; Glutathione ; blood ; Humans ; Methemoglobin ; metabolism ; Oxidative Stress ; drug effects ; Oximetry ; methods ; Oxygen ; blood ; Thiobarbituric Acid Reactive Substances ; metabolism ; tert-Butylhydroperoxide ; toxicity

BACKGROUNDBone marrow hematopoietic function suppression is one of the most common side effects of chemotherapy. After chemotherapy, the bone marrow structure gets destroyed and the cells died, which might cause the hematopoietic function suppression. Heme oxygenase-1 (HO-1) is a key enzyme of antioxidative metabolism that associates with cell proliferation and resistance to apoptosis. The aim of this study was to restore or resist the bone marrow from the damage of chemotherapy by the HO-1 expression of mouse mesenchymal stem cells (mMSCs) homing to the mice which had the chemotherapy-induced bone marrow suppression.

METHODSOne hundred and sixty female Balb/c mice (6-8-weeks old) were randomly divided into four groups. Each group was performed in 40 mice. The control group was intraperitoneally injected for 5 days and tail intravenously injected on the 6th day with normal saline. The chemotherapy-induced bone marrow suppression was established by intraperitoneally injecting cyclophosphamide (CTX) into the mice which performed as the chemotherapy group. The mMSCs were tail intravenously injected into 40 chemotherapically damaged mice which served as the mMSCs group. The difference between the HO-1 group and the mMSCs group was the injected cells. The HO-1 group was tail intravenously injected into the mMSCs that highly expressed HO-1 which was stimulated by hemin. The expression of HO-1 was analyzed by Western blotting and RT-PCR. Cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Histopathologic examinations were performed 1 week after injection.

RESULTSCompared with the control group, the expression levels of HO-1 mRNA and protein were significantly higher in the HO-1 group (all P < 0.05), even obviously than the mMSCs group. CTX treatment induced apoptosis and inhibited proliferation. After injected, the white blood cell (WBC), red blood cell (RBC) and platelet (PLT) declined fast and down to the bottom at the 7th day. The bone marrow structure was destroyed incomplete. In vitro, the survival rate of cells in chemotherapy group was less than 50% after 24 hours. In contrast, mMSCs could do a favor to the cellular cleavage and proliferation. They slowed down the cell mortality and more than 50% cells survived after 24 hours. The effects of blocking apoptosis and bone marrow recovery could be more effective in the HO-1 group. In the HO-1 group, it had observed that the bone marrow structure became complete and the hemogram closed to normal at 7th day.

CONCLUSIONSHO-1 played an important role in promoting the recovery of CTX-induced hematopoietic damage. We suggest that HO-1 is able to restore the functions of chemotherapy-induced hematopoietic damage.

Animals ; Apoptosis ; drug effects ; Blood Platelets ; drug effects ; Blotting, Western ; Bone Marrow ; drug effects ; enzymology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclophosphamide ; toxicity ; Erythrocytes ; drug effects ; Female ; Heme Oxygenase-1 ; genetics ; metabolism ; Leukocytes ; drug effects ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; enzymology ; physiology ; Mice ; Mice, Inbred BALB C ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: Normal erythrocyte is deformable and this facilitates blood flow in the capillaries. Oxidative stress reduces the deformability of erythrocytes, and influences on blood flow in microcirculation. The objective of this study was to investigate the deformability of erythrocytes exposed to oxidative stress, the protective effects of verapamil and ascorbic acid against oxidative damages in erythrocytes, and the value of the microfluidic ektacytometer, RheoScan-D (RheoMeditech, Korea) in clinical application. METHODS: Effects of oxidative stress on erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Before exposure to tBHP, the erythrocytes were pretreated with verapamil and ascorbic acid to examine their protective effect against oxidative damages. The deformability of erythrocytes was measured by the microfluidic ektacytometer, RheoScan-D. RESULTS: When treated with tBHP, the deformability of erythrocytes was decreased (P<0.01) and methemoglobin (metHb) formation and mean corpuscular volume (MCV) of erythrocytes were increased (P<0.01, P<0.05) compared to those of the untreated control cells. Compared to the tBHP treated cells, pretreatment with verapamil increased the deformability of erythrocytes (P<0.01) and decreased metHb formation (P<0.01) and MCV (P<0.05). Likewise, pretreatment with ascorbic acid increased the deformability of erythrocytes (P<0.01) and decreased metHb formation (P<0.01). CONCLUSIONS: Oxidative stress reduces the deformability of erythrocytes and the deformability could be one of markers for oxidative damage. Verapamil and ascorbic acid have protective role against tBHP induced oxidative stress. The ektacytometer, RheoScan-D used in this study is convenient for clinical measurement and could be used in various fields of clinical medicine.
Adult ; Antioxidants/pharmacology ; Ascorbic Acid/*pharmacology ; Calcium Channel Blockers/pharmacology ; Erythrocyte Deformability/*drug effects ; Erythrocytes/drug effects/physiology ; Humans ; Male ; Microfluidic Analytical Techniques/*instrumentation ; Oxidative Stress ; Statistics, Nonparametric ; Verapamil/*pharmacology ; tert-Butylhydroperoxide/*pharmacology