Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
Adult ; Caspases/metabolism* ; Cell Death ; Enzyme Activation ; Humans ; Male ; Mitochondria/pathology* ; Necrosis ; Nitrosative Stress/physiology* ; Permeability ; Peroxynitrous Acid/pharmacology* ; Phosphatidylserines/metabolism* ; Spermatozoa/ultrastructure*

Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.
Apoptosis ; Blood Platelets ; cytology ; metabolism ; Caspase 3 ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Phosphatidylserines ; metabolism ; Protein Kinase C ; metabolism

Phosphatidylserine (PS) is an amphiphilic phospholipid ubiquitously present in the inside of the membrane of prokaryotic and eukaryotic cells. In mammalian cells, there are two synthetic pathways for PS that are different from those of bacterial biosynthesis. The translocation of sperm PS from the inner to the outer leaflet of the plasma membrane is considered to be associated with sperm apoptosis and male infertility. The level of PS externalization in human sperm is used as an indicator for the evaluation of sperm quality. Fast separation of PS-externalized sperm at the molecular level by flow cytometry or magnetic activated cell sorting can effectively improve the quality of sperm and the success rate of assisted reproductive technology. This paper reviews the structure properties, distribution, biological activity and synthesis of PS, as well as its association with male reproduction.
Animals ; Humans ; Infertility, Male ; metabolism ; Male ; Phosphatidylserines ; biosynthesis ; metabolism

OBJECTIVETo investigate the expression and procoagulant activity of phosphatidylserine (PS) on the normal peripheral blood cells of adults.

METHODSNormal peripheral blood samples were collected from 10 healthy volunteers (5 ml from each volunteer), platelets, neutrophils, lymphocytes and erythrocytes were isolated. The expression and procoagulant activity of PS on normal blood cells were identified by flow cytometry, inhibition test with lactadherin as PS probe and coagulation anticoagulant, respectively.

RESULTSThere was PS expression on a few normal blood cells (9.1%, 5.4%, 3.9% and 3.2% in platelets, neutrophils, lymphocytes and erythrocytes, respectively). The PS on these normal blood cells in vitro showed significant procoagulant activity. The plasma recalcification time was shortened by 47%, 36.5%, 25% and 12.5% by platelets, neutrophils, lymphocytes and erythrocytes, respectively; the formation of factor Xa (through both intrinsic and extrinsic pathways) and thrombin was also increased by 13% - 26% by platelets, neutrophils, lymphocytes and erythrocytes, respectively.

CONCLUSIONThe PS on normal blood cells in vivo may play a crucial role in the coagulation cascade.

Adult ; Blood Cells ; metabolism ; physiology ; Blood Coagulation Tests ; Female ; Flow Cytometry ; Humans ; Male ; Phosphatidylserines ; metabolism

BACKGROUNDUp to date, there is few satisfactory pharmacotherapy, except for aspirin and heparin, to stop the preeclampsia progression. Although the mechanism of preeclampsia is poorly understood, it has been proven to be associated with coagulation activation. Researches on prophylactic and therapeutic application of anticoagulants may benefit the clinical aspects of preeclampsia individuals. This study aimed to evaluate the effects of Danshensu on maternal syndrome in phosphatidylserine/phosphatidylcholine (PS/PC) microvesicle induced-mouse model.

METHODSSixty-six preeclampsia-like pregnant mice, induced by PS/PC microvesicle administration, were randomly divided into six groups. From days 5.5 to 16.5 of pregnancy, each group was respectively treated as follows: a) mice in group C (n = 12, control group) were injected with 100 microl of filtered phosphate-buffered saline into the tail vein every day; b) group PE (n = 15, preeclampsia model group) were injected in the same way with 100 microl of filtered PS/PC vesicle suspension; c) group H (n = 9, group treated with heparin) were injected with 1 unit heparin together with PS/PC vesicle suspension; d) group A (n = 10, group treated with aspirin) were injected with 20 microg/g aspirin-DL lysine as well; e) group LD (n = 10, group treated with low-dose Danshensu) were injected with 10 microg/g Danshensu; and f) group HD (n = 10, group treated with high-dose Danshensu) were injected with 30 microg/g Danshensu. Systolic blood pressure, total urinary protein levels, blood tests for some hemostatic function parameters (mean platelet counts, plasma antithrombin III activity (AT-III), D-D dimmer levels, and thrombin time), fibrin deposition by phosphotungstic acid hematoxylin staining, and thrombomodulin expression by immunohistochemistry staining in placentas were examined as indices for maternal syndrome.

RESULTSHeparin showed significant effects on maternal syndrome of preeclampsia such as hypertension and proteinuria, and different doses of Danshensu also presented the certain effects. High-dose Danshensu and aspirin all demonstrated better effects than low-dose Danshensu on decreasing blood pressure to normal level, while high-dose Danshensu demonstrated better effects than aspirin and low-dose Danshensu on decreasing proteinuria to normal level. As to Danshensu's effects on hemostatic function, high- and low-dose Danshensu's marked effects on increasing the plasma AT-III activity were the same as that of aspirin and inferior to that of heparin. High-dose Danshensu's better effect on elevating the platelet counts was superior to low-dose Danshensu and aspirin. Low-dose Danshensu's obvious effect on decreasing D-D levels was close to heparin and superior to high-dose Danshensu and aspirin. High- and low-dose Danshensu's significant effects on reduced thrombin time level are same to heparin. Different anticoagulants all played improvement roles in placental fibrin depositions, but heparin and high-dose Danshensu's roles on lowering thrombomodulin expression in placentas were superior to low-dose Danshensu and aspirin. However, anticoagulant function of high-dose Danshensu was still inferior to heparin. We found long-term use of heparin and aspirin, in spite of low-dose administration, could raise the risk of bleeding such as placental abruption and intestinal hemorrhage. But no any side effect was observed in mice treated with different doses of Danshensu in our study.

CONCLUSIONSDanshensu has proven to be effective and safe in ameliorating the prognosis of maternal syndrome in a preeclampsia mouse model. We suggest long-term provision of low-dose Danshensu in pregnancy, leading to an improvement of preeclampsia syndrome with considerable maternal safety.

Animals ; Anticoagulants ; therapeutic use ; Aspirin ; therapeutic use ; Disease Models, Animal ; Female ; Heparin ; therapeutic use ; Lactates ; therapeutic use ; Mice ; Mice, Inbred ICR ; Phosphatidylcholines ; adverse effects ; Phosphatidylserines ; adverse effects ; Placenta ; metabolism ; Pre-Eclampsia ; chemically induced ; prevention & control ; Pregnancy ; Random Allocation ; Thrombomodulin ; metabolism

The role of surfactant protein A (SP-A) in the recognition and clearance of apoptotic cells is well established, but to date, it is still not clear which surface molecules of apoptotic cells are involved in the process. Here we present evidence that phosphatidylserine (PS) is a relevant binding molecule for human SP-A. The binding is Ca(2+)-dependent and is not inhibited by mannose, suggesting that the sugar-binding site of the carbohydrate recognition domain (CRD) of SP-A is not involved. Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells, and this was consistent for Jurkat cells and neutrophils. Supporting these data, confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells. However, we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface, as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.
Annexin A5 ; metabolism ; Apoptosis ; Carboxy-Lyases ; metabolism ; Flow Cytometry ; Humans ; Jurkat Cells ; Microscopy, Confocal ; Neutrophils ; physiology ; Phosphatidylserines ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism

BACKGROUNDThe side effects of cyclosporine therapy include thromboembolic complications. However, the mechanisms underlying the hypercoagulable state induced by cyclosporine are not fully understood. Cyclosporine binds to red blood cells (RBCs) with a high affinity in circulation and alters the membranes of RBCs. Therefore, we propose that such alterations in RBCs membranes play a role in cyclosporine-induced coagulopathy and this disorder may be rectified by lactadherin, a phosphatidylserine binding protein.

METHODSRBCs from healthy adults were treated with various concentrations of cyclosporine. Procoagulant activity of the RBC membrane was measured by the single stage recalcification time and confirmed by detection of tenase and thrombin assembly through enzymatic assays. Inhibition assays of coagulation were carried out in the presence of lactadherin, annexin V or antitissue factor. Phosphatidylserine exposure was detected by flow cytometry and confocal microscopy through binding with fluorescein isothiocyanate (FITC)-labeled lactadherin as well as FITC annexin V.

RESULTSRBCs treated with cyclosporine demonstrated increased procoagulant activity. Cyclosporine treatment markedly shortened the clotting time of RBCs ((305 +/- 10) seconds vs (366 +/- 15) seconds) and increased the generation of intrinsic factor Xase ((7.68 +/- 0.99) nmol/L vs (2.86 +/- 0.11) nmol/L) and thrombin ((15.83 +/- 1.37) nmol/L vs (4.88 +/- 0.13) nmol/L). Flow cytometry and confocal microscopy indicated that cyclosporine treatment induced an increased expression of phosphatidylserine on the RBC membrane. Lactadherin was more sensitive in detecting phosphatidylserine exposure of the RBC membrane than annexin V. The modulating effect of procoagulant activity was concomitant with and dependent on phosphatidylserine exposure. Blocking of phosphatidylserine with lactadherin effectively inhibited over 90% of FXa generation and prothrombinase activity and prolonged coagulation time.

CONCLUSIONSProcoagulant properties of RBCs membranes resulting from phosphatidylserine exposure may play an important role in cyclosporine-induced thrombosis. Lactadherin can be used as a sensitive probe for phosphatidylserine detection. Its high affinity for phosphatidylserine may provide a new approach for the treatment of cyclosporine induced thrombogenic properties.

Adult ; Animals ; Annexin A5 ; chemistry ; Cattle ; Cell Membrane ; drug effects ; metabolism ; Cells, Cultured ; Cyclosporine ; pharmacology ; Erythrocytes ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Membrane Glycoproteins ; chemistry ; Microscopy, Confocal ; Milk Proteins ; chemistry ; Phosphatidylserines ; chemistry ; metabolism ; Thrombosis ; chemically induced ; metabolism

AIMTo determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.

METHODSAcrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.

RESULTSAlthough sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.

CONCLUSIONThe ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Acrosome ; drug effects ; physiology ; Acrosome Reaction ; drug effects ; Annexin A5 ; metabolism ; Anti-Bacterial Agents ; pharmacology ; Calcimycin ; pharmacology ; Ethidium ; Flow Cytometry ; Fluorescent Dyes ; Humans ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Pancreatic Elastase ; metabolism ; Phosphatidylserines ; metabolism ; Phospholipases A2, Secretory ; metabolism ; Semen ; cytology ; drug effects ; Spermatozoa ; drug effects ; enzymology

Though high concentration of glucose can benefit the survival of lyophilized human red blood cells, the high concentration of glucose can result in serious damage of red blood cells. This study was aimed to investigate the inhibitory effect of trehalose on damage of red blood cells induced by high concentration of glucose. After incubation with the high concentration of glucose buffer containing different concentrations of trehalose for three hours at 37 degrees C, the phosphatidylserine exposure and the osmotic fragility of cells were analyzed by flow cytometry and the lipid peroxidation of membrane was evaluated by TBA method. The results showed that the high concentration of glucose could lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage which were dependent on the glucose concentrations and incubation temperature. However, trehalose could effectively prevent the phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage induced by high concentration glucose. With increase of the trehalose concentrations. As the trehalose concentration increases, the phosphatidylserine exposure, maloni-aldehyde concentration and cell debris rate decreased gradually. In conclusion, the high concentration of glucose can lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage of red blood cells. However, trehalose can inhibit the damaging effects of high concentration of glucose on red blood cells, which may be useful for the application of sugars to lyophilization of red blood cells.
Blood Preservation ; methods ; Erythrocyte Membrane ; drug effects ; metabolism ; Erythrocytes ; drug effects ; Flow Cytometry ; Glucose ; adverse effects ; Humans ; Lipid Peroxidation ; drug effects ; Membrane Lipids ; metabolism ; Osmotic Fragility ; drug effects ; Phosphatidylserines ; pharmacology ; Trehalose ; pharmacology

This study was aimed to investigate the mechanism of phosphatidylserine exposure of human erythrocytes induced by high concentrated glucose. After exposure to high concentrated glucose, the phosphatidylserine (PS) exposure and forward scatter value were analyzed by flow cytometry; the activities of caspase-3 and caspase-8 were detected; The inhibitory effect of leupeptin on cell PS exposure induced by high concentrated glucose was observed by flow cytometry and fluorescent microscopy. The results showed that the high concentrated glucose could induce PS exposure of erythrocytes and this inducing efficiency was dependent on the glucose concentrations. With increase of the glucose concentrations, the percentages of cells with exposed PS also increased. When the glucose concentration was 0.8 mol/L, the PS exposure was over 80%. However, caspase-3 and caspase-8 were not activated during PS exposure of cells induced by high concentrated glucose, but leupeptin could significantly inhibit PS exposure and volume shrinkage induced by high concentrated glucose. With increase of the leupeptin concentrations, the percentage of cells with exposed PS decreased and the cell volume increased. It is concluded that the high concentrated glucose can result in serious PS exposure, which does not depend on caspase. It can be hypothesized that the PS exposure of erythrocytes induced by high concentrated glucose may be controlled by an unknown pathway sensitive to leupeptin.
Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Erythrocytes ; drug effects ; metabolism ; Flow Cytometry ; Glucose ; administration & dosage ; pharmacology ; Humans ; Phosphatidylserines ; biosynthesis