Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with annexin V

BACKGROUND: Annexin V binding to platelets (PLTs) is considered the gold standard for monitoring phosphatidylserine (PS) exposure. However, recent comparison of annexin V with the new calcium-independent PS probe lactadherin revealed that annexin V requires a certain threshold of PS exposure (2%-8%) for binding to occur. The aim of this study was to compare annexin V and lactadherin labeling of PLTs in PLT concentrates (PCs).
STUDY DESIGN AND METHODS: Optimal labeling conditions for lactadherin and annexin V were established and then compared in either resting or calcium ionophore (CI)-activated PLTs from normal whole blood. Furthermore, 40 PCs (20 apheresis-derived and 20 pooled buffy coat–derived) were stored under standard blood bank conditions and PLT activation was monitored by measuring PS exposure with annexin V and lactadherin along with CD42b, CD61, and CD62P by flow cytometry on Days 1, 3, 5, and 7.
RESULTS: Lactadherin reported a higher exposure of PS than did annexin V in normal PLTs at submaximal doses of CI. PLTs from both types of concentrate, as expected, demonstrated evidence of increased activation during storage using annexin V, lactadherin, CD42b, or CD62P. However, a significantly higher percentage of PS-positive PLTs was found with lactadherin than annexin V.
CONCLUSION: PS exposure on the surface of stored PLTs has been previously underestimated due to the wide use of annexin V. Lactadherin provides a truer reflection of the degree of PS exposure and offers a new calcium-independent approach to studying PLT activation and/or apoptosis.     

重组膜联蛋白Ⅱ表达载体构建及其促纤溶特性研究

膜联蛋白Ⅱ(annexinⅡ)是纤溶酶原和组织型纤溶原激活物的共同受体。本研究旨在探讨annexinⅡ引起急性白血病及其它肿瘤患原发性纤溶亢进的分子病理机制，阐明annexinⅡ促纤溶活性，为探讨annexinⅡ在凝血障碍中的作用提供理想的载体模型。用逆转录聚合酶链反应(RT—PCR)扩增annexinⅡ基因片段，经纯化及连接，用脂质体转染入HL－60细胞中表达；用多光子激发的共聚焦显微镜观察其表达产物的胞内分布及结构特性；用流式细胞术及Western印迹对转染的细胞表达进行定量及定性分析，并揭示其对纤溶酶活性的影响。结果显示：成功地构建了pZeoSV2(＋)／ANNⅡ真核表达质粒，经转染ⅡL—60细胞后，可表达annexinⅡ活性蛋白，荧光检测显示其表达蛋白分布在转染细胞的膜表面；FCM检测annexinⅡ在转染后48小时呈高表达；annexinⅡ可显增加纤溶酶的活性，且瞬时表达及稳定表达具有相同的结果。annexinⅡ反义寡核苷酸与单抗均可显抑制annexinⅡ的促纤溶活性(P＜0.01)。结论：annexinⅡ在纤维蛋白溶解中具有重要作用，用本研究构建的annexinⅡ重组载体可为今后的相关研究提供实验基础，并为防治出血及血栓性疾病研究有可能提供新的思路和开辟新的途径。     

Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity

BACKGROUND: Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity. OBJECTIVE: To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DeltaPsi(m)), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression. METHODS: Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca(2+)-ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DeltaPsi(m) collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. RESULTS AND CONCLUSIONS: Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DeltaPsi(m) collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.     

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor,Histone H2B,to the macrophage surface

Summary. Background: Plasminogen (Plg) binding to cell surface Plg receptors (Plg‐Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg‐R and its cell surface expression is up‐regulated when monocytes are differentiated to macrophages via a pathway dependent on L‐type Ca²⁺ channels and intracellular Ca²⁺. Objectives: We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on the macrophage surface. Methods: THP‐1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outer membrane exposure of phosphatidylserine (PS). Results: H2B interacted directly with PS via an electrostatic interaction. Anti‐PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L‐type Ca²⁺ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions: These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L‐type Ca²⁺ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to.     

Diannexin,an annexin A5 homodimer,binds phosphatidylserine with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation

Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half‐life. Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet‐mediated events during thrombus formation. Methods: The affinities of Diannexin and annexin A5 for PS‐containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet‐fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser‐induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results: Diannexin and annexin A5 bind PS with K_D values of 0.6 and 5 nm , respectively, and both bind to the same subpopulation of PS‐exposing platelets. Diannexin inhibited thrombin generation and platelet‐fibrin clot formation in vitro at 10 nm (P < 0.05–0.001 compared with control), and reduced platelet accumulation at 1 μg g^?1 (P < 0.05) and activation at 0.25 μg g^?1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g^?1 (P < 0.01). Conclusions: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation.     

99mTc-AnnexinB1的制备及其探测细胞凋亡的实验研究

目的 制备99mTc-Annexin B1并对其体外、体内生物分布以及探测体内细胞凋亡的潜力进行评价.方法 应用99mTc直接标记蛋白质的方法制备99mTc-Annexin B1.采用与活化人血小板结合实验检测99mTc-Annexin B1的体外生物活性.在正常小鼠体内进行生物分布研究.采用地塞米松诱导小鼠胸腺凋亡的动物模型和anti-Fas单抗诱导小鼠肝脏凋亡的动物模型检测99mTc-Annexin B1探测体内细胞凋亡的潜力,并用TUNEL染色证实细胞凋亡.结果 直接标记法制备的99mTc-Annexin B1具有很高的放射化学纯度和很好体外稳定性.与活化人血小板的结合实验表明,99mTc-Annexin B1具有与PS结合的体外生物活性.99mTc-Annexin B1在体内具有较快的清除特性,主要聚集于肾脏.地塞米松诱导18 h后,小鼠胸腺对99mTc-Annexin B1的摄取显著高于对照小鼠胸腺的摄取.Anti-Fas诱导后2 h时后,小鼠肝脏对99mTc-AnnexinB1的摄取显著高于为对照动物的肝脏摄取.结论 99mTc-Annexin B1具有与PS结合的体外生物活性和探测体内细胞凋亡的潜力,是一种新的细胞凋亡显像剂.     

Allyl‐isatin suppresses cell viability,induces cell cycle arrest,and promotes cell apoptosis in hepatocellular carcinoma HepG2 cells

The anticancer effect of the newly synthesized isatin derivative, N‐allyl‐isatin (Allyl‐I), was evaluated in vitro with human hepatocellular carcinoma HepG2 cells. Cell viability was detected by cell counting kit‐8 (CCK8) assay. Acridine orange (AO)/ethidium bromide (EB) double staining was used to observe the cell morphology. Flow cytometry was used to assess the effects of Allyl‐I on the cell cycle, apoptosis rate, and mitochondrial membrane potential (MMP). Western blot analysis was performed to detect the influence of Ally1‐I on the expression of cytochrome c (cyt c), Bax, Bcl‐2, and cleaved caspase‐3. Allyl‐I significantly inhibited HepG2 cell viability in a time‐ and dose‐dependent manner. Allyl‐I can induce cell cycle arrest in HepG2 cells at the G2/M phase. Apoptotic nuclear morphological changes were observed after AO/EB double staining. Fluorescein isothiocyanate‐conjugated Annexin V (Annexin V‐FITC) and propidium iodide (PI) double staining showed that the apoptotic rates significantly increased in the presence of Allyl‐I. Rhodamine 123 staining indicated that Allyl‐I can decrease the MMP. Allyl‐I also altered the expression of mitochondrial apoptosis‐related proteins. Protein levels of cyt c and cleaved caspase‐3 were upregulated following Allyl‐I treatment. By contrast, the Bcl‐2/Bax ratio decreased. Results suggest that Allyl‐I suppresses cell viability, induces cell cycle arrest, and promotes cell apoptosis in HepG2 cells. Furthermore, the induction of apoptosis might be correlated with the mitochondrial pathway.     

Development of Antibody-Carrying Microbubbles Based on Clinically Available Ultrasound Contrast Agent for Targeted Molecular Imaging: A Preliminary Chemical Study

Purpose

The purpose of the study was to examine the feasibility of an antibody-carrying targeted-bubble preparation using clinically available phosphatidylserine (PS)-containing perfluorobutane-filled microbubbles for molecular ultrasound imaging.

Procedures

Firstly, we examined whether PS on the surface of perfluorobutane-filled microbubbles could be detected by means of flow cytometry (fluorescence activated cell sorting (FACS)) using annexin V. After conjugation with fluorescein isothiocyanate (FITC)-labeled annexin V (up to 50 ??L) for 15 min on ice, microbubbles were assessed using a FACSCalibur. Secondly, we examined whether phycoerythrin (PE)-labeled streptavidin could be attached onto PS-containing perfluorobutane-filled microbubbles through the intermediacy of biotinylated annexin V. Microbubbles conjugated with biotinylated annexin V were incubated with PE?Cstreptavidin for 30 min on ice, then FACS analysis was performed. Finally, we examined whether attachment of biotinylated IgG onto PS-containing perfluorobutane-filled microbubbles could be accomplished using biotinylated annexin V and avidin?Cbiotin binding. Microbubbles with avidin?Cbiotin complexes were incubated with Alexa488-labeled biotinylated IgG for 30 min on ice.

Results

FITC-positive microbubbles could be detected after conjugation with FITC?Cannexin V. Additionally, the mean fluorescence intensity of Sonazoid bubbles increased in a dose-dependent manner (0 ??L, 3.3 vs. 50 ??L, 617.1). The PE signal of microbubbles in the presence of biotinylated annexin V was higher than that in the absence of biotinylated annexin V (mean fluorescence intensity, 327.1 vs. 14.8). Significant amplification of the Alexa488-signal was accomplished through the intermediation of biotinylated annexin V and streptavidin.

Conclusions

Our results support the feasibility of an antibody-carrying targeted-bubble preparation based on clinically available PS-containing perfluorobutane-filled microbubbles. Although further study is needed, this technique could be applicable for in vivo molecular ultrasound imaging.     

Effects of apoptosis and lipid peroxidation on T-lymphoblastoid phospholipid-dependent procoagulant activity

Summary.  Background:  Coagulation has an absolute requirement for macromolecular complexes to be assembled on a negatively charged phospholipid (PL) surface. Previously, we reported that malignant T-lymphoblastoid cells have the ability to support procoagulant activity (PCA) independently of tissue factor by providing such a surface. Objective:  To explore the effect of two pathophysiologic processes, apoptosis and lipid peroxidation (LP), on this PL-dependent PCA. Methods:  Three different assays for PL-dependent PCA (factor IXa-initiated FXa and thrombin generation and prothrombinase activity) were used to investigate this PCA after exposing three T-lymphoblastoid cell lines to either apoptotic stimuli (1 μ m staurosporine) or oxidative stress (4 m m H₂O₂ and 40 μ m CuSO₄). Surface exposure of anionic PL was measured by flow cytometry using annexin  A5^FITC and an antibody (3G4) specific for native, but not oxidized, phosphatidylserine (PS). Results and Conclusions:  Both apoptosis and LP significantly enhanced the PCA of cells, to a level that was greater than that observed following calcium ionophore treatment, suggesting that the increased activity was not solely due to anionic PL exposure. Whereas cells undergoing apoptosis bound both annexin  A5^FITC and 3G4, only annexin  A5^FITC bound to cells undergoing LP. This implies that apoptosis increases PCA by causing the translocation of oxidized/native PS to the outer membrane, whereas LP appears to increase the PCA, possibly due to malondialdehyde adducts altering the net charge on the cell surface, which allows PLs other than PS to participate in thrombin generation.     

Dual-colour (near-infrared/visible) emitting annexin V for fluorescence imaging of tumour cell apoptosis in vitro and in vivo

Indocyanine green (ICG) labelled recombinant annexin V proteins (ICG–EGFP–Annexin V and ICG–mPlum–Annexin V) were synthesized for dual-colour fluorescence imaging of tumour cell apoptosis in vitro and in vivo. The ICG-labelled fluorescent annexin V proteins showed dual (near-infrared and visible) fluorescence emissions with binding ability to phosphatidylserines on the plasma membranes of apoptotic cells. Although several types of fluorescence labelled annexin V (e.g. FITC–annexin V, Cy3- and Cy5-annexin V) have been reported, there are no dual-colour (near-infrared/visible) emitting apoptosis-detection probes which can be used in vitro and in vivo. In this paper, the utilities of the dual-colour fluorescent annexin V are demonstrated for in vitro and in vivo fluorescence imaging of the apoptosis of human breast tumour cells induced by an antibody–drug conjugate, Kadcyla. The results suggest that the present annexin V probes will be useful to visualize the action of anti-cancer drugs against tumours both at the cellular and whole-body level.

Indocyanine green labeled recombinant annexin V probes (ICG–EGFP–Annexin V and ICG–mPlum–Annexin V) were synthesized for near-infrared and visible fluorescence imaging of tumor cell apoptosis both in vitro and in vivo.     

抗血小板整合素β3抗体诱导内皮细胞凋亡机制的研究

目的:探讨抗血小板整合素β3抗体对人脐静脉血管内皮细胞(HUVEC)的损伤及其相关机制.方法:收集36例慢性免疫性血小板减少症患者的血清,通过流式细胞术和单克隆抗体特异性俘获血小板抗原技术(MAIPA)筛选出含抗整合素β3抗体的患者血清.用抗整合素β3血清处理HUVEC后,乳酸脱氢酶(LDH)活性测定法检测HUVEC损...     

Localization of annexin V in rat normal kidney and experimental glomerulonephritis

The localization of annexin V, a calcium binding protein, was immunochemically and immunohistologically studied in experimental rat glomerulonephritis using annexin V polyclonal antibody. Plasma and urinary annexin V levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Urinary annexin V level, which was correlated with urinary l-lactate dehydrogenase activity, N-acetyl-β-d-glucosaminidase activity and protein level, increased time-dependently after the injection of nephritogenic antigen (bovine glomerular basement membrane), progressively increasing to attain a peak level at 4 weeks of 51.5±11.3 ng/h. However, plasma annexin V level showed no increase during the study period. Normal kidneys showed strong staining for annexin V in distal tubules, being particularly strong in tubules of the inner stripe of the outer medulla, but could not be detected in proximal tubules. Annexin V was seen in visceral epithelial cells, Bowman’s capsule of the glomerulus, the vascular endothelium of arterioles and interlobular arteries, and vascular smooth muscle. In nephritis, the lumen of distal tubules and the luminal cell membrane were deeply stained, with leakage of annexin V being observed from tubular cells. In the present study, renal annexin V was markedly excreted into urine, and its urinary level reflected the severity of damage of renal tissue and the progression of nephritis. These changes of annexin V in the distal tubule and visceral epithelial cells may be of significance in cell injury of the kidney.     

Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia

Summary. Background: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As₂O₃). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. Methods: Lactadherin, a milk protein with stereospecific binding to phosphatidyl‐L‐serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. Results: Plasma procoagulant activity of NB4 and APL cells increased approximately 15‐fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As₂O₃. Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As₂O₃ and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80–85% of intrinsic FXase, FVIIa‐tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low‐level PS exposure. Conclusions: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.     

The presence of antiphospholipid antibodies is not related to increased levels of annexin A5 in plasma

Summary.  Annexin A5 has been proposed to be important for shielding of negatively charged phospholipids from blood, thereby preventing the binding of clotting factors. It has been suggested that antiphospholipid antibodies can disrupt the binding of annexin A5 from negatively phospholipid-containing surfaces, resulting in uncontrolled coagulation. If this hypothesis is correct, than the plasma levels of annexin A5 will be increased in patients with antiphospholipid antibodies. Therefore, we have measured plasma levels of annexin A5 of 175 patients with systemic lupus erythematosus (SLE), of which 104 had antiphospholipid antibodies and 23 patients had primary antiphospholipid syndrome. The annexin A5 levels were compared with the annexin A5 plasma levels measured in 23 patients with diabetes mellitus type 2 and 35 healthy volunteers. We found a significant increase of annexin A5 plasma levels in patients with SLE (median 6.7 ng mL⁻¹) and primary antiphospholipid syndrome (median 7.1 ng mL⁻¹) as compared to patients with diabetes mellitus type 2 (median 3.3 ng mL⁻¹) and healthy volunteers (median 3.9 ng mL⁻¹). However, no correlation was found with the presence of antiphospholipid antibodies or with a history of thromboembolic complications. Based on these observations, we conclude that displacement of annexin A5 from cellular surfaces by antiphospholipid antibodies is not a common mechanism in patients with antiphospholipid antibodies.     

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence.

BACKGROUND: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. OBJECTIVE: To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. METHODS: Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. RESULTS: Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. CONCLUSIONS: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.     

Characterization of the cell-surface procoagulant activity of T-lymphoblastoid cell lines

Summary.  The procoagulant activity (PCA) of four T-lymphoblastoid cell lines (CEM-CCRF, Jurkat, Molt-4 and A3.01) at different stages of differentiation has been characterized and compared with that of a monocytoid cell line (THP-1). Four assay systems were employed; the activated partial thromboplastin time (APTT); prothrombin time/tissue factor (TF) activity; a purified factor (F)Xa generation system and cancer procoagulant. High levels of TF activity were seen only with the monocytic cells. However the more differentiated of the T-lymphoblastoid cells (Molt-4 and A3.01) were more active than monocytic cells in supporting FXa generation. This pattern was not repeated for the APTT assay, which was related to cell-surface TF activity, since it was partially inhibited by antiTF antibody. Annexin V totally inhibited the activity observed in all three assay systems, indicating that the PCA of T-lymphoblastoid cells is primarily due to expression of negatively charged phospholipids. However, antiphosphatidylserine antibody even at a high concentration gave only partial inhibition of the activity observed in the APTT and FXa generation systems for the cells compared with almost total inhibition for the phospholipid standard, suggesting either that cellular phosphatidylserine (PS) is less accessible to the antibody, or that PS is not the sole negatively charged phospholipid responsible for this activity. Flow cytometry studies using propidium iodide and annexin V showed that the PCA, although linked to PS exposure, was not the result of apoptosis.     

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

BACKGROUND: That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. OBJECTIVES: In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. METHODS: The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. RESULTS: TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. CONCLUSION: C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.     

Microbubble-Assisted Ultrasound-Induced Transient Phosphatidylserine Translocation

Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells. Sonopermeabilization induced transient membrane permeabilization, which is positively correlated with reversible PS externalization. This membrane disorganization was temporary and not associated with loss of cell viability. Sonopermeabilization did not induce PS externalization via activation of the scramblase. We hypothesize that acoustically induced membrane pores may provide a new pathway for PS migration between both membrane leaflets. During the membrane-resealing phase, PS asymmetry may be re-established by amino-phospholipid flippase activity and/or endocytosis, along with exocytosis processes.     

The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis.

The pathogenic role of antiendothelial cell antibodies (AECA) remains unclear. They are frequently associated with antibodies to anionic phospholipids (PL), such as phosphatidylserine (PS), which is difficult to reconcile with the distribution of PL molecular species within the plasma membrane. Since it is already known that PS is transferred to the outer face of the membrane as a preclude to apoptosis, the possibility exists that apoptosis is initiated by AECA. AECA-positive/anti-PL antibody-negative sera from eight patients with systemic sclerosis (SS) and 21 control patients were evaluated. Endothelial cells (EC) were incubated with AECA and the exposure of PS was established through the binding of annexin V. Hypoploid cell enumeration, DNA fragmentation, and optical and ultrastructural analyses of EC were used to confirm apoptosis. Incubation of EC with AECA derived from six of eight patients with SS led to the expression of PS on the surface of the cells. This phenomenon was significantly more frequent in SS (P < 0.04) than in control diseases. The redistribution of plasma membrane PS preceded other events associated with apoptosis: hypoploidy, DNA fragmentation, and morphology characteristic for apoptosis. Apoptosis-inducing AECA did not recognize the Fas receptor. We conclude that AECA may be pathogenic by inducing apoptosis.     

Removal of Plasmodium falciparum–infected red blood cells from whole blood by leukoreduction filters

BACKGROUND: There has been an unexplained decrease in the incidence of transfusion‐transmitted malaria in recent years. The decrease in incidence has paralleled the increasing use of leukoreduction filters. Malaria‐infected red blood cells (RBCs) share surface characteristics of hemoglobin S–containing cells. Because units collected from donors with sickle trait do not filter optimally due to adherence of RBCs to the filters, the possibility that malaria‐infected RBCs may also adhere to filters was investigated. STUDY DESIGN AND METHODS: Malaria‐infected whole blood or calcium ionophore (A25187)‐treated and control RBCs were filtered with leukoreduction filters. Quantitation of malaria‐infected RBCs before and after filtration was performed by flow cytometry to determine the presence of DNA within RBCs, indicating malaria infection. Annexin V binding was also determined before and after filtration of RBCs treated with A25187. Immediately after filtration, filters were fixed and examined by scanning and transmission electron microscopy. RESULTS: There were at least three configurations of adherence of malaria‐infected RBCs demonstrated within the filters. The first was direct adherence of infected RBCs to filter fibers; the second involved adherence of malaria‐infected RBCs to platelets, which were adherent to filter fibers; and the third was adherence of infected RBCs to other RBCs. Filtration also resulted in preferential removal of phosphatidylserine (PS)‐expressing cells as seen by the reduction of annexin V binding after filtration. This was further confirmed by electron micrographic examination of the filters in which untreated RBCs sit within the filter resting on top of filter fibers; however, calcium ionophore–treated RBCs are seen to cling tightly to the fibers. CONCLUSIONS: PS expression by RBCs leads to their adherence within leukoreduction filters. Malaria‐infected RBCs are retained via more than one mechanism. The efficiency of removal requires further study.