Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

Tissue factor de-encryption: ionophore treatment induces changes in tissue factor activity by phosphatidylserine-dependent and -independent mechanisms.

Coagulation is initiated on tissue-factor-bearing cells when factor VIIa complexes with membrane-bound tissue factor and activates factors X and IX. Cellular tissue factor activity does not correlate with tissue factor antigen; treatment with calcium ionophore rapidly increases tissue factor activity without increasing tissue factor antigen. Our study examined the effect of calcium ionophore A23187 on tissue factor activity of freshly isolated, lipopolysaccharide-stimulated monocytes and non-transformed human dermal fibroblasts. A23187 increased tissue factor activity on monocytes and fibroblasts in a dose-dependent fashion between 0.1 and 50 micromol/l ionophore. This increase in activity was proportional to an increase in intracellular calcium in monocytes. The increase in tissue factor activity was partially attributable to an increase in phosphatidylserine expression, as measured by increased prothrombinase activity (1.1- to 4-fold) on ionophore-treated cells. The phosphatidylserine-binding protein annexin V decreased tissue factor activity on both ionophore-treated and untreated cells, reflecting the role of phosphatidylserine in tissue factor activity. However, even in the presence of saturating concentrations of annexin V, the tissue factor activity of ionophore-treated cells was 1.3- to 11.3-fold higher than that of untreated cells, indicating that the increase in tissue factor activity did not result solely from increased expression of phosphatidylserine. A23187 increased tissue-factor-dependent activation of factors IX and X 1.4- to 7-fold on both cell types, indicating that ionophore treatment did not alter factor VIIa/tissue factor substrate specificity. We conclude that the mechanism by which calcium ionophore increases tissue factor activity is not unique to monocytoid or transformed cells. Furthermore, the ionophore-induced increase in activity is not solely the result of increased exposure to phosphatidylserine. Finally, tissue factor de-encryption by A23187 does not alter factor VIIa/tissue factor substrate specificity.     

In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with β-thalassaemia major

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with β-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly ( P  < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation ( P  < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelarate thrombin generation in vivo which, in turn, triggers platelet activation.     

Role of FcRgamma and factor XIIIA in coated platelet formation

Platelet activation in response to dual stimulation with collagen and thrombin results in the formation of a subpopulation of activated platelets known as coated platelets. Coated platelets are characterized by high surface levels of alpha-granule proteins and phosphatidylserine, which support the assembly of procoagulant protein complexes. Using murine models, we tested the hypothesis that the collagen receptor-associated molecule FcRgamma and the transglutaminase factor XIIIA are required for the formation of coated platelets. Following dual stimulation with the collagen receptor agonist convulxin and thrombin, 68% of platelets from C57BL/6 mice acquired the coated platelet phenotype, defined by high surface levels of fibrinogen and von Willebrand factor and decreased binding of the alphaIIbbeta3 activation-dependent antibody PE-JON/A. In FcRgamma-/- mice, only 10% of platelets became "coated" after dual stimulation with convulxin plus thrombin (P < .05 vs C57BL/6 platelets). Decreased coated platelet formation in FcRgamma-/- platelets was accompanied by decreased annexin V binding (P < .01) and decreased platelet procoagulant activity (P < .05). Platelets from FXIIIA-/- mice did not differ from control platelets in coated platelet formation or annexin V binding. We conclude that FcRgamma, but not factor XIIIA, is essential for formation of highly procoagulant coated platelets following dual stimulation with collagen and thrombin.     

Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity

The activation of human platelets by thrombin is mediated primarily by protease-activated receptors (PARs). PAR1 and PAR4 are present on human platelets and are activated by the hexapeptides SFLLRN and GYPGQV, respectively. To further characterize the involvement of PAR1 and PAR4 in platelet activation, the ability of SFLLRN or GYPGQV to generate annexin V binding to newly exposed phospholipids on the platelet surface and generate procoagulant activity has been examined. Exposure of phosphatidylserine and phosphatidylethanolamine on platelets, as determined by an increase in annexin V binding, was strongly stimulated by SFLLRN, thrombin, and collagen, but only to a minor extent by GYPGQV. In a clotting assay initiated with factor VIIa, soluble tissue factor, and calcium, the clotting time in the absence of platelets was >5 min. In the presence of unstimulated platelets, the clotting time was 200 +/- 20 sec. In the presence of platelets activated with SFLLRN or collagen, the clotting time decreased to 100 +/- 10 sec. This shortening of the clotting time is equivalent to about a 5-fold increase in coagulant activity when stimulated platelets are compared with unstimulated platelets and activated platelets are used as a reference. These results indicate that thrombin initiates a very strong response in platelets through PAR1, leading to exposure of anionic phospholipids that support blood clotting. The response mediated by PAR4, however, was limited to platelet aggregation and similar to that triggered in platelets by weaker agonists such as ADP or epinephrine.     

抗内皮细胞抗体介导的内皮细胞损伤机制

目的探讨抗内皮细胞抗体（antiendothelial cell antibody，AECA）对内皮细胞凋亡的影响及重组α-烯醇化酶对内皮细胞凋亡的阻断作用。方法以EA．hy926细胞（内皮细胞永生细胞）提取物蛋白为抗原，采用免疫印迹法检测并筛选47000-AECA阳性患者血清，用蛋白G亲和层析法纯化IgG，在体外诱导内皮细胞凋亡，观察重组α-烯醇化酶对凋亡的阻断作用。结果含47000-AECA IgG在体外可诱导内皮细胞凋亡，荧光染色可见明显的核形态变化及典型的凋亡小体；含47000-AECA系统性红斑狼疮患者的IgG作用于EA．hy926细胞24、48和72小时后，凋亡率均明显高于相同剂量正常人kG作用EA．hy926细胞的凋亡率；而经重组α-烯醇化酶预处理后EA．hy926细胞凋亡率则明显降低。含47000-AECA IgG作用于EA．hy926细胞8小时后，AnnexinV^＋细胞数明显增加，并随IgG作用时间和浓度的增加而升高；而经重组α-烯醇化酶预处理的含47000-AECA IgG作用于EA．hy926细胞后，AnnexinV^＋细胞有所减少。结论AECA在体外可诱导内皮细胞凋亡，引起内皮细胞损伤；重组α-烯醇化酶可部分阻断47000-AECA体外诱导内皮细胞凋亡的作用，α-烯醇化酶是AECA识别的自身抗原之一。     

Platelets significantly modify procoagulant activities in haemophilia A

Summary. Haemophilia A replacement therapy is dosed according to patient’s weight and plasma FVIII activity (FVIII:C). The FVIII interacts with platelet membrane but limited data on the impact of platelet procoagulant activity (PCA) are available in haemophilia A. Our aim was to characterize individual PCA in vitro in 20 adult haemophilia A patients at various FVIII:C levels. We detected thrombin generation in platelet‐poor (PPP) and platelet‐rich plasma (PRP) using: (i) calibrated automated thrombography (CAT) triggered with tissue factor, (ii) adhesion‐induced PCA upon collagen and (iii) annexin V binding, expression of P‐selectin and active glycoprotein (GP) IIbIIIa on platelets after stimulation of GPVI with collagen‐related peptide. The FVIII:C levels varied between <1% and 37%. Thrombin generation was individual and strongly enforced by platelets and associated within the three methods. Range of thrombin generation was maximal (up to 30‐fold) at FVIII:C levels 1–5%, underlining the impact of platelets in the presence of traces of replacement therapy. At FVIII:C > 5% platelet contribution in the variance faded. Platelet PCA and P‐selectin exposure lead to a fivefold variation. Intriguingly, at FVIII:C < 1% thrombin generation in PPP associated negatively with platelet GPVI activation, suggestive of a regulatory interplay between plasma and platelets. In haemophilia A, the variability in thrombin generation is partially related to plasma FVIII:C, but mainly dependent on platelet procoagulant capacity. Annexin V binding and PCA in response to activation by collagen receptors contribute to this variability. In all, platelet PCA at least following collagen interaction significantly impacts thrombin generation in haemophilia A.     

Sodium salicylate-triggered apoptosis in HL-60 cells depends on caspase-8 activation

For investigation of the killing and proapoptotic effects of sodium salicylate (Na-Sal) on HL-60 cells, the cytotoxic activity of Na-Sal was measured by means of MTT assay. Apoptosis was identified and analyzed with the help of transmission electron microscopy, annexin V staining, and DNA gel electrophoresis, and the association of caspase-8 activation with apoptosis was determined with the specific protease inhibitor IETD-fmk. After exposure of HL-60 cells to increasing concentrations of Na-Sal (0.5, 1, 3, 5, and 7 mmol/L) for 24 hours, the mean cell viability gradually dropped to 92%, 83%, 68%, 50%, and 42%. With treatment of target cells with 5-mmol/L (IC50) Na-Sal for 6, 12, 24, or 36 hours, the mean cell survival tapered to 91%, 81%, 48% (P <.05 versus control), and 14% (P <.05 versus control). Again incubated with 5-mmol/L Na-Sal for 12 or 24 hours, HL-60 cells displayed clear early or late signs of apoptosis, including (1) notable enhancement of phosphatidylserine externalization, (2) cell shrinkage, membrane blebbing, and eventual disintegration into numerous apoptotic bodies, and (3) formation of ladder DNA. The viability of HL-60 cells increased significantly during 24 or 36 hours of coculture with 100-micromol/L IETD-fmk in combination with 5-mmol/L Na-Sal compared with the viability when 5-mmol/L Na-Sal was used alone (P < .05). Moreover, the target cells showed a considerable decrease in phosphatidylserine exposure and DNA fragmentation after coincubation for 12 or 24 hours performed as described above. The findings presented herein strongly suggest that Na-Sal can exert potent killing and proapoptotic activity against HL-60 cells, and this effect appears to depend on caspase-8 activation.     

Key role of platelet procoagulant activity in tissue factor-and collagen-dependent thrombus formation in arterioles and venules in vivo differential sensitivity to thrombin inhibition

OBJECTIVE: Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation. METHODS: Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels. RESULTS: Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI-mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phosphatidylserine abolished thrombus formation in arterioles and venules, while mutant M1234-annexin A5 was ineffective. Arterial and venous thrombus formations were only slightly affected in mice carrying the factor V Leiden mutation, suggesting insensitivity to factor Va inactivation. CONCLUSIONS: In this microvascular model, the formation of both arterial and venous thrombi relies on collagen-induced platelet activation and tissue factor-induced thrombin generation. Activated, phosphatidylserine-exposing platelets play a key role in thrombus growth in arterioles and venules.     

A soluble tissue factor-annexin V chimeric protein has both procoagulant and anticoagulant properties

Tissue factor (TF) initiates blood coagulation, but its expression in the vascular space requires a finite period of time. We hypothesized that targeting exogenous tissue factor to sites of vascular injury could lead to accelerated hemostasis. Since phosphatidylserine (PS) is exposed on activated cells at sites of vascular injury, we cloned the cDNA for a chimeric protein consisting of the extracellular domain of TF (called soluble TF or sTF) and annexin V, a human PS-binding protein. Both the sTF and annexin V domains had ligand-binding activities consistent with their native counterparts, and the chimera accelerated factor X activation by factor VIIa. The chimera exhibited biphasic effects upon blood coagulation. At low concentrations it accelerated blood coagulation, while at higher concentrations it acted as an anticoagulant. The chimera accelerated coagulation in the presence of either unfractionated or low-molecular-weight heparins more potently than factor VIIa and shortened the bleeding time of mice treated with enoxaparin. The sTF-annexin V chimera is a targeted procoagulant protein that may be useful in accelerating thrombin generation where PS is exposed to the vasculature, such as may occur at sites of vascular injury or within the vasculature of tumors.     

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.     

The role of activated human platelets in prothrombin and factor X activation

The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.     

Lipid rafts are necessary for tonic inhibition of cellular tissue factor procoagulant activity

A fraction of total cellular tissue factor procoagulant activity remains masked or "encrypted" in intact cells. Decryption of this activity partly involves the extracellular exposure of anionic phospholipids such as phosphatidylserine. Because of the potential association of tissue factor and phospholipid scramblase activity with lipid rafts, we have explored the role of lipid rafts in regulating factor VIIa/tissue factor activity. In HEK293 cells, tissue factor antigen was not stably associated with lipid rafts, yet disruption of rafts with methyl-beta-cyclodextrin resulted in a 3-fold stimulation of tissue factor procoagulant activity. Treatment with methyl-beta-cyclodextrin was not associated with cytotoxicity and did not result in the exposure of additional tissue factor antigen. Factor VIIa/tissue factor activity decrypted with methyl-beta-cyclodextrin was quantitatively similar to that obtained by using lytic concentrations of octyl glucoside but more sensitive to inhibition by cell surface tissue factor pathway inhibitor and the phospholipid binding protein, annexin V. Partial decryption of tissue factor was achieved with methyl-beta-cyclodextrin prior to complete disruption of lipid rafts, suggesting the role of an enzyme localized to lipid rafts in the transbilayer transport of phosphatidylserine. We conclude that lipid rafts are required for the maintenance of cellular tissue factor in an encrypted state.     

In vivo detection and imaging of phosphatidylserine expression during programmed cell death

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.     

Anti-endothelial cell antibodies from lupus patients bind to apoptotic endothelial cells promoting macrophage phagocytosis but do not induce apoptosis

OBJECTIVE: Anti-endothelial cell antibodies (AECA) have been reported to induce apoptosis. We investigated the induction of apoptosis by these autoantibodies and their involvement in the removal of apoptotic cells. METHODS: AECA isolated from patients with active systemic lupus erythematosus (SLE) were incubated with human umbilical vein endothelial cells (HUVECs). AECA-positive sera were identified using a cell-based ELISA. Apoptosis was measured by morphology and phosphatidylserine externalization using flow cytometry with fluorescein isothiocyanate (FITC)-conjugated annexin V. Flow cytometry was used to investigate AECA binding to apoptotic cells using FITC-conjugated anti-human immunoglobulin G (IgG). Apoptotic endothelial cells were stained with a red dye (PKH26) and co-cultured with macrophages, and phagocytosis was visualized under phase contrast microscopy. RESULTS: AECA from patients with SLE did not induce apoptosis compared with normal IgG (nIgG) at any time point, as assessed by morphology (at 24 h, P = 0.167) or phosphatidylserine externalization (at 24 h, P = 0.098). However, there was increased binding of AECA to apoptotic endothelial cells (48.8 +/- 11.9 compared with 25.8 +/- 6.7% AECA binding to freshly isolated cells, P< 0.001). These opsonized endothelial cells showed greater phagocytosis by macrophages (mean phagocytic index 24.9 +/- 4.5%) when cells opsonized with nIgG were compared with AECA (34.8 +/- 3.4% n = 5, P = 0.01). CONCLUSION: In conclusion, AECA bind to apoptotic endothelial cells but do not induce endothelial cell apoptosis. Macrophage phagocytosis is increased by opsonization of apoptotic endothelial cells by AECA, a proinflammatory mechanism of cell removal.     

Platelet procoagulant complex assembly in a tissue factor-initiated system

Summary. The aim of this study was to examine the assembly of the factor IXa/VIIIa (Xase) and factor Xa/Va (IIase) complexes on the platelet surface in a system designed to mimic tissue factor-initiated coagulation. The experimental system contained tissue factor-bearing monocytes, unactivated platelets, and plasma concentrations of factors V, VIII, IX, X, prothrombin, tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and small amounts of factor VIIa. The time courses of platelet activation, coagulation factor binding and thrombin generation were compared. In this system, thrombin generation by the combination of monocytes and platelets was synergistic compared to each cell type alone. Platelet activation and thrombin generation were minimal in the absence of prothrombin or factor X. After a lag period, platelet activation began, followed by progressive binding of factors Va and VIIIa. This was followed by factor IXa and Xa binding and the onset of thrombin generation. Unexpectedly, a transient early increase in platelet-associated factor IX and X was also seen, that was due to release from platelets. The amount of factor IX bound to isolated activated platelets was increased by addition of factor VIIIa, or by activation of factor IX to IXa. In contrast, factor VIIIa binding was not altered by the presence of factor IX or IXa. We conclude that in a tissue factor-initiated system, assembly of the procoagulant complexes on the platelet surface begins after platelet activation occurs. Platelet activation requires thrombin generation in the vicinity of the tissue factor bearing cells. The cofactors Va and VIIIa bind to the platelets and facilitate subsequent binding of factors IXa and Xa to form functional procoagulant complexes.     

Melatonin induces apoptosis in human neuroblastoma cancer cells

Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK-N-MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6-diamidino-2-phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase-3 activation. Cell death was dose and time-dependent; the lowest effective concentration of melatonin was 100 microm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.     

Determinants of Prothrombinase Activity and Modification of Prothrombin Conversion by Thrombin-Treated Factor V

S ummary . The development of an intrinsic two-stage assay for factor V using highly purified protein components is described. Phospholipid, activated factor X, calcium ion and factor V are incubated together to form a prothrombinase. After addition of prothrombin, sequential aliquots are removed and tested for thrombin activity using fibrinogen as a substrate. The rate of thrombin generation is linear for at least 10 min and increases with increasing concentrations of factor V or activated factor X. At a fixed concentration of activated factor X, a linear relationship is observed between the logarithm of the initial rate of thrombin generation and the logarithm of the factor V concentration. Oligomeric forms of factor V generate parallel but non-identical concentration curves indicating differing abilities to form prothrombinase.
The rate of thrombin generation eventually decreases and a maximum thrombin concentration is attained. This phenomenon is not attributable to substrate exhaustion or to decay of prothrombinase activity. Evidence for the formation of an inhibitor during prothrombin conversion, which is directed against both prothrombinase and the reaction of thrombin with fibrinogen, is offered.
Activation of factor V by thrombin is demonstrable in the two-stage intrinsic system and in a modified extrinsic sequential technique. In both systems the time course and extent of activation are similar to those observed using a one-stage extrinsic assay.
Thrombin formation began immediately with or without prior exposure of factor V to thrombin. The 'lag time' observed by some previous workers is shown to be an artifact due to inappropriate extrapolation of the standard thrombin curve.     

Doxorubicin induces Fas-mediated apoptosis in human thyroid carcinoma cells.

Doxorubicin remains the most extensively used drug in the chemotherapy of thyroid cancer. However, drug resistance often limits the efficacy of chemotherapy in clinical practice. Several anticancer drugs exert their cytotoxic effect by triggering Fas-mediated apoptosis in some cell types. However, no investigations have been conducted to determine whether doxorubicin causes apoptosis in thyroid carcinomas. In the present study, we assessed the cytotoxic and apoptotic effects of doxorubicin on two thyroid cancer cell lines (FTC 238 and FTC 133). Cytotoxic effects of doxorubicin were evaluated by a 3-(4,5 dimethylthiazol-2yl) 2-5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by fluorescein isothiocyanate-conjugated annexin V/flow cytometric analysis and by DNA fragmentation. Fas expression was measured by flow cytometric analysis. After a 24-hour incubation, doxorubicin induces a dose-dependent cytotoxicity in the two cell lines. Treatment with doxorubicin (0.5 and 1 microM) for 24 hours induced cell apoptosis and upregulated Fas expression. A significant correlation was found between the fluorescence intensity values obtained with annexin V staining and those observed for Fas expression (r = 0.996; p < 0.001 or r = 0.957; 0.02 < p < 0.05 for FTC 238 or FTC 133 cells, respectively). In conclusion, doxorubicin exerts its cytotoxic effects, at least partly, through Fas-mediated apoptosis in thyroid cancer cells. These results may have clinical implications for thyroid cancer therapy.