ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling.

Rapid Ca2+-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca2+ ionophores in megakaryoblastic HEL cells. Ca2+ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca2+, whereas exogenous Ca2+ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca2+ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms.

Mechanism of factor IXa inhibition by antithrombin in the presence of unfractionated and low molecular weight heparins and fucoidan.

Heparin exerts its anticoagulant activity by catalysing the inhibition of coagulation proteases by antithrombin (AT). Its main target is thrombin but it also catalyses the inhibition of the other serine-proteases of the coagulation cascade, such as factor IXa (fIXa). The aim of this study was to compare the catalysis of inhibition of blood fIXa by antithrombin in the presence of several sulfated polysaccharides with anticoagulant activity, i.e. heparin, three widely used in therapeutics low molecular weight heparins (LMWH) and fucoidan. Plots of the second-order rate constants of the fIXa-antithrombin reaction vs. the concentration of added heparin and LMWH are bell-shaped and fit the kinetic model established for thrombin-antithrombin reaction by Jordan R., Beeler D., Rosenberg R. (1979) J. Biol. Chem., 254, 2902-2913. In the ascending branch, the catalyst (C) binds quickly to the inhibitor (I) to form a catalyst-inhibitor (CI) complex which is more reactive towards the enzyme (E) than the free inhibitor, leading to the formation of an inactive enzyme-inhibitor complex (EI) and the release of free catalyst, in a rate-limiting second step. After a maximum corresponding to an optimal catalyst concentration, the decrease in the reaction rate was in keeping with the formation of a catalyst-enzyme (CE) complex, whose inactivation by the CI complex was slower than that of the free enzyme. Maximum second-order rate constants for the inhibition of fIXa by AT were 105, 6.8, 12.24 and 22 microM-1 min-1 with heparin, Enoxaparin, Fraxiparin and Fragmin, respectively, leading to 3500-, 225-, 405- and 728-fold increases in the inhibition rate in the absence of polysaccharide, respectively. Fucoidan yielded 23-fold increase in the fIXa-antithrombin interaction rate. The kinetic profiles obtained with this polysaccharide exhibited ascending branch which correlated well with the kinetic model based on the formation of binary complexes (CI or CE). Fucoidan was covalently conjugated with a fluorescent probe (DTAF) and used in conjunction with fluorescence anisotropy to follow its binding to antithrombin, heparin cofactor II (HCII), thrombin and fIXa. The binding of fucoidan to these proteins occurred with low affinities when compared to heparin and LMWH. Fucoidan had higher affinity for the inhibitor HCII compared to antithrombin and enzymes. These data suggest that binding of heparins and fucoidan to the inhibitor (CI) is required for the polysaccharide-dependent enhancement in the rate of neutralization of the enzyme by the inhibitor.

Comparison between the loss of platelet membrane asymmetry, microvesiculation and the tyrosine phosphorylation of proteins.

Platelet activation by agents such as the Ca2+-ionophore A23187 or Ca2+-ATPase inhibitors leads to the generation of a procoagulant surface and the formation of microparticles. These responses are late events of platelet activation and readily detected by flow cytometry using annexin V-FITC as an aminophospholipid probe. One Ca2+-ATPase inhibitor, 2,5-di-(tertbutyl)-1,4-benzohydroquinone induced aminophospholipid exposure without microparticle formation. Previous work has shown that microparticle formation is strictly linked to the activation of calpain, a thiol-protease that modifies the platelet cytoskeleton and some signal transduction enzymes. We now report how the detection of platelet tyrosine phosphorylation by western-blotting clearly shows that when platelet activation and aminophospholipid exposure are accompanied by microparticle formation there is a decrease in the tyrosine phosphorylation of proteins.

Procoagulant activity and active calpain in platelet-derived microparticles.

Microparticles are released during in vitro platelet activation and have been detected in vivo in several pathologies. Their characterization is of interest as they may play a potential role in hemostasis. Here, we report the formation of microparticles as the result of increases in intracellular Ca2+ brought about by inhibition of Ca(2+)-ATPases. They were isolated following centrifugation of the activated platelet suspension over a sucrose layer. Flow cytometric studies using annexin V-FITC as a probe for aminophospholipids, prothrombinase activity measurements and annexin V inhibition experiments enabled us to evaluate the procoagulant activity of microparticles prepared in this way. The efficiency of the annexin V inhibition (IC50 = 10-20 nM) of this activity confirmed significant anticoagulant properties for this protein. Microparticles also contained the glycoprotein IIb-IIIa complex, detected in flow cytometry at a density higher than on the remnant platelets. The activation of calpain, a Ca(2+)-dependent protease, in platelets was shown to be more efficient under conditions of a sudden Ca2+ influx. The microparticles contained only the active form of calpain detected by Western blotting using a monoclonal antibody able to recognize both the unactivated and the activated catalytic subunit of the enzyme. However, flow cytometry failed to find significant amounts of active calpain on the microparticle or on the platelet surface. Our results, while confirming the procoagulant activity of microparticles, also document for the first time the exclusive presence of the activated form of calpain, inferring a possible role for this protease in microparticle-mediated functions.

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release.

Intracellular amyloid beta-peptide (A beta) accumulation is considered to be a key pathogenic factor in sporadic Alzheimer's disease (AD), but the mechanisms by which it triggers neuronal dysfunction remain unclear. We hypothesized that gradual mitochondrial dysfunction could play a central role in both initiation and progression of sporadic AD. Thus, we analyzed changes in mitochondrial structure and function following direct exposure to increasing concentrations of A beta(1--42) and A beta(25--35) in order to look more closely at the relationships between mitochondrial membrane viscosity, ATP synthesis, ROS production, and cytochrome c release. Our results show the accumulation of monomeric A beta within rat brain and muscle mitochondria. Subsequently, we observed four different and additive modes of action of A beta, which were concentration dependent: (i) an increase in mitochondrial membrane viscosity with a concomitant decrease in ATP/O, (ii) respiratory chain complexes inhibition, (iii) a potentialization of ROS production, and (iv) cytochrome c release.

Aprotinin can inhibit the proteolytic activity of thrombin. A fluorescence and an enzymatic study.

Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow-up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human alpha-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x 10(-6) mol.l-1 was determined. Aprotinin can displace p-aminobenzamidine, a fluorescent-probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H-D-phenylalanylpipecolylarginine-p-nitroanalidehydrochloride++ + (S-2238). With S-2238, double-reciprocal plots show that the inhibition is competitive with a Ki of 61 microM and a Km of 1.72 microM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 +/- 0.12 and an apparent dissociation constant of 57.0 +/- 13.1 microM (mean +/- SEM). Thus, the inhibition of thrombin-induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin-induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.

Simultaneous detection of changes in cytoplasmic Ca(2+), aminophospholipid exposure and micro-vesiculation in activated platelets.

We have used flow cytometry to compare the temporal relationship between cytoplasmic Ca(2+)-fluxes and micro-vesiculation during platelet activation. Changes in fluorescence of the Ca(2+)-dye, fluo-3, and in forward light scatter as a measure of the decrease in platelet size that accompanies micro-vesiculation, were assessed simultaneously. In other experiments, changes in Ca(2+) levels and aminophospholipid exposure were assessed using fura-red, which is a long wavelength range indicator, and FITC-annexin V. Results obtained using the ionophore A23 187 and the ATPase inhibitor, thapsigargin, showed that micro-vesiculation is a relatively late event compared with intracellular Ca(2+) elevation. The relatively slow binding kinetics of annexin V prevented the establishment of a temporal relationship between increases in intracellular Ca(2+) and aminophospholipid exposure. Nevertheless, the combined use of fura-red and annexin V highlighted the heterogeneous response seen on some occasions with thapsigargin and always with a thrombin plus collagen mixture, and confirmed that individual platelets that bound annexin V were also those with elevated intracellular Ca(2+) levels.

Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen.

Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for mu-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.

Calcium influx is a determining factor of calpain activation and microparticle formation in platelets.

We have related the release of procoagulant microparticles from platelets to calcium movement and the activation of the Ca(2+)-dependent protease calpain. The effects of the Ca(2+)-ATPase inhibitors thapsigargin, cyclopiazonic acid and 2.5-di-(t-butyl)-1,4-benzohydroquinone were compared with those of the Ca2+ ionophore A23187. Whereas all three Ca(2+)-ATPase inhibitors induced aminophospholipid exposure on platelets, only thapsigargin and cyclopiazonic acid promoted microparticle formation and only when strong Ca2+ influx, calpain activation and proteolysis of cytoskeletal proteins occurred concomitantly. Preincubation with dibutylbenzohydroquinone inhibited the responses to thapsigargin and cyclopiazonic acid but not to A23187. When platelets were suspended in a Ca(2+)-free medium, calpain activation and microparticle formation were not observed, even with maximum mobilisation of internal Ca2+ stores by A23187. Incubation of fluo-3-loaded plateters with A23187 in 0.1 mM EGTA followed by the sequential addition of 25 microM Ca2+ increments to the medium showed that calpain activation occurred when the intraplatelet [Ca2+] reached 3-8 microM. To assess the physiologic significance of these results, the subpopulation of platelets that expressed procoagulant activity after stimulation by a thrombin/collagen mixture was isolated by means of annexin-V-coupled magnetic beads. Subsequent western blotting experiments confirmed that this subpopulation contained activated calpain. Overall, our results provide evidence that microparticle formation and calpain activation require an elevated intraplatelet [Ca2+] that is brought about by influx across the plasma membrane.

Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium.

In pathological conditions such as sickle cell disease, falciparum malaria and diabetes, an abnormal adherence of erythrocytes to endothelium is concomitant with loss of phospholipid asymmetry resulting in phosphatidylserine (PS) exposure. We have investigated the involvement of PS in this interaction by studying adhesion of human erythrocytes, treated with Ca2+-ionophore A23187 in combination with N-ethylmaleimide, to human umbilical vein endothelial cells in a flow-based assay. Results showed that erythrocytes which exposed PS, massively adhered to HUVEC in a Ca2+-dependent manner. This adhesion was inhibited by PS liposomes and by annexin V, giving clear evidence of the PS dependence of these interactions.

Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors.

The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1, 4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure. The Ca(2+)-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 microM (n = 11) for thapsigargin (3 microM), 19.6 +/- 2.2 microM (n = 8) for cyclopiazonic acid (100 microM), and 0.619 +/- 0.137 microM (n = 8) for 2,5-di-tert-butyl-1,4-benzohydroquinone (100 microM). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t1/2 = 133 +/- 14 s) was more rapid than that induced by TG (t1/2 = 280 +/- 30 s), although the rate-limiting step in the assay was the binding of annexin V to activated platelets (t1/2 = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca(2+)-induced calpain activation and a fusion process inhibited by annexin V.

Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics.

The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium-ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine-labelled proteins in platelets treated with thrombin or a thrombin + collagen mixture and in particular the phosphorylation of a 40 kD band were severely reduced. Furthermore, inhibition of thiol-containing enzymes. including tyrosine-phosphatases, by N-ethyl maleimide did not lead to aminophospholipid exposure in the patient's platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.

Significance of capacitative Ca2+ entry in the regulation of phosphatidylserine expression at the surface of stimulated cells.

The transverse redistribution of plasma membrane phosphatidylserine is one of the hallmarks of cells undergoing apoptosis and also occurs in cells fulfilling a more specialized function, such as platelets after appropriate activation. Although an increase in intracellular Ca2+ is required to trigger the remodeling of the plasma membrane, little information regarding intracellular signals leading to phosphatidylserine externalization has been provided. Scott syndrome is an extremely rare inherited disorder of the migration of phosphatidylserine toward the exoplasmic leaflet of the plasma membrane of stimulated blood cells. We have studied here the intracellular Ca2+ mobilization and Ca2+ entry involved in tyrosine phosphorylation in Epstein Barr virus (EBV)-infected B cells derived from a patient with Scott syndrome, her daughter, and control subjects. An alteration of Ca2+ entry through the plasma membrane and subsequent tyrosine phosphorylation induced by Ca2+ were observed in Scott EBV-B cells, but the release of Ca2+ from intracellular stores was normal. Furthermore, phosphatidylserine externalization at the surface of stimulated cells does not depend on tyrosine kinases. These results suggest that the defect of phosphatidylserine exposure in Scott syndrome cells is related to the alteration of a particular way of Ca2+ entry, referred to as capacitative Ca2+ entry, although some differences may be related to the cell type. Hence, this genetic mutant testifies to the prime significance of Ca2+ signaling in the regulation of phosphatidylserine expression at the surface of stimulated cells.

Physiopathological significance of catalytic phospholipids in the generation of thrombin.

Thrombin generation is the culminating event of the coagulation cascade. It is initiated after the expression of tissue factor by endothelial cells and monocytes exposed to thrombogenic stimuli. Anionic phospholipids, chiefly phosphatidylserine, are necessary for the optimal activity of tissue factor and completion of the clotting process. They display a catalytic potential by allowing the formation of the characteristic enzyme complexes at the membrane surface. Platelets are viewed as the main source of procoagulant phospholipid referred to as platelet factor 3. The plasma membrane of resting cells presents an asymmetrical distribution of phospholipids, aminophospholipids being sequestered in the inner leaflet. Procoagulant phospholipids become available at the outer surface after cell stimulation. The collapse of the membrane asymmetry is thought to promote a phospholipid scrambling accompanied by the shedding of microparticles. The plasma membranes of such vesicles bear irreversibly externalized procoagulant phosphatidylserine and contain glycoproteins that testify to their tissue origin. Hence, microparticles could disseminate a dual procoagulant and adhesive potential. Thrombin autoamplification is exerted through feedback activation loops involving either coagulation factors or platelets. This article details the mechanisms by which procoagulant phospholipids promote the generation of an excess of thrombin. A new pharmacological approach of thrombosis is presented, based on the control of the exposure of procoagulant phospholipids and membrane microparticle shedding.

Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide.

Endotoxin-stimulated monocytes can elicit a dual procoagulant response. They express tissue factor and expose phosphatidylserine in the outer leaflet of the plasma membrane. Tissue factor, a membrane glycoprotein, is the cellular trigger of blood coagulation reactions. Phosphatidylserine is an essential anionic phospholipid for surface amplification of thrombin generation. In this study the distribution of these two procoagulant entities between activated monocytes and derived microparticles was assessed after stimulation by LPS. The presence of CD14, CD11a, and CD18, and possible associated adhesion potential were examined, particularly on microparticles. Tissue factor was evidenced by using a specific functional assay and flow cytometry. Phosphatidylserine exposure was monitored through its catalytic activity in a thrombin generation assay and by flow cytometry with the use of FITC-conjugated annexin V, a protein probe of anionic phospholipids. CD14, CD11a, and CD18 were detected by flow cytometry. The interaction of microparticle CD11a/CD18 with intracellular adhesion molecule-1 was demonstrated by using immobilized recombinant intracellular adhesion molecule-1 fusion protein. The major part of tissue factor and phosphatidylserine-dependent procoagulant activity was associated with microparticles after LPS stimulation. This was confirmed by flow cytometry. The presence of functional CD11a/CD18, and CD14 on microparticles testifies to an associated adhesion potential. These results show that membrane vesiculation could be responsible for dissemination of inducible monocyte procoagulant activities and suggest that derived microparticles could also participate in endothelium stimulation. This emphasizes the role of monocyte as a central element in the coupling between inflammation/infection and thrombosis.

Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups.

Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 > thrombin plus collagen > collagen > thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.

Caspase activation is an early event in anthracycline-induced apoptosis and allows detection of apoptotic cells before they are ingested by phagocytes.

An increasing number of methods are being described to detect apoptotic cells. However, attempts to detect apoptotic cells in clinical samples are rarely successful. A hypothesis is that apoptotic cells are cleared from the circulation by phagocytosis before they become detectable by conventional morphological or cytometric methods. Using LR73 adhering cells as phagocytes in a model of in vitro phagocytosis, we found that phagocytosis of daunorubicin (DNR)-treated U937, HL60, or K562 leukemia cell lines occurred prior to phosphatidylserine externalization, DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mitochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis assay to detect caspase activity in ceramide- or DNR-treated cells (fluorogenic substrate for caspase), we found that caspase activity increased in apoptosis-committed cells before they were detected by flow cytometry or recognized by phagocytes. Similarly a caspase activity increase was detected in circulating mononuclear cells of luekemic patients 15 h after the beginning of anthracyclin treatment. We suggest that recent findings on enzymatic events (caspase activation) occurring in the early events of apoptosis must now allow the development of new markers for apoptosis, irrespective of the morphological features or internucleosomal fragmentation which are late events in apoptosis.