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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 ofin vitrophagocytosis, 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 leukemic 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.

Changes in the fluorescence of factors IX and II in the presence of calcium at procoagulant phospholipid interfaces suggest a conformational change of the proteins upon binding. Shifts of transition temperature of phosphatidylcholine—phosphatidylserine mixtures towards those of the pure lecithin component when calcium and factors are added suggest that lateral phase separation does exist. The higher resonance-energy transfer efficiency with charged pyrene-labelled phospholipids leads to the conclusion that within the membrane, factor II and IX binding sites are domains of phosphatidylserine. Calcium-independent phospholipid binding, selective for phosphatidylserine, is well documented and is proposed to be mediated by electrostatic forces between the arginine and lysine residues of the proteins and the negative charges of the phosphatidylserine. The binding of heparin or the synthetic anticoagulant polymer PSSO 2 —Glu with antithrombin or thrombin induces different fluorescence changes in these proteins which traduce local changes in the tryptophan residues of the proteins upon binding. The interaction of heparin with various mixtures of thrombin and antithrombin indicates that heparin does not modify the structure of the preformed thrombin—antithrombin complex, as far as can be seen by spectrofluorimetry.