Published online before print February 27, 2008
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,2
* Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Univesität Düsseldorf, Düsseldorf, Germany; and
Institut für Pharmakologie Universität Duisburg-Essen, and
Klinik für Kardiologie, Universitätsklinikum Essen, Essen, Germany
2Correspondence: Institut für Pharmakologie, Universität Duisburg-Essen, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. E-mail: artur.weber{at}uk-essen.de
ABSTRACT
Monocyte apoptosis is an important determinant of atherothrombosis. Two major mechanisms for apoptosis-associated thrombogenicity have been described: exposure of negatively charged membrane phospholipids and up-regulation of tissue factor (TF). However, the relative importance of these mechanisms is unclear. Thus, procoagulant functions (thrombin generation) of apoptotic (staurosporine, 2 µM, 24 h) U937 cells versus cell-derived microparticles (MPs) were studied. In apoptotic U937 cells, a significant increase in TF mRNA (real-time PCR), surface expression of TF (flow cytometry), and total cellular amount of TF (Western blotting) was observed. Control cells only minimally triggered thrombin generation (endogenous thrombin potential), and apoptotic cells were highly procoagulant. However, addition of negatively charged membranes completely restored the thrombin generation capacity of control U937 cells to the levels of apoptotic cells. MPs (defined as CD45+ particles of subcellular size), derived from apoptotic U937 cells, were highly procoagulant but did not exhibit an increased TF expression or annexin V binding. Taken together, our data support the concept that the membrane environment, independent of TF expression, determines the extent of thrombin formation triggered by apoptosis of monocytic cells. Externalization of negatively charged phospholipids represents the most important mechanisms for whole cells. Additional yet unknown mechanisms appear to be involved in the procoagulant actions of MPs derived from apoptotic monocytes.
Key Words: U937 cells • monocytes • staurosporine
Apoptosis not only controls proliferative cell functions [1 ] but is also an important determinant of atherothrombosis [2 , 3 ]. Two major mechanisms for apoptosis-associated thrombogenicity have been described: exposure of negatively charged membrane phospholipids and up-regulation of tissue factor (TF) [3 ]. However, the relative importance of these mechanisms is unclear. In addition, during apoptosis, procoagulant microparticles (MPs) are released [4 ]. Elevated levels of circulating monocyte-derived MPs are associated with poor clinical outcome [5 ]. Similar to apoptotic cells, these MPs are also characterized by the externalization of negatively charged phospholipids, mainly phosphatidylserine [5 , 6 ]. TF has also been found on the surface of MPs, providing an additional, important procoagulant mechanism [7 ].
Interestingly, in a most recent study, procoagulant activity of endothelial MPs, released upon cisplatin-induced apoptosis, was shown to be independent of TF activity [8 ]. Phospholipid composition and surface protein expression of MPs are known to vary with the parental cell type and the mechanism of MP release. We have, therefore, used a monocytic cell line (U937) to study the effects of apoptosis on cell- versus MP-dependent thrombin generation by measuring the endogenous thrombin potential (ETP).
Apoptosis of U937 cells was induced by staurosporine (2 µM) for 24 h and was verified by the demonstration of chromatin condensation, caspase-3 processing, and poly(ADP-ribose)polymerase (PARP) cleavage, respectively (Fig. 1A ). Cells and cell-derived MPs were first characterized by flow cytometry (Fig. 1B) , real-time PCR, and Western blotting, respectively (Fig. 1C) . As expected, apoptotic cells bound annexin V (flow cytometry). Upon apoptosis induction, a significant increase in TF mRNA (real-time PCR), surface expression of TF (flow cytometry), and total cellular amount of TF (Western blotting) was observed (Fig. 1C) .
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Figure 1. Procoagulant functions of monocytic cells (U937) undergoing apoptosis. (A) Detection of apoptosis by demonstration of nuclear condensation, caspase-3 (casp-3) processing, and PARP cleavage (con, control; stauro, stauosporin; 2 µM, 24 h). (B) Original flow cytometry dot-plots demonstrating the gating procedure for detection of cells versus MPs and for the determination of the percentage TF+/CD45+ events. SS, Side-scatter; FS, forward-scatter; FITC, fluoroscein isothiocyanate. (C) Analysis of annexin V binding, TF mRNA, TF-positive events, and total cellular TF expression in control versus apoptotic (stauro) U937 cells. (D) Analysis of counts (MPs/100 cells), annexin V binding, TF-positive events, and total cellular TF expression in MPs derived from control versus apoptotic (stauro) U937 cells. (E) Representative tracings of the ETP triggered by control versus apoptotic (stauro) U937 cells. (F) Quantitative analysis of the ETP triggered by control versus apoptotic (stauro) U937 cells in the absence [–phospholipid (–PL)] and presence (+PL) of exogenous phospholipid membranes. (G) Quantitative analysis of the ETP triggered by MPs derived from control versus apoptotic (stauro) U937 cells in the absence and presence of exogenous phospholipid membranes. Original graphs are representative for n = 4–6 experiments. Quantitative data are given as means ± SEM of n = 4–6 independent experiments. *, P < 0.05, versus control (unpaired t-test or ANOVA/Bonferroni for multiple comparisons, as appropriate). Chromatin condensation of cells treated with staurosporine was visualized by HOE33342 staining (fluorescence microscopy, BX-50 Olympus, Japan). Caspase-3 (Cell Signaling Technology, Beverly, MA, USA; #9662, 1:500), PARP (Cell Signaling Technology; #9542, 1:500), and TF (American Diagnostica, Stamford, CT, USA; #4501, 1:2000) were detected by Western blotting as described [9
, 10
]. cDNA was synthesized for each sample from 1 µg total RNA. PCR was performed on a Real-Time PCR System 7300 with TaqMan® Universal PCR Master Mix with 50 ng cDNA as described in ref. [11
] (Hs00175225_m1). Data were analyzed with Sequence Detection Software v1.2.3, and comparative threshold values were normalized to GAPDH (Hs99999905_m1). All products were supplied by Applied Biosystems (Foster City, CA, USA; Applera, Norwalk, CT, USA). Flow cytometry was performed as described [12
] using the following antibodies: CD45-PE (Coulter, Miami, FL, USA; #A07785), TF-FITC (American Diagnostica, #4508 CJ), or annexin V-PE (Cell Signaling Technology). The cut-off values for CD45 positivity were set at 1% using the appropriate PE-conjugated isotypic control antibodies (Coulter, clone 15H6). EDTA (5 mM) was used as a negative control for annexin V binding (cut-off value for annexin V positivity, 1%). Detectors were set to logarithmic amplification, and 10,000 events were measured. ETP was measured as described [9
]. In brief, 60 µL cell suspension or cell supernatants were incubated with 10 µL phospholipid membranes and 20 µL recalcification buffer (2 mmol/L Ca2+, 2 mmol/L Mg2+; final concentrations) containing the fluorogenic substrate Z-Gly-Gly-Arg-7-amino-4-methylcoumarin (Bachem, Bubendorf, Switzerland). The reaction was started by addition of 20 µL citrated human plasma, and thrombin generation was monitored for 60 min.
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1.5-fold) increase in MP counts (Fig. 1D)
. As the release of MPs is known to be associated with a loss of membrane asymmetry, even in the absence of apoptosis [13
], it was not surprising that annexin V binding was not different in MPs derived from apoptotic versus control cells (Fig. 1D)
. Interestingly, in contrast to apoptotic cells, expression of TF on the surface of MPs, derived from apoptotic cells, was similar to MPs, spontaneously released from control cells (Fig. 1D)
. This was confirmed by Western blotting of MPs isolated by ultracentrifugation (100,000 g, 1 h; Fig. 1D
). Next, cells and MPs were functionally characterized by measuring the ETP. These experiments were performed in the absence and the presence of exogenous, negatively charged platelet membranes. In the absence of these membranes, control cells only minimally triggered thrombin generation, and apoptotic cells were highly procoagulant (Fig. 1E) . Addition of membranes did not further increase thrombin generation triggered by apoptotic cells (Fig. 1F) . However, negatively charged membranes completely restored the thrombin generation capacity of control U937 cells to levels of apoptotic cells. Thus, despite marked differences in cell surface expression levels of TF, the procoagulant function of control versus apoptotic cells was identical, provided that negatively charged phospholipids are present.
These data suggest that even minute amounts of TF appear to be sufficient to stimulate thrombin formation in the presence of an adequate membrane environment.
To further substantiate this hypothesis, the procoagulant properties of MPs were also determined. In these experiments, it became evident that despite similar annexin V binding and TF expression, MPs derived from apoptotic cells were highly procoagulant, even in the absence of exogenous phospholipid membranes (Fig. 1G) .
In contrast, MPs derived from control U937 cells did not significantly trigger thrombin generation (Fig. 1G) . Interestingly, unlike with whole cells, addition of exogenous membranes only partially (<50%) restored the full thrombin-generating capacity, as observed with MPs derived from apoptotic cells (Fig. 1G) .
Our study has several limitations. First, only in vitro experiments with a cell line were performed. Second, thrombin generation, as measured by the ETP, might be insensitive to low concentrations of TF. However, the data provide a mechanistic explanation for apoptosis-associated thrombogenicity that might be of biological importance.
It is concluded that apoptosis of monocytic cells results in a marked increase in procoagulant functions at the cellular and MP level. Evidence is provided to support the concept [8 ] that the membrane environment (e.g., the externalization of negatively charged surfaces), independent of TF expression, determines the extent of thrombin formation triggered by apoptosis of monocytic cells. Additional, yet unknown mechanisms appear to be involved in the procoagulant actions of MPs derived from apoptotic monocytes.
FOOTNOTES
1 These authors contributed equally to this work. 
Received December 19, 2007; revised February 1, 2008; accepted February 2, 2008.
REFERENCES
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