In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant

Abstract

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Figure 1

Flow cytometric quantitation of EMPs. MPs (Gate A) were discriminated by size on an FSC/SSC cytogram (a). Only events included within gate A were further analyzed for fluorescence associated with irrelevant (b) and specific (c) labeling. Gate A was defined by excluding the first FSC channel that contained most of the background noise and by using 0.8-μm latex beads. The gate was defined to include the beads in its upper 33%. In this example EMP quantitation was done using FITC-annexin V labeling, and EDTA buffer was used as a negative control. (a) Determination of forward scatter (FSC) and side-scatter (SSC) characteristics of MPs in suspension. A similar approach was used to analyze MPs in platelet-free plasma. The 2 gates represent the pattern of 0.8- and 3-μm latex beads (A and B, respectively). (b) Determination of the limit for negative fluorescence, performed in the presence of EDTA as a negative control for annexin V. (c) Detection of PS on MPs through annexin V-FITC binding (FL1), expressed in relation to structure (SSC).

Figure 2

Morphology of HUVECs and EMPs. First-passage monolayers of resting (a and b) and TNF-stimulated (c and d) HUVECs were analyzed by scanning electron microscopy. (a and c) ×1,500; (b and d) ×3,000. (e and f) High-power magnification (×24,000 and ×36,000) of EMPs shed from TNF-stimulated HUVECs. Both resting and stimulated ECs showed surface blebs (arrows) or detached vesicles (arrowhead in d). Scale bars: 10 μm (a–d), 1 μm (e and f). P, filter pore.

Figure 3

Effect of TNF on EMP shedding. HUVECs were incubated with either medium alone or varying concentrations of recombinant human TNF (a). A neutralizing anti-TNF antibody was added 1 hour before stimulation with the highest TNF concentration or was tested alone (b). Results are expressed in numbers of MPs labeled with annexin V-FITC, extracted from culture supernatants of 10³ ECs. Bars represent SD of 3 determinations in 4 experiments.

Figure 4

Distribution of endothelial antigens on resting or TNF-stimulated HUVECs and their derived MPs. HUVECs were cultured for 24 hours in the presence or absence of TNF (100 ng/mL), detached and analyzed for mAb binding by flow cytometry (top). MPs derived from these ECs were labeled with the same mAb (bottom). For each antigen studied, the antibody binding was expressed as mean fluorescence intensity (MFI) of the positive population for HUVECs and as number of positive events for MPs, in view of the low intensity of labeling of the latter. For each mAb, MFI of cells and number of their derived MPs are shown under resting (left) and stimulated (right) conditions. Irrelevant mAb’s (both IgG1 and IgG2a) led to identical background staining.

Figure 5

Flow cytometric analysis of EMPs under resting and stimulated conditions. MPs were obtained from EC supernatants and stained with mAb’s, as described in Methods. The cytograms (FL1/SSC) shown here are representative graphs of mAb binding on MPs, counted using 3-μm latex beads (gate B of Figure 1a) as an internal standard. In resting conditions (a), constitutive antigens such as PECAM-1, αvβ3, TM, and ICAM-1 were present on MPs, whereas inducible antigens such as TF and E-selectin were detectable only upon TNF stimulation (b). The horizontal bars represent the level of irrelevant mAb binding, used as control.

Figure 6

Plasma clotting time in the presence of EMPs. MPs were extracted by ultracentrifugation from the culture supernatant of unstimulated HUVECs (open circles), HUVECs incubated with anti-TNF antibody alone (filled triangles), anti-TNF antibody before TNF (100 ng/mL) (filled squares), and TNF (100 ng/mL) alone (filled diamonds). Various numbers of EMPs in suspension were added (10 μL corresponded to the number of MPs derived from 10⁵ cells) to a chronometric test of normal (a) and factor VII–deficient (b) plasma clotting (Howell time). Data are expressed in absolute clotting times (measured in seconds).

Figure 7

Use of PECAM-1 and αvβ3 coexpression to delineate the endothelial origin of MPs: in vitro setup and ex vivo detection. For in vitro studies, the release of MPs in EC supernatants was induced by TNF, as described. (a) Setup of PECAM-1 and αvβ3 double labeling of MPs generated in vitro. (b) SDS-PAGE and Western blot analysis of HUVECs (E) and MP lysates (M) under nonreducing conditions, revealed with control isotype mAb, PECAM-1, and αvβ3. Ex vivo, double labeling of MPs was performed in normal human plasma (c–f). (c) Labeling with isotype control mAb’s, allowing the setting of the background noise position on the subsequent cytograms. (d) Positive double labeling of plasma MPs with FITC–anti-αvβ3 and either PE–anti-PECAM-1, PE–anti-CD41 (e), or PE–anti-CD14 (f).

Figure 8

Transmission electron and confocal laser microscopy of MPs. Transmission electron microscopy appearance of MPs generated in vitro (a) or in vivo (e), i.e., isolated from normal plasma (final magnification: ×25,000). In confocal laser microscopy, MPs appeared as elements with a diameter less than 1 μm and stained for αvβ3 (c) and E-selectin (d) in vitro. In vivo–generated MPs expressed αvβ3 (g) and CD41/GPIIb-IIIa (h). In this case, the majority of the elements were positive for CD41 (h), whereas only about 10% were positive for αvβ3 (g), confirming that they originate from ECs (final magnification: ×1,000). (b and f) Negative staining using isotope control IgG1.

Figure 9

Flow cytometric analysis of EMPs in the plasma from healthy donors (n = 30; open triangles) and LA patients (n = 30; all circles) was performed using FITC–anti-αvβ3 labeling. EMPs were quantitated in plasma as described in Methods. Among the LA patients 15 had a history of thrombosis (filled circles). The difference between groups was analyzed by the Mann-Whitney U test.