In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant
Valéry Combes, … , José Sampol, Françoise Dignat-George
Valéry Combes, … , José Sampol, Françoise Dignat-George
Published July 1, 1999
Citation Information: J Clin Invest. 1999;104(1):93-102. https://doi.org/10.1172/JCI4985.
View: Text | PDF
Article

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, αvβ3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with αvβ3 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.

Authors

Valéry Combes, Anne-Christine Simon, Georges-Emile Grau, Dominique Arnoux, Laurence Camoin, Florence Sabatier, Murielle Mutin, Marielle Sanmarco, José Sampol, Françoise Dignat-George

×

Figure 1

Options: View larger image (or click on image) Download as PowerPoint
Flow cytometric quantitation of EMPs. MPs (Gate A) were discriminated by...
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).