Abstract

Microparticles (MPs), shed from cell membranes upon activation or apoptosis, circulate in the blood of healthy donors and are elevated in various pathologies. MPs are associated with inflammatory disease, vascular damage and hypercoagulability states. The aim of this study was to evaluate the procoagulant and apoptotic effects of MPs on endothelial cells.

The procoagulant level of monocyte cell line (THP-1) MPs was characterized and their influence on human umbilical vain endothelial cell (HUVEC) was evaluated by flow cytometry (FACS), confocal and inverted microscopy, ELISA and proteomics. In order to isolate and characterize MPs, THP-1 cells were stimulated with LPS/Ca-ionophore. Isolated MPs expressed higher level of tissue factor (TF) antigen than their parent cells. In order to evaluate the interaction between MPs and HUVEC, endothelial cells (EC) and MPs were incubated for increased time periods. The results demonstrated that MPs bind to HUVEC via CD18 - the lymphocyte function-associated antigen-1 (LFA-1). MPs were incorporated into the EC membrane and cytoplasm resulting in cell network interruption and morphologic changes characterized by cell elongation and formation of inter-cell gaps. EC disruption resulted in blobbing; leading to shedding of additional MPs which may thereby intensify MPs effects. This interaction was virtually diminished when cells were incubated at 4oC. It can be concluded that MPs-EC interaction leads to morphologic changes of cells.

Stimulation of HUVEC with MPs was further applied for evaluation of the apoptotic effect of MPs. Membrane ‘flip-flop’ - early stage of apoptosis - was depicted by FACS and inverted microscopy after 2–4 hours of stimulation. Nucleuses degradation was observed after a longer exposure. Cells labeled with Annexin V-FITC or with propidium iodide (PI), demonstrated significant increase of the labeled cells - from 2.31±1.13% and 2.43±1.15%, respectively in the non-stimulated cells to 28.54±6.79% and 58.63±22.12% after 20 hours of stimulation (p=0.0005; p=0.003). Proteomics analysis presented a 7% increase of the proteins, while 21% of the proteins were decreased. One of the increased proteins was identified by mass spectrometry as Galactin, which is known to mediate and induce apoptosis in tumor cells. One of the decreased proteins was identified as Vimentin. Cleavage of Vimentin by caspases 3, 6 and 7 resulted in enhanced apoptosis. It may be concluded that MPs stimulation leads to HUVEC apoptosis, a process that is intensified as stimulation time exposure increases.

Thrombotic effects of MPs on HUVEC were evaluated by measuring coagulation proteins expression in the cells using FACS and confocal microscopy. HUVEC demonstrated an increase in TF through incorporation of MPs’ TF into the cell membrane as well as an increase of intrinsic TF. TF expression was significantly elevated from 3.17±0.94% of labeled non-stimulated cells to 28.11±12.43% in labeled cells after 20 hours of stimulation (p=0.0004). Additionally, a decrease in the anticoagulants - tissue factor pathway inhibitor and thrombomodulin - was recorded, from 23.65±11.69% and 52.88±6.6%, respectively in non-stimulated cells to 8.62±4.5% and 25.18±16.43% after 20 hours of MPs stimulation (p=0.042, p=0.013).

In conclusion, this study demonstrated that interaction of procoagulant monocyte MPs with HUVEC result in cell morphologic disruption, apoptosis and increased thrombogenicity. These physiologic effects of MPs on endothelial cells may be relevant to vascular and inflammation disorders.

Disclosure: No relevant conflicts of interest to declare.

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