Phospholipid composition of in vitro endothelial microparticles and their in vivo thrombogenic properties

Hussein, M. N. Abid; Böing, Anita N.; Bíró, Éva; Hoek, Frans J.; Vogel, G; Meuleman, D.G.; Sturk, A.; Nieuwland, Rienk

doi:10.1016/j.thromres.2007.08.005

Cited by 87 publications

(68 citation statements)

References 36 publications

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“…1D). This may be due to the lower proportions of incorporated phosphatidylserine as seen at 45 min (supplemental Table 2), which is a prerequisite for the procoagulant activity of TF and is in agreement with previous reports (23). In contrast, alanine-substitution of Ser 253 (TF Ala253 ) suppressed the release of TF antigen and activity.…”

Section: Resultssupporting

confidence: 90%

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

Collier

Ettelaie

2011

Journal of Biological Chemistry

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The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser 253 and Ser 258 , were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. Increased levels of circulating endothelial cell-derived microparticles are recognized as a cause and prognostic marker for vascular disease and injury (1-3). Moreover, these microparticles have been shown to be able to support the induction of thrombosis through a TF-dependent mechanism (3, 4). The mechanisms that regulate the incorporation and release of TF 2 into cell-derived microparticles are as yet unidentified. Furthermore, it appears that cellular activation (5) for example through activation of PKC (6) can induce microparticle release although this is not completely understood. The proteolytic activation of PAR2 by enzymes such as factor Xa and trypsin is known to result in the translocation of PKC␣ to the membrane (7). It has previously been shown that the incubation of cells with trypsin can induce the release of TF into membrane-derived vesicles (8). Furthermore, PKC␣ is known to phosphorylate the cytoplasmic domain of TF at Ser 253 (7, 9), whereas Ser 258 is part of a proline-directed kinase consensus sequence (10), which becomes phosphorylated separately (11) initiated through the phosphorylation of Ser 253 (9). The cytoplasmic domain of TF is not required for the procoagulant activity of TF or de-encryption of this protein (12, 13). Moreover, it has been reported that substitution of the cytoplasmic domain of TF with the cytoplasmic domain of decayaccelerating factor does not alter the release of TF in cells that are capable of releasing TF-containing microparticles without activation (14). However, it is known that cells spontaneously release decay-accelerating factor under standard culture conditions (15). In this study, by aspartate substitution (to mimic phosphoserine) (16) or alanine substitution (to prevent phosphorylation) of two of the serine residues within the cytoplasmic domain of TF, we investigated the contribution of these residues to the regulation of TF incorporation and release into cell-derived microparticles. EXPERIMENTAL PROCEDURESPlasmid Vectors-The pCMV-XL5-TF plasmid for the expression of full-length human TF was obtained from OriGene. Aspartate and alanine substitutions at Ser 253 and Ser 258were carried out to produce mutated constructs (pCMV-XL5-TF Asp253 , pCMV-XL5-TF Ala253 , pCMV-XL5-TF Asp258 , and pCMV-XL5-TF Ala258 ) ...

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Section: Resultssupporting

confidence: 90%

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

Collier

Ettelaie

2011

Journal of Biological Chemistry

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“…For the construction of standard curves phospholipids of defined composition were used (33% PS and 67% PC). Interestingly, recent studies showed that the phospholipid composition of circulating procoagulant MPs varies depending on type of originating cell and type of stimulation [36][37][38]. Therefore, great variations of the PS and PC distribution on the surface of circulating MPs of different origin may be possible.…”

Section: Discussionmentioning

confidence: 99%

Circulating procoagulant microparticles in cancer patients

Thaler

Weinstabl

et al. 2010

Ann Hematol

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Accumulating evidence indicates that microparticles (MPs) are important mediators of the interaction between cancer and the hemostatic system. We conducted a large prospective cohort study to determine whether the number of circulating procoagulant MPs is elevated in cancer patients and whether the elevated MP levels are predictive of occurrence of venous thrombembolism (VTE). We analyzed plasma samples of 728 cancer patients from the ongoing prospective observational Vienna Cancer and Thrombosis Study (CATS). Study endpoint was the occurrence of symptomatic VTE. Sixty-five age-and sex-matched healthy controls were recruited for defining the cut-off point for elevated MPs (4.62 nanomolar phosphatidylserine nM PS ), which was set at the 95th percentile of MP levels in healthy controls. The measurement of MPs was performed after capture onto immobilized annexin-V, and determination of their procoagulant activity was quantified with a prothrombinase assay. During a median observation period of 710 days 53 patients developed VTE. MP levels (nM PS) were significantly higher in cancer patients than in healthy controls (median 25th-75th percentile : 3.95 1.74-7.96 vs. 1.19 0.81-1.67 , p<0.001). Multivariate analysis including age, sex, surgery, chemo-and radiotherapy showed no statistically significant association of the hazard ratio of elevated MPs with VTE (0.95 [95% CI: 0.55-1.64], p=0.856). In conclusion, MP levels were elevated in cancer patients compared to healthy individuals in this study. However, elevated MP levels were not predictive of VTE.

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“…The thrombogenic activity of EMP was then confirmed by the demonstration that EMP triggered TF-dependent thrombin formation in vitro and thrombus formation in vivo. 52 Moreover, TF-positive EMP expressing endothelial adhesive molecules can bind to other cell types, such as monocytes, and transfer bioactive TF in vitro. 53 A possible TF transfer from TF-bearing EMP to activated platelets could be also involved in this procoagulant response, because such a mechanism was reported for TF-exposing leukocyte MP.…”

Section: Multifaceted Roles Of Emp Effects Of Emp In Thrombosis and Imentioning

confidence: 99%

The Many Faces of Endothelial Microparticles

Dignat-George

Boulanger

2011

ATVB

563

495

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Abstract-Endothelial microparticles (EMP) are complex vesicular structures shed from activated or apoptotic endothelial cells. They play a remarkable role in coagulation, inflammation, endothelial function, and angiogenesis and thus disturb the vascular homeostasis, contributing to the progression of vascular diseases. As a cause or a consequence, elevated levels of EMP were found in plasma from patients with vascular diseases, where they serve as a surrogate marker of endothelial function. More recent data challenged the presumed deleterious role of EMP because they could promote cell survival, exert antiinflammatory effects, counteract coagulation processes, or induce endothelial regeneration. This review focuses on the ambivalent role of EMP in vascular homeostasis. (Arterioscler Thromb Vasc Biol. 2011;31:27-33.)Key Words: apoptosis Ⅲ atherosclerosis Ⅲ endothelial function Ⅲ endothelium Ⅲ thrombin Ⅲ thrombosis Ⅲ vascular biology Ⅲ microparticles V ascular endothelial cells, like most cells, release different types of membrane vesicles, including microparticles (MP) and exosomes, in response to cellular activation or apoptosis. In addition, apoptotic bodies might be generated during the final steps of programmed cell death. These different vesicles are distinguished from one another on the basis of their subcellular origin, their size, their content, the mechanisms leading to their formation, and, from a practical point of view, how they are obtained. Endothelial Microparticle CharacteristicsEndothelial microparticles (EMP) (Ϸ100 nm to 1 m in diameter) result from endothelial plasma membrane blebbing and carry endothelial proteins such as vascular endothelial cadherin, platelet endothelial cell adhesion molecule-1, intercellular cell adhesion molecule (ICAM)-1, endoglin, E-selectin, S-endo or ␣v integrin (Figure 1). 1 Endothelial NO synthase and vascular endothelial growth factor receptor (VEGF-R2) have also been identified on EMP, 2 but there is so far no evidence on whether or not MP endothelial nitric oxide synthase is capable of generating nitric oxide; furthermore, endothelial nitric oxide synthase may also be present on platelet or red blood cell-derived MP. E-selectin (CD62E) is expressed by activated endothelial cells but can equally be found on EMP generated following either tumor necrosis factor-␣ (TNF-␣) activation or growth factor deprivationinduced apoptosis. 3 Identification of endothelial origin of circulating MP relies on the use of specific markers for flow cytometry analysis (see below); unfortunately, except E-selectin and vascular endothelial cadherin, most of them lack exclusive endothelial expression. Endoglin (CD105) is also expressed by activated monocytes/macrophages and bone marrow cell subsets; platelet endothelial cell adhesion molecule-1 (CD31) is present on activated platelets, platelet MP, and leukocyte subsets; S-endo (CD146) has been found on pericytes, tumor cells, and activated T-cells; ICAM-1 (CD54) is also expressed by leukocytes; and ␣v integrin (CD51) is present on monocy...

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Phospholipid composition of in vitro endothelial microparticles and their in vivo thrombogenic properties

Cited by 87 publications

References 36 publications

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

Circulating procoagulant microparticles in cancer patients

The Many Faces of Endothelial Microparticles

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