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

doi:10.1016/j.thromres.2007.08.005

Thrombosis Research

Volume 121, Issue 6, 2008, Pages 865-871

https://doi.org/10.1016/j.thromres.2007.08.005 Get rights and content

Abstract

Introduction

Microparticles from activated endothelial cells (EMP) are well known to expose tissue factor (TF) and initiate coagulation in vitro. TF coagulant activity is critically dependent on the presence of aminophospholipids, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), but it is unknown whether or not TF-exposing EMP are enriched in such aminophospholipids. Furthermore, despite the fact that EMP have been reported in several pathological conditions, direct evidence for their (putative) coagulant properties in vivo is still lacking. We investigated the phospholipid composition of endothelial MP (EMP) and their thrombogenic properties in vivo.

Materials and methods

Human umbilical vein endothelial cells (HUVEC; n = 3) were incubated with or without interleukin (IL)-1α (5 ng/mL; 0–72 h). Phospholipid composition of EMP was determined by high-performance thin layer chromatography. The association between EMP, TF antigen and activity was confirmed in vitro (ELISA, Western blot and thrombin generation). Thrombogenic activity of EMP in vivo was determined in a rat venous stasis model.

Results

Levels of TF antigen increased 3-fold in culture medium of IL-1α-treated cells (P < 0.0001). This TF antigen was associated with EMP and appeared as a 45–47 kDa protein on Western blot. In addition, EMP from activated cells were enriched in both PS (P < 0.0001) and PE (P < 0.0001), and triggered TF-dependent thrombin formation in vitro and thrombus formation in vivo. In contrast, EMP from control cells neither initiated coagulation in vitro nor thrombus formation in vivo.

Conclusions

EMP from activated endothelial cells expose coagulant tissue factor and are enriched in its cofactors PS and PE.

Introduction

Tissue factor (TF), a 45–47 kDa transmembrane receptor, initiates coagulation [1], triggers cell migration [2] and trafficking of mononuclear phagocytes across the endothelium [3], regulates angiogenic properties of tumor cells [4], acts as a chemotactic factor for vascular smooth muscle cells [5], and protects endothelial cells from apoptosis [6], [7]. TF is widely distributed within the body. Extravascular cell types constitutively express TF [8], [9], and cells at the blood interface (endothelial cells) or circulating within the blood (monocytes) inducibly express TF [10], [11], [12], [13].

TF can also be present on cell-derived microparticles (MP) in vivo. MP isolated from pericardial wound blood [14], synovial fluid [15] or venous blood from a patient with meningococcal septic shock complicated by fulminant disseminated intravascular coagulation [16] initiate TF-dependent thrombin generation in vitro. In addition, we demonstrated that MP from (pericardial) wound blood trigger TF-mediated thrombus formation in vivo [17]. As yet, other MP have not been demonstrated to have such activity in vivo.

Endothelial cell-derived MP (EMP) from TNFα-or LPS-activated endothelial cells expose procoagulant TF in vitro [18], [19], but whether such EMP have any biological activity in vivo is unknown. This question is becoming increasingly relevant since elevated numbers of EMP are now known to occur in various pathological conditions, including systemic lupus erythematosus [20], thrombotic thrombocytopenia purpura [21], vasculitis of the young [22], paroxysmal nocturnal haemoglobinuria [23] and multiple sclerosis [24]. EMP in healthy subjects were reported to correlate with the serum triglyceride concentration, suggesting that EMP may reflect endothelial dysfunction or injury [25].

Aminophospholipids like phosphatidylserine (PS) and phosphatidylethanolamine (PE) are well established cofactors for the procoagulant activity of membrane-exposed TF [26], [27], [28]. Recently, we showed that the phospholipid composition of platelet-derived MP changes upon activation [29]. Whether or not the phospholipid composition of EMP changes during activation of endothelial cells, however, is unknown.

The aims of the present study were to study the presumed procoagulant properties of EMP in vivo and to determine whether phospholipid composition changes during endothelial cell activation may support this TF activity.

Section snippets

Reagents and assays

Medium M199, penicillin, streptomycin, amphotericin B and l-glutamine were obtained from GibcoBRL, Life Technologies (Paisley, Scotland). IgG₁-FITC and IgG₁-PE (clone × 40) were obtained from Becton Dickenson ((BD) San Jose, CA). Annexin V-(allophycocyanin; APC) was from Caltag Laboratories (Burlingame, CA). Human serum albumin (HSA) and monoclonal antibodies (MoAbs), directed against factor VIIa (VII-1 [1.46 mg/mL], VII-15 [0.53 mg/mL]) and anti-factor XII (OT-2 [0.71 mg/mL), were from Sanquin

TF ELISA

TF in conditioned media was determined by ELISA (American Diagnostica Inc.; Greenwich, CT).

EMP from IL-1α-treated endothelial cells expose TF and trigger coagulation in vitro

After 72 h the numbers of EMP in conditioned medium from control (unstimulated) cultures had increased gradually about 6-fold compared to (conditioned medium from) 3 h control cultures (Fig. 1A). In contrast, upon activation with IL-1α, the numbers of EMP increased already about 13-fold after 12 h of culture compared to the 3 h time interval, and these numbers remained virtually constant up to 72 h of culturing. In IL-1α-treated cultures, the overall increase of EMP numbers in time differed

Discussion

Previous studies demonstrated that EMP from TNF-α or LPS-treated endothelial cells expose TF and trigger thrombin generation in vitro [18], [19]. Similarly, our present data show that also EMP from IL-1α-activated endothelial cells expose TF and trigger thrombin generation in vitro. More interestingly, however, is that such EMP become enriched in both PS and PE, and trigger thrombus formation in vivo by a TF-initiated pathway.

In this study we present data that TF exposed by EMP from activated

References (37)

Y. Nemerson
Tissue factor and hemostasis
Blood
(1988)
G.J. Randolph et al.
Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro
Blood
(1998)
B.B. Sorensen et al.
Antiapoptotic effect of coagulation factor VIIa
Blood
(2003)
R. Nieuwland et al.
Cellular origin and procoagulant properties of microparticles in meningococcal sepsis
Blood
(2000)
E. Biró et al.
Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner
J Thromb Haemost
(2003)
H. Kagawa et al.
Expression of functional tissue factor on small vesicles of lipopolysaccharide-stimulated human vascular endothelial cells
Thromb Res
(1998)
M.N. Abid Hussein et al.
Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo
J Thromb Haemost
(2003)
E.F. Smeets et al.
Contribution of different phospholipid classes to the prothrombin converting capacity of sonicated lipid vesicles
Thromb Res
(1996)
S.A. Smith et al.
Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI)
J Thromb Haemost
(2004)
E. Biró et al.
The phospholipid composition and cholesterol content of platelet-derived microparticles: a comparison with platelet membrane fractions
J Thromb Haemost
(2005)

G.M. Vogel et al.

Comparison of two experimental thrombosis models in rats effects of four glycosaminoglycans

Thromb Res

(1989)

M.N. Abid Hussein et al.

Cell-derived microparticles contain caspase 3 in vitro and in vivo

J Thromb Haemost

(2005)

A.M. Weerheim et al.

Phospholipid composition of cell-derived microparticles determined by one-dimensional high-performance thin-layer chromatography

Anal Biochem

(2002)

J.J. Jimenez et al.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Thromb Res

(2003)

I. Del Conde et al.

Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation

Blood

(2005)

W. Ruf et al.

Tissue factor signaling

Thromb Haemost

(1999)

Y. Zhang et al.

Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice

J Clin Invest

(1994)

Y. Sato et al.

Tissue factor induces migration of cultured aortic smooth muscle cells

Thromb Haemost

(1996)

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Regular ArticlePhospholipid composition of in vitro endothelial microparticles and their in vivo thrombogenic properties

Abstract

Introduction

Materials and methods

Results

Conclusions

Introduction

Section snippets

Reagents and assays

TF ELISA

EMP from IL-1α-treated endothelial cells expose TF and trigger coagulation in vitro

Discussion

Blood

Blood

Blood

Blood

J Thromb Haemost

Thromb Res

J Thromb Haemost

Thromb Res

J Thromb Haemost

J Thromb Haemost

Thromb Res

J Thromb Haemost

Anal Biochem

Thromb Res

Blood

Tissue factor signaling

Thromb Haemost

Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice

J Clin Invest

Tissue factor induces migration of cultured aortic smooth muscle cells

Thromb Haemost

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