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