TRAIL/Apo2L Mediates the Release of Procoagulant Endothelial Microparticles Induced by Thrombin In Vitro

Abstract

Abstract-Microparticles are small vesicles playing a crucial role in cell communication by promoting prothrombotic and proinflammatory responses. However, the molecular mechanisms underlying their release are still elusive. We previously established that thrombin promoted the generation of endothelial microparticles (EMPs). In the present study, gene profiling identified TRAIL/Apo2L, a cytokine belonging to the tumor necrosis factor-␣ superfamily, as a target of thrombin. Thrombin increased the expression of cell-associated and soluble forms of TRAIL (sTRAIL) in HMEC-1 cells and human umbilical vein endothelial cells (HUVECs). Blocking TRAIL by specific antibodies or by small interfering RNA reduced both the number and the procoagulant activity of EMPs released by thrombin. Consistent with an involvement of sTRAIL in thrombin-induced EMP release, we showed that (1) exogenously added sTRAIL generated procoagulant EMPs; (2) supernatants from thrombin-stimulated endothelial cells induced EMP release by HMEC-1 cells and HUVECs, whereas those recovered from TRAIL knockdown endothelial cells displayed no effect. TRAIL/ TRAIL-R2 complex mediated EMP release by initiating the recruitment of adaptor proteins and the activation of nuclear factor B. Moreover, sTRAIL modulated intercellular adhesion molecule-1 and interleukin-8 expression induced by thrombin by a downstream pathway involving nuclear factor B activation. Our data reveal a novel mechanism controlling EMP release and identify TRAIL as a key partner in the pathway linking coagulation and inflammation elicited by thrombin. Key Words: endothelium Ⅲ vesiculation Ⅲ cell signaling Ⅲ inflammation Ⅲ coagulation Ⅲ thrombosis T hrombin is a serine protease that plays a key role in the pathogenesis of vascular diseases. It is generated at sites of vessel damage and controls the formation of a hemostatic plug. Thrombin plays a central role in the crosstalk between inflammation and coagulation by inducing the synthesis of proinflammatory cytokines that activates the coagulation cascade 1 and by impairing anticoagulant mechanisms. 2 It also exerts pleiotropic effects on endothelium. 3 Recent data from our laboratory indicated that thrombin induces the release of microparticles by endothelial cells (endothelial microparticles [EMPs]), an effect mediated by the receptor PAR-1. 4 Microparticles (MPs) are a heterogeneous population of small membrane-coated vesicles resulting from disruption of the membrane phospholipid asymmetry, leading to phosphatidylserine exposure in response to cell activation or apoptosis. Their capacity to carry surface antigens, cytoplasmic proteins, or nucleic acids from their parent cells confers to MPs a major role in cell communication by binding to their targets or facilitating cell-cell interactions. 5 In theory, all cells have the capacity to form MPs and endothelial cells generate EMPs that provide procoagulant surfaces able to trigger coagulation activation. 6 Indeed, once available at the surface of the MP, phosphatidylserine exposure pro...

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

“…A role for Pyk2 in atherosclerosis has also been reported previously (18). Because thrombin is produced at the site of vascular injury (6,7) and it causes endothelial cell barrier disruption (19) and inflammation (20), we asked the question of whether it also influences the migration of monocytes/macrophages and, if so, whether it requires Pyk2 activation. In the present study, we report that 002067.2), ASO1 (5Ј-GGGCUUTGCTCTCCTCCATCCG-GUU-3Ј), ASO2 (5Ј-UCCACTTCCTCCGCTCCGAC-3Ј); hGNA12 (NM_007353.2), ASO1 (5Ј-GGUGGUGAAGTGGT-GGAAGAGUGG-3Ј), ASO2 (5Ј-GCCAGAATCCCTCCAGA-GUGCGCU-3Ј); hPyk2 (NM_004103), ASO1 (5Ј-GGUCUGT-ACTTAGGTCGGCUGGGC-3Ј), ASO2 (5Ј-CCUGUGTCCA-TAGCCCAGAGUCCC-3Ј), ASO3 (5Ј-GUCCUCCACCATC-TGCUUCCU-3Ј); hGab1 (NM_207123), ASO1 (5Ј-GUUCCG-CTTCTCACCATCUUUCCU-3Ј), ASO2 (5Ј-GGUCUGTAC-TTAGGTCGGCUGGGC-3Ј), ASO3 (5Ј-CATGCATAACGC-TTCAACT-3Ј); hp115 RhoGEF (NM_199002), ASO1 (5Ј-UUCCUCCTCCAACTCCUCCA-3Ј), ASO2 (5Ј-CCUUGTC-CTTAGTCACCCGC-3Ј), ASO3 (5Ј-GCUUCACCTGGCTC-TUGGGC-3Ј); hRac1 (NM_006908), ASO1 (5Ј-UCCGUCTC-CCACCACCACACACUU-3Ј), ASO2 (5Ј-UUUCUCTTCCT-CTTCUUCAC-3Ј); hRhoA (NM_001664), ASO1 (5Ј-ACUCU-ATCCTGCTTTCCATCCACCU-3Ј), ASO2 (5Ј-UGGUGTG-TCAGGTGGGAGUG-3Ј), ASO3 (5Ј-ACCUCTCTCACTCC-AUCUUU-3Ј); hPak2 (NM_002577), ASO1 (5Ј-UCAUCATC-ATCCTCCTCCUCUGUC-3Ј), ASO2 (5Ј-GGUGCTTCTGT-TCCCUUGGC-3Ј), ASO3 (5Ј-GTCCCACAAATCCCTTCCT-3Ј).…”

“…A large body of evidence suggests that thrombin and its receptors (PARs), particularly PAR1, play a role in endothelial barrier disruption, promoting the sticking of leukocytes and monocytes/macrophages to the endothelium and their infiltration into the subendothelial space (4,6,20,21), which are the hallmarks of atherogenesis (57). An enhanced expression of PAR1 in the regions of inflammation associated with macrophage influx, smooth muscle cell proliferation, and mesenchymal-like intimal cells further supports a role for its involvement in atherosclerosis (58 -60).…”

Microparticles, Vascular Function, and Atherothrombosis

“…12,13 Caspase activities have been identified in different MPs, and thus could represent an attempt for cells to escape cellular apoptosis. 14 -16 Non-standard Abbreviations and Acronyms HUVEC 177 Nomura et al 178 Stampfuss et al 179 … Thrombin Sapet et al, 18 Simoncini et al 20 Barry et al, 60 Dale et al, 180 Chang et al, 181 … … Collagen … Barry et al, 60 Boilard et al, 182 Chang et al, 181 … … Homocysteine Sekula et al 183 Olas 187 Curtis et al, 19 Abid Hussein et al, 15 Wang et al, 22 Nomura et al, 178 Piguet et al 188 Schecter et al 189 Jungel et al 190 Oxidative stress Vince et al, 191 Endothelial MP formation and release have received significant attention over the past recent years, and different signaling pathways have been identified depending on the stimuli (Table). 17 Clearly, endothelial MP shedding can occur independently of endothelial apoptosis.…”

“…First, thrombin binds to its proteaseactivated receptor-1, followed by Rho kinase II activation. Second, a later pathway involves TRAIL/Apo2L, a cytokine that belongs to the TNF-␣ superfamily, 20 followed by interleukin (IL)-1 release and IL-1 receptor activation. 21 The second phase is characterized by an amplification loop based on the release by endothelial cells stimulated by thrombin of soluble forms of TRAIL and of IL-1 that act in an autocrine or paracrine manner on endothelial cells and stimulate MP shedding.…”

Les microparticules endothéliales

“…Des équipes tentent actuellement de déterminer les processus intracellulaires à l'origine de la libération de microparticules [12]. Le TNF-α (tumor necrosis factor), un puissant inducteur de la libération de microparticules endothéliales [1], pourrait être contrôlé par les biothérapies anti-TNF-α.…”