Subcellular localization of tissue factor and human coronary artery smooth muscle cell migration.

Summary. Background: Tissuefactor(TF)isthemostrelevantphysiological trigger of thrombosis. Additionally TF is atransmembrane receptor with cell signaling functions. Objec-tives: The aim of this study was to investigate TF subcellularlocalization, function and signaling in human coronary arterysmooth musclecell migration. Methods: Coronary arteries andprimary cultures of vascular smooth muscle cells (HVSMC)wereobtainedfromhumanexplantedhearts.WoundrepairandBoyden chamber assays were used to measure migration invitro. TF-pro-coagulant activity (TF-PCA) was measured inextracted cellular membranes. Analysis of TF distribution wasperformed by confocal microscopy. A nucleofector device wasusedforTFandproteaseactivatedreceptor2(PAR2)silencing.mRNAlevelswereanalyzedbyRT-PCR. Results: InmigratingHVSMC TF translocates to the leading edge of the cellsshowinganintensepatch-likestaininginthelamellipodia.Inthemigrating front TF colocalizes with filamin (FLN) in thepolarized lipid rafts. TF-PCA was increased in migrating cells.Silencing of the TF gene inhibits RSK-induced FLN-Ser-2152phosphorylation, down-regulates CDC42, RhoA, and Rac1protein expression and significantly inhibits cell migration.Silencing PAR2 also inhibits cell migration; however, silencingboth TF and PAR2 induces a significantly higher effect onmigration. Smooth muscle cells expressing TF have beenidentified in non-lipid-rich human coronary artery atheroscle-rotic plaques. Conclusions: TF translocates to the cell front inassociation with cytoskeleton proteins and regulates HVSMCmigrationbymechanismsdependentandindependentoffactor(F)VIIa/PAR2.TheseresultsextendthefunctionalroleofTFtosmooth muscle cell trafficking in vessel wall remodeling.Keywords: atherosclerosis,filamin,smoothmuscle,thrombosis,tissue factor.IntroductionTissue factor (TF) is currently considered the single mostrelevant physiological trigger of blood coagulation in responseto vascular injury [1–3]; however, TF has also shown to beinvolved in various cellular processes associated with vascularsmooth muscle cell function [4,5]. Under physiological condi-tions TF antigen is not detected in the intima or media ofhuman vessels [6] with its expression restricted to the adventitia[7,8]. However, TF is widely found in atherosclerotic lesionsassociated with the lipid core and infiltrated inflammatory cells[9]. Vascular smooth muscle cells have been shown to expressTF upon induction by growth factors, vasoactive agonists andclotting factors [4,10]. The plasticity of VSMCs for undergoingphenotypic changes is well known. In vascular remodelingprocesses, from atherogenesis to restenosis, VSMCs migratefrom the media to the intima in a process that involves genereprogramming and protein synthesis. Filamin A (FLN) isessential for mammalian cell locomotion. FLN is important inthe stabilization of the cortical skeleton of cells and mightinfluence the processing and expression of many membraneproteins. Previous studies reported that cells lacking FLN haveimpaired locomotion [11,12].TF, a member of the cytokine receptor family, is a 47-kDaglycosylated transmembrane protein [13,14] whose activity ismodulated by a variety of factors, including its membranedomain association, the phosphatidylserine transmigration tothe outer leaflet of the plasma membrane [15,16] and thesphingomyelin levels [10]. TF activity is also modulated by theredox state of its membrane proximal cysteine pair (Cys186/Cys209) [17], as oxidation of this cysteine pair increases theprocoagulant activity of TF. The extracellular domain of TFbinds the multi domain coagulation serine protease factor(F)VII with sub-nanomolar affinity leading to its allostericactivation (FVIIa) [18] and to the resulting TF–FVIIa complexthat activates FIX [19] and FX [20]. Ligand binding not onlyinitiates the coagulation cascade but also induces cell signalingthroughthecytoplasmicdomainofTFbyactivatingG-proteincoupled protease-activated receptors (PAR) [21,22]. PARactivation causes the mobilization of cytosolic Ca