Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal-Regulated Kinase Phosphorylation and...

Thrombomodulin Prolongs Thrombin-Induced ExtracellularSignal–Regulated Kinase Phosphorylation and Nuclear

Retention in Endothelial CellsJean-Marc Olivot, Eva Estebanell, Monique Lafay, Brigitte Brohard, Martine Aiach, Francine Rendu

Abstract—On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, andto protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cellproliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellularsignal–regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin andthrombin receptor–activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, aninhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies ofpERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-acti-vated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in thepresence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombinin the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs areinvolved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERKnuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of bloodcoagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferationin response to thrombin.(Circ Res. 2001;88:681-687.)

Key Words: thrombin n thrombomodulinn human endothelial cellsn mitogen-activated protein kinase

Thrombin is a multifunctional serine protease generated atsites of vascular injury. Thrombin plays a key role in

blood coagulation and thrombotic disorders. It acts as thecentral enzyme of the coagulation cascade by cleavingfibrinogen into fibrin and favoring its own production byactivating several coagulation factors by limited proteolysis.Thrombin also regulates its own formation after binding tothrombomodulin (TM), an integral membrane-bound glyco-protein expressed on endothelial cells. TM acts as a cofactorof thrombin to activate protein C, a serine protease ensuringproteolytic inactivation of 2 coagulation factors, factor Vaand factor VIIIa. Thrombin also interacts with a variety ofcells mediating inflammatory and proliferative responses tovascular injury.1 For all protein and cellular interactions,thrombin has a recognition site and a catalytic active site. Bythe former, called the anion-binding exosite (ABE1), throm-bin binds to specific negatively charged sequences. By thecatalytic site, thrombin exerts its proteolytic activity.2

On vascular endothelial cells, thrombin ABE1 binds toTM. This binding occurs through the epithelial growth factor

(EGF)-like domains 4 and 5 of TM.3 Thrombin ABE1 alsobinds to the typical heptahelical thrombin receptor, the firstmember of protease-activated G protein–coupled receptorfamily (PAR1).4 Thrombin catalytic site cleaves the amino-terminal tail of PAR1 that flips over to effect receptoractivation. Endothelial cell responses to thrombin includeprostacyclin, von Willebrand factor secretion, and cell pro-liferation.5 Synthetic peptides that mimic this new aminoter-minus, so-called thrombin receptor–activating peptides(TRAPs), are full agonists for receptor activation, even afterthrombin cleavage.6

Comparison of thrombin and TRAP as to their ability toinduce proliferation has led to the postulation that TMmodulates PAR1 activation. Overexpression of TM onsmooth muscle cells downregulates proliferation (estimatedby 3H thymidine incorporation into the DNA) induced byboth TRAP and thrombin, and the degree by which cellproliferation was blocked is directly correlated with the levelof TM expressed on the cell surface.7 On endothelial cells, theinhibition of thrombin binding to TM by a monoclonal

Original received October 6, 2000; revision received January 18, 2001; accepted February 9, 2001.From the Unité INSERM 428, Faculte de Pharmacie, Université René Descartes, Paris, France. Present address for J.-M.O. is Service de Neurologie,

Hôpital Tenon, Paris, France. Present address for E.E. is Servicio de hemoterapia y hemostasia, Hospital Clinic, Barcelona, Spain.Correspondence to Dr Francine Rendu, U428 INSERM, Faculté de Pharmacie, 4 avenue de l’Observatoire, F-75270, Paris, Cedex, France. E-mail

[email protected]© 2001 American Heart Association, Inc.

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antibody 3E2, directed against the fourth EGF-like domain,8

resulted in an increase of thrombin mitogenic effect.9 In awide range of tumoral cells, TM seems to exert an inhibitoryeffect on cell proliferation10 independently of its thrombincofactor activity. Thus, TM not only acts as a cofactor forprotein C activation to inhibit thrombin generation but alsoseems to modulate the mitogenic signal triggered by PAR1activation.

The mitogenic signal induced by many stimuli occursthrough mitogen-activated protein kinases (MAPKs), amongwhich are the two extracellular signal–regulated kinases(ERKs) p44ERK1 and p42ERK2. ERK1 and ERK2 are ubiquitouscytosolic serine-threonine kinases involved in cellular growthand proliferation. Their activation requires phosphorylationof their tyrosine and threonine residues.11 Phosphorylation ofERKs promotes their dimerization and nuclear transloca-tion.12,13Once in the nucleus, phosphorylated ERKs (pERKs)activate various transcription factors. Duration of ERK phos-phorylation and nuclear translocation are thus determinant forcell responses.14 In fibroblasts, thrombin and TRAP activateERKs to induce their proliferation.15 In endothelial cells,pERKs activate cytosolic phospholipase A2 implicated inprostacyclin secretion.16,17

With this as background, we studied the TM-modulatingrole on thrombin-induced nuclear translocation of pERKs.For this purpose, we used an antibody anti-TM to preventthrombin binding to TM and a thrombin mutant with nocatalytic activity but the ability to bind TM. Our studyindicates that in human umbilical vein endothelial cells(HUVECs), PAR stimulation leads to ERK phosphorylationand nuclear translocation and TM downregulates PAR signalto enhance pERK nuclear retention.

Materials and Methods

MaterialsHuman purified thrombin was purchased from Diagnostica Stago.The synthetic TRAP (SFLLRN) and the anti-TM antibody 3E2 werefrom Serbio. S195A was provided by Dr B. Le Bonniec18 (Faculté dePharmacie, Paris, France). Polyclonal anti-ERK antibodies werepurchased from New England Biolabs. PD98059 was from Calbio-chem. Peroxidase coupled and biotinylated anti-rabbit antibodies and[3H]-thymidine were purchased from Amersham. All culture mediumand material were purchased as previously described.9 Texas Redstreptavidin was from Jackson ImmunoResearch, Interchim. Thenucleus marker 4,6-diamidino-2-phenylindol (DIAP) Vectashieldwas from Vector Laboratories.

Cell CultureHUVECs were obtained as originally described by Jaffe et al.19 Cellswere maintained in a humidified atmosphere of 5% CO2 at 37°C, andthe culture medium was changed every 48 hours until cells wereconfluent. Cells were detached by 0.5% trypsin-EDTA and wereseeded either on 6-well plates for immunoblotting studies andmitogenesis assay or on glass coverslips for immunocytochemicalstudies. Cells were used at first and second passages.

3H-Thymidine Incorporation Into the DNAThe DNA synthesis was evaluated by measuring the incorporation of59-[3H]-thymidine in the insoluble material precipitated with trichlo-roacetic acid, as previously described.9 PD98059 was added 15minutes before agonists.

ImmunoblottingCells that had reached 80% confluence were serum-starved (0.5%FCS) for 18 hours. The agonists thrombin (10 nmol/L), TRAP (100mmol/L), and S195A (200 nmol/L) and the 3E2 antibody (50mg/mL)were added in the culture medium without FCS. At the end of thestimulation, cells were lysed, and proteins were separated by gelelectrophoresis. Proteins were transferred on nitrocellulose, and thephosphorylated forms of p44ERK1 and p42ERK2 were identified withthe anti–phospho-ERK antibody (1:1000 in Tris-buffered saline[TBS]) according to the manufacturer’s instructions (New EnglandBiolabs). The amount of p44ERK1 and p42ERK2 proteins within eachsample was controlled after stripping of the blot that was reprobedwith a polyclonal anti-ERK1 and anti-ERK2 antibody (1:1000 inTBS) to ensure that equivalent amounts of ERKs were present.

Immunocytochemical AnalysisThe localization of pERKs was studied as previously described forfibroblasts.20 HUVECs were seeded on gelatin-coated glass cover-slips and activated as above. HUVEC activation was stopped byremoval of the medium and washed in TBS (Tris 0.2 mol/L and NaCl0.8 mol/L, pH 7.6). The cells were fixed in 50% vol/vol methanoland 50% vol/vol acetone at 20°C for 8 minutes. After 6 washes inTBS, cells were incubated overnight at 4°C with the anti–phospho-ERK antibody (1:200 in TBS containing 3% vol/vol BSA). Theincubation was stopped by 6 washes in TBS, and cells wereincubated with biotinylated anti-rabbit IgG for 1 hour. After 6washes in TBS, cells were incubated with Texas Red streptavidin for1 hour. After the last washing step, cells were fixed for 30 secondsin absolute ethanol, washed in TBS and covered with mountingmedium with DIAP, and examined with excitation-emission filtersfor rhodamine and for DIAP.

Statistical AnalysisResults of mitogenic assay were compared using Student’st test. Forimmunocytochemical analysis, at least 80 to 150 cells from at least3 separate experiments were examined to quantitate the amount ofpERK visualized. Statistical analysis was performed using Student’sunpairedt test.

ResultsEffect of MEK Inhibitor PD98059 on3H-Thymidine Incorporation Into DNA Induced byTRAP or ThrombinTo establish whether ERKs are involved in HUVEC prolif-eration induced by thrombin or TRAP, we studied the effectof the MAPK kinase (MEK) inhibitor PD98059.21 Cellproliferation was estimated by [3H]-thymidine incorpora-tion.22 As previously shown, HUVEC proliferation is moreimportant in response to TRAP than to thrombin, at optimalconcentrations, ie, 10 nmol/L and 100mmol/L for thrombinand TRAP, respectively (Figure 1).9 Increasing concentra-tions of PD98059 resulted in significant decrease in DNAsynthesis induced by thrombin or TRAP. The inhibitoryeffect of 10mmol/L PD98059 was.80% both with thrombinand TRAP. This concentration was ascertained to inhibitthrombin- and TRAP-induced ERK phosphorylation but notglobal tyrosin phosphorylation in HUVECs (data not shown).The results show that DNA synthesis induced by thrombinand TRAP involves the phosphorylation of ERKs inHUVECs.

Kinetics of ERK Phosphorylation Induced byThrombin and TRAPThe phosphorylation of ERKs was analyzed by Westernblotting using an antibody specific for the phosphorylated

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forms of p44ERK1 and p42ERK2. After the addition of TRAP orthrombin, the two proteins were rapidly and heavily phos-phorylated after 15 minutes (Figure 2, top). The maximumphosphorylation level was identical with the two agonists andwas reached between 60 and 90 minutes (top left). After 3hours of activation, ERKs were slightly less phosphorylatedwith TRAP than with thrombin, a difference even morepronounced after 4 and 5 hours (top right). The dephosphor-ylation of ERKs back to the basal level was complete after 5hours with TRAP and 6 hours with thrombin (not shown). Animmunoblot anti-ERK1 and anti-ERK2 was performed ateach time of activation to ensure that equal amounts of totalERKs were deposited on the gel before the blot analysis(Figure 2, bottom).

ERK Phosphorylation and Localization Triggeredby TRAP or ThrombinPhosphorylation of MAPKs is essential for their activationand nuclear translocation.11,13 Moreover, MAPK nuclearretention duration is critical for the determination of theresponse.14 We therefore studied the intracellular localizationby immunofluorescence (Figure 3). For this purpose, thepERK1 and pERK2 were visualized by Texas Red, and thenucleus was visualized by coloring with blue. Before theaddition of the inducers (Figure 3A), pERKs were at basallevel and could not be detected at a significant level in thecytoplasm nor in the nucleus. After the addition of TRAP and

thrombin, the two proteins were rapidly phosphorylated to thesame level with the two agonists after 15 minutes (Figures 3Cand 3D) and the pERKs were scattered in both the nucleusand the cytosol, with an important amount in the nucleus.After 4 hours of activation, ERKs were less phosphorylatedthan at short times and more retained in the nucleus ofHUVECs with thrombin (Figure 3F) than with TRAP (Figure3E). Thus, 27% of thrombin-stimulated cells exhibited high-level pERKs in their nucleus (Figure 3F) compared with 7%in TRAP-stimulated cells (P50.04). The results suggest thatin HUVECs the amount of DNA synthesis generated byPAR1 stimulation is inversely correlated with the duration ofnuclear pERK retention.

Regulation of the PAR1-Generated ActivationSignal by ThrombomodulinIn view of the modulating effect of TM on PAR1 activationthat we described on DNA synthesis in HUVECs,9 wesearched for a modulating effect of TM on ERK phosphory-lation duration. For this purpose, we used 2 different tools:S195A, a mutant thrombin that binds to TM and has noproteolytic activity,18 and a specific anti-TM antibody. Re-sults obtained with the mutant thrombin S195A are presentedin Figure 4. The thrombin mutant S195A (200 nmol/L),which does not stimulate DNA synthesis,9 induced a lowphosphorylation of ERKs (Figure 4B) compared with control(Figure 4A). The addition of S195A simultaneously withTRAP resulted in a higher ERK phosphorylation after 4 hours(Figure 4D) than in the presence of TRAP alone (Figure 4C).Moreover, S195A retained the pERKs in the nucleus (Figure4D), whereas phospho-ERKs were no longer present after 4hours of stimulation by TRAP alone (Figure 4C). The numberof cells exhibiting a high level of pERK was increased from7% with TRAP alone to 18% when S195A was added toTRAP.

We next prevented thrombin binding to TM with themonoclonal antibody 3E2 that we previously described toincrease thrombin-induced DNA synthesis.9 Comparedwith control (Figure 5A), anti-TM by itself induced a slight

Figure 1. MEK inhibitor, PD98059, inhibits3H-thymidine incorporation in HUVECs.After addition of 10 nmol/L thrombin or100 mmol/L TRAP in the presence of 10mmol/L PD98059 or vehicle, 3H-thymidineincorporation into endothelial cells wasmeasured. Results are presented asmedian of 3 different experiments6SEM.*Student’s unpaired t test; P,0.05. Inset,Effect of increasing PD98059 concentra-tions on 3H-thymidine incorporationinduced by thrombin (solid lines) or byTRAP (dashed lines).

Figure 2. Kinetics of phosphorylation of ERKs after thrombin-and TRAP-induced stimulation of HUVECs. Cells were eitherunstimulated or stimulated by 10 nmol/L thrombin or 100mmol/L TRAP for the indicated times. Cells were lysed, andsamples were separated on SDS-PAGE. Blots were probed withanti–phospho-ERKs (top). Blots were stripped and reprobedwith anti-general ERK antibody (bottom).

Olivot et al Thrombomodulin and ERK Activation 683



increase of ERK phosphorylation (Figure 5B), as did,however, an irrelevant antibody (Figure 5C). After 4 hoursof stimulation by thrombin, the amount of pERKs in thenucleus (Figure 5D) was decreased in the presence ofthe anti-TM (Figure 5E), suggesting that it accelerated thedephosphorylation. Moreover, in the presence of anti-TM,in 30% of the cells the pERKs remained at the edge of thenucleus membrane (red annulus around the blue nucleus)within the cytoplasm (Figure 5E) compared with,3% inthe absence of the antibody. Thus, the number of thrombin-stimulated cells exhibiting a high level of pERKs in theirnucleus was significantly reduced from 27% to 14% in thepresence of the anti-TM antibody (P50.02). These resultssuggest that thrombin binding to TM contributes to themaintenance of pERKs and nuclear retention.

DiscussionThe activation of ERKs is an important step in cell stimulus–response coupling, and their localization is determinant oftheir function. In the present study, we found that afterstimulation of HUVECs by thrombin or TRAP, ERK1 andERK2 phosphorylation duration and nuclear retention wereinversely correlated to the DNA synthesis. TRAP, a strongmitogen for HUVECs, induced a short-lasting phosphoryla-tion and nuclear retention of pERKs; thrombin, which in-duced a lower DNA synthesis than did TRAP, induced along-lasting phosphorylation and nuclear retention of pERKs.These results additionally demonstrate the regulatory role ofTM in thrombin-induced PAR1 activation: TM, known toreduce HUVEC proliferation in response to thrombin, ex-tended pERK nuclear retention.7,9

The activation of ERKs is dependent on a dual-specificityMAPK kinase, MEK.14,23 ERKs are phosphorylated on aTXY motif, both on threonine and tyrosine residues, andthese phosphorylations are essential for activity.11 The stim-ulation of HUVECs by thrombin and TRAP led to DNAsynthesis through activation of the ERK pathway. First,proliferation of DNA synthesis was strongly and dose-dependently inhibited by PD98059. PD98059 is a semiselec-tive inhibitor of MEK1 and MEK2 and thus inhibits thephosphorylation of MEK substrates, the MAPKs. Second,thrombin and TRAP induced the phosphorylation of 2 mem-bers of the MAPK family, p44ERK1 and p42ERK2. This phos-phorylation was rapid and reached a plateau of approximatelysimilar amplitude with thrombin and TRAP. After longer

Figure 3. Immunolocalization of pERKs after thrombin- andTRAP-induced stimulation of HUVECs. Cells were eitherunstimulated (A and B) or stimulated by 100 mmol/L TRAP (Cand E) or 10 nmol/L thrombin (D and F). After 15 minutes (Cand D) or 4 hours (E and F) of stimulation, cells from dupli-cate wells were fixed and examined by fluorescence micros-copy after staining with the same anti–phospho-ERK anti-body, a second biotinylated antibody, and Texas Redstreptavidin and after staining with DIAP (blue) to visualizethe nucleus. Unstimulated cells were incubated with eithersaline (A) or an irrelevant antibody (B) and stained as above.Bar530 mm.

Figure 4. Phosphorylation and immunolocalization of ERKs afterTRAP-induced stimulation of HUVECs in the presence of S195A.Cells were either unstimulated (A) or stimulated by 200 nmol/LS195A (B), 100 mmol/L TRAP (C), or S195A plus TRAP simulta-neously (D) for 4 hours before fixation and examination as inFigure 3. The corresponding Western blots obtained with theanti–phospho-ERK antibody as in Figure 2 are shown at thebottom. Bar530 mm.




times (.2 hours), phosphorylation decreased, and the de-phosphorylation was more rapid with TRAP than with throm-bin. The duration of ERK phosphorylation and activation iscritical for the cell signaling decision: cells use transient orsustained activation of ERKs, depending on the response.14 Inthe present study, we showed that in HUVECs, activation ofPAR1 induced transient ERK phosphorylation and led toDNA synthesis.

The MAPK pathway that ultimately leads to cell prolifer-ation or differentiation provides a mechanism that transmitsthe information to the nucleus.24 The nuclear translocationstep concerns activated pERKs.13 Once in the nucleus, ERKsphosphorylate and activate transcriptional factor.25,26 InHUVECs, using immunocytochemical analysis, we showedthat pERKs were translocated in the nucleus after stimulationby TRAP and by thrombin. However, the retention time in thenucleus was different with the two agonists. After 4 hours ofstimulation, pERKs were still present in the nucleus ofthrombin-activated cells, whereas they were barely detectablein TRAP-stimulated cells. Thus, pERK retention in thenucleus correlated with the protein phosphorylation state.

Thrombin and TRAP may activate different PAR recep-tors. At least 3 PAR receptors have already been described onendothelial cells (PAR1, PAR2, and PAR3), of which PAR1and PAR2 are involved in endothelial cell proliferation.27,28

Moreover, TRAP is able to activate PAR1 even after throm-bin cleavage. On the other hand, HUVEC proliferation

induced by thrombin is downregulated by thrombin bindingto TM.9 Overexpression of TM on smooth muscle cell surfacedecreases the TRAP-induced proliferation.7 Surface TM ex-pression downregulates melanoma cell proliferation indepen-dently of its thrombin cofactor activity.10 In the strategy tospecifically isolate the effect of thrombin binding to TM, weused a thrombin analogue devoid of any catalytic activity,S195A, and a specific monoclonal anti-TM antibody, 3E2.Hence, using these 2 different tools, we demonstrated thatTM regulated the pERK nuclear retention. First, in thepresence of anti-TM antibody that inhibits thrombin bindingto TM, pERK nuclear retention was reduced. Second, theaddition of an inactive thrombin mutant that binds to TMtogether with TRAP prolonged pERK nuclear retention.These data suggest that thrombin binding to TM triggers asignal that enhances pERK nuclear retention. Thus, in addi-tion to its role of cofactor to activate protein C, TM seems toact as a new cell receptor for thrombin.

The mechanism required for the nuclear translocation hasbeen elucidated for ERK2: phosphorylation creates charge-charge interactions that result in ERK dimerization, anddimers are actively imported into the nucleus.13 The transientphosphorylation of ERKs in stimulated HUVECs suggeststhat the proteins are under the control of phosphatases. Manycytosolic and nuclear MAPK phosphatases dephosphorylateERKs in their respective cell compartments.23,29 Similarphosphatase-controlled phosphorylation regulation could oc-cur in HUVECs. The results described above also suggest thatTM could govern nuclear pERK retention. Thrombomodulincould trigger a signal to inhibit a nuclear MAPK phosphatase(MKP), such as MKP1 (Figure 6). MKP1 is implicated in theinitiation of the cell cycle and activated by pERKs via afeedback control.30 It is not unlikely that TM controls MKP1activation by pERKs.

Thrombin-induced endothelial cell response is of specialimportance during ischemia consecutive to arterial embolicdisorders. The latter corresponds to pathological situations inwhich the endothelium integrity is relatively preserved, incontrast to local thrombosis occurring at the site of anatherosclerotic plaque rupture. The thrombin generated by anembolic clot comes into contact with endothelial cells andenhances edema, cell proliferation, and fibrosis.1 The in-volvement of TM, on the one hand, and pERK signaling, onthe other hand, has been emphasized in arterial thromboticdisorders. ERK activation leads to deleterious effects duringfocal brain or myocardial ischemia.31–33Although myocardialinfarction is most frequently the consequence of local throm-bosis, ischemic strokes more frequently result from embolicdisorders.34 TM expression has been demonstrated in brainvessels, because TM mRNA expression is present in theblood-brain barrier35 and protein C is activated in jugular veinblood during carotid artery occlusion in humans.36 Theendothelial TM expression in brain is constitutively down-regulated by astrocytes and acutely downregulated by patho-genic mediators of ischemic stroke, such as interleukin-1band tumor necrosis factor-a.35 Interestingly, astrocyte-induced downregulation of TM endothelial expression occursthrough transforming growth factor-b secretion,37 known toenhance endothelial cell proliferation.38 Overall, that TM

Figure 5. Phosphorylation and immunolocalization of ERK1 andERK2 after thrombin-induced stimulation of HUVECs in thepresence of an anti-TM antibody. Cells were either unstimulated(A through C) or stimulated by 10 nmol/L thrombin alone (D) or10 nmol/L thrombin in the presence of 50 mg/mL anti-TM anti-body 3E2 (E) for 4 hours before fixation and examination as inFigure 3. Unstimulated cells were incubated with an irrelevantantibody (B) or the anti-TM alone (C) and stained as in panel A.The corresponding Western blots obtained with the anti–phospho-ERK antibody as in Figure 2 are shown at the bottom.Bar530 mm.




expression and cell proliferation are inversely related fits withour results, showing a control of DNA synthesis by TM.

In conclusion, we show that TM controls the thrombin-induced ERK pathway in endothelial cells. Thus, TM, whichplays a key role in the regulation of blood coagulation, couldalso be critically involved in the endothelial response tothrombosis. The latter would be of important clinical rele-vance in acute arterial ischemia, as recently suggested byepidemiological studies on myocardial infarction.39,40 It isthus worthwhile to study the implication of TM in ischemicstroke.

AcknowledgmentsThis work was supported by Association pour la Recherche sur leCancer (grant 9545 to F.R.). J.-M.O. was a recipient of a grant fromthe Fondation pour la Recherche Médicale. We are indebted to Dr B.Le Bonniec for providing us with the mutated thrombin S195A andto Drs G. Keryer and J. Badet for their helpful advice for theimmunofluorescence study.

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Francine RenduJean-Marc Olivot, Eva Estebanell, Monique Lafay, Brigitte Brohard, Martine Aiach and

Phosphorylation and Nuclear Retention in Endothelial CellsRegulated Kinase−Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal

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