Activation of PAR4 Induces a Distinct Actin Fiber Formation via p38 MAPK in Human Lung Endothelial Cells
Department of Molecular Pathology, Nippon Medical School, Graduate School of Medicine, Institute of Gerontology, Kanagawa, Japan
Correspondence to: Oichi Kawanami, MD, PhD, Department of Molecular Pathology, Nippon Medical School, Graduate School of Medicine, Institute of Gerontology, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, Japan. E-mail: kawanami{at}nms.ac.jp
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Summary |
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Key Words: pulmonary endothelial cells G proteincoupled receptors protease-activated receptor thrombin actin p38 MAPK
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Introduction |
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Stimulation of PARs is known to induce a variety of cellular effects in many types of cells. In the endothelial cells, for example, PAR1 upregulated cyclooxygenase-2 expression (Houliston et al. 2002) and intercellular adhesion molecule-1 transcription (Rahman et al. 2002
), and PAR2 induced tissue factor expression and von Willebrand factor release (Langer et al. 1999
). These results indicate the multifunctionality of these G proteincoupled receptors and show the functional involvement of receptors in a number of events taken place in endothelial cell. However, functional analysis of PAR4 has been limited to the studies in platelets, smooth muscle cells, and epithelial cells (Bretschneider et al. 2001
; Asokananthan et al. 2002
; Covic et al. 2002
; Henriksen and Hanks 2002
), but not in endothelial cells.
Previously, we have shown the preferential expression of PAR4 in human lung vascular endothelial cells in vitro (Fujiwara et al. 2004). In PARs signaling, activation of mitogen-activated protein kinase (MAPK) takes an important part in endothelial cell function (Marin et al. 2001
; Kataoka et al. 2003
). Among the MAPK family, p38 MAPK was shown to regulate actin cytoskeletal remodeling in pulmonary microvascular endothelial cells on intercellular adhesion molecule-1 ligation (Wang and Doerschuk 2001
). Furthermore, remodeling of actin fibers was deeply involved in the major functions of endothelial cells, such as permeability (Kouklis et al. 2004
), endothelium-dependent relaxation (Hamilton et al. 2001
), cell migration (Vasanji et al. 2004
), microtubele integrity (Bayless and Davis 2004
), and leukocyte adherence (Vergnolle et al. 2002
).
In this study, we investigated whether PAR4 and PAR1 play different roles in actin reorganization in human pulmonary artery endothelial cells (HPAEC) and human microvascular endothelial cells from lung (HMVEC-L), and whether the actin formation by elicitation of PAR4 or PAR1 is p38 MAPK-dependent in these human lung endothelial cells. Furthermore, we examined if PAR4-induced actin fibers display different morphology from the PAR1-induced actin fibers. The results indicated that the functional role of PAR4 in lung endothelial cells involved actin fiber formation and that the resulting morphology of the fibers differed from that derived from PAR1 activation through a distinct signaling pathway.
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Materials and Methods |
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Endothelial Cell Culture and Tissue Sections
HPAEC and HMVEC-L obtained from Clonetics (Walkersville, MD) were cultured in endothelial cell basal medium (EBM) (Clonetics) supplemented with endothelial growth medium-microvascular singlequots. Cells were plated onto gelatin-coated flasks, and grown under 5% CO2 at 37C.
Formalin-fixed, paraffin-embedded tissue sections from human lung and lymph node were obtained from the surgical pathology division.
Immunohistochemistry
Tissue sections (4 µm) were deparaffinized in xylene and rehydrated in ethanol series. Endogenous peroxidase activity in the sections was blocked by 0.3% (v/v) hydrogen peroxide in distilled water for 10 min. To retrieve antigens, the sections were placed in an antigen retrieval medium (Immunosaver, Nissin EM Co., Ltd, Tokyo, Japan) and heated at 98C for 1 hr. After cooling the sections to the room temperature, nonspecific binding sites were inhibited by incubation with 1:10 normal rabbit serum for 30 min. The slides were then incubated overnight with a 1:50 dilution of polyclonal goat anti-PAR4 antibody. The reaction product was visualized using the labeled streptavidin-biotin system (DAKO; Kyoto, Japan) and 3, 3'diaminobenzidine as a chromogen, and the sections were counterstained with hematoxylin. Negative control sections were processed by substitution of the primary antibody with normal goat serum. The intensity of immunostaining was semiquantitated as: , negative; +, weak; ++, moderate; and +++, strong.
Semiquantitative RT-PCR
Cultured endothelial cells were washed and then lysed in guanidine thiocyanate-containing buffer and total RNAs were extracted using the RNeasy Mini Kit (Qiagen; Hilden, Germany) with DNase I treatment. Semiquantitative RT-PCR for PAR4 was performed as previously described (Fujiwara et al. 2004). The PCR was performed at 94C for 45 sec, 58C for 45 sec, and 72C for 2 min. After 24 cycles of amplification, cDNA products were visualized with SYBR Green I (Molecular Probes; Eugene, OR) and band images were captured using Molecular Imager FX. Signal intensity of PAR4 was then quantitated by PDQuest software (Bio-Rad; Hercules, CA) and normalized to or against glyceraldehyde-3-phosphate dehydrogenase signal intensity. Independent experiments were conducted three times. RT-PCR reaction with no SuperScript RNase H reverse transcriptase did not show any PCR products.
Western Blot Analysis for PAR4 and p38 MAPK Activations
Endothelial cells were plated in 60-mm dishes to reach subconfluence. After treatments, cells were washed and lysed either in radio immuno precipitation assay buffer for PAR4 (PBS, 1% [v/v] NP-40, 0.5% [w/v] sodium deoxycholate, 0.1% [w/v] SDS, 0.1 mg/ml PMSF, 50 µg/ml aprotinin) or in buffer for p38 MAPK and Hsp27 analysis (50 mM HEPES, pH7.4, 50 mM NaCl, 5 mM EDTA, 1% [v/v] Triton X-100, 10% [v/v] glycerol, 1 mM Na3VO4, 100 mM NaF, 10 mM sodium pyrophosphate, and 34 µg/ml Aprotinin). SDS-PAGE was performed by loading samples (100 µg/lane for PAR4 and 30 µg/lane for p38 MAPK and Hsp27) in 515% gradient gels. Protein was transferred electrophoretically to a polyvinyl difluoride membrane for 1 hr. The membrane was incubated in TBS (10 mM Tris HCl, pH 8.0, and 150 mM NaCl) containing 10% FBS for 1 hr and with TBS containing 0.05% (v/v) Tween 20, 10% FBS, and each primary polyclonal antibody (1:100 for PAR4, 1:300 for p38 MAPK, and 1:500 for Hsp27). After three washes with TBS containing 0.05% (v/v) Tween 20, the binding of the anti-PAR4 antibody was detected with biotinylated anti-goat IgG and avidin:biotinylated enzyme complex (Vector; Burlingame, CA). Immunoreactive bands for p38 MAPK and Hsp27 were visualized by chemiluminescence (ECL Plus; Amersham Pharmacia, Piscataway, NJ) with anti-rabbit or mouse antibody-horseradish peroxidase (1:4000). Intensity of bands was analyzed by NIH Image, performed on a Macintosh computer using the public domain NIH Image program developed at the US National Institutes of Health, which were available at http://rsb.info.nih.gov/nih-image/. After quantifying the bands, signal intensities of phosphorylated p38 MAPK (pp38 MAPK) were normalized to or against p38 MAPK signal intensities and fold increase in phosphorylation was calculated.
Actin Fluorescence Staining
HPAEC and HMVEC-L (2x104 cells/1.7 cm2) were cultured in Lab-Tek II chamber slides (NUNC; Rochester, NY) coated with gelatin and grown for 34 days to attain confluence. Cells were serum starved for 18 hr before addition of -thrombin (Birukova et al. 2004
; Fujiwara et al. 2004
), SFLLRN (Vouret-Craviari et al. 1998
; Fujiwara et al. 2004
), GYPGQV, or dH2O in EBM medium (0.25% [w/v] BSA) with 5 µM amastatin. After exposure to the experimental conditions for the indicated period, the cells were washed with HBSS and fixed with 10% (v/v) neutralized formalin solution and then permeabilized with 0.1% (v/v) Triton X-100. The actin filaments of cells were stained with Alexa 488-phalloidin (Molecular Probes) for 30 min at room temperature, washed three times with PBS, mounted with FluoroGuard (Bio-Rad), and examined under a confocal laser scanning microscopy equipped with x20 and x40 objective lenses (Leica; Wetzlar, Germany). Independent experiments were conducted three times.
p38 MAPK Inhibition
In the p38 MAPK inhibitory experiments, cells were first pretreated with 2.5 µM SB203580 for 15 min. At 2.5 µM of concentration, SB203580 clearly suppressed GYPGQV (PAR4 activator)-induced actin fibers and thus this dose was used in the present study. After the pretreatment, various agents were applied to the cells in the presence of SB203580 (2.5 µM) or vehicle for the indicated periods. The analyses were done by actin fluorescence staining and Western blot as described previously.
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Results |
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PAR4 Activation and Induction of Actin Fiber Formation
When HPAEC were exposed to various concentrations of the PAR4-activating peptide, GYPGQV (20, 100, and 500 µM), for 30 min (Figure 3), only subtle effects in the width of actin bundles were observed at 20 µM and 100 µM. However, the reaction at the concentration of 500 µM resulted in the formation of dense bundles of long actin filaments. Compared with the control cells treated with vehicle, actin bundles were thick and densely localized at the cell boundary.
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Discussion |
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As determined by immunohistochemistry, PAR4 was strongly expressed in the endothelial cells of human lung tissue, whereas lymph node tissue was unreactive. This is consistent with the previous results showing a high expression of PAR4 mRNA in human lung tissues and lack of detections in lymph node tissue (Xu et al. 1998). In addition, we found that PAR4 was also expressed in the cultured cells of HPAEC and HMVEC-L, being higher in HMVEC-L. Because our previous comparative study on PAR4 mRNA expression among human endothelial cells from pulmonary artery, aorta (Fujiwara et al. 2004
), and dermal microvessel (data not shown) also showed a preferential expression of PAR4 mRNA in the lung endothelial cells, a functionally important role for PAR4 was implicated in the lung endothelial cells, especially in the capillary endothelial cells.
The differences between PAR4 and PAR1 have been reported from diverse viewpoints. At the genome structure level, human PAR4 was shown to be localized at 19p12, whereas other human PARs, PAR13, formed a gene cluster at 5q13 (Kahn et al. 1998a). At the signaling cascade level, differential couplings of PAR1 and PAR4 to G proteins have been suggested (Faruqi et al. 2000
; Asokananthan et al. 2002
). Likewise, at the phenotypical level, we showed differences between PAR4- and PAR1-induced actin fiber formations. The morphology of rearranged PAR4-induced actin fibers was more broadened compared with tightened actin fibers induced by PAR1 activation. It should be noted that our study has demonstrated the morphological consequences of PAR1 activation in ECs and we have not directly measured the expression levels of PAR1 mRNA and protein in parallel, although the data could be confirmatory to our findings.
The morphological difference between PAR4 and PAR1 in rearranged actin fibers points out distinct activation kinetics of PAR4 from PAR1 in endothelial cells. Consistent with our findings, different kinetics of PAR4 were reported by others (Kahn et al. 1998b; Shapiro et al. 2000
). For example, Shapiro et al. indicated that signals of PAR4 shut off more slowly than PAR1 in human platelets. Also, in the platelets, Kahn et al. indicated the requirement of higher concentration of thrombin for the activation of PAR4. This higher concentration requirement was also observed in our experiments (i.e., 500 µM of PAR4-activating peptide [GYPGQV] was needed to induce dense and thickened bundles of long filaments, whereas only 100 µM of PAR1 activating peptide [SFLLRN] was required to induce ringlike actin structures). These differences in activation kinetics indicated the unique and different roles of PAR4, which was not provided by PAR1.
The observation that PAR4 induced actin fiber formation was highly sensitive to p38 MAPK inhibitor, SB203580, raised a hypothesis that p38 MAPK could be the principal factor that controls the diverse PAR actin traffic pathways. In cardiomyocytes, PAR4 was shown to activate p38 MAPK via Src, an important upstream signaling factor for actin polymerization, whereas PAR1 was unable to activate Src, indicating the involvement of a different signal activation cascade in p38 MAPK (Sabri et al. 2003). Additionally, for example, PAR1-activated p38 MAPK was reported to induce cell proliferation in microglia (Suo et al. 2002
) and in smooth muscle cells (Ghosh et al. 2002
), showing MAPK activity toward the cell proliferation. These differences in PAR-p38 MAPK signaling events may explain the differences in PAR4- and PAR1-induced actin fiber formation. Interestingly, expression level of Hsp27, a factor downstream of p38 MAPK, was suppressed in GYPGQV-treated cells, whereas control and SFLLRN-treated cells showed similar expression of Hsp27. Thus our results strongly implied a distinct role for PAR4 in lung microvascular endothelial cells represented by alveolar capillaries.
Similar forms of PAR4-induced actin fibers were reported under such stimuli as mechanical stretch (Birukov et al. 2003), intercellular adhesion molecule-1 cross-linking (Wang and Doerschuk 2001
), and vascular endothelial growth factor treatments (Rousseau et al. 1997
), which exhibited actin fibers with thick bundles in endothelial cells. The actin fibers formed by these stimuli were involved in cell barrier, neutrophil adherence, and cell migration, respectively. Concurrently, the cytoskeletal remodeling was revealed to be p38 MAPK-dependent in these experiments, reinforcing a link between PAR4/p38 MAPK-induced actin fibers and the physiological events. These evidences further indicate the importance of PAR4 activation in endothelial cell functions, although quantitative morphometric analysis of actin remodeling in EC cultures, preferably by image analysis, could lend support to our findings.
The importance of PAR4 in lung endothelial cells could be hypothesized from the fact that PAR4 expression is upregulated in response to inflammatory stimuli, tumor necrosis factor-, and interleukin-1
(Hamilton et al. 2001
). These factors are highly expressed and are central mediators in the pathogenesis during pulmonary fibrosis (Raines et al. 1989
; Piguet et al. 1993
). Thus PAR4 might participate, at least in part, in the induction of pulmonary diseases. In these processes, PAR4-induced actin fiber may play significant roles in permeability control (Kiemer et al. 2002
), neutrophil migration (Rousseau et al. 1997
), adherence (Wang and Doerschuk 2001
), and vascular relaxation (Hamilton et al. 2001
).
In conclusion, we have demonstrated the expression of PAR4 in lung vascular endothelial cells and its functional effect on actin fiber formation. The morphology of PAR4-induced actin fiber was distinct from that of PAR1-induced actin fiber. The PAR4-induced actin fiber formation was highly p38 MAPK-dependent, whereas the inhibition of p38 MAPK had little effect on PAR1-induced actin formation. These results indicated that PAR4 might provide unique capabilities that could not be contributed by PAR1. Further attempts will be required to elucidate the physiological and pathological role of PAR4 in the lung vascular endothelial cells.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Asokananthan N, Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS, Thompson PJ, et al. (2002) Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol 168:35773585
Bahou WF, Kutok JL, Wong A, Potter CL, Coller BS (1994) Identification of a novel thrombin receptor sequence required for activation-dependent responses. Blood 84:41954202
Bayless KJ, Davis GE (2004) Microtubule depolymerization rapidly collapses capillary tube networks in vitro and angiogenic vessels in vivo through the small GTPase Rho. J Biol Chem 279:1168611695
Birukova AA, Birukov KG, Smurova K, Adyshev D, Kaibuchi K, Alieva I, Garcia JG, et al. (2004) Novel role of microtubules in thrombin-induced endothelial barrier dysfunction. FASEB J 18:18791890
Birukov KG, Jacobson JR, Flores AA, Ye SQ, Birukova AA, Verin AD, Garcia JG (2003) Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch. Am J Physiol Lung Cell Mol Physiol 285:L785L797
Bretschneider E, Kaufmann R, Braun M, Nowak G, Glusa E, Schror K (2001) Evidence for functionally active protease-activated receptor-4 (PAR-4) in human vascular smooth muscle cells. Br J Pharmacol 132:14411446[CrossRef][Medline]
Carney DH, Mann R, Redin WR, Pernia SD, Berry D, Heggers JP, Hayward PG, et al (1992) Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides. J Clin Invest 89:14691477[Medline]
Covic L, Singh C, Smith H, Kuliopulos A (2002) Role of the PAR4 thrombin receptor in stabilizing platelet-platelet aggregates as revealed by a patient with Hermansky-Pudlak syndrome. Thromb Haemost 87:722727[Medline]
Faruqi TR, Weiss EJ, Shapiro MJ, Huang W, Coughlin SR (2000) Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function. J Biol Chem 275:1972819734
Fujiwara M, Jin E, Ghazizadeh M, Kawanami O (2004) Differential expression of protease-activated receptors 1, 2, and 4 on human endothelial cells from different vascular sites. Pathobiology 71:5258[CrossRef][Medline]
Gerszten RE, Chen J, Ishii M, Ishii K, Wang L, Nanevicz T, Turck CW, et al. (1994) Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 368:648651[CrossRef][Medline]
Ghosh SK, Gadiparthi L, Zeng ZZ, Bhanoori M, Tellez C, Bar-Eli M, Rao GN (2002) ATF-1 mediates protease-activated receptor-1 but not receptor tyrosine kinase-induced DNA synthesis in vascular smooth muscle cells. J Biol Chem 277:2132521331
Hamilton JR, Frauman AG, Cocks TM (2001) Increased expression of protease-activated receptor-2 (PAR2) and PAR4 in human coronary artery by inflammatory stimuli unveils endothelium-dependent relaxations to PAR2 and PAR4 agonists. Circ Res 89:9298
Henriksen RA, Hanks VK (2002) PAR-4 agonist AYPGKF stimulates thromboxane production by human platelets. Arterioscler Thromb Vasc Biol 22:861866
Houliston RA, Keogh RJ, Sugden D, Dudhia J, Carter TD, Wheeler-Jones CP (2002) Protease-activated receptors upregulate cyclooxygenase-2 expression in human endothelial cells. Thromb Haemost 88:321328[Medline]
Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, et al. (1997) Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386:502506[CrossRef][Medline]
Kahn ML, Hammes SR, Botka C, Coughlin SR (1998a) Gene and locus structure and chromosomal localization of the protease-activated receptor gene family. J Biol Chem 273:2329023296
Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese RV Jr, et al. (1998b) A dual thrombin receptor system for platelet activation. Nature 394:690694[CrossRef][Medline]
Kataoka H, Hamilton JR, McKemy DD, Camerer E, Zheng YW, Cheng A, Griffin C, et al. (2003) Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells. Blood 102:32243231
Kawabata A, Kuroda R, Nakaya Y, Kawai K, Nishikawa H, Kawao N (2001) Factor Xa-evoked relaxation in rat aorta: involvement of PAR-2. Biochem Biophys Res Commun 282:432435[CrossRef][Medline]
Kiemer AK, Weber NC, Furst R, Bildner N, Kulhanek-Heinze S, Vollmar AM (2002) Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability. Circ Res 90:874881
Kouklis P, Konstantoulaki M, Vogel S, Broman M, Malik AB (2004) Cdc42 regulates the restoration of endothelial barrier function. Circ Res 94:159166
Lan RS, Stewart GA, Henry PJ (2000) Modulation of airway smooth muscle tone by protease activated receptor-1,-2,-3 and -4 in trachea isolated from influenza A virus-infected mice. Br J Pharmacol 129:6370[CrossRef][Medline]
Langer F, Morys-Wortmann C, Kusters B, Storck J (1999) Endothelial protease-activated receptor-2 induces tissue factor expression and von Willebrand factor release. Br J Haematol 105:542550[CrossRef][Medline]
Marin V, Farnarier C, Gres S, Kaplanski S, Su MS, Dinarello CA, Kaplanski G (2001) The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment. Blood 98:667673
Molino M, Barnathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie JA, et al. (1997) Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 272:40434049
Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J (1995) Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem 232:8489[Abstract]
Nystedt S, Emilsson K, Wahlestedt C, Sundelin J (1994) Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91:92089212
Piguet PF, Ribaux C, Karpuz V, Grau GE, Kapanci Y (1993) Expression and localization of tumor necrosis factor-alpha and its mRNA in idiopathic pulmonary fibrosis. Am J Pathol 143:651655[Abstract]
Rahman A, True AL, Anwar KN, Ye RD, Voyno-Yasenetskaya TA, Malik AB (2002) Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells. Circ Res 91:398405
Raines EW, Dower SK, Ross R (1989) Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science 243:393396[Medline]
Rousseau S, Houle F, Landry J, Huot J (1997) p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15:21692177[CrossRef][Medline]
Sabri A, Guo J, Elouardighi H, Darrow AL, Andrade-Gordon P, Steinberg SF (2003) Mechanisms of protease-activated receptor-4 actions in cardiomyocytes. Role of Src tyrosine kinase. J Biol Chem 278:1171411720
Shapiro MJ, Weiss EJ, Faruqi TR, Coughlin SR (2000) Protease-activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin. J Biol Chem 275:2521625221
Suo Z, Wu M, Ameenuddin S, Anderson HE, Zoloty JE, Citron BA, Andrade-Gordon P, et al. (2002) Participation of protease-activated receptor-1 in thrombin-induced microglial activation. J Neurochem 80:655666[CrossRef][Medline]
Vasanji A, Ghosh PK, Graham LM, Eppell SJ, Fox PL (2004) Polarization of plasma membrane microviscosity during endothelial cell migration. Dev Cell 6:2941[CrossRef][Medline]
Vergnolle N, Derian CK, D'Andrea MR, Steinhoff M, Andrade-Gordon P (2002) Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4. J Immunol 169:14671473
Vouret-Craviari V, Boquet P, Pouyssegur J, Van Obberghen-Schilling E (1998) Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function. Mol Biol Cell 9:26392653
Vu TK, Hung DT, Wheaton VI, Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:10571068[CrossRef][Medline]
Wang Q, Doerschuk CM (2001) The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation. J Immunol 166:68776884
Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, et al. (1998) Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95:66426646
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