Thrombin-induced Rapid Geranylgeranylation of RhoA as an Essential Process for RhoA Activation in Endothelial Cells

Abstract

RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 ± 8 and 69 ± 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA. RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 ± 8 and 69 ± 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA. Thrombin regulates various responses in the vasculature including endothelial cell (EC) 1The abbreviations used are: EC, endothelial cell; GGPP, geranylgeranylpyrophosphate; GGTase I, geranylgeranyltransferase I; GEF, guanine-nucleotide exchange factor; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; ANOVA, analysis of variance. activation, vascular constriction, platelet aggregation, and activation of the coagulation cascade, all of which contribute to the pathogenesis of coronary atherosclerosis and acute coronary syndrome (1Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar, 2Sonel A. Sasseen B.M. Fineberg N. Bang N. Wilensky R.L. Circulation. 2000; 102: 1107-1113Crossref PubMed Scopus (35) Google Scholar, 3Ardissino D. Merlini P.A. Bauer K.A. Bramucci E. Ferrario M. Coppola R. Fetiveau R. Lucreziotti S. Rosenberg R.D. Mannucci P.M. Blood. 2001; 98: 2726-2729Crossref PubMed Scopus (70) Google Scholar). 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Geranylgeranylpyrophosphate (GGPP) is an isoprenoid that is a substrate for geranylgeranylation of the unprocessed GDP-bound form of RhoA (GDP-RhoA) catalyzed by geranylgeranyltransferase I (GGTase I) (16Moores S.L. Schaber M.D. Mosser S.D. Rands E. O'hara M.B. Garsky V.M. Marshall M.S. Pompliano D.L. Gibbs J.B. J. Biol. Chem. 1991; 266: 14603-14610Abstract Full Text PDF PubMed Google Scholar, 17Begum N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google Scholar). Activation, i.e. GTP loading of geranylgeranylated mature GDP-RhoA, occurs through the activity of a guanine-nucleotide exchange factor (GEF), which can bind to and be activated by the α-subunit of G12/G13 (18Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (677) Google Scholar, 19Majumdar M. Seasholtz T.M. Buckmaster C. Toksoz D. Brown J.H. J. Biol. 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Thrombin rapidly activates RhoA in ECs and other cells within 1 min via p115RhoGEF (20Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Previously, we demonstrated the unusually acute effect of a statin, namely the rapid inhibition of lysophosphatidylcholine-induced RhoA activation in ECs by a statin (22Yokoyama K. Ishibashi T. Ohkawara H. Kimura J. Matsuoka I. Sakamoto T. Nagata K. Sugimoto K. Sakurada S. Maruyama Y. Circulation. 2002; 105: 962-967Crossref PubMed Scopus (64) Google Scholar). The effect of statin was mediated through its action on GGPP metabolism, because it was reversed by the addition of GGPP. However, little is known regarding how rapidly geranylgeranylation of RhoA is regulated and how important geranylgeranylation is in the regulation of RhoA activity. In this study, we investigated the rapid effect of thrombin on geranylgeranylation of RhoA, the role of geranylgeranylation in thrombin-induced RhoA activation, and the effects of a statin and a GGTase I inhibitor on geranylgeranylation and RhoA activation in ECs. Materials—The sources of most of the conventional reagents were described previously (22Yokoyama K. Ishibashi T. Ohkawara H. Kimura J. Matsuoka I. Sakamoto T. Nagata K. Sugimoto K. Sakurada S. Maruyama Y. Circulation. 2002; 105: 962-967Crossref PubMed Scopus (64) Google Scholar, 24Nagata K. Ishibashi T. Sakamoto T. Nakazato K. Seino Y. Yokoyama K. Ohkawara H. Teramoto T. Maruyama Y. J. Hypertens. 2001; 19: 775-783Crossref PubMed Scopus (35) Google Scholar). Thrombin was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Atorvastatin was kindly provided by Pfizer (New York, NY). GGPP was purchased from Sigma, and GGTase I inhibitor GGTI-286 was from Calbiochem. Endothelial Cell Culture—Human aortic ECs were cultured according to the supplier's instructions (Clonetics, Inc., Walkersville, MD, and Sanko Junyaku Co. Ltd., Tokyo, Japan) as described previously (22Yokoyama K. Ishibashi T. Ohkawara H. Kimura J. Matsuoka I. Sakamoto T. Nagata K. Sugimoto K. Sakurada S. Maruyama Y. Circulation. 2002; 105: 962-967Crossref PubMed Scopus (64) Google Scholar). They were used for experiments after 5–10 passages. Separation of Unprocessed and Geranylgeranylated RhoA Protein— Unprocessed RhoA and geranylgeranylated RhoA were separated by the Triton X-114 partition method as described previously (25Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar, 26Gutierrez L. Magree A.I. Marshall C.J. Hancock J.F. EMBO J. 1989; 8: 1093-1098Crossref PubMed Scopus (331) Google Scholar). Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in a buffer containing 50 mmol/liter Tris-HCl, pH 7.5, 150 mmol/liter NaCl, 5 mmol/liter MgCl2, 200 μmol/liter GDP, 1 mmol/liter dithiothreitol, 1 mmol/liter Pefabloc, 20 μmol/liter each of leupeptin, aprotonin, and soybean trypsin inhibitor and then sonicated. Triton X-114 (11% (w/v)) was added to the lysates to a final concentration of 1% (v/v). The lysates were mixed for 10 min at 4 °C and centrifuged at 15,000 × g at 4 °C for 30 min to remove insoluble materials. The supernatant was warmed at 37 °C for 2 min until it became cloudy and then centrifuged at 400 × g for 4 min at room temperature to separate the upper (unprocessed) phase from the lower (processed) phase. Both phases were adjusted to 1% (v/v) Triton X-114 on ice, and protein concentrations were determined by the Bradford method. The level of Rho protein in each phase was determined by Western blotting followed by densitometric analysis as described previously (22Yokoyama K. Ishibashi T. Ohkawara H. Kimura J. Matsuoka I. Sakamoto T. Nagata K. Sugimoto K. Sakurada S. Maruyama Y. Circulation. 2002; 105: 962-967Crossref PubMed Scopus (64) Google Scholar, 27Ishibashi T. Sakamoto T. Ohkawara H. Nagata K. Sugimoto K. Sakurada S. Sugimoto N. Watanabe A. Yokoyama K. Sakamoto N. Kurabayashi M. Takuwa Y. Maruyama Y. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 681-687Crossref PubMed Scopus (40) Google Scholar). A mouse monoclonal antibody to RhoA (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:500 was used for immunoblotting. The from were with In ECs were with 5 μmol/liter for min and stimulated with or thrombin for 1 min followed by Triton X-114 The was by after and the Western blotting was to the was to RhoA. of activity of RhoA was determined by a as described previously (22Yokoyama K. Ishibashi T. Ohkawara H. Kimura J. Matsuoka I. Sakamoto T. Nagata K. Sugimoto K. Sakurada S. Maruyama Y. Circulation. 2002; 105: 962-967Crossref PubMed Scopus (64) Google Scholar, 27Ishibashi T. Sakamoto T. Ohkawara H. Nagata K. Sugimoto K. Sakurada S. Sugimoto N. Watanabe A. Yokoyama K. Sakamoto N. Kurabayashi M. Takuwa Y. Maruyama Y. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 681-687Crossref PubMed Scopus (40) Google Scholar, H. Takuwa N. T. Sugimoto N. Sakurada S. H. Takuwa Y. Cell. Biol. 2000; PubMed Scopus Google Scholar, Y. Takuwa N. Sugimoto N. Sakurada S. S. H. Takuwa Y. Circ. Res. 2002; PubMed Scopus Google Scholar). ECs were washed with ice-cold and lysed in a buffer mmol/liter pH mmol/liter NaCl, 10 mmol/liter MgCl2, 1% Triton 20 20 leupeptin, 1 μmol/liter at × g at 4 °C for 10 the were at 4 °C for min with with protein to RhoA activity. RhoA proteins were by Western blotting as described GGTase I activity of GGTase I was determined as described previously N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google Scholar, J. I. K. R. M. B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). ECs were with 1 thrombin for 1 min in the or of GGTI-286 (3 and then lysed in a buffer containing 150 mmol/liter NaCl, 5 mmol/liter MgCl2, 1 mmol/liter 1 mmol/liter dithiothreitol, 1 mmol/liter 1 mmol/liter 1% Triton 20 μmol/liter aprotonin, 20 μmol/liter leupeptin, and 50 mmol/liter pH containing GGTase I from ECs with or thrombin in the or of GGTI-286 were at 37 °C for min with protein and The was by the addition of 1 of 1 in and the was through a and protein was with a Tokyo, Japan). In Western blotting of GGTase was a antibody to GGTase (Santa Cruz of of stress was determined in cells in a as described previously A. Cell. 1995; 81: Full Text PDF PubMed Scopus Google Scholar). ECs were washed with PBS, in for 10 and for 5 min at room temperature in Triton in The ECs then were for 30 min with 1 tetramethylrhodamine in in PBS, were cells were in the until were by Tokyo, Japan). into a Tokyo, the of in Western were of The of each was for each was from the densitometric for each were with A of p < was are as the ± S.D. of Unprocessed and Geranylgeranylated of 1 the of the unprocessed and geranylgeranylated forms of RhoA in cultured human aortic ECs, as determined by the Triton X-114 partition method. The of the unprocessed and the geranylgeranylated forms of RhoA in ECs were 31 ± 8 and 69 ± respectively (n = 6, p < of Thrombin and Atorvastatin on Geranylgeranylation and GTP of with 1 thrombin for 1 min stimulated the conversion of most of the unprocessed form of RhoA into its geranylgeranylated form rapid geranylgeranylation was by acute with 10 μmol/liter atorvastatin for min thrombin GGPP to ECs not the of geranylgeranylated RhoA from the the of geranylgeranylated RhoA, which was by antibody in Western blotting in the phase in to thrombin within 1 min is evidence that thrombin rapidly the level of RhoA in ECs. analysis that thrombin the of the GTP-bound form of RhoA within 1 pretreatment with atorvastatin also thrombin-stimulated GTP loading of RhoA by ± = p < that depletion of the geranylgeranylated form of RhoA inhibited thrombin-induced RhoA of thrombin-induced GTP loading of RhoA by atorvastatin = p < was reversed by 5 μmol/liter GGPP = p < that this effect of atorvastatin was to the depletion of GGPP. The GTP loading was not by the addition of GGPP not The that the effect of atorvastatin on geranylgeranylation of RhoA was after min that the cellular GGPP was rapidly by results indicate that thrombin rapidly stimulated geranylgeranylation of the unprocessed form of RhoA as well as exchange in RhoA and that the cellular level of the geranylgeranylated form of RhoA a on thrombin-stimulated GTP loading of RhoA. In atorvastatin thrombin stimulation of RhoA, at by its of GGTase I in to the activity of GGTase I, an for geranylgeranylation of unprocessed in thrombin-stimulated ECs. Thrombin the activity of GGTase I within 1 which for at 5 min = p < in to thrombin within 1 min was by pretreatment with μmol/liter GGTI-286 for min = p < However, atorvastatin not thrombin stimulation of GGTase I activity after min or 5 and = p < of Thrombin and GGPP on Geranylgeranylation and GTP in ECs with cellular GGPP ECs were pretreated with 10 μmol/liter atorvastatin that the of the unprocessed and geranylgeranylated forms of RhoA in ECs pretreated with atorvastatin were ± and ± respectively (n = p < Atorvastatin pretreatment the of unprocessed RhoA with of cells 1 and ± 31 ± p < Moreover, the thrombin-induced in the geranylgeranylated form of RhoA was in ECs by pretreatment with atorvastatin with ECs. thrombin a small in the geranylgeranylated form of RhoA from ± total RhoA to ± in statin p < all of the unprocessed form of RhoA was converted into geranylgeranylated form in ECs Importantly, the addition of 5 μmol/liter GGPP to ECs thrombin-stimulated GTP loading within 1 min with thrombin as determined by analysis and = p < results provided evidence that geranylgeranylation of unprocessed RhoA is required for thrombin-induced full activation of RhoA. of GGTI-286 on Thrombin-induced RhoA that pretreatment of ECs with μmol/liter GGTI-286 for min thrombin-induced geranylgeranylation of unprocessed RhoA, which was to the effect of atorvastatin We the effect of GGTI-286 on thrombin-induced GTP loading of RhoA. analysis demonstrated that pretreatment with μmol/liter GGTI-286 for min inhibited thrombin-induced RhoA activation These with thrombin stimulation of GGTase I indicate that thrombin-induced rapid geranylgeranylation of RhoA is mediated via stimulation of GGTase I and to the that thrombin-induced RhoA activation is on rapid geranylgeranylation of RhoA. of GGTI-286 and on of shown in 6, thrombin stress in ECs within 1 min and this was by pretreatment with GGTI-286 for min 6, and Atorvastatin also a effect as GGTI-286 The addition of GGPP to ECs pretreated with atorvastatin the of stress in to thrombin with thrombin stimulation 6, and evidence that, in to the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which is converted into GTP-bound RhoA in human aortic ECs. has been that, in and vascular total cellular RhoA is in the geranylgeranylated mature form N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google Scholar, J. I. K. R. M. B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P.M. B. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, I. M. B. Circ. Res. 2000; 87: PubMed Scopus Google Scholar). In the of RhoA is in the geranylgeranylated form L. R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). However, little is known regarding the of the unprocessed and geranylgeranylated forms of RhoA in ECs. we determined the amounts of the unprocessed and geranylgeranylated forms of RhoA in cultured human aortic ECs the Triton X-114 partition method. The cellular geranylgeranylated form of RhoA the GDP-bound and active GTP-bound results that was twice as of the geranylgeranylated form of RhoA ± 8% total cellular the unprocessed form 31 ± with atorvastatin an in the of the unprocessed form of RhoA from 31 ± ± is with the by L. R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google that with for the of geranylgeranylated RhoA to the form in We and (4van Nieuw Amerongen G.P. van Delft S. Vermeer M.A. Collard J.G. van Hinsbergh V.W.N. Circ. Res. 2000; 87: 335-340Crossref PubMed Google Scholar, 7Eto M. Barandier C. Rathgeb L. Kozai T. Joch H. Yang Z. Luscher T.F. Circ. Res. 2001; 89: 583-590Crossref PubMed Scopus (155) Google Scholar, 20Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (138) Google demonstrated that thrombin activated RhoA in ECs within min and activity within 1 min in cells (20Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, H. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). (20Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (138) Google that thrombin p115RhoGEF in human ECs within 1 However, not thrombin stimulated the geranylgeranylation of unprocessed RhoA. We the effect of thrombin on geranylgeranylation of the unprocessed form of RhoA as determined by the Triton X-114 partition with ECs with and GGTase I Thrombin rapidly stimulated geranylgeranylation of unprocessed RhoA with a rapid in cellular GGTase I in total conversion of the unprocessed form into the geranylgeranylated form of RhoA within 1 min A and and Thrombin also the level of the GTP-bound active form of RhoA within 1 min of the unprocessed form into the mature geranylgeranylated form was for thrombin-induced full activation of RhoA, because either depletion of GGPP by atorvastatin or inhibition of GGTase I with of which inhibited thrombin-induced of the geranylgeranylated form from the unprocessed form, thrombin-induced GTP loading of RhoA A and and These results suggest that the unprocessed form of RhoA (GDP-RhoA) is for the exchange through its geranylgeranylation in to was by the that the of GGPP in ECs in which cellular GGPP was reversed the to thrombin with to RhoA activation the addition of GGPP to ECs with atorvastatin in which GGPP was reversed the thrombin stimulation of GTP loading and stress fiber in a short (3 min) and These observations indicate that thrombin rapidly geranylgeranylation of the unprocessed form of RhoA, in an in the of the geranylgeranylated form, which is converted into the GTP-bound active form by the stimulated activity of a regarding the cellular of the unprocessed form of RhoA and GGPP in ECs. with thrombin a rapid conversion of all of the unprocessed form of RhoA into the geranylgeranylated form in ECs. of cellular GGPP by a statin and inhibition of GGTase I by GGTI-286 with for short (3 min) reversed the that the cellular of unprocessed RhoA and GGPP may be small in ECs. Previously, we that, within 1 rapidly GTP-bound RhoA in in cultured human aortic ECs. was inhibited by a statin, and GGPP reversed the effect of a statin on RhoA activation by A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). with (4van Nieuw Amerongen G.P. van Delft S. Vermeer M.A. Collard J.G. van Hinsbergh V.W.N. Circ. Res. 2000; 87: 335-340Crossref PubMed Google Scholar, 7Eto M. Barandier C. Rathgeb L. Kozai T. Joch H. Yang Z. Luscher T.F. Circ. Res. 2001; 89: 583-590Crossref PubMed Scopus (155) Google Scholar, 17Begum N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google Scholar, 20Holinstat M. Mehta D. Kozasa T. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 28793-28798Abstract Full Text Full Text PDF PubMed Scopus (138) Google results suggest that, in ECs, rapid activation of unprocessed occurs through stimulation of geranylgeranylation of RhoA and exchange in RhoA. with the rapid conversion of unprocessed RhoA into the geranylgeranylated form in to we that thrombin the activity of GGTase I in ECs for to 5 min with atorvastatin the of the unprocessed form of RhoA by GGPP However, atorvastatin not the activity of GGTase I with or thrombin stimulation with a effect of of GGPP on GGTase I activity. In Western blotting that thrombin not the amounts of GGTase I within 5 min with not These results suggest that thrombin the activity of GGTase I, in the of in ECs. results also demonstrated of GGTI-286 and statin inhibition of geranylgeranylation the statin the isoprenoid for geranylgeranylation, GGTI-286 inhibited the that N. Sandu O.A. Duddy N. Diabetes. 2002; 51: 2256-2263Crossref PubMed Scopus (54) Google that thrombin GGTase I activity and the of geranylgeranylated RhoA by in However, not the of unprocessed and geranylgeranylated forms of RhoA in to In this study, thrombin the of the geranylgeranylated form of RhoA from 69 to 1 and A and with GGTI-286 for min the rapid geranylgeranylation and inhibited thrombin-induced GTP loading of RhoA, to atorvastatin A and These observations suggest that geranylgeranylation is a in thrombin-induced activation of unprocessed in ECs. We also the of conversion of unprocessed RhoA into the geranylgeranylated form and GTP min of thrombin the mature geranylgeranylated RhoA at a the level of active GTP form of RhoA with 1 min of stimulation not with the of the GGTase I suggest that thrombin-induced geranylgeranylation of RhoA is and that the in level may as a of thrombin The also may suggest that is stimulation of at within min in to thrombin in ECs. However, the of activity to be Cytoskeleton analysis demonstrated that acute with thrombin the of stress and that this was reversed by the inhibition of geranylgeranylation with GGTI-286 or atorvastatin for min that rapid activation of unprocessed RhoA may an important role in the which to endothelial In we provide evidence that unprocessed is rapidly activated in to thrombin in ECs. We suggest that inhibition of this rapid activation may be a for endothelial dysfunction in cardiovascular