Thrombomodulin Is a Clock-controlled Gene in Vascular Endothelial Cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events. Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events. Thromboembolic events such as pulmonary embolism (PE), 3The abbreviations used are: PE, pulmonary embolism; AMI, acute myocardial infarction; TM, thrombomodulin; PAI-1, plasminogen activator inhibitor-1; ZT, Zeitgeber time; HUVECs, human umbilical vein endothelial cells; BAEC, bovine aortic endothelial cells; GFP, green fluorescent protein; ChIP, chromatin Immunoprecipitation; clk, clock. cerebral infarction, and acute myocardial infarction (AMI) are major causes of death in developed countries. The onset of PE, cerebral infarction, and AMI shows apparent circadian variation and frequently occurs in the morning (1Colantonio D. Casale R. Abruzzo B.P. Lorenzetti G. Pasqualetti P. Am. J. Cardiol. 1989; 64: 403-404Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 2Muller J.E. Stone P.H. Turi Z.G. Rutherford J.D. Czeisler C.A. Parker C. Poole W.K. Passamani E. Roberts R. Robertson T. Sobel B.E. Willerson J.T. Braunwald E. N. Engl. J. Med. 1985; 313: 1315-1322Crossref PubMed Scopus (1663) Google Scholar, 3Marler J.R. Price T.R. Clark G.L. Muller J.E. Robertson T. Mohr J.P. Hier D.B. Wolf P.A. Caplan L.R. Foulkes M.A. Stroke. 1989; 20: 473-476Crossref PubMed Scopus (493) Google Scholar). Thus, elucidation of the mechanisms of circadian variation of these disorders may lead to the development of methods that prevent the onset of thromboembolic events. The circadian variation of the onset of thromboembolic events is thought to be related to the biological clock. Recently, the molecular mechanisms of the biological clock have been elucidated (4Gekakis N. Staknis D. Nguyen H.B. Davis F.C. Wilsbacher L.D. King D.P. Takahashi J.S. Weitz C.J. Science. 1998; 280: 1564-1569Crossref PubMed Scopus (1566) Google Scholar). This system is composed of several clock genes, including clock, period, bmal, and cry. The heterodimer of CLOCK and BMAL binds to the E-box sites upstream of the period and cry genes and transactivates these genes. The PERIOD and CRY proteins inhibit their own transactivation by CLOCK and BMAL, resulting in the formation of a negative feedback loop. This negative feedback loop persists for ∼24 h, which corresponds to circadian rhythm (5Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2391) Google Scholar). The biological clock regulates the expression of downstream target genes, clock-controlled genes, and modulates the cellular responses to these downstream genes. The central biological clock is located in the suprachiasmatic nucleus in the hypothalamus (6Sassone-Corsi P. Nature. 1998; 392: 871-874Crossref PubMed Scopus (81) Google Scholar). Furthermore, we and other researchers have reported that clock genes are also expressed in peripheral organs and cells and that their expression shows circadian oscillation, suggesting the existence of a peripheral clock (7Balsalobre A. Damiola F. Schibler U. Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1572) Google Scholar, 8Oishi K. Sakamoto K. Okada T. Nagase T. Ishida N. Neurosci. Lett. 1998; 256: 117-119Crossref PubMed Scopus (100) Google Scholar, 9McNamara P. Seo S.P. Rudic R.D. Sehgal A. Chakravarti D. FitzGerald G.A. Cell. 2001; 105: 877-889Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Approximately 8–10% of the total number of genes expressed in mouse heart and liver were found to show a circadian expression pattern (10Storch K.F. Lipan O. Leykin I. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1252) Google Scholar). The peripheral clock is thought to regulate the expression of organ-specific, clock-controlled genes directly or indirectly through the expression of core clock genes such as clock and bmal (10Storch K.F. Lipan O. Leykin I. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1252) Google Scholar). The circadian expression of clock genes in the cardiovascular system has been demonstrated as well (11Durgan D.J. Hotze M.A. Tomlin T.M. Egbejimi O. Graveleau C. Abel E.D. Shaw C.A. Bray M.S. Hardin P.E. Young M.E. Am. J. Physiol. Heart Circ. Physiol. 2005; 289: 1530-1541Crossref PubMed Scopus (174) Google Scholar, 12Maemura, K., de la Monte, S. M., Chin, M. T., Layne, M. D., Hsieh, C. M., Yet, S. F., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36847–36851Google Scholar, 13Nonaka H. Emoto N. Ikeda K. Fukuya H. Rohman M.S. Raharjo S.B. Yagita K. Okamura H. Yokoyama M. Circulation. 2001; 104: 1746-1748Crossref PubMed Scopus (159) Google Scholar, 14Portman M.A. Circ. Res. 2001; 89: 1084-1086Crossref PubMed Scopus (21) Google Scholar, 15Rudic R.D. McNamara P. Reilly D. Grosser T. Curtis A.M. Price T.S. Panda S. Hogenesch J.B. FitzGerald G.A. Circulation. 2005; 112: 2716-2724Crossref PubMed Scopus (123) Google Scholar, 16Young M.E. Razeghi P. Taegtmeyer H. Circ. Res. 2001; 88: 1142-1150Crossref PubMed Scopus (217) Google Scholar). However, the biological significance of this expression remains to be elucidated. Previously, we identified a clock gene in vascular endothelial cells, CLIF, which is also termed BMAL2 (12Maemura, K., de la Monte, S. M., Chin, M. T., Layne, M. D., Hsieh, C. M., Yet, S. F., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36847–36851Google Scholar). Subsequently, we found that CLOCK and BMAL2 in peripheral tissue may directly regulate the circadian expression of the plasminogen activator inhibitor-1 (PAI-1) gene, contributing to the morning onset of AMI (17Andreotti F. Kluft C. Chronobiol. Int. 1991; 8: 336-351Crossref PubMed Scopus (139) Google Scholar, 18Braunwald E. Circulation. 1995; 91: 1604-1606Crossref PubMed Scopus (46) Google Scholar, 19Maemura K. Layne M.D. Watanabe M. Perrell M.A. Nagai R. Lee M.E. Ann. N. Y. Acad. Sci. 2001; 947: 398-402Crossref PubMed Scopus (33) Google Scholar, 20Yamamoto K. Saito H. Int. J. Hematol. 1998; 68: 371-385Crossref PubMed Google Scholar). Thus, in this study we screened for clock-controlled genes in vascular endothelial cells using microarray technology. A total of 229 genes were up-regulated by CLOCK and BMAL2. Among them, we focused on the endothelial membrane protein, thrombomodulin (TM), since TM plays an important role in the regulation of blood coagulation processes by exerting anti-coagulant effects through the activation of protein C (21Esmon C.T. FASEB J. 1995; 9: 946-955Crossref PubMed Scopus (361) Google Scholar, 22Sadler J.E. Thromb. Haemostasis. 1997; 78: 392-395Crossref PubMed Scopus (170) Google Scholar, 23Wouwer M. Collen D. Conway E.M. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1374-1383Crossref PubMed Scopus (313) Google Scholar). Our results demonstrate that TM mRNA and protein expression have a circadian pattern and that a heterodimer of CLOCK and BMAL2 binds to the E-box upstream of the TM promoter and transactivates promoter activity. In addition, these results suggest that the circadian rhythm of TM gene expression is under the control of circadian clock molecules. Animals—This study was approved by the Animal Committee of University of Tokyo. Clock mutant mice in BALB/c and C57BL/6J backgrounds were supplied by J. S. Takahashi (Northwestern University, Evanston, IL) (24Vitaterna M.H. King D.P. Chang A.M. Kornhauser J.M. Lowrey P.L. McDonald J.D. Dove W.F. Pinto L.H. Turek F.W. Takahashi J.S. Science. 1994; 264: 719-725Crossref PubMed Scopus (1332) Google Scholar). Clock mutant mice carry an internal deletion of 51 amino acids in the C-terminal activation domain of the CLOCK protein, which results in a dominant negative mutation. Thus, Clock homozygotes fail to express persistent circadian rhythms when maintained in constant darkness. A breeding colony was established by further backcrossing with Jcl:ICR mice as described previously (25Oishi K. Miyazaki K. Ishida N. Biochem. Biophys. Res. Commun. 2002; 298: 198-202Crossref PubMed Scopus (84) Google Scholar). Male BALB/c mice of 8–12 weeks of age were maintained under a 12-h:12-h light-dark cycle. Zeitgeber time (ZT) 0 refers to light on, and ZT 12 refers to light off. ZT 0–12 is the subjective light phase, and 12–24 h ZT is the subjective dark phase. Under the restrictive feeding condition, mice were to for h to for K. M. Ishida N. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). of and TM to was as described previously S. H. F. M. K. J. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. T. H. K. K. S. PubMed Scopus Google Scholar). The to was by deletion of the was using and which to or and which to is to that the E-box to was to The expression for and CLOCK were as described previously (12Maemura, K., de la Monte, S. M., Chin, M. T., Layne, M. D., Hsieh, C. M., Yet, S. F., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36847–36851Google Scholar). were as described previously K. S. Layne M.D. Y. S. M.A. Lee M.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (51) Google and green fluorescent protein or BMAL2 were as or umbilical vein endothelial cells were A was aortic endothelial cells were as and were in was using the or using was as described previously K. S. Layne M.D. Y. S. M.A. Lee M.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (51) Google Scholar). tissue protein was using under and to The used was thrombomodulin was used to for in protein was using as described previously (12Maemura, K., de la Monte, S. M., Chin, M. T., Layne, M. D., Hsieh, C. M., Yet, S. F., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36847–36851Google Scholar). for each are as the was using human as described previously J. M. Y. J.M. PubMed Scopus Google Scholar, G. T. Lee J.C. J.M. J. M.S. D. D. D. Science. 1999; PubMed Scopus Google Scholar). were with CLOCK and or total was and to microarray and were to the The of each was by of the with a an for each data were using expression were demonstrated as the of the green and This was in with A of a was used for In and and and gel shift were as described previously N. K. Y. T. D. T. I. Nagai R. Circ. Res. 2004; PubMed Scopus Google Scholar). The of the the E-box was the of the TM promoter to to of was used for the were as described previously CLOCK and BMAL2 of I. J. 2001; PubMed Scopus Google Scholar). was of the TM promoter the E-box was using chromatin with the of and the negative of the of the TM gene was with the of and A of as a control for was used for as described previously M.E. Razeghi P. Taegtmeyer H. Circ. Res. 2001; 88: 1142-1150Crossref PubMed Scopus (217) Google Scholar). and were and used to the The of was in each to control for in are expressed as for or of was used for of was to of the of Clock in we the existence of an clock system in endothelial the circadian expression of clock genes in (7Balsalobre A. Damiola F. Schibler U. Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1572) Google Scholar). Thus, we a of to of cells and the expression of clock genes. the circadian expression of clock genes, suggesting that vascular endothelial cells have an clock system Although results were with the of the oscillation was This may be to the of and several for of in the downstream target genes of the peripheral clock in endothelial cells, we screened for genes expression was up-regulated by CLOCK and BMAL2 using microarray with and and a we used that been with was on microarray of human using The expression of a total of 229 genes was up-regulated including and which have been identified previously as clock genes or clock-controlled genes. The 229 identified genes genes that genes that membrane and genes that 229 genes are in the Among these genes we that TM expression was up-regulated by CLOCK and BMAL2. TM is expressed primarily in vascular endothelial cells and plays an important role in the blood coagulation M. Collen D. Conway E.M. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1374-1383Crossref PubMed Scopus (313) Google Scholar). further examined the molecular mechanisms by which TM is up-regulated by Clock and BMAL2. The of other genes be described K. and R. in mRNA and by CLOCK and that TM expression is under the control of CLOCK and we using and demonstrated that the of CLOCK and BMAL2 in an in TM mRNA A and This was to be because by the of control not in an the TM mRNA results that TM mRNA is up-regulated by CLOCK and BMAL2 in vascular endothelial TM mRNA expression shows circadian oscillation, we using total the mouse heart and TM mRNA in the mouse lung and heart apparent circadian expression with the expression ZT and the expression ZT C and TM protein expression in the lung also showed circadian oscillation not TM protein expression in the heart by which may be to the of TM mRNA in the heart with that of the of CLOCK and or the further the by which CLOCK and BMAL2 the TM mRNA we used a that a of the human TM This was with the human CLOCK expression and the human or BMAL2 expression or or BMAL2 not TM promoter However, the of CLOCK and or of CLOCK and BMAL2 TM promoter by and BMAL2 showed with to TM promoter found E-box E-box and E-box the the TM promoter which as sites of CLOCK and or BMAL2. of to in the TM promoter the of TM promoter by CLOCK and suggesting that the E-box but not the E-box is important for transactivation of the TM promoter CLOCK and BMAL2 the TM promoter by to E-box we a mutant TM promoter with and to A this was of the E-box abolished the transactivation of the TM promoter by CLOCK and BMAL2 of CLOCK and BMAL2 to the E-box of the TM the heterodimer of CLOCK and BMAL2 binds directly to we gel shift using in CLOCK and BMAL2 proteins and a the E-box In the of the CLOCK and BMAL2 was was by the of an E-box but not by an E-box with The in BMAL2 protein was using The of of in this with of the heterodimer further the of CLOCK and BMAL2 with the the E-box of the TM gene in we using CLOCK and BMAL2 or as a control was using and by of total immunoprecipitation of or the negative the of the TM gene that E-box was The the E-box of the TM gene was in a with on chromatin cells CLOCK and BMAL2 protein but not control data suggest that a heterodimer of CLOCK and BMAL2 with the the E-box of the TM gene in vivo. of mRNA in Clock TM expression is regulated by CLOCK in we the of TM expression h a period in mice and clock mutant mice, which have an clock system. In mice, the TM mRNA showed clear However, in clock mutant mice, the oscillation of TM mRNA expression was suggesting that TM gene expression is regulated by clock in the of TM mRNA in the feeding was found to a of circadian gene expression in peripheral the phase of gene expression in the suprachiasmatic nucleus F. N. N. F. Schibler U. PubMed Scopus Google Scholar). TM gene expression is regulated by the peripheral clock, we examined the of restrictive feeding on TM Under the mice the the of clock genes and TM mRNA was However, under restrictive the of circadian expression of and TM were suggesting that TM gene expression is regulated by the peripheral clock In this study we searched for clock-controlled genes using microarray and found that TM is a clock-controlled gene expressed in vascular endothelial The central biological clock is located in the suprachiasmatic nucleus in the hypothalamus (6Sassone-Corsi P. Nature. 1998; 392: 871-874Crossref PubMed Scopus (81) Google Scholar). demonstrated the existence of a peripheral biological clock in each peripheral tissue, including the heart and The central clock regulates a peripheral clock in each or tissue through a U. Nature. PubMed Scopus Google Scholar). However, the biological relevance of the peripheral clock remains to be elucidated. the biological significance of the peripheral clock, genes expression is regulated by the peripheral clock to be In addition, we to the peripheral are altered under and the biological clock is with the development of diseases M.E. Am. J. Physiol. Heart Circ. Physiol. PubMed Scopus Google Scholar). Previously, as the to these we examined several genes as of clock-controlled genes and found that expression is under the control of the clock genes, Clock and (12Maemura, K., de la Monte, S. M., Chin, M. T., Layne, M. D., Hsieh, C. M., Yet, S. F., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36847–36851Google Scholar). plays a role in the circadian oscillation of the of thromboembolic events in the morning (17Andreotti F. Kluft C. Chronobiol. Int. 1991; 8: 336-351Crossref PubMed Scopus (139) Google Scholar, 18Braunwald E. Circulation. 1995; 91: 1604-1606Crossref PubMed Scopus (46) Google Scholar, 19Maemura K. Layne M.D. Watanabe M. Perrell M.A. Nagai R. Lee M.E. Ann. N. Y. Acad. Sci. 2001; 947: 398-402Crossref PubMed Scopus (33) Google Scholar, 20Yamamoto K. Saito H. Int. J. Hematol. 1998; 68: 371-385Crossref PubMed Google Scholar). Among cardiovascular vascular endothelial has been well to show circadian oscillation H. J.M. A. C. J.P. U. Circulation. PubMed Scopus (170) Google Scholar). in the morning M.E. A. S. M. Circulation. 2004; PubMed Scopus Google Scholar). endothelial cells prevent and the of thromboembolic including pulmonary and AMI, morning (1Colantonio D. Casale R. Abruzzo B.P. Lorenzetti G. Pasqualetti P. Am. J. Cardiol. 1989; 64: 403-404Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 2Muller J.E. Stone P.H. Turi Z.G. Rutherford J.D. Czeisler C.A. Parker C. Poole W.K. Passamani E. Roberts R. Robertson T. Sobel B.E. Willerson J.T. Braunwald E. N. Engl. J. Med. 1985; 313: 1315-1322Crossref PubMed Scopus (1663) Google Scholar). the expression of the vascular endothelial gene shows circadian oscillation, the of a circadian rhythm in processes in S. Y. H. H. S. S. H. Res. Google Scholar). The internal biological clock is thought to an important role in circadian oscillation of endothelial the mechanisms are not elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells and demonstrated that TM expression is under the control of the clock genes, Clock and The oscillation of TM mRNA expression was abolished in clock mutant mice, suggesting that TM expression is regulated by CLOCK in vivo. the phase of circadian expression of TM was by restrictive TM gene expression was regulated by the peripheral clock. as well as a heterodimer with CLOCK and transactivates the promoter of clock-controlled genes by to the E-box of their promoter The of or BMAL2 on clock-controlled gene expression in is thought to on the tissue or Cardiovascular including heart and blood show circadian the molecular the cardiovascular system has own internal clock system (12Maemura, K., de la Monte, S. M., Chin, M. T., Layne, M. D., Hsieh, C. M., Yet, S. F., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36847–36851Google Scholar, 13Nonaka H. Emoto N. Ikeda K. Fukuya H. Rohman M.S. Raharjo S.B. Yagita K. Okamura H. Yokoyama M. Circulation. 2001; 104: 1746-1748Crossref PubMed Scopus (159) Google Scholar). In the the clock system cardiovascular R.D. McNamara P. Reilly D. Grosser T. Curtis A.M. Price T.S. Panda S. Hogenesch J.B. FitzGerald G.A. Circulation. 2005; 112: 2716-2724Crossref PubMed Scopus (123) Google Scholar). However, in a such as the circadian expression of several clock genes and genes including is M.E. Razeghi P. Taegtmeyer H. Circ. Res. 2001; 88: 1142-1150Crossref PubMed Scopus (217) Google Scholar). data suggest that the peripheral clock is in Furthermore, the clock system plays an important role in a in the clock gene show circadian rhythm (24Vitaterna M.H. King D.P. Chang A.M. Kornhauser J.M. Lowrey P.L. McDonald J.D. Dove W.F. Pinto L.H. Turek F.W. Takahashi J.S. Science. 1994; 264: 719-725Crossref PubMed Scopus (1332) Google Scholar). In a clock mutant mice were to be to a that in F.W. C. A. E. G. E. A. S. A. Takahashi J.S. J. Science. 2005; PubMed Scopus Google Scholar). results suggest that an biological clock is with the development of using mice that have a of the clock system the of the peripheral clock, that of the central clock. the circadian expression of the TM gene, the phase of circadian TM protein expression is to that of mRNA TM protein is thought to be of the TM gene K. K. H. J. S. H. I. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). However, the of TM protein is h in M. J.E. 1991; PubMed Google Scholar). in to the circadian regulation of may be other mechanisms such as processes that for the oscillation of TM protein the biological significance of circadian TM The of the and expression of the TM gene were to of and to by formation as well as the TM an to by the of the of TM and protein which has an on (21Esmon C.T. FASEB J. 1995; 9: 946-955Crossref PubMed Scopus (361) Google Scholar, 22Sadler J.E. Thromb. Haemostasis. 1997; 78: 392-395Crossref PubMed Scopus (170) Google Scholar, 23Wouwer M. Collen D. Conway E.M. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1374-1383Crossref PubMed Scopus (313) Google Scholar). is that TM may the the of In of a in the TM gene showed an of thromboembolic diseases including and AMI Thromb. Haemostasis. 1999; PubMed Scopus Google Scholar, C.J. G. H. Thromb. Haemostasis. 1998; PubMed Scopus Google Scholar). of TM to an and and may thromboembolic events. In TM may blood coagulation through of the of since the protein as the activator of under the of a on the vascular J. Thromb. Haemostasis. PubMed Scopus Google Scholar, J. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are to the role of circadian expression of TM in cardiovascular In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene and the circadian oscillation of TM expression may contribute to the circadian variation of cardiovascular events. of the biological relevance of the peripheral clock of the heart and the development as well as the of cardiovascular J. S. Takahashi (Northwestern University, Evanston, IL) for the of Clock mutant are to for with