人類凝血酶調節素(thrombomodulin, TM) 是血管內皮細胞表面的一種醣蛋白(glycoprotein)，其分布包括動脈、靜脈、微血管以及淋巴管，當TM與凝血酶(thrombin)形成複合體時，可改變thrombin的受質特異性，使thrombin由促凝特性轉變為抑制凝血的功能。且當兩者結合之後，thrombin可增加對protein C的活化作用，被活化的protein C可更進一步將血流內被活化的Va和VIIIa凝血因子分解，藉此達到抑制凝血的作用。原子力顯微鏡在不需要複雜樣本前處理下，顯影方面擁有奈米級的解析度，因此特別適合用於生物樣本上的量測，此外與一般顯微鏡不同的是，能夠對利用其中力-位移曲線(force-distance curve)的功能，量測樣本表面的機械性質。本實驗利用原子力顯微鏡來量測凝血酶調節素基因與其重組基因轉殖後，人類黑色素腫瘤細胞表面的機械性質變化，並且配合有限元素分析來探討在細胞不同厚度的區域量測上的誤差性。發現，在經由TMG基因轉殖後，細胞表面有ruffle的產生，且在ruffle的表面楊氏係數約為15.0±4.19 kPa，明顯比其他區域高。對只有轉殖螢光蛋白控制組細胞GFP來說，經TMG轉殖後的細胞整體來講表面楊氏係數較高。轉殖TMG(△C)的細胞，型態上呈現較為紡錘形態，且在周圍邊緣的楊氏係數分布上，於離細胞核較遠的兩端點區域較高，靠近細胞核兩邊的區域較低。使用的量測原理Sneddon model，在有限元素模擬中發現實驗誤差會隨著細胞絕對厚度的下降而上升。

Human thrombomodulin(TM) is a kind of glycoprotein distributed on endothelial cell of blood vessel including artery, vein, capillary and lymph. By forming complex with thrombin, TM alters the procoagulant activity of thrombin and acts as a cofactor in thrombin-catalyzed activation of protein C. Activated protein C can proteolytically inactive coagulation factor Va and VIIIa which in turn shutdown the generation of thrombin. Atomic force microscope (AFM) has high resolution in imaging, easy sample preparation so it’s suitable for imaging biological sample under liquid. Besides, it differs from other optical microscopy is that it could be used to measure mechanical properties of sample surface by using force-distance curve. In this study, the AFM is employed to measure the differences of surface elasticity of living human melanoma cell after TM and it recombinant gene transfection under liquid. Finally, finite element analysis is used to analyze the error in the measurement of different region thickness of cell. We found that after TMG transfection, there is significant ruffle formation on the leading edge and has relatively high Young’s modulus than other region, 15.0±4.19 kPa. As refer to GFP transfection, TMG has higher Young’s modulus then GFP ones and TMG (ΔC) exhibits a spindle-like shape. Furthermore, the Young’s modulus around cell edge represents a higher value at the opposite site far from nucleus and lower one near nucleus. The error occurred as using the principle in this study would be increased as the thickness decreased by finite element simulation.

[1] Maruyama I. and Majerus P. W., The turnover of thrombin thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin, Journal of Biological Chemistry, 260, 15432-15438, 1985.
[2] Wong V. L., Hofman F. M., Ishii H., and Fisher M., Regional distribution of thrombomodulin in human brain, Brain Research, 556, 1-5, 1991.
[3] Ishii H. and Majerus P. W., Thrombomodulin is present in human plasma and urine, Journal of Clinical Investigation, 76, 2178-2181, 1985.
[4] Esmon C. T., The regulation of natural anticoagulant pathways, Science, 235, 1348-1352, 1987.
[5] Esmon C. T., Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface, FASEB Journal, 9, 946-955, 1995.
[6] Wen D. Z., Dittman W. A., Ye R. D., Deaven L. L., Majerus P. W., and Sadler J. E., Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene, Biochemistry, 26, 4350-4357, 1987.
[7] Shirai T., Shiojiri S., Ito H., Yamamoto S., Kusumoto H., Deyashiki Y., Maruyama I., and Suzuki K., Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C. Journal of Biochemistry, 103, 281-285, 1988.
[8] Esmon C. T., Esmon N. L., and Harris K. W., Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation, Journal of Biological Chemistry, 257, 7944-7947, 1982.
[9] Esmon N. L., Carroll R. C., and Esmon C. T., Thrombomodulin blocks the ability of thrombin to activate platelets, Journal of Biological Chemistry, 258, 12238-12242, 1983.
[10] Salem H. H., Maruyama I., Ishii H., and Majerus P. W., Isolation and characterization of thrombomodulin from human placenta, Journal of Biological Chemistry, 259, 12246-12251, 1984.
[11] Jackman R. W., Beeler D. L., VanDeWater L., and Rosenberg R. D., Characterization of a thrombomodulin cDNA reveals structural similarity to the low density lipoprotein receptor, Proceedings of the National Academy of Sciences of the United States of America, 83, 8834-8838, 1986.
[12] Patthy L., Detecting distant homologies of mosaic proteins. Analysis of the sequences of thrombomodulin, thrombospondin complement components C9, C8 alpha and C8 beta, vitronectin and plasma cell membrane glycoprotein PC-1. Journal of Molecular Biology, 202, 689-696, 1988.
[13] Lu R. L., Esmon N. L., Esmon C. T., and Johnson A. E., The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface. Journal of Biological Chemistry, 264, 12956-12962, 1989.
[14] Edward M., Conway E. M., Pollefeyt S., Collen D., and Steiner-Mosonyi M., The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis, Blood, 89, 652-661, 1997.
[15] Kurosawa S., Galvin J. B., Esmon N. L., and Esmon C. T., Proteolytic formation and properties of functional domains of thrombomodulin, Journal of Biological Chemistry, 262, 2206-2212, 1987.
[16] Zushi M., Gomi K., Yamamoto S., Maruyama I., Hayashi T., and Suzuki K., The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity, Journal of Biological Chemistry, 264, 10351-10353, 1989.
[17] Honda G., Masaki C., Zushi M., Tsuruta K., and Sata M., The roles played by the D2 and D3 domains of recombinant human thrombomodulin in its function, Journal of Biochemistry, 118, 1030-1036, 1995.
[18] Dittman W. A. and Majerus P. W., Structure and function of thrombomodulin: a natural anticoagulant, Blood, 75, 329-336, 1990.
[19] Dittman W. A., Kumada T., Sadler J. E., and Majerus P. W.. The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells, Journal of Biological Chemistry, 263, 15815-15822, 1988.
[20] Alberts, Johnson, Lewis, Raff, Roberts, and Walter, Molecular Biology of the Cell, 3rd Ed., GARLAND, 796-834, 2003.

[21] Park Scientific Instruments Corp., Users Guide to Autoprobe CP, Part II, http://www.park.com.
[22] NT-MDT Corp., SPM introduction, http://www.ntmdt.ru.
[23] Digital Instruments Corp., Data Sheets, http://www.di.com.
[24] Piner R. D., Hong S., and Mirkin C. A., A new tool for studying the in-situ growth processes for self-assembled monolayers under ambient conditions, Langmuir, 15, 5457-5460, 1999.
[25] Zammaretti P., Fakler A., Zaugg F., and Spichiger-Keller U. E., Atomic force microscope: a tool for studying ionophores, Analytical Chemistry, 72, 3689-3695, 2000.
[26] Park Scientific Instruments Corp., A Practical Guide to Scanning Probe Microscopy.
[27] Sneddon, I. N., The relation between load and penatration in the cxisymmetric boussinesq problem for a punch of arbitrary profile, J. Engng. Sci., 3, 47-57, 1965.
[28] Hertz H., Über die Berührung fester elastischer Körper, J. Reine Angew. Mathematik, 92, 156-171, 1882.
[29]http://www.di.com/movies/movies_inhance/appnotes/forcevol/fvmain.html
[30] Jan D. and Silke D.¨h., Substrate dependent differences in morphology and elasticity of living osteoblasts investigated by atomic force microscopy, Colloids and Surfaces B: Biointerfaces, 19, 367–379, 2000.
[31] Sinha R. K., Morris F., and Shah S. A., Surface composition of orthopaedic implant metals regulates cell attachment, spreading, and cytoskeletal organization of primary human osteoblasts in vitro, Clinical orthopaedics and related research, 305, 258-272, 1994.
[32] Jorgensen N. R., Geist S. T., Civitelli R., and Steinberg T. H., ATP- and Gap junction-dependent intercellular calcium signaling in osteoblastic cells, The Journal of Cell Biology, 139, 497-506, 1997.
[33] Glogauer M., Ferrier J., and McCulloch C. A., Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts, American Journal of Physiology Cell Physiology, 269, 1093-1104, 1995.
[34] Charras G. T., Lehenkari P. P., Horton M. A., Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions, Ultramicroscopy, 86, 85-95, 2001.
[35] Shao Z., Mou J., Czajkowsky D. M., Yang J., and Yuan J.-Y., Biological atomic force microscopy: what is achieved and what is needed. Advances in Physics, 45, 1-86, 1996.
[36] Le Grimellec C., Lesniewska E., Giocondi M.-C., Finot E., Vie V., and Goudonnet T.-P., Imaging of the surface of living cells by low-force contact-mode atomic force microscopy, Biophysical Journal, 76, 1101-1111, 1998.
[37] Domke J., Radmacher M., Measuring the Elastic Properties of Thin Polymer Films with the Atomic Force Microscope, Langmuir, 14, 3320-3325, 1998.
[38] Anja V. and Giorgio S., Measuring elasticity of biological materials by atomic force microscopy FEBS Letters, 430, 12-16, 1998.