Turnover of Thrombomodulin at the Cell Surface Occurs at a Similar Rate to Receptors that Are Not Actively Internalized

Summary

Loss of thrombomodulin (TM) from the endothelial cell surface is thought to contribute to thrombosis encountered in malignant and inflammatory disease. Internalization or endocytosis of TM from the cell surface has been proposed to be one mechanism by which TM levels are reduced. Previous work has led to a view that TM is rapidly internalized using a non-conventional pathway, under the direction of a signal motif in its extracellular domain. This is contrary to the clathrin-dependent route taken by most rapidly internalized receptors that possess signals in their cytoplasmic domains. However the internalization rate of TM has never been directly compared to known internalizing or non-internalizing molecules. Here we show that the rate and amount of uptake of TM (5-10% per hour) is indistinguishable from the non-internalizing influenza virus haemagglutinin (HA), and is considerably less than the actively internalizing low density lipoprotein receptor, which reaches a steady state of approximately 50% internalized in 15 min. The low level rate of TM and HA internalization observed is comparable to the rate of normal plasma membrane turnover. Furthermore, this uptake of TM does not require a signal in its extracellular domain.

• References

• 1 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-52.
• 2 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalysed activation of protein C. J Biol Chem 1982; 257: 859-64.
• 3 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-42.
• 4 Kisiel W, Canfield WM, Ericsson LH, Davie EW. Anticoagulant properties of bovine plasma protein C following activation. Biochemistry 1977; 16: 5824-31.
• 5 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-10.
• 6 Walker FJ. Regulation of activated protein C by a new protein. J Biol Chem 1980; 255: 5521-4.
• 7 Suzuki K, Kusumoto H, Deyashiki Y, Nishioka J, Maruyama I, Zushi M, Kawahara S, Honda G, Yamamoto S, Horiguchi S. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 1987; 6: 1891-7.
• 8 Petersen TE. The amino-terminal domain of thrombomodulin and pancreatic stone protein are homologous with lectins. Feb 1988; 231: 51-3.
• 9 Conway EM, Pollefeyt S, Collen D, Steiner-Mosonyi M. The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 1997; 89: 652-61.
• 10 Parkinson JF, Garcia JGN, Bang NU. Decreased thrombin affinity of cell-surface thrombomodulin following treatment of cultured endothelial cells with b-D-xyloside. Biochem Biophys Res Comm 1990; 169: 177-83.
• 11 Parkinson JF, Vlahos CJ, Yan SCB, Bang NU. Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain. Biochem J 1992; 283: 151-7.
• 12 Lin JH, McLean K, Morser J, Young TA, Wydro RM, Andrews WH, Light DR. Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin. J Biol Chem 1995; 269: 25021-30.
• 13 Ye J, Esmon CT, Johnson AE. The chondroitin sulfate moiety of thrombomodulin binds a second molecule of thrombin. J Biol Chem 1993; 268: 2373-9.
• 14 Maruyama I, Majerus PW. The turnover of thrombin-Thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. J Biol Chem 1985; 260: 15432-8.
• 15 Maruyama I, Majerus PW. Protein C inhibits endocytosis of thrombinThrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells. Blood 1987; 69: 1481-4.
• 16 Beretz A, Freyssinet J-M, Gauchy J, Schmitt DA, Klein-Soyer C, Edgell C-JS, Cazenave J-P. Stability of the thrombin-thrombomdulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926. Biochem J 1989; 259: 35-40.
• 17 Brisson C, Archipoff G, Haratmann M-L, Hanau D, Beretz A, Freyssinet J-M, Cazenave J-P. Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells. Thromb Haemost 1992; 68: 737-43.
• 18 Conway EM, Boffa M-C, Nowakowski B, Steiner-Mosonyi M. An ultra-structural study of Thrombomodulin endocytosis: Internalization occurs via clathrin-coated and non-coated pits. J Cell Physiol 1992; 151: 604-12.
• 19 Horvat R, Palade GE. Thrombomodulin and thrombin localization on the vascular endothelium; their internalization and transcytosis by plasmalemmal vesicles. Eur J Cell Biol 1993; 61: 299-313.
• 20 Conway EM, Nowakowski B, Steiner-Mosonyi M. Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis. J Cell Physiol 1994; 158: 285-98.
• 21 Teasdale MS, Bird CH, Bird P. Internalization of the anticoagulant thrombomodulin is constitutive and does not require a signal in the cytoplasmic domain. Immunol Cell Biol 1994; 72: 480-8.
• 22 Goldstein JL, Brown MS, Anderson RGW, Russell DW, Schneider WJ. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol 1985; 1: 1-39.
• 23 Roth MG, Doyle C, Sambrook J, Gething M-J. Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway. J Cell Biol 1986; 102: 1271-83.
• 24 Gibson J, Nelson M, Brown R, Salem H, Kronenberg H. Autoantibodies to thrombomodulin: Development of an enzyme immunoassay and a survey of their frequency in patients with the lupus anticoagulant. Thromb Haemost 1992; 67: 507-9.
• 25 Wen D, Dittman WA, Ye RD, Deaven LL, Majerus PW, Sadler JE. Human thrombomodulin: Complete cDNA sequence and chromosome localization of the gene. Biochem 1987; 26: 4350-7.
• 26 Madison EL, Bird P. A vector, pSHT, for the expression and secretion of protein domains in mammalian cells. Gene 1992; 121: 179-80.
• 27 Cockerill GW, Meyer G, Noack L, Vadas MA, Gamble JR. Characterization of a spontaneously transformed human endothelial cell line. Lab Invest 1994; 71: 497-509.
• 28 Lazarovits J, Roth M. A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits. Cell 1988; 53: 743-52.
• 29 Aroeti B, Mostov KE. Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids. EMBO J 1994; 13: 2297-304.
• 30 Laemmli UK. Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 1970; 227: 680-5.
• 31 Bretscher MS, Thomson JN, Pearse BMF. Coated pits act as molecular filters. Proc Natl Acad Sci USA 1980; 77: 4156-9.
• 32 Lemansky P, Fatemi SH, Gorican B, Meyale S, Rossero R, Tartakoff AM. Dynamics and longevity of the glycolipid-anchored membrane protein, Thy-1. J Cell Biol 1990; 110: 1525-31.
• 33 Mellman I. Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 1996; 12: 575-625.
• 34 Davis CG, Lehrman MA, Russell DW, Anderson RGW, Brown MS, Goldstein JL. The J D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell 1986; 45: 15-24.
• 35 Davis CG, van Driel IR, Russell DW, Brown MS, Goldstein JL. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J Biol Chem 1987; 262: 4075-82.
• 36 Lehrman MA, Goldstein JL, Brown MS, Russell DW, Schneider WJ. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell 1985; 41: 735-43.
• 37 Bamezai A, Goldmacher VS, Rock KL. Internalization of glycosyl-phosphidylinositol (GPI)-anchored lymphocyte proteins II. GPI-anchored and transmembrane molecules internalize through distinct pathways. Eur J Immunol 1992; 22: 15-21.
• 38 Moran P, Raab H, Kohr WJ, Caras IW. Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site. J Biol Chem 1991; 266: 1250-7.
• 39 Moran P, Caras IW. A nonfunctional sequence can be converted to a signal for glycophosphatidylinositol membrane attachment. J Cell Biol 1991; 115: 329-36.
• 40 Moran P, Caras IW. Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment. J Cell Biol 1991; 115: 1595-600.
• 41 Trowbridge IS, Collawn JF, Hopkins CR. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 1993; 9: 129-61.
• 42 Raposo G, Dunia I, Delavier-Klutchko C, Kaveri S, Strosberg AD, Benedetti EL. Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles. Eur J Cell Biol 1989; 50: 340-52.
• 43 Montesano R, Roth J, Robert A, Orci L. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxin. Nature 1982; 296: 651-3.
• 45 Illinger D, Poindron P, Fonteneau P, Modollel M, Kuhry J-G. Internalization of the lipophilic flourescent probe trimethylamino-diphenylhexatriene follows the endocytosis and recycling of the plasma membrane in cells. Biochim Biophys Acta 1990; 1030: 73-81.
• 46 Illinger D, Kuhry J-G. The kinetic aspects of intracellular fluorescence labelling with TMA-DPH support the maturation model for endocytosis in L929 cells. J Cell Biol 1994; 125: 783-94.
• 47 Cupers P, Veithen A, Kiss A, Baudhuin P, Courtoy PJ. Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts. J Cell Biol 1994; 127: 725-35.
• 48 Gonda Y, Hirata S, Saitoh K, Aoki Y, Mohri M, Gomi K, Sugihara T, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Antithrombotic effect of recombinant human soluble thrombomodulin on endotoxin-induced disseminated intravascular coagulation in rats. Thromb Res 1993; 71: 325-35.
• 49 Aoki Y, Tkei R, Mohri M, Gonda Y, Sugihara T, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Antithrombotic effects of recombinant human thrombomodulin (rhs-TM) on arteriovenous shunt thrombosis in rats. Am J Hematol 1994; 47: 162-6.
• 50 Bird P. Activation and function of protein C and protein S occur as part of a complex network regulating hemostasis and inflammation. Hematol Rev 1997; 9: 251-74.
• 51 Moore KL, Esmon CT, Esmon NL. Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture. Blood 1989; 73: 159-65.
• 52 Conway EM, Rosenberg RD. Tumor necrosis factor suppresses transcription of the Thrombomodulin gene in endothelial cells. Mol Cell Biol 1988; 8: 5588-92.
• 53 Lentz SR, Tsiang M, Sadler JE. Regulation of thrombomodulin by tumor necrosis factor-alpha: Comparison of transcriptional and postranscriptional mechanisms. Blood 1991; 77: 542-50.