Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Apr 2;133(2):293–304. doi: 10.1083/jcb.133.2.293

Ligand-induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor- dependent coagulation pathway

PMCID: PMC2120798  PMID: 8609163

Abstract

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low- density, detergent-insoluble microdomains that inefficiently support TF- VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ameri A., Kuppuswamy M. N., Basu S., Bajaj S. P. Expression of tissue factor pathway inhibitor by cultured endothelial cells in response to inflammatory mediators. Blood. 1992 Jun 15;79(12):3219–3226. [PubMed] [Google Scholar]
  2. Anderson R. G. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10909–10913. doi: 10.1073/pnas.90.23.10909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behrendt N., Rønne E., Ploug M., Petri T., Løber D., Nielsen L. S., Schleuning W. D., Blasi F., Appella E., Danø K. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J Biol Chem. 1990 Apr 15;265(11):6453–6460. [PubMed] [Google Scholar]
  4. Bom V. J., van Hinsbergh V. W., Reinalda-Poot H. H., Mohanlal R. W., Bertina R. M. Extrinsic activation of human coagulation factors IX and X on the endothelial surface. Thromb Haemost. 1991 Sep 2;66(3):283–291. [PubMed] [Google Scholar]
  5. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  6. Broze G. J., Jr, Girard T. J., Novotny W. F. Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry. 1990 Aug 21;29(33):7539–7546. doi: 10.1021/bi00485a001. [DOI] [PubMed] [Google Scholar]
  7. Callander N. S., Rao L. V., Nordfang O., Sandset P. M., Warn-Cramer B., Rapaport S. I. Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa-tissue factor complexes in the absence of factor Xa. J Biol Chem. 1992 Jan 15;267(2):876–882. [PubMed] [Google Scholar]
  8. Carson S. D., Perry G. A., Pirruccello S. J. Fibroblast tissue factor: calcium and ionophore induce shape changes, release of membrane vesicles, and redistribution of tissue factor antigen in addition to increased procoagulant activity. Blood. 1994 Jul 15;84(2):526–534. [PubMed] [Google Scholar]
  9. Chun M., Liyanage U. K., Lisanti M. P., Lodish H. F. Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11728–11732. doi: 10.1073/pnas.91.24.11728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conkling P. R., Patton K. L., Hannun Y. A., Greenberg C. S., Weinberg J. B. Sphingosine inhibits monocyte tissue factor-initiated coagulation by altering factor VII binding. J Biol Chem. 1989 Nov 5;264(31):18440–18444. [PubMed] [Google Scholar]
  11. Contrino J., Hair G. A., Schmeizl M. A., Rickles F. R., Kreutzer D. L. In situ characterization of antigenic and functional tissue factor expression in human tumors utilizing monoclonal antibodies and recombinant factor VIIa as probes. Am J Pathol. 1994 Dec;145(6):1315–1322. [PMC free article] [PubMed] [Google Scholar]
  12. Drake T. A., Cheng J., Chang A., Taylor F. B., Jr Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis. Am J Pathol. 1993 May;142(5):1458–1470. [PMC free article] [PubMed] [Google Scholar]
  13. Drake T. A., Ruf W., Morrissey J. H., Edgington T. S. Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line. J Cell Biol. 1989 Jul;109(1):389–395. doi: 10.1083/jcb.109.1.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ellis V., Pyke C., Eriksen J., Solberg H., Danø K. The urokinase receptor: involvement in cell surface proteolysis and cancer invasion. Ann N Y Acad Sci. 1992 Dec 4;667:13–31. doi: 10.1111/j.1749-6632.1992.tb51591.x. [DOI] [PubMed] [Google Scholar]
  15. Enjyoji K., Miyata T., Kamikubo Y., Kato H. Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor: a segment, Gly212-Phe243, of the third Kunitz domain is a heparin-binding site. Biochemistry. 1995 May 2;34(17):5725–5735. doi: 10.1021/bi00017a004. [DOI] [PubMed] [Google Scholar]
  16. Fra A. M., Williamson E., Simons K., Parton R. G. Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae. J Biol Chem. 1994 Dec 9;269(49):30745–30748. [PubMed] [Google Scholar]
  17. Fujimoto T., Nakade S., Miyawaki A., Mikoshiba K., Ogawa K. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol. 1992 Dec;119(6):1507–1513. doi: 10.1083/jcb.119.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gemmell C. H., Broze G. J., Jr, Turitto V. T., Nemerson Y. Utilization of a continuous flow reactor to study the lipoprotein-associated coagulation inhibitor (LACI) that inhibits tissue factor. Blood. 1990 Dec 1;76(11):2266–2271. [PubMed] [Google Scholar]
  19. Geoghegan W. D., Ackerman G. A. Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J Histochem Cytochem. 1977 Nov;25(11):1187–1200. doi: 10.1177/25.11.21217. [DOI] [PubMed] [Google Scholar]
  20. Girard T. J., Warren L. A., Novotny W. F., Likert K. M., Brown S. G., Miletich J. P., Broze G. J., Jr Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature. 1989 Apr 6;338(6215):518–520. doi: 10.1038/338518a0. [DOI] [PubMed] [Google Scholar]
  21. Gorodinsky A., Harris D. A. Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J Cell Biol. 1995 May;129(3):619–627. doi: 10.1083/jcb.129.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gupta M., Doellgast G. J., Cheng T., Lewis J. C. Expression and localization of tissue factor-based procoagulant activity (PCA) in pigeon monocyte-derived macrophages. Thromb Haemost. 1993 Dec 20;70(6):963–969. [PubMed] [Google Scholar]
  23. Hamamoto T., Yamamoto M., Nordfang O., Petersen J. G., Foster D. C., Kisiel W. Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces. J Biol Chem. 1993 Apr 25;268(12):8704–8710. [PubMed] [Google Scholar]
  24. Hanada K., Nishijima M., Akamatsu Y., Pagano R. E. Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes. J Biol Chem. 1995 Mar 17;270(11):6254–6260. doi: 10.1074/jbc.270.11.6254. [DOI] [PubMed] [Google Scholar]
  25. Kirchhofer D., Tschopp T. B., Hadváry P., Baumgartner H. R. Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors. J Clin Invest. 1994 May;93(5):2073–2083. doi: 10.1172/JCI117202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krishnaswamy S., Field K. A., Edgington T. S., Morrissey J. H., Mann K. G. Role of the membrane surface in the activation of human coagulation factor X. J Biol Chem. 1992 Dec 25;267(36):26110–26120. [PubMed] [Google Scholar]
  27. Kurzchalia T. V., Dupree P., Monier S. VIP21-Caveolin, a protein of the trans-Golgi network and caveolae. FEBS Lett. 1994 Jun 6;346(1):88–91. doi: 10.1016/0014-5793(94)00466-8. [DOI] [PubMed] [Google Scholar]
  28. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  29. Le D. T., Rapaport S. I., Rao L. V. Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces. J Biol Chem. 1992 Aug 5;267(22):15447–15454. [PubMed] [Google Scholar]
  30. Li S., Okamoto T., Chun M., Sargiacomo M., Casanova J. E., Hansen S. H., Nishimoto I., Lisanti M. P. Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J Biol Chem. 1995 Jun 30;270(26):15693–15701. doi: 10.1074/jbc.270.26.15693. [DOI] [PubMed] [Google Scholar]
  31. Lisanti M. P., Scherer P. E., Vidugiriene J., Tang Z., Hermanowski-Vosatka A., Tu Y. H., Cook R. F., Sargiacomo M. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol. 1994 Jul;126(1):111–126. doi: 10.1083/jcb.126.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mayor S., Rothberg K. G., Maxfield F. R. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science. 1994 Jun 24;264(5167):1948–1951. doi: 10.1126/science.7516582. [DOI] [PubMed] [Google Scholar]
  33. Mulder A. B., Hegge-Paping K. S., Magielse C. P., Blom N. R., Smit J. W., van der Meer J., Hallie M. R., Bom V. J. Tumor necrosis factor alpha-induced endothelial tissue factor is located on the cell surface rather than in the subendothelial matrix. Blood. 1994 Sep 1;84(5):1559–1566. [PubMed] [Google Scholar]
  34. Müller M., Flössel C., Haase M., Luther T., Albrecht S., Nawroth P. P., Zhang Y. Cellular localization of tissue factor in human breast cancer cell lines. Virchows Arch B Cell Pathol Incl Mol Pathol. 1993;64(5):265–269. doi: 10.1007/BF02915121. [DOI] [PubMed] [Google Scholar]
  35. Narahara N., Enden T., Wiiger M., Prydz H. Polar expression of tissue factor in human umbilical vein endothelial cells. Arterioscler Thromb. 1994 Nov;14(11):1815–1820. doi: 10.1161/01.atv.14.11.1815. [DOI] [PubMed] [Google Scholar]
  36. Narita M., Bu G., Olins G. M., Higuchi D. A., Herz J., Broze G. J., Jr, Schwartz A. L. Two receptor systems are involved in the plasma clearance of tissue factor pathway inhibitor in vivo. J Biol Chem. 1995 Oct 20;270(42):24800–24804. doi: 10.1074/jbc.270.42.24800. [DOI] [PubMed] [Google Scholar]
  37. Nawroth P. P., Stern D. M. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med. 1986 Mar 1;163(3):740–745. doi: 10.1084/jem.163.3.740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Palade G. E., Bruns R. R. Structural modulations of plasmalemmal vesicles. J Cell Biol. 1968 Jun;37(3):633–649. doi: 10.1083/jcb.37.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Parton R. G., Joggerst B., Simons K. Regulated internalization of caveolae. J Cell Biol. 1994 Dec;127(5):1199–1215. doi: 10.1083/jcb.127.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Parton R. G., Simons K. Digging into caveolae. Science. 1995 Sep 8;269(5229):1398–1399. doi: 10.1126/science.7660120. [DOI] [PubMed] [Google Scholar]
  41. Rapaport S. I., Rao L. V. The tissue factor pathway: how it has become a "prima ballerina". Thromb Haemost. 1995 Jul;74(1):7–17. [PubMed] [Google Scholar]
  42. Raposo G., Dunia I., Delavier-Klutchko C., Kaveri S., Strosberg A. D., Benedetti E. L. Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles. Eur J Cell Biol. 1989 Dec;50(2):340–352. [PubMed] [Google Scholar]
  43. Raposo G., Dunia I., Marullo S., André C., Guillet J. G., Strosberg A. D., Benedetti E. L., Hoebeke J. Redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands. Biol Cell. 1987;60(2):117–123. doi: 10.1111/j.1768-322x.1987.tb00551.x. [DOI] [PubMed] [Google Scholar]
  44. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  45. Ruf W., Edgington T. S. An anti-tissue factor monoclonal antibody which inhibits TF.VIIa complex is a potent anticoagulant in plasma. Thromb Haemost. 1991 Nov 1;66(5):529–533. [PubMed] [Google Scholar]
  46. Ruf W., Edgington T. S. Structural biology of tissue factor, the initiator of thrombogenesis in vivo. FASEB J. 1994 Apr 1;8(6):385–390. [PubMed] [Google Scholar]
  47. Ruf W. Factor VIIa residue Arg290 is required for efficient activation of the macromolecular substrate factor X. Biochemistry. 1994 Sep 27;33(38):11631–11636. doi: 10.1021/bi00204a026. [DOI] [PubMed] [Google Scholar]
  48. Ruf W., Miles D. J., Rehemtulla A., Edgington T. S. Mutational analysis of receptor and cofactor function of tissue factor. Methods Enzymol. 1993;222:209–224. doi: 10.1016/0076-6879(93)22015-8. [DOI] [PubMed] [Google Scholar]
  49. Ruf W., Rehemtulla A., Edgington T. S. Antibody mapping of tissue factor implicates two different exon-encoded regions in function. Biochem J. 1991 Sep 15;278(Pt 3):729–733. doi: 10.1042/bj2780729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ruf W., Rehemtulla A., Morrissey J. H., Edgington T. S. Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem. 1991 Feb 5;266(4):2158–2166. [PubMed] [Google Scholar]
  51. Røttingen J. A., Enden T., Camerer E., Iversen J. G., Prydz H. Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem. 1995 Mar 3;270(9):4650–4660. doi: 10.1074/jbc.270.9.4650. [DOI] [PubMed] [Google Scholar]
  52. Sandset P. M., Abildgaard U., Larsen M. L. Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res. 1988 Jun 15;50(6):803–813. doi: 10.1016/0049-3848(88)90340-4. [DOI] [PubMed] [Google Scholar]
  53. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Schnitzer J. E., McIntosh D. P., Dvorak A. M., Liu J., Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins. Science. 1995 Sep 8;269(5229):1435–1439. doi: 10.1126/science.7660128. [DOI] [PubMed] [Google Scholar]
  55. Schnitzer J. E., Oh P., Pinney E., Allard J. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol. 1994 Dec;127(5):1217–1232. doi: 10.1083/jcb.127.5.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schroeder R., London E., Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12130–12134. doi: 10.1073/pnas.91.25.12130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Simionescu M., Simionescu N., Palade G. E. Preferential distribution of anionic sites on the basement membrane and the abluminal aspect of the endothelium in fenestrated capillaries. J Cell Biol. 1982 Nov;95(2 Pt 1):425–434. doi: 10.1083/jcb.95.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Smart E. J., Ying Y. S., Mineo C., Anderson R. G. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10104–10108. doi: 10.1073/pnas.92.22.10104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stahl A., Mueller B. M. The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae. J Cell Biol. 1995 Apr;129(2):335–344. doi: 10.1083/jcb.129.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Takahasi K., Sawasaki Y. Rare spontaneously transformed human endothelial cell line provides useful research tool. In Vitro Cell Dev Biol. 1992 Jun;28A(6):380–382. doi: 10.1007/BF02634037. [DOI] [PubMed] [Google Scholar]
  61. Tschopp J., Podack E. R., Müller-Eberhard H. J. Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7474–7478. doi: 10.1073/pnas.79.23.7474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
  63. Warn-Cramer B. J., Maki S. L. Purification of tissue factor pathway inhibitor (TFPI) from rabbit plasma and characterization of its differences from TFPI isolated from human plasma. Thromb Res. 1992 Aug 15;67(4):367–383. doi: 10.1016/0049-3848(92)90267-e. [DOI] [PubMed] [Google Scholar]
  64. Warshawsky I., Broze G. J., Jr, Schwartz A. L. The low density lipoprotein receptor-related protein mediates the cellular degradation of tissue factor pathway inhibitor. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6664–6668. doi: 10.1073/pnas.91.14.6664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wesselschmidt R., Likert K., Huang Z., MacPhail L., Broze G. J., Jr Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin. Blood Coagul Fibrinolysis. 1993 Oct;4(5):661–669. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES