Interaction of the Fibrinolytic Receptor, Annexin II, with the Endothelial Cell Surface

Abstract

Endothelial cells express a cell surface co-receptor for plasminogen and tissue plasminogen activator (t-PA) which we recently identified as annexin II (Hajjar, K. A., Jacovina, A. T., and Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197). This protein enhances the catalytic efficiency of t-PA-dependent plasmin generation by 60-fold (Cesarman, G. M., Guevara, C. A., and Hajjar, K. A. (1994) J. Biol. Chem. 269, 21198-21203). Here, we demonstrate that annexin II is constitutively translocated to the endothelial cell surface within 16 h of biosynthesis, and that cell surface annexin II comprises 4.3 ± 1.0% of the total cellular pool. Exogenous 125I-annexin II bound to EGTA-washed endothelial cells with high affinity (Kd 49 n) and in a calcium-dependent (I50 = 3 µ), phospholipid-sensitive manner. Peptides KASMKGLGTDED and YDSMKGKGTRDK, mimicking the calcium-binding “endonexin” motif (KGXGT) of annexin II, blocked its interaction with endothelial cells. Recombinant annexin II, bearing the calcium-binding site substitution D161A of core repeat 2, failed to compete with binding of the wild type protein to the cell surface, while E246A and D321A mutants, corresponding to core repeats 3 and 4, behaved as effective competitors. These data suggest that translocated annexin II interacts with cell surface phospholipid via a high affinity calcium-dependent binding site that includes residues 118-122 (KGLGT) and the coordinating Asp161 of core repeat 2. Thus, calcium-regulated expression of annexin II on the endothelial cell surface may play a central role in control of plasmin-mediated processes. Endothelial cells express a cell surface co-receptor for plasminogen and tissue plasminogen activator (t-PA) which we recently identified as annexin II (Hajjar, K. A., Jacovina, A. T., and Chacko, J. (1994) J. Biol. Chem. 269, 21191-21197). This protein enhances the catalytic efficiency of t-PA-dependent plasmin generation by 60-fold (Cesarman, G. M., Guevara, C. A., and Hajjar, K. A. (1994) J. Biol. Chem. 269, 21198-21203). Here, we demonstrate that annexin II is constitutively translocated to the endothelial cell surface within 16 h of biosynthesis, and that cell surface annexin II comprises 4.3 ± 1.0% of the total cellular pool. Exogenous 125I-annexin II bound to EGTA-washed endothelial cells with high affinity (Kd 49 n) and in a calcium-dependent (I50 = 3 µ), phospholipid-sensitive manner. Peptides KASMKGLGTDED and YDSMKGKGTRDK, mimicking the calcium-binding “endonexin” motif (KGXGT) of annexin II, blocked its interaction with endothelial cells. Recombinant annexin II, bearing the calcium-binding site substitution D161A of core repeat 2, failed to compete with binding of the wild type protein to the cell surface, while E246A and D321A mutants, corresponding to core repeats 3 and 4, behaved as effective competitors. These data suggest that translocated annexin II interacts with cell surface phospholipid via a high affinity calcium-dependent binding site that includes residues 118-122 (KGLGT) and the coordinating Asp161 of core repeat 2. Thus, calcium-regulated expression of annexin II on the endothelial cell surface may play a central role in control of plasmin-mediated processes. INTRODUCTIONEndothelial cells express abundant binding sites for the fibrinolytic zymogen plasminogen and its serine protease activator, tissue plasminogen activator (t-PA) 1The abbreviations used are: t-PAtissue plasminogen activatorHBSHepes-buffered salineHUVEChuman umbilical vein endothelial cellsIPBimmunoprecipitation bufferPLGplasminogen, PBS phosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisELISAenzyme-linked immunosorbent assay. (1Hajjar K.A. Thromb. Haemostasis. 1995; 74: 294-301Google Scholar). Binding of plasminogen to endothelial cells enhances the catalytic efficiency of its activation by tissue plasminogen activator by at least 12-fold (2Hajjar K.A. Harpel P.C. Jaffe E.A. Nachman R.L. J. Biol. Chem. 1986; 261: 11656-11662Google Scholar). Cell surface plasmin activity may contribute to several endothelial cell functions including thromboresistance, angiogenesis, and activation of growth and differentiation factors (1Hajjar K.A. Thromb. Haemostasis. 1995; 74: 294-301Google Scholar). Recently, we identified annexin II as an endothelial cell surface co-receptor for plasminogen (PLG) and t-PA (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). When overexpressed in a renal epithelial cell line, this protein was translocated to the cell surface, conferred the capacity to bind both t-PA and PLG, and enhanced cell surface plasmin generation (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). Upon treatment of human endothelial cells with either EGTA, polyclonal anti-annexin II IgG, or antisense oligonucleotides directed against annexin II mRNA, binding of both t-PA and PLG decreased by approximately 50%, suggesting that annexin II accounts for about one-half of available endothelial cell binding sites for these ligands (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). Purified annexin II, moreover, specifically enhanced the catalytic efficiency of t-PA-dependent PLG activation by 60-fold in a cell-free system (4Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar).The annexins represent a family of some 13 calcium-dependent phospholipid-binding proteins, at least 10 of which occur in mammals (5Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Google Scholar, 6Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Google Scholar). All annexins possess a variable amino-terminal “tail” domain followed by a conserved “core” region that imparts membrane-binding capability (7Smith P.D. Moss S.E. Trends Genet. 1994; 10: 241-246Google Scholar). The latter consists of either four or eight homologous “endonexin” motifs which contain the general consensus sequence (L/M)-K-G-X-G-T-(38 residues)-(D/E) (8Geisow M.J. Fritsche U. Hexham J.M. Dash B. Johnson T. Nature. 1986; 320: 636-638Google Scholar).Recent crystallographic studies have served to identify potential calcium-dependent phospholipid-binding sites of annexin V and annexin I (9Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Luecke H. J. Mol. Biol. 1992; 223: 683-704Google Scholar, 10Weng X. Luecke H. Song I.S. Kang D.S. Kim S. Huber R. Protein Sci. 1993; 2: 448-458Google Scholar). The pentapeptide sequence K-G-X-G-T within the endonexin motif constitutes a loop connecting the first and second α-helices of a given repeat within the core region (11Gerke V. Moss S.E. The Annexins. Portland Press Ltd., London1992: 47-59Google Scholar). In addition, an acidic amino acid, located 38 residues downstream and within the junction of the fourth and fifth α-helices, coordinates with KGXGT motifs forming a potential calcium-binding site (11Gerke V. Moss S.E. The Annexins. Portland Press Ltd., London1992: 47-59Google Scholar). Based on such studies, the major calcium binding domains of annexin V appear to reside within repeats 1, 2, and 4, while those of annexin I are located in repeats 1, 2, and 3 (9Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Luecke H. J. Mol. Biol. 1992; 223: 683-704Google Scholar, 10Weng X. Luecke H. Song I.S. Kang D.S. Kim S. Huber R. Protein Sci. 1993; 2: 448-458Google Scholar). Similarly, Gerke and co-workers have used site-directed mutagenesis to locate three potential calcium-binding sites within repeats 2, 3, and 4 of annexin II, as well as a weaker site within repeat 1. Half-maximal saturation of these sites occurs at 5-10 µ and 200-300 µ Ca2+, respectively (12Jost M. Thiel C. Weber K. Gerke V. Eur. J. Biochem. 1992; 207: 923-930Google Scholar, 13Jost M. Weber K. Gerke V. Biochem. J. 1994; 298: 553-559Google Scholar).In the present study, we examined the mechanism by which annexin II associates with the endothelial cell surface. The data indicate that this interaction is an equilibrium-based, calcium-dependent binding event, with an absolute requirement for endonexin repeat 2. These findings suggest that calcium-mediated binding of annexin II to the cell membrane may play a central role in the control of cell surface plasminogen activation.DISCUSSIONThis study demonstrates for the first time that cell surface association of the fibrinolytic receptor, annexin II, is a high affinity binding event that specifically requires endonexin repeat 2. The data indicate that annexin II is synthesized by cultured endothelial cells and translocated within hours to the cell surface in a calcium-dependent interaction. By quantitative ELISA, cell surface annexin II appears to represent approximately 4-5% of the total endothelial cell pool. Cell surface annexin II was not detected on hepatoma cells or renal epithelial cells, but did appear on the surface of human smooth muscle cells.As a class, the annexins were originally identified in intracellular locations and shown to be involved in membrane fusion events (5Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Google Scholar, 6Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Google Scholar). Recently, however, several annexins have been found to be localized on cell surfaces or within extracellular compartments (33Muesch A. Hartmann E. Rohde K. Rubartelli A. Sitia R. Rapoport T.A. Trends Biochem Sci. 1990; 15: 86-88Google Scholar). Annexin II, for example, has been identified on the surface of human colon adenocarcinoma cells (34Yeatman T.J. Updyke T.V. Kaetzel M.A. Dedman J.R. Nicolson G.L. Clin. Exp. Metastasis. 1993; 11: 37-44Google Scholar), murine large cell lymphoma cells (35Tressler R.J. Updyke T.V. Yeatman T. Nicolson G.L. J. Cell. Biochem. 1993; 53: 265-276Google Scholar), and endothelial cells (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar, 24Wright J.F. Kurosky A. Wasi S. Biochem. Biophys. Res. Commun. 1994; 198: 983-989Google Scholar, 25Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Google Scholar). Annexin II, as well as annexin V, is released in microvesicles from chondrocytes during bone mineralization (36Takahashi S. Reddy S.V. Chirgwin J.M. Devlin R. Haipek C. Anderson J. Roodman G.D. J. Biol. Chem. 1994; 269: 28696-28701Google Scholar, 37Genge B.R. Wu L.N.Y. Adkisson IV, H.D. Wuthier R.E. J. Biol. Chem. 1991; 266: 10678-10685Google Scholar, 38Genge B.R. Cau X. Wu L.N.Y. Buzzi W.R. Showman R.W. Arsenault A.L. Ishikawa Y. Wuthier R.E. J. Bone Miner. Res. 1992; 7: 807-819Google Scholar), and from skin keratinocytes (39Ma A.S.P. Bell D.J. Mittal A.A. Harrison H.H. J. Cell Sci. 1994; 107: 1973-1984Google Scholar). Annexin I is selectively secreted in high concentrations by the human prostate (40Christmas P. Callaway J. Fallon J. Jones J. Haigler H.T. J. Biol. Chem. 1991; 266: 2499-2507Google Scholar) and by inflammatory cells (41Pepinsky R.B. Sinclair L.K. Browning J.L. Mattaliano R.J. Smart J.E. Chow E.P. Falbel T. Ribolini A. Garwin J. Wallner B.P. J. Biol. Chem. 1986; 261: 4239-4246Google Scholar). Annexin V is found in plasma, amniotic fluid, and post-culture medium from endothelial cells and endometrium (42Flaherty M.J. West S. Heimark R.L. Fujikawa K. Tait J.F. J. Lab. Clin. Med. 1990; 115: 174-181Google Scholar). The specific stimuli for these redistribution events have yet to be determined, and their elucidation may provide insight into the regulation of pericellular protease activity.In the present study, several lines of evidence support an annexin II-cell surface interaction which is absolutely dependent upon micromolar free Ca2+. First, surface-associated immunoreactive material was efficiently eluted upon chelation of extracellular Ca2+ with EGTA (Fig. 2). Second, 36-kDa cross-reactive material that comigrated with authentic annexin II was recovered in EGTA eluates of endothelial cells both in vitro and in situ (Fig. 3). Third, metabolically labeled annexin II was eluted from HUVEC cell surface with EGTA, but not HBS (Fig. 4). Fourth, calcium-dependent rebinding of annexin II to EGTA-treated endothelial cells was demonstrated (Fig. 5). The half-saturating calcium concentration (pCa 5.5 or ∼3 µ) agreed closely with that of the high affinity calcium-binding sites within repeats 2, 3, and 4 of the core region of annexin II (Kd 5-10 µ Ca2+) (13Jost M. Weber K. Gerke V. Biochem. J. 1994; 298: 553-559Google Scholar), and with the calcium dependence of annexin II binding to phosphatidylserine-containing vesicles (Kd 2-5 µ Ca2+) (32Powell M.A. Glenney J.R. Biochem. J. 1987; 247: 321-328Google Scholar, 43Glenney J. J. Biol. Chem. 1986; 261: 7247-7252Google Scholar). Since plasma free Ca2+ is ∼1 m, this interaction would be supported in vivo.To further define the cell surface binding domain of annexin II, two types of experiments were conducted. In the first, peptides mimicking the four potential endonexin repeats of annexin II were tested for their ability to block binding of the parent molecules to cultured endothelial cells. Of these peptides, two, corresponding to repeats 2 and 4 and sharing the K-G-X-G-T motif (KGLGT and KGKGT), specifically blocked binding (Fig. 8). The essential nature of this pentapeptide sequence was further underscored by the fact that reversal of the initial K and final T residues rendered the peptides almost completely inactive. Given the similarity between these two peptides (KG(L/K)GT), it is not possible to determine whether repeat 2 and 4 peptides were blocking repeat 2, repeat 4, or both.To further refine the analysis, therefore, recombinant forms of annexin II mutated at the coordinating downstream acidic residue (D or E) of repeat 2, 3, or 4 were examined for their ability to compete with the wild type protein for binding to the cell surface. Because only the mutant corresponding to repeat 2 failed to block binding (Fig. 9), the data suggest that repeat 2 plays a dominant role in mediating the cell surface interaction. This result is also consistent with our previous observation that cell surface annexin II is subject to proteolytic cleavage at Lys307-Arg308. This modification creates a carboxyl-terminal lysine residue, enabling binding of plasminogen via lysine-binding “kringle” structures (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). Cleavage at Lys307-Arg308 would release a carboxyl-terminal fragment (Arg308-Asp328) that contains Asp321, thereby inactivating the Ca2+-binding site of repeat 4. Thus, carboxyl-terminal modification of annexin II is consistent with the hypothesis that repeat 2 is primarily responsible for Ca2+-mediated cell surface binding of annexin II.The binding isotherm depicted in Fig. 5 indicates a high affinity interaction between annexin II and the endothelial cell surface (Kd 49 n). The dissociation constant is reasonably close to Kd values reported for the interaction of annexin V with platelets (7 n) (44Thiagarajan P. Tait J.F. J. Biol. Chem. 1990; 265: 17420-17423Google Scholar), ovarian carcinoma cells (9 n) (45Rao L.V.M. Tait J.F. Hoang A.D. Thromb. Res. 1992; 67: 517-531Google Scholar), or endothelial cells (16 n) (46Van Heerde W.L. Poort S. Van Veer C. Reutelingsperger C.P.M. De Groot P.G. Biochem. J. 1994; 302: 305-312Google Scholar). The slightly higher affinity for annexin V, compared with annexin II, is consistent with the former's relatively more avid binding to phospholipid (47Blackwood R.A. Ernst J.D. Biochem. J. 1990; 266: 195-200Google Scholar). The preferential inhibition of binding of annexin II to endothelial cells by anionic phospholipid over neutral phospholipid most likely reflects greater affinity for phosphatidylserine over phosphatidylcholine.In summary, the present data indicate that expression of annexin II on the endothelial cell surface results from translocation of newly synthesized annexin II, and its calcium-dependent association with outer leaflet phospholipid. This interaction requires the high affinity calcium-binding site of repeat 2. Although the mechanism of annexin II transport by the endothelial cell is unknown, several other proteins including fibroblast growth factor-1, interleukin-1β, thioredoxin, and lectin L-29 that lack typical signal peptides, are known to undergo leaderless secretion in response to specific stimuli (48Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Google Scholar, 49Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Google Scholar, 50Rubartelli A. Cozzolino F. Talio M. Sitia R. EMBO J. 1990; 9: 1503-1510Google Scholar, 51Rubartelli A. Bajetto A. Allavena G. Wollman E. Sitia R. J. Biol. Chem. 1992; 267: 24161-24164Google Scholar, 52Lindstedt R. Apodaca G. Barondes S.H. Mostov K.E. Leffler H. J. Biol. Chem. 1993; 268: 11750-11757Google Scholar). Highly regulated, nonclassical release of these proteins may represent a newly recognized form of host defense (33Muesch A. Hartmann E. Rohde K. Rubartelli A. Sitia R. Rapoport T.A. Trends Biochem Sci. 1990; 15: 86-88Google Scholar, 53Kuchler K. Thorner J. Curr. Opin. Cell Biol. 1990; 2: 617-624Google Scholar). Since annexin II represents a major fibrinolytic receptor that co-binds plasminogen and tissue plasminogen activator and augments the efficiency of plasmin generation, it is reasonable to hypothesize that regulation of secretion and/or binding of annexin II to cell surfaces may play a central role in the control of plasmin-mediated processes. INTRODUCTIONEndothelial cells express abundant binding sites for the fibrinolytic zymogen plasminogen and its serine protease activator, tissue plasminogen activator (t-PA) 1The abbreviations used are: t-PAtissue plasminogen activatorHBSHepes-buffered salineHUVEChuman umbilical vein endothelial cellsIPBimmunoprecipitation bufferPLGplasminogen, PBS phosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisELISAenzyme-linked immunosorbent assay. (1Hajjar K.A. Thromb. Haemostasis. 1995; 74: 294-301Google Scholar). Binding of plasminogen to endothelial cells enhances the catalytic efficiency of its activation by tissue plasminogen activator by at least 12-fold (2Hajjar K.A. Harpel P.C. Jaffe E.A. Nachman R.L. J. Biol. Chem. 1986; 261: 11656-11662Google Scholar). Cell surface plasmin activity may contribute to several endothelial cell functions including thromboresistance, angiogenesis, and activation of growth and differentiation factors (1Hajjar K.A. Thromb. Haemostasis. 1995; 74: 294-301Google Scholar). Recently, we identified annexin II as an endothelial cell surface co-receptor for plasminogen (PLG) and t-PA (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). When overexpressed in a renal epithelial cell line, this protein was translocated to the cell surface, conferred the capacity to bind both t-PA and PLG, and enhanced cell surface plasmin generation (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). Upon treatment of human endothelial cells with either EGTA, polyclonal anti-annexin II IgG, or antisense oligonucleotides directed against annexin II mRNA, binding of both t-PA and PLG decreased by approximately 50%, suggesting that annexin II accounts for about one-half of available endothelial cell binding sites for these ligands (3Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Google Scholar). Purified annexin II, moreover, specifically enhanced the catalytic efficiency of t-PA-dependent PLG activation by 60-fold in a cell-free system (4Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Google Scholar).The annexins represent a family of some 13 calcium-dependent phospholipid-binding proteins, at least 10 of which occur in mammals (5Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Google Scholar, 6Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Google Scholar). All annexins possess a variable amino-terminal “tail” domain followed by a conserved “core” region that imparts membrane-binding capability (7Smith P.D. Moss S.E. Trends Genet. 1994; 10: 241-246Google Scholar). The latter consists of either four or eight homologous “endonexin” motifs which contain the general consensus sequence (L/M)-K-G-X-G-T-(38 residues)-(D/E) (8Geisow M.J. Fritsche U. Hexham J.M. Dash B. Johnson T. Nature. 1986; 320: 636-638Google Scholar).Recent crystallographic studies have served to identify potential calcium-dependent phospholipid-binding sites of annexin V and annexin I (9Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Luecke H. J. Mol. Biol. 1992; 223: 683-704Google Scholar, 10Weng X. Luecke H. Song I.S. Kang D.S. Kim S. Huber R. Protein Sci. 1993; 2: 448-458Google Scholar). The pentapeptide sequence K-G-X-G-T within the endonexin motif constitutes a loop connecting the first and second α-helices of a given repeat within the core region (11Gerke V. Moss S.E. The Annexins. Portland Press Ltd., London1992: 47-59Google Scholar). In addition, an acidic amino acid, located 38 residues downstream and within the junction of the fourth and fifth α-helices, coordinates with KGXGT motifs forming a potential calcium-binding site (11Gerke V. Moss S.E. The Annexins. Portland Press Ltd., London1992: 47-59Google Scholar). Based on such studies, the major calcium binding domains of annexin V appear to reside within repeats 1, 2, and 4, while those of annexin I are located in repeats 1, 2, and 3 (9Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Luecke H. J. Mol. Biol. 1992; 223: 683-704Google Scholar, 10Weng X. Luecke H. Song I.S. Kang D.S. Kim S. Huber R. Protein Sci. 1993; 2: 448-458Google Scholar). Similarly, Gerke and co-workers have used site-directed mutagenesis to locate three potential calcium-binding sites within repeats 2, 3, and 4 of annexin II, as well as a weaker site within repeat 1. Half-maximal saturation of these sites occurs at 5-10 µ and 200-300 µ Ca2+, respectively (12Jost M. Thiel C. Weber K. Gerke V. Eur. J. Biochem. 1992; 207: 923-930Google Scholar, 13Jost M. Weber K. Gerke V. Biochem. J. 1994; 298: 553-559Google Scholar).In the present study, we examined the mechanism by which annexin II associates with the endothelial cell surface. The data indicate that this interaction is an equilibrium-based, calcium-dependent binding event, with an absolute requirement for endonexin repeat 2. These findings suggest that calcium-mediated binding of annexin II to the cell membrane may play a central role in the control of cell surface plasminogen activation.