The Role of Annexin II Tetramer in the Activation of Plasminogen
1998; Elsevier BV; Volume: 273; Issue: 8 Linguagem: Inglês
10.1074/jbc.273.8.4790
ISSN1083-351X
AutoresGeetha Kassam, Kyu‐Sil Choi, Jaspinder Ghuman, Hyoung-Min Kang, Sandra L. Fitzpatrick, Tracy Zackson, Saul L. Zackson, Mikayo Toba, Aya Shinomiya, David M. Waisman,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoAnnexin II tetramer (AIIt) is a major Ca2+-binding protein of endothelial cells which has been shown to exist on both the intracellular and extracellular surfaces of the plasma membrane. In this report, we demonstrate that AIIt stimulates the activation of plasminogen by facilitating the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. Fluid-phase AIIt stimulated the rate of activation of [Glu]plasminogen about 341-fold compared with an approximate 6-fold stimulation by annexin II. AIIt bound to [Glu]plasminogen(S741C-fluorescein) with a Kd of 1.26 ± 0.04 μm (mean ± S.D.,n = 3) and this interaction resulted in a large conformational change in [Glu]plasminogen. Kinetic analysis established that AIIt produces a large increase of about 190-fold in the kcat, app and a small increase in theKm,app which resulted in a 90-fold increase in the catalytic efficiency (kcat/Km) of t-PA for [Glu]plasminogen. AIIt also stimulated the t-PA-dependent activation of [Lys]plasminogen about 28-fold. Furthermore, other annexins such as annexin I, V, or VI did not produce comparable activation of t-PA-dependent conversion of [Glu]plasminogen to plasmin. The stimulation of the activation of [Glu]plasminogen by AIIt was Ca2+-independent and inhibited by ε-aminocaproic acid. AIIt bound to human 293 cells potentiated t-PA-dependent plasminogen activation. AIIt that was bound to phospholipid vesicles or heparin also stimulated the activation of [Glu]plasminogen 5- or 11-fold, respectively. Furthermore, immunofluorescence labeling of nonpermeabilized HUVEC revealed a punctated distribution of AIIt subunits on the cell surface. These results therefore identify AIIt as a potent in vitroactivator of plasminogen. Annexin II tetramer (AIIt) is a major Ca2+-binding protein of endothelial cells which has been shown to exist on both the intracellular and extracellular surfaces of the plasma membrane. In this report, we demonstrate that AIIt stimulates the activation of plasminogen by facilitating the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. Fluid-phase AIIt stimulated the rate of activation of [Glu]plasminogen about 341-fold compared with an approximate 6-fold stimulation by annexin II. AIIt bound to [Glu]plasminogen(S741C-fluorescein) with a Kd of 1.26 ± 0.04 μm (mean ± S.D.,n = 3) and this interaction resulted in a large conformational change in [Glu]plasminogen. Kinetic analysis established that AIIt produces a large increase of about 190-fold in the kcat, app and a small increase in theKm,app which resulted in a 90-fold increase in the catalytic efficiency (kcat/Km) of t-PA for [Glu]plasminogen. AIIt also stimulated the t-PA-dependent activation of [Lys]plasminogen about 28-fold. Furthermore, other annexins such as annexin I, V, or VI did not produce comparable activation of t-PA-dependent conversion of [Glu]plasminogen to plasmin. The stimulation of the activation of [Glu]plasminogen by AIIt was Ca2+-independent and inhibited by ε-aminocaproic acid. AIIt bound to human 293 cells potentiated t-PA-dependent plasminogen activation. AIIt that was bound to phospholipid vesicles or heparin also stimulated the activation of [Glu]plasminogen 5- or 11-fold, respectively. Furthermore, immunofluorescence labeling of nonpermeabilized HUVEC revealed a punctated distribution of AIIt subunits on the cell surface. These results therefore identify AIIt as a potent in vitroactivator of plasminogen. One of the major physiological functions of the proteolytic enzyme plasmin is the degradation and solubilization of fibrin, the major constituent of blood clots. Plasmin has a broad trypsin-like specificity and the production of plasmin from its precursor plasminogen is precisely regulated (reviewed in Refs. 1Lijnen H.R. Collen D. Bailliere's Clin. Haematol. 1995; 8: 277-290Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 2Plow E.F. Herren T. Redlitz A. Miles L.A. Hoover-Plow J.L. FASEB J. 1995; 9: 939-945Crossref PubMed Scopus (380) Google Scholar, 3Magnusson S. Sottrup-Jensen L. Petersen T.E. Dudek-Wojciechowska G. Claeys H. Ribbons D.W. Brew K. Proteolysis and Physiological Regulation 1976. 11. Academic Press, New York1996: 203-238Google Scholar, 4Markus G. Fibrinolysis. 1996; 10: 75-85Crossref Scopus (41) Google Scholar, 5Hajjar K.A. Thromb. Haemostasis. 1995; 74: 294-301Crossref PubMed Scopus (94) Google Scholar). One way in which plasmin activity is localized to the fibrin clot involves a fibrin-specific mechanism for the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA). 1The abbreviations used are: t-PA, tissue plasminogen activator; AIIt, annexin II tetramer; p11, p11 light chain of annexin II tetramer; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline. 1The abbreviations used are: t-PA, tissue plasminogen activator; AIIt, annexin II tetramer; p11, p11 light chain of annexin II tetramer; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline. For example, recent studies have shown that by virtue of its ability to bind both t-PA and plasminogen, fibrin acts as a template that promotes the formation of a t-PA·fibrin·plasminogen ternary complex. The catalytic efficiency of t-PA-dependent conversion of plasminogen to plasmin is determined by the stability of the ternary complex (6Horrevoets A.J.G. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1997; 272: 2183-2191Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Thus, fibrin is both a substrate of plasmin and a template for plasmin production. Fibrin also plays a role in the plasmin-dependent stimulation of plasmin formation. For example, the partial proteolysis of fibrin results in the transient generation of new carboxyl-terminal lysine residues that act as high affinity binding sites for the lysine-binding sites of plasminogen (7Suenson E. Lutzen O. Thorsen S. Eur. J. Biochem. 1984; 140: 513-522Crossref PubMed Scopus (195) Google Scholar, 8de Vries C. Veerman H. Koornneef E. Pannekoek H. J. Biol. Chem. 1990; 265: 13547-13552Abstract Full Text PDF PubMed Google Scholar). The partially proteolyzed fibrin, but not intact fibrin, also stimulates the plasmin-dependent conversion of [Glu]plasminogen to [Lys]plasminogen (9Suenson E. Bjerrum P. Holm A. Lind B. Meldal M. Selmer J. Petersen L.C. J. Biol. Chem. 1990; 265: 22228-22237Abstract Full Text PDF PubMed Google Scholar). Since [Lys]plasminogen is more rapidly converted by t-PA to plasmin than [Glu]plasminogen, this results in a substantial enhancement in plasmin formation. Other proteins that interact with the lysine-binding sites of plasminogen such as the histidine-proline-rich glycoprotein or certain proteins of the extracellular matrix have also been shown to stimulate plasminogen activation (10Moser T.L. Enghild J.J. Pizzo S.V. Stack M.S. J. Biol. Chem. 1993; 268: 18917-18923Abstract Full Text PDF PubMed Google Scholar, 11Stack S. Gonzalez-Gronow M. Pizzo S.V. Biochemistry. 1990; 29: 4966-4970Crossref PubMed Scopus (68) Google Scholar, 12Borza D.-B. Morgan W.T. J. Biol. Chem. 1997; 272: 5718-5726Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).Recently, the endothelial cell-surface Ca2+-binding protein, annexin II, has also been shown to stimulate the t-PA-dependent formation of plasmin from plasminogen (13Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar,14Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar). Annexin II was originally identified as an intracellular Ca2+- and phospholipid-binding protein and subsequent studies suggested that this protein was potentially involved in the regulation of membrane trafficking events such as exocytosis or endocytosis (reviewed in Ref. 15Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). Annexin II can exist in cells as both a monomer or as a heterotetramer. The heterotetramer, called annexin II tetramer (AIIt) consists of two annexin II molecules and two molecules of an 11-kDa regulatory subunit referred to as the p11 light chain. The binding of the p11 light chain regulates many of thein vitro activities of annexin II and the biochemical properties of AIIt are distinct from the annexin II monomer (16Drust D.S. Creutz C.E. Nature. 1988; 331: 88-91Crossref PubMed Scopus (333) Google Scholar, 17Kang H.M. Kassam G. Jarvis S.E. Fitzpatrick S.L. Waisman D.M. Biochemistry. 1997; 36: 2041-2050Crossref PubMed Scopus (41) Google Scholar). In many cells such as Madin-Darby canine kidney cells, bovine intestinal epithelial cells, and calf pulmonary arterial endothelial cells, 90–95% of the total cellular annexin II is present in the heterotetrameric form (18Gerke V. Weber K. EMBO J. 1984; 3: 227-233Crossref PubMed Scopus (385) Google Scholar, 19Nilius B. Gerke V. Prenen J. Szücs G. Heinke S. Weber K. Droogmans G. J. Biol. Chem. 1996; 271: 30631-30636Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Annexin II and AIIt have been shown to exist on the extracellular surface of many cells although the relative extracellular distribution of the two forms of the protein has not been quantified (13Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar, 20Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar, 21Wright J.F. Kurosky A. Wasi S. Biochem. Biophys. Res. Commun. 1994; 198: 983-989Crossref PubMed Scopus (79) Google Scholar, 22Tressler R.J. Updyke T.V. Yeatman T. Nicolson G.L. J. Cell. Biochem. 1993; 53: 265-276Crossref PubMed Scopus (94) Google Scholar, 23Yeatman T.J. Updyke T.V. Kaetzel M.A. Dedman J.R. Nicolson G.L. Clin. Exp. Metastasis. 1993; 11: 37-44Crossref PubMed Scopus (92) Google Scholar).In the present report, we have compared the kinetics of annexin II and AIIt-dependent activation of t-PA-mediated plasminogen activation. These experiments establish the presence of AIIt on the HUVEC surface and that AIIt is a potent in vitro activator of t-PA-mediated plasminogen activation.DISCUSSIONThe most dramatic changes in the structure and function of annexin II occur upon the binding of the p11 light chains to annexin II and hence the formation of the heterotetrameric form of the protein, AIIt. For example, the formation of the heterotetramer redistributes the protein from the cytosol to the plasma membrane and also decreases theKd (Ca2+) for the binding of annexin II to biological membranes (reviewed in Ref. 15Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). Although both annexin II and AIIt have been shown to exist on the extracellular surface of many cells and AIIt has been shown to comprise 90–95% of the total pool of annexin II present in endothelial, epithelial, and Madin-Darby canine kidney cells (18Gerke V. Weber K. EMBO J. 1984; 3: 227-233Crossref PubMed Scopus (385) Google Scholar, 19Nilius B. Gerke V. Prenen J. Szücs G. Heinke S. Weber K. Droogmans G. J. Biol. Chem. 1996; 271: 30631-30636Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the extracellular distribution of the two forms of the protein has not been extensively investigated. Furthermore, detailed characterization of the various in vitro activities of these proteins has established that annexin II and AIIt are functionally distinct (17Kang H.M. Kassam G. Jarvis S.E. Fitzpatrick S.L. Waisman D.M. Biochemistry. 1997; 36: 2041-2050Crossref PubMed Scopus (41) Google Scholar). As shown in Fig. 8, A and B, annexin II and p11 co-localize to the surface of endothelial cells. Furthermore, after surface biotinylation of intact living HUVEC, anti-annexin II antibody coprecipitated p11, therefore confirming the presence of AIIt on the surface of HUVEC (Fig. 8 D). These observations differ with a previous report suggesting that p11 is not present on the extracellular surface of HUVEC (38Hajjar K.A. Guevara C.A. Lev E. Dowling K. Chacko J. J. Biol. Chem. 1996; 271: 21652-21659Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, this conclusion was based on the observation that Western blots of a cellular extract prepared by the washing of HUVEC with EGTA failed to stain for p11. However, as shown in Fig. 8 B, cells washed with EGTA stain positive for annexin II and p11. This suggests, as has been reported by others that a portion of the binding of annexin II to membranes is Ca2+-independent (39Harder T. Kellner R. Parton R.G. Gruenberg J. Mol. Biol. Cell. 1997; 8: 533-545Crossref PubMed Scopus (186) Google Scholar, 40Jost M. Zeuschner D. Seemann J. Weber K. Gerke V. J. Cell Sci. 1997; 110: 221-228Crossref PubMed Google Scholar).The annexin II present on the extracellular surface of endothelial cells has been shown to bind t-PA, plasminogen, and plasmin and to stimulate the activation of plasminogen (13Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar). As much as 40% of the total plasminogen binding capacity of HUVEC has been suggested to be due to annexin II (14Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar). Considering our observation that a significant amount of annexin II is present on the extracellular surface of HUVEC as AIIt we were interested in establishing whether or not AIIt could also activate plasminogen. We found that compared with annexin II, AIIt was an extremely potent activator of plasminogen. Therefore, in the present study we have characterized the features of the AIIt-dependent activation of plasminogen in detail. The major findings of this study are summarized as follows.First, the plots of A405 nm versustime squared for the activation of plasminogen by annexin II and AIIt are linear for the initial rates of plasminogen activation. This establishes that annexin II and AIIt have an immediate, intrinsic stimulatory effect on plasminogen activation and do not require processing by either t-PA or plasmin. Many other plasminogen activators such as fibrin or fibrinogen fragments exhibit a lag period during which the internal lysine residues are exposed by plasmin to form new plasminogen-binding sites that accelerate the plasminogen activation process (7Suenson E. Lutzen O. Thorsen S. Eur. J. Biochem. 1984; 140: 513-522Crossref PubMed Scopus (195) Google Scholar, 8de Vries C. Veerman H. Koornneef E. Pannekoek H. J. Biol. Chem. 1990; 265: 13547-13552Abstract Full Text PDF PubMed Google Scholar, 9Suenson E. Bjerrum P. Holm A. Lind B. Meldal M. Selmer J. Petersen L.C. J. Biol. Chem. 1990; 265: 22228-22237Abstract Full Text PDF PubMed Google Scholar). Alternatively, the stimulation of plasminogen activation by the histidine-proline-rich glycoprotein does not require processing since this protein contains the prerequisite carboxyl-terminal lysine residue (41Saez C.T. Jansen G.J. Smith A. Morgan W.T. Biochemistry. 1995; 34: 2496-2503Crossref PubMed Scopus (17) Google Scholar). Considering that annexin II does not possess a carboxyl-terminal lysine residue but activates plasminogen by a lysine-dependent mechanism (13Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar), this suggests that annexin II or the annexin II subunit of AIIt may utilize internal lysine residues to activate plasminogen.Second, AIIt stimulates plasminogen activation by increasing the apparent kcat. Previous studies have shown that annexin II enhances plasminogen activation by decreasing theKm. Other activators of plasminogen such as fibrin, or the histidine-proline-rich glycoprotein also have been shown to decrease the Km. However, several extracellular matrix proteins such as laminin and type IV collagen activate plasminogen by a mechanism involving both a decrease in theKm and an increase in thekcat (10Moser T.L. Enghild J.J. Pizzo S.V. Stack M.S. J. Biol. Chem. 1993; 268: 18917-18923Abstract Full Text PDF PubMed Google Scholar, 11Stack S. Gonzalez-Gronow M. Pizzo S.V. Biochemistry. 1990; 29: 4966-4970Crossref PubMed Scopus (68) Google Scholar). Considering our observation that the binding of [Glu]plasminogen to AIIt results in a large conformational change in [Glu]plasminogen (Fig. 6), it is reasonable to propose that a significant component of the effect of AIIt on thekcat of t-PA-dependent activation of [Glu]plasminogen is due to an AIIt induced conformational change in [Glu] plasminogen. It is likely that when [Glu]plasminogen is converted to the new AIIt-dependent conformation that [Glu]plasminogen is more readily proteolyzed by t-PA. Since both t-PA and [Glu]plasminogen bind to AIIt (Table II), the formation of a ternary complex between t-PA, [Glu]plasminogen, and AIIt may also contribute to the rate enhancement.Third, the AIIt-dependent stimulation of plasminogen activation is Ca2+-independent and inhibited by ε-aminocaproic acid. Our observation that the enhancement of the rate of [Glu]plasminogen activation by AIIt was Ca2+-independent was unexpected. To date all of thein vitro activities reported for AIIt have been shown to be Ca2+-dependent (15Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). For example, AIIt bundles F-actin, binds heparin, and aggregates chromaffin granules at physiological Ca2+ concentrations in vitro. Furthermore, the activation of catecholamine secretion in chromaffin cells by AIIt has also been shown to be Ca2+-dependent (42Burgoyne R.D. Morgan A. Biochem. Soc. Trans. 1990; 18: 1101-1104Crossref PubMed Scopus (16) Google Scholar, 43Ali S.M. Geisow M.J. Burgoyne R.D. Nature. 1989; 340: 313-315Crossref PubMed Scopus (235) Google Scholar, 44Chasserot-Golaz S. Vitale N. Sagot I. Delouche B. Dirrig S. Pradel L.A. Henry J.P. Aunis D. Bader M.F. J. Cell Biol. 1996; 133: 1217-1236Crossref PubMed Scopus (101) Google Scholar, 45Liu L. Fisher A.B. Zimmerman U.J.P. Biochim. Biophys. Acta. 1995; 1259: 166-172Crossref PubMed Scopus (29) Google Scholar, 46Sarafian T. Pradel L.A. Henry J.P. Aunis D. Bader M.F. J. Cell Biol. 1991; 114: 1135-1147Crossref PubMed Scopus (149) Google Scholar). Our observation that ε-amino-n-caproic acid inhibits the AIIt-dependent stimulation of [Glu]plasminogen activation (Fig. 4 B) suggests that the lysine residues involved in the interaction of AIIt with t-PA and [Glu]plasminogen are fully exposed and are not influenced by Ca2+-dependent conformational changes in AIIt.Fourth, both fluid-phase and solid-phase AIIt activate plasminogen. Although our examination of the activation of plasminogen by fluid-phase AIIt has allowed us to establish the mechanism for activation of plasminogen, it is important to stress that in vivo, AIIt is bound to the extracellular surface of the plasma membrane. It has been shown that the annexin II bound to the extracellular surface of the endothelial plasma membrane participates in the enhancement rate of [Glu]plasminogen activation (13Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar), however, it has not been established how annexin II or AIIt binds to the surface of cells. As shown in Fig. 7, the binding of AIIt to 293 cells results in the potentiation of t-PA-dependent plasminogen activation. Conceivably, AIIt could bind to plasma membrane phospholipid, glycosaminoglycan, or specific protein receptors. Since the biochemical properties of fluid-phase AIIt could be distinct from those of plasma membrane-bound AIIt, we examined the ability of either phospholipid-bound or heparin-bound AIIt to enhance the rate of [Glu]plasminogen activation. Our observation that AIIt complexed with either phospholipid (Fig. 5) or heparin (Fig. 4 D) activates [Glu]plasminogen, establishes that fluid-phase and solid-phase AIIt are both capable of the activation of [Glu]plasminogen. Whether or not the kinetics of plasminogen activation by fluid-phase or heparin-bound AIIt are similar remains to be established.Our observation that under our assay conditions (5.6 nmt-PA and 0.11 μm [Glu]plasminogen) 94% of [Glu]plasminogen and 49% of t-PA bound to the AIIt·heparin complex (Table II) suggests that these proteins bind to AIIt-heparin with nanomolar dissociation constants. Since we measured aKd of 1.26 μm for the interaction of AIIt with [Glu]plasminogen (Fig. 6 B), and also observed that 0.11 μm [Glu]plasminogen was almost totally bound to AIIt-heparin (Table II) it is reasonable to propose that [Glu]plasminogen binds to AIIt-heparin with much higher affinity than to AIIt alone.Fifth, the binding of AIIt to [Glu]plasminogen(S741C-fluorescein) results in a conformational change in the microenvironment of the active site of the zymogen. The Kd of 1.26 μm for the interaction of AIIt with [Glu]plasminogen was stronger than the Kd of 30 μmestimated for the binding of [Glu]plasminogen to fibrin but similar to the Kd of 1.2 μm estimated for the binding of [Lys]plasminogen to fibrin (47Fischer B.E. Blood Coagul. Fibrinolysis. 1992; 3: 197-204PubMed Google Scholar). Native [Glu] plasminogen exhibits a tight spiral structure which results in the occlusion of the activation cleavage site (Arg561-Val562) from attack by t-PA. It is therefore possible that the AIIt-dependent conformational change in [Glu]plasminogen results in exposure of the activation cleavage site of [Glu]plasminogen to t-PA.Last, of the five annexins tested, AIIt is by far the most potent activator of plasminogen (Table I). For example, 5 μmannexin II stimulated the rate of [Glu]plasminogen conversion about 7-fold compared with 341-fold for AIIt. Furthermore, although annexin I, V, and VI have been visualized on the extracellular surface of various cells (23Yeatman T.J. Updyke T.V. Kaetzel M.A. Dedman J.R. Nicolson G.L. Clin. Exp. Metastasis. 1993; 11: 37-44Crossref PubMed Scopus (92) Google Scholar, 48Tressler R.J. Yeatman T. Nicolson G.L. Exp. Cell Res. 1994; 215: 395-400Crossref PubMed Scopus (25) Google Scholar), the low enhancement of the rate of [Glu]plasminogen activation by these proteins, compared with AIIt suggests that a functional domain unique to AIIt is probably involved in the activation of [Glu]plasminogen.At present the physiological significance of our observations must remain speculative. Although AIIt is present on the surface of endothelial cells, the nature of the AIIt-binding sites on the cellular surface are not known Considering the high affinity of AIIt for heparin it is tempting to speculate that AIIt may associate with heparan sulfate glycosaminoglycan on the cell surface. One popular theory is that the interactions of plasminogen with cell surface heparan sulfate glycosaminoglycan functions to elevate local plasminogen concentrations on the cell surface. It is therefore reasonable to suspect that cell surface heparan sulfate glycosaminoglycan binding may be a mechanism for colocalizing and concentrating both plasminogen and AIIt on the endothelial cell surface. One of the major physiological functions of the proteolytic enzyme plasmin is the degradation and solubilization of fibrin, the major constituent of blood clots. Plasmin has a broad trypsin-like specificity and the production of plasmin from its precursor plasminogen is precisely regulated (reviewed in Refs. 1Lijnen H.R. Collen D. Bailliere's Clin. Haematol. 1995; 8: 277-290Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 2Plow E.F. Herren T. Redlitz A. Miles L.A. Hoover-Plow J.L. FASEB J. 1995; 9: 939-945Crossref PubMed Scopus (380) Google Scholar, 3Magnusson S. Sottrup-Jensen L. Petersen T.E. Dudek-Wojciechowska G. Claeys H. Ribbons D.W. Brew K. Proteolysis and Physiological Regulation 1976. 11. Academic Press, New York1996: 203-238Google Scholar, 4Markus G. Fibrinolysis. 1996; 10: 75-85Crossref Scopus (41) Google Scholar, 5Hajjar K.A. Thromb. Haemostasis. 1995; 74: 294-301Crossref PubMed Scopus (94) Google Scholar). One way in which plasmin activity is localized to the fibrin clot involves a fibrin-specific mechanism for the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA). 1The abbreviations used are: t-PA, tissue plasminogen activator; AIIt, annexin II tetramer; p11, p11 light chain of annexin II tetramer; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline. 1The abbreviations used are: t-PA, tissue plasminogen activator; AIIt, annexin II tetramer; p11, p11 light chain of annexin II tetramer; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline. For example, recent studies have shown that by virtue of its ability to bind both t-PA and plasminogen, fibrin acts as a template that promotes the formation of a t-PA·fibrin·plasminogen ternary complex. The catalytic efficiency of t-PA-dependent conversion of plasminogen to plasmin is determined by the stability of the ternary complex (6Horrevoets A.J.G. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1997; 272: 2183-2191Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Thus, fibrin is both a substrate of plasmin and a template for plasmin production. Fibrin also plays a role in the plasmin-dependent stimulation of plasmin formation. For example, the partial proteolysis of fibrin results in the transient generation of new carboxyl-terminal lysine residues that act as high affinity binding sites for the lysine-binding sites of plasminogen (7Suenson E. Lutzen O. Thorsen S. Eur. J. Biochem. 1984; 140: 513-522Crossref PubMed Scopus (195) Google Scholar, 8de Vries C. Veerman H. Koornneef E. Pannekoek H. J. Biol. Chem. 1990; 265: 13547-13552Abstract Full Text PDF PubMed Google Scholar). The partially proteolyzed fibrin, but not intact fibrin, also stimulates the plasmin-dependent conversion of [Glu]plasminogen to [Lys]plasminogen (9Suenson E. Bjerrum P. Holm A. Lind B. Meldal M. Selmer J. Petersen L.C. J. Biol. Chem. 1990; 265: 22228-22237Abstract Full Text PDF PubMed Google Scholar). Since [Lys]plasminogen is more rapidly converted by t-PA to plasmin than [Glu]plasminogen, this results in a substantial enhancement in plasmin formation. Other proteins that interact with the lysine-binding sites of plasminogen such as the histidine-proline-rich glycoprotein or certain proteins of the extracellular matrix have also been shown to stimulate plasminogen activation (10Moser T.L. Enghild J.J. Pizzo S.V. Stack M.S. J. Biol. Chem. 1993; 268: 18917-18923Abstract Full Text PDF PubMed Google Scholar, 11Stack S. Gonzalez-Gronow M. Pizzo S.V. Biochemistry. 1990; 29: 4966-4970Crossref PubMed Scopus (68) Google Scholar, 12Borza D.-B. Morgan W.T. J. Biol. Chem. 1997; 272: 5718-5726Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Recently, the endothelial cell-surface Ca2+-binding protein, annexin II, has also been shown to stimulate the t-PA-dependent formation of plasmin from plasminogen (13Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar,14Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar). Annexin II was originally identified as an intracellular Ca2+- and phospholipid-binding protein and subsequent studies suggested that this protein was potentially involved in the regulation of membrane trafficking events such as exocytosis or endocytosis (reviewed in Ref. 15Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). Annexin II can exist in cells as both a monomer or as a heterotetramer. The heterotetramer, called annexin II tetramer (AIIt) consists of two annexin II molecules and two molecules of an 11-kDa regulatory subunit referred to as the p11 light chain. The binding of the p11 light chain regulates many of thein vitro activities of annexin II and the biochemical properties of AIIt are distinct from the annexin II monomer (16Drust D.S. Creutz C.E. Nature. 1988; 331: 88-91Crossref PubMed Scopus (333) Google Scholar, 17Kang H.M. Kassam G. Jarvis S.E. Fitzpatrick S.L. Waisman D.M. Biochemistry. 1997; 36: 2041-2050Crossref PubMed Scopus (41) Google Scholar). In many cells such as Madin-Darby canine kidney cells, bovine intestinal epithelial cells, and calf pulmonary arterial endothelial cells, 90–95% of the total cellular annexin II is present in the heterotetrameric form (18Gerke V. Weber K. EMBO J. 1984; 3: 227-233Crossref PubMed Scopus (385) Google Scholar, 19Nilius B. Gerke V. Prenen J. Szücs G. Heinke S. Weber K. Droogmans G. J. Biol. 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