Modulation of Fibrinolysis by Thrombospondin" DEANE F. MOSHER,b~CTINA M. M1SENHEIMEKb JOHAN STENFL0,t AND PHILIP J. HOGGd bDepavtmenaofMdUane and Biomoleculcrr Chemistry U n M t y of Wijumrin 4459Merticalscienurctnar 1300 Unipmity h u e U i c o n , Wh&n 53706 CDcprwtment of Clinical Cbemr;mY U n M t y of Lund Malmi General Ha~pi~al Malmi s-21401, & S dDeparhnenofofIlkematoloBy The Prince of W a h Harpiarl Randwick, N.S.W 2031 Amtnzlia Thrombospondin (TSP)is a 450 000 dalton trimer of identical 150 000 dalton subunits. 1,2 The subunits contain b u r different types of well-recognized modules and are connected near the amino-terminus by disulfide linkages. Platelet TSP is secreted from a-granules upon activation and is normally present at very low concentration (approximately 0.1 pg/ml) in plasma.' Normal and transfbrmed cells in culture also produce TSP. Platelet TSP is composed of different domains that mediate binding of TSP to cells, platelets, and numerous proteins including extracellular matrix proteins such as collagen, fibronectin, heparin sulfite proteoglycan, and laminin and plasma proteins such as fibrinogen, plasminogen and histidine-rich glycoprotein. Recently, a second gene fbr TSP was described in the mouse3 and chicken.4 Two genes also are present in humans.3 The expression of the second gene (Thbs2) in various tissues of the mouse differs from that of the Thbs 1 gene3 and is not subject to as much upregulation in cultured cells exposed to serum growth f i ~ t o r sCompar.~ ison of the 2 TSPs reveals a gradient of sequence identity in which the aminoterminal regions are less homologous than the carboxyterminal regions. For instance, mouse TSP 1 (platelet TSP)is 38% identical to mouse TSP 2 in the aminoterminal heparinbinding domain and 65-75% identical in the type 1 properdin-like modules.3 The functions of both brms of TSP remain to be elucidated. TSP complexes to 6brin-d and copolymerizeswith fibrin in ~ l o t s .A~ number ,~ a Supported by HL29586 from the National Institutes of Hcalth, the Swedish Medical Research Council, and the Albert Pahlsson's Foundation. Author to whom comspondencc should be a d h d ; Tcl.: (608) 262-1576; FAX: (608) 262-2327.
64
MOSHER
ct
af.: THROMBOSPONDIN
IN FXBRINOLYSIS
65
of observations, mostly made with one or more of the proteins adsorbed to micmtitration plates, indicate that TSP enhances the generation of plasmin through its interactions with various proteins of the fibrinolyticsystem (TABLE1). TSP has been demonstrated to form bimolecular complexes with plasminogen and plasmin9J0 and a trimolecular complex with plasminogen and histidine-rich glycoprotein.11 Activation of plasminogen by tissue plasminogen activator (tPA) is facilitated by these interactions, and most of the plasmin generated remains bound in the complex and is pmtected from inactivation by c~2-antiplasmin.12The binding of plasminogen to TSP is enhanced by tPA and urokinase plasminogen activator ( u P A ) . ~This ~ enhancement is dependent on the generation of plasmin, which is postulated to enzymatically modify TSP and plasminogen and thereby enhance the formation of the TSP-plasminogen complexes. TSP can also form a complex with uPA, but not tPA; the TSP-uPA complex has plasminogen-activating activity equivalent to fluid phase uPA, but is protected from inhibition by plasminogen activator inhibitor type 1 (PAI-l).14 We have found that TSP in solution has an opposite effect, that is, it inhibits fibrinolysis.15 Platelet TSP is slow tight-binding inhibitor of plasmin as determined by the loss of plasmin's amidolytic, fibrinolytic and fibrinogenolytic activities.15 The rate constant for inhibition is approximately 6.3 x 103 M-1 sec-1, roughly comparable to the rate constant for inhibition of thrombin by antithrombin in the absence of heparin, and the stoichiometry of the inhibition is approximately one mol ofGlu- or Lys-plasmin to one mol trimeric TSP.Plasmin in complex with streptokinase or ~-aminocaproicacid is protected from inhibition by thrombospondin, thereby implicating the lysine-binding kringles of plasmin in the inhibition process. However, the protease part of plasmin must be of primary importance, because thrombospondin is a p o d inhibitor of vypsin (P. J. Hog, manuscript in preparation). Thrombospondin also inhibits uPA albeit much more slowly than it inhibits plasmin. It has no effect on thrombin or factor Xa and has a stimulatory effect on the amidolytic activity of tissue plasminogen activator. TSP released from activated platelets and incorporated into the fibrin clot may play an important role in fibrinolysis. The kinetics of activation of plasminogen by tPA are unfavorable in circulation, but upon binding of tPA and plasminogen to fibrin, plasmin is readily formed and clot lysis can occur.16 Platelet-rich clots, however, have been observed to be much more resistant to lysis with tPA than erythrocyte-rich clots for reasons that are unclear.17 It is likely that this resistance is due, at least partially, to
TSP.
TABLE2 describes experiments in which 60 pg/ml TSP inhibited lysis of plasma clots activated with uPA, reproducing results obtained with purified proteins.15 Remarkably, the effect is seen at a TSP concentration only shghtly above the concentration in whole serum (about 20 pg/ml) and in the presence of the full plasma concentrations of a2-antiplasmin and plasminogen activator inhibitors. Because plasmin activity is important for phenomena such as inflammation and tumor cell implantation and migration that are unrelated to lysis of fibrin, TSP has the potential to play important roles in many processes.18 For instance, TSP binds to the extracellular mauix19 and may protect plasmin-sensitive fibronectin matrix20 from plasmin generated around it. WI-38 fibroblasts generated from embryonic lung have been observed to secrete substantial quantities of plasminogen activator and have been documented to lyse clots formed over the cell layer without dqpting the cell rnat r k 2 1 This could be due to the inhibition of plasmin by the TSP bound to the matrix. Urokinase and TSP colocalize to invading mammary m i n o m a cells.14 TSP may control plasmin generation thought to be important for metastasis.18 Further studies
199014
Binding is Plg-dependent Complexed Pn protected from az-antiplasmin tPA or uPA enhance binding of Plg to TSP Enhancement mediated by plasmin K-Plg enhanced more than EPlg Substrate-bound TSP cleaved by plasmin to 160, 120, 70, 47,22, and 18K h g m e n n Adsorbed TSP binds l-chain or 2-chain uPA tchain uPA in complex protected from PAI-1 Complexed l-chain uPA cleaved by plasmin TSP and uPA colocalize in normal and malignant breast tissue
ELISA Plg activation assay Fluoromemc uPA substrate Immunohistochemistry
Fluorometric Pn substrate ELISA, aflinity beads ELISA ELISA SDSPAGE
M e t immunoelectrophoresis ELISA Fibrin plate assay Fibrin plate assay Fibrin plate assay Fluorometric Pn substrate Ligand blotting of TSP to Plg and hgmented Plg and vice versa Inhibition studies ELISA anity chromatography ELISA ELISA plate assay Fluoromemic Pn substrate Fluoromemc Pn substrate Fluoromemc Pn substrate ELISA Rmket immunoelecaophoresis AfFinity bead
Methods
ABBRJNIATTONS:
TSP, thrombospondin; Plg, plasminogcn; cACA, epsilon aminoCaproic acid; tPA, tissue plasminogen activator; Pn, plasmin; K5, w e 5; CAM, carboxyamidomethyl;HRGP, histidine-rich glycoprotein; K, lys; E, Glu; uPA, urokinase plasminogen activator; PAI-1, plasminogen activator inhibitor 1.
~ a r p ect l al.,
~
Silverstein ct al. 198613
Siverstein et al., 198512
Pn generated from bound complexes More PN generated from bound TSP-Plg-HRGP than TSP-Plg Substrate-bound TSP lowers K,, of fluid phase Plg for tPA Plg, tPA, and TSP form a complex
TSP or CAM-TSP forms a aimolecular complex with Plg and HRGP TSP-HRGPcomplex formation not blocked by eACA TSP alone or TSP in complexes binds heparin
Lysine-binding sites of Plg not directly involved in TSP-Plg binding
TSP binds to K5 of Plg
Depoli ct al., 19891°
Siverstein ct al., 1985l'
TspPlg complex formation (& = 35 nM) (Not Ca++-sensitive;blocked by E-ACA [KI = 200 pM]) TSP blocks l y ~ kof fibrin by tPA-Plg TSP blocks activation of Plg by tPA TSP does not inhibit Pn
Finding
Siverstein ct al., 19M9
Reference
TABLB 1. Summary of Published Interaction among TSP and Components of the Fibrinolytic System
R
R
8
5
1
FI
4
zE
8
MOSHER ct ul.: THROMBOSPONDIN IN FIBRINOLYSIS
TABLE 2.
67
Effect of TSP on Lysis of Clotted Whole Plasma
Condition
Lysis Area (%)
No TSP With TSP
72 f 3%
Po01
Human plasma without or with added TSP, 60 pgcglml, was clotted in small wells of tissue culture plates with thrombin and Ca2+. After clotting, lysis was initiated by addition of urokinase to the center of the well. Lysis zones were measured 16 h later. The methods were similar to those used to show the effects of TSP on lysis of plasminogen-containing clots made with purified fibrinogen.15 Multiple wells were assayed in 4 separate experiments. TSP caused decreased lysis zones in all 4 experiments, although the lysis areas varied somewhat from experiment to experiment. Results are expressed as mean % of control [no TSP] f SEM.
on the inhibition of plasmin activity by TSP, therefore, are vital for both an understanding of fibrinolysis and for an understanding of other plasmin-related processes. At least ten families of protein inhibitors of serine proteases have been identified, most of which follow the “standard mechanism” of inhibition.22 The mechanism involves the binding of the inhibitor to the protease fbllowed by the tbrmation of a stable complex. During complex formation, a peptide bond at the reactive site of the inhibitor is hydrolyzed very slowly by the protease. Although the o v e d sequence of the inhibitors is not conserved, the inhibitors share a common loop structure at their reactive centers.23 The overall structures of the inhibitors are distinct within each family, but the small inhibitors have numerous disulfide bonds that, along with hydrophobic interactions, stabilize the tertiary structure of the proteins and provide a “scaffold” for the loop containing the reactive center. None of the protein modules within TSPlJ: the procollagen, properdin (type l),epidermal growth factor (type 2) and calcium binding (type 3) modules have previously been implicated in inhibition of serine protease.22.23 A leech protein, antistasin, which shares a short sequence homology with part of the properdin modules of TSP,a has recently been identified as a potent inhibitor of Factor Xa and exhibits many functional similarities to other serine protease inhibitors.25 The properdin domain in TSP, however, does not contain the lysine residue identified as the reactive site for Factor Xa in antistasin,25 nor does it contain any other lysine.or arginine residue in an obvious reactive site. Moreover, the homology between antistasin and the properdin modules does not extend to all of the cysteines in the properdin modules, making it unlikely that the properdin modules can adopt an antistasin-like “scaffold.” Owing to the one-to-one stoichiometry of TSP to plasrnin,l5 the most likely candidate for the inhibitory domain of TSP is the procollagen domain, because it is located adjacent to the intermolecular disulfide bridges of TSP, or the bridge region itself. Interestingly, the bridge region, which is thought to be stabilized by the three subunits coming together in an a-helical coiled-coil,26 is different between the two forms of TSP in that five extra residues are inserted between the end of the presumptive coiled-coil and the procollagen repeat to TSP 2.334
SUMMARY Thrombospondin is a large, trimeric glycoprotein secreted by activated platelets and growing cells. Thrombospondin copolymerizes with fibrin during blood coagulation and deposits in extracellular matrix. We tbund that thrombospondin is a slow (rate
68
ANNALS NEW YORK ACADEMY OF SCIENCES
constant approximately 6.3 x lo3 M-1 sec-l), Wt-binding (& < M)inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to degrade fibrinogen, and decreased lysis wnes in fibrin plate assays (Biochmrktty 31: 265469,1992). Thrombospondin also slowly inhibits wkinase plasminogen activator. The lysis zone when urokinase is put on fibrin plates made from whole plasma is less if thrombospondin is present. The stoichiometry of inhibition is approximately one mole plasmin:one mole thrombospondin trimer, a somewhat surprising result considering the trimeric nature of thrombospondin. These results indicate that thrombospondin is an important regulatorof fibrinolysis and degradation of extracellularmatrix, particularly when these processes are initiated by urokinase and even when other inhibitors of fibrinolysis are present. REFERENCES 1. MOSHER,D. F. 1990. Physiology of thrombospondin. Annu. Rev. Med. 41: 85-97. 2. FRAZIER, W. A. 1991. Thrombospondins. Cum. Opinion Cell. Biol. 3: 792-799. 3. BORNSTEIN,P., K. O’RDURYB,K. WIKSTROM,F. W. WOLF, R Urn, P. LI & V. M. DIXIT.1991. A second, expressed thrombospondin gene (Thh2)exists in the mouse genome. J. Biol. Chem. 266: 12821-12824. 4. LAWLER, J., M. DUQUETTE & P. FBRRO.1991. Cloning and sequencing of chicken thrombospondin. J. Biol. Chem. 266: 8039-8043. 5. BORNSTEIN, P., S. DEVABAYALU, P.LI, C. M.DISTECHE & P. FMMSON.1991. A second thrombospondin gene in the mouse is similar in organization to thrombospondin 1 but does not mpond to serum.Proc. Natl. Acad. Sci. USA 88: 8636-8640. 6. LEUNG,L. L. K. & R L. NACHMAN.1982. Complex formation of platelet thrombospondin with fibrinogen. J. Clin. Invest. 70: 542-549. 7. BALE,M. D.,L. G. WESTRICK & D.F. MOSHER.1985. Incorporation of thrombospondin into fibrin clots. J. Biol. Chem. 260: 7502-7508. J. E. & D. F. MOSHER.1985. Localization ofthrombospondin in clots 8. MURPHY-ULLRICH, formed in riru. Blood 66. 1098-1104. R L., L. L. K. LEUNG,P.C. HARPEL& R L. NACHMAN.1984. Complex 9. SILVERSTEIN, 10. 11. 12. 13.
formation of platelet thrombospondin with plasminogen: Modulation of activation by tissue activator. J. Clin. Invest. 7 4 1625-1633. DEPOLI,P.,T. BACON-BAGULEY, S. KENDRA-FRANCZAK, M. T. CEDERHOLM & D. A. WALZ.1989. Thrombospondin interaction with plasminogen. Evidence for binding to a specific region of the kringle structure of plasminogcn. Blood 73: 976-982. SILVERSTEIN, R L., L. L. K. LEUNG,P. C. HARPEL& R L. NACHMAN.1985. Platelet thrombospondin forms a trimolecular complex with plasminogen and histidine-rich glycoprotein. J. Clin. Invest. 75: 2065-2073. SILVERSTEIN, R L., R L. NACHMAN,L. L. K. LEIJNG& P.C. HARPEL.1985. Activation of immobilized plasminogen by tissue activator: Multimolecular complex formation. J. Biol. Chem. Mo: 10346-10352. SILVERSTEIN, R L., P.C. HARPEL& R L. NACHMAN.1986. Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin. J. Biol. Chem.
261: 9959-9965. 14. HARPEL,P. C., R L. SILVERSTEIN, R PANNELL, V. GUREWICH & R L. NACHMAN. 1990.
Thrombospondin forms complexes with singlexhain and rwo-chain forms of urokinase. J. Biol. Chem. 265: 11289-11294. & D. F. MOSHER.1992. Thrombospondin is a slow tight-binding 15. HOGG,P.J., J. STENPLO inhibitor of plasmin. Biochemistry. 31: 265-269. R L. & R L. NACHMAN.1987. Thrombospondin-plasminogen interactions: 16. SILVERSTEIN, Modulation of plasmin generation. Sem. Thromb. Hemostasis 13: 335-342. 17. JANG, I. K., H. K. GOLD,A. A. ZISKIND,J. T. FALMN,R E. HOLT, R C. LEINBACH, J. W. MAY& D. COLLEN.1989. Dfirential sensitivity of erythrocyte-rich and platelet-
MOSHER ct al.: THROMBospONDIN IN FIBRINOLYSIS
18. 19. 20.
21. 22. 23.
69
rich arterial thrombi to lysis with recombinant tissue-type plasminop activator: A possible explanation for resistance to coronary thrombolysis. Circulation 79: 920-928. SAKSELA, 0.& D. B. RIFKIN. 1988. Ccll-associated plasminogen activator: Regulation and physiological function. Annu. Rev. CCU Biol. 4: 93-126. MCKEOWN-LONGO, P. J., R HANNING & D. F. MOSHER.1984. Binding and degradation of platelet thrombospondin by cultured fibroblasts. J. C c U Biol. 98: 22-28. PETERSEN,T. E., H. C. THWERSEN, K. S K O ~ N G M R DK., VIBE PEDERSEN,P. SAHL, L. SOTTRUP-JENSEN & S. MAGNUSON.1983. Partial primary structure of bovine plasma fibronectin: Three types of internal homology. PIUC.Natl. Acad. Sci. USA 8 0 137-141. MOSHER, D. F., 0. SAKSELA & A. VAHERI. 1977. Synthesis and secretion of alpha-2macroglobulin by cultured adherent lung cells: Comparison with cell strains derived from other tissues. J. Clin. Invest. 60: 10361045. LASKOWSKI, M., JR. & I. KATO. 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 4 9 593-626. CARRELL,R W., P. A. PEMBERTON & D. R BOSWELL. 1987. The serpins: Evolution and adaptation in a family of protease inhibitors. Cold Spring Harbor Symp. Quant. Biol.
:527-535. 24. HOLT,G. D., H. C. KRIVAN,G. J. GASIC& V. GINSBURG.1989. Antistasin, an inhibitor of coagulation and metastasis, binds to sulhtide (Gal(3-!Xl&31-1Cer) and has a sequence homoiogy with other proteins that bind sulfated glycoconjugtes. J. Biol. Chem: 264: 12138-12140. 25. DUNWIDDIE, C., N. A. THORNBERRY, H. G. BULL,M. SARDANA,P. A. FRIEDMAN,J. W. JACOBS & E. SIMPSON.1989. Antistasin, a Ieech-derived inhibitor of factor Xa: Kinetic
analysis of enzyme inhibition and identification of the reactive site. J. Biol. Chem. 264: 16694-16699. 26. SOTITLE,J., J. SELEGUE& D. F. MOSHER.1991. Synthesis of truncated amino-terminal trimers of thrombospondin. Biochemistry 30: 6556-6562.
64
MOSHER
ct
af.: THROMBOSPONDIN
IN FXBRINOLYSIS
65
of observations, mostly made with one or more of the proteins adsorbed to micmtitration plates, indicate that TSP enhances the generation of plasmin through its interactions with various proteins of the fibrinolyticsystem (TABLE1). TSP has been demonstrated to form bimolecular complexes with plasminogen and plasmin9J0 and a trimolecular complex with plasminogen and histidine-rich glycoprotein.11 Activation of plasminogen by tissue plasminogen activator (tPA) is facilitated by these interactions, and most of the plasmin generated remains bound in the complex and is pmtected from inactivation by c~2-antiplasmin.12The binding of plasminogen to TSP is enhanced by tPA and urokinase plasminogen activator ( u P A ) . ~This ~ enhancement is dependent on the generation of plasmin, which is postulated to enzymatically modify TSP and plasminogen and thereby enhance the formation of the TSP-plasminogen complexes. TSP can also form a complex with uPA, but not tPA; the TSP-uPA complex has plasminogen-activating activity equivalent to fluid phase uPA, but is protected from inhibition by plasminogen activator inhibitor type 1 (PAI-l).14 We have found that TSP in solution has an opposite effect, that is, it inhibits fibrinolysis.15 Platelet TSP is slow tight-binding inhibitor of plasmin as determined by the loss of plasmin's amidolytic, fibrinolytic and fibrinogenolytic activities.15 The rate constant for inhibition is approximately 6.3 x 103 M-1 sec-1, roughly comparable to the rate constant for inhibition of thrombin by antithrombin in the absence of heparin, and the stoichiometry of the inhibition is approximately one mol ofGlu- or Lys-plasmin to one mol trimeric TSP.Plasmin in complex with streptokinase or ~-aminocaproicacid is protected from inhibition by thrombospondin, thereby implicating the lysine-binding kringles of plasmin in the inhibition process. However, the protease part of plasmin must be of primary importance, because thrombospondin is a p o d inhibitor of vypsin (P. J. Hog, manuscript in preparation). Thrombospondin also inhibits uPA albeit much more slowly than it inhibits plasmin. It has no effect on thrombin or factor Xa and has a stimulatory effect on the amidolytic activity of tissue plasminogen activator. TSP released from activated platelets and incorporated into the fibrin clot may play an important role in fibrinolysis. The kinetics of activation of plasminogen by tPA are unfavorable in circulation, but upon binding of tPA and plasminogen to fibrin, plasmin is readily formed and clot lysis can occur.16 Platelet-rich clots, however, have been observed to be much more resistant to lysis with tPA than erythrocyte-rich clots for reasons that are unclear.17 It is likely that this resistance is due, at least partially, to
TSP.
TABLE2 describes experiments in which 60 pg/ml TSP inhibited lysis of plasma clots activated with uPA, reproducing results obtained with purified proteins.15 Remarkably, the effect is seen at a TSP concentration only shghtly above the concentration in whole serum (about 20 pg/ml) and in the presence of the full plasma concentrations of a2-antiplasmin and plasminogen activator inhibitors. Because plasmin activity is important for phenomena such as inflammation and tumor cell implantation and migration that are unrelated to lysis of fibrin, TSP has the potential to play important roles in many processes.18 For instance, TSP binds to the extracellular mauix19 and may protect plasmin-sensitive fibronectin matrix20 from plasmin generated around it. WI-38 fibroblasts generated from embryonic lung have been observed to secrete substantial quantities of plasminogen activator and have been documented to lyse clots formed over the cell layer without dqpting the cell rnat r k 2 1 This could be due to the inhibition of plasmin by the TSP bound to the matrix. Urokinase and TSP colocalize to invading mammary m i n o m a cells.14 TSP may control plasmin generation thought to be important for metastasis.18 Further studies
199014
Binding is Plg-dependent Complexed Pn protected from az-antiplasmin tPA or uPA enhance binding of Plg to TSP Enhancement mediated by plasmin K-Plg enhanced more than EPlg Substrate-bound TSP cleaved by plasmin to 160, 120, 70, 47,22, and 18K h g m e n n Adsorbed TSP binds l-chain or 2-chain uPA tchain uPA in complex protected from PAI-1 Complexed l-chain uPA cleaved by plasmin TSP and uPA colocalize in normal and malignant breast tissue
ELISA Plg activation assay Fluoromemc uPA substrate Immunohistochemistry
Fluorometric Pn substrate ELISA, aflinity beads ELISA ELISA SDSPAGE
M e t immunoelectrophoresis ELISA Fibrin plate assay Fibrin plate assay Fibrin plate assay Fluorometric Pn substrate Ligand blotting of TSP to Plg and hgmented Plg and vice versa Inhibition studies ELISA anity chromatography ELISA ELISA plate assay Fluoromemic Pn substrate Fluoromemc Pn substrate Fluoromemc Pn substrate ELISA Rmket immunoelecaophoresis AfFinity bead
Methods
ABBRJNIATTONS:
TSP, thrombospondin; Plg, plasminogcn; cACA, epsilon aminoCaproic acid; tPA, tissue plasminogen activator; Pn, plasmin; K5, w e 5; CAM, carboxyamidomethyl;HRGP, histidine-rich glycoprotein; K, lys; E, Glu; uPA, urokinase plasminogen activator; PAI-1, plasminogen activator inhibitor 1.
~ a r p ect l al.,
~
Silverstein ct al. 198613
Siverstein et al., 198512
Pn generated from bound complexes More PN generated from bound TSP-Plg-HRGP than TSP-Plg Substrate-bound TSP lowers K,, of fluid phase Plg for tPA Plg, tPA, and TSP form a complex
TSP or CAM-TSP forms a aimolecular complex with Plg and HRGP TSP-HRGPcomplex formation not blocked by eACA TSP alone or TSP in complexes binds heparin
Lysine-binding sites of Plg not directly involved in TSP-Plg binding
TSP binds to K5 of Plg
Depoli ct al., 19891°
Siverstein ct al., 1985l'
TspPlg complex formation (& = 35 nM) (Not Ca++-sensitive;blocked by E-ACA [KI = 200 pM]) TSP blocks l y ~ kof fibrin by tPA-Plg TSP blocks activation of Plg by tPA TSP does not inhibit Pn
Finding
Siverstein ct al., 19M9
Reference
TABLB 1. Summary of Published Interaction among TSP and Components of the Fibrinolytic System
R
R
8
5
1
FI
4
zE
8
MOSHER ct ul.: THROMBOSPONDIN IN FIBRINOLYSIS
TABLE 2.
67
Effect of TSP on Lysis of Clotted Whole Plasma
Condition
Lysis Area (%)
No TSP With TSP
72 f 3%
Po01
Human plasma without or with added TSP, 60 pgcglml, was clotted in small wells of tissue culture plates with thrombin and Ca2+. After clotting, lysis was initiated by addition of urokinase to the center of the well. Lysis zones were measured 16 h later. The methods were similar to those used to show the effects of TSP on lysis of plasminogen-containing clots made with purified fibrinogen.15 Multiple wells were assayed in 4 separate experiments. TSP caused decreased lysis zones in all 4 experiments, although the lysis areas varied somewhat from experiment to experiment. Results are expressed as mean % of control [no TSP] f SEM.
on the inhibition of plasmin activity by TSP, therefore, are vital for both an understanding of fibrinolysis and for an understanding of other plasmin-related processes. At least ten families of protein inhibitors of serine proteases have been identified, most of which follow the “standard mechanism” of inhibition.22 The mechanism involves the binding of the inhibitor to the protease fbllowed by the tbrmation of a stable complex. During complex formation, a peptide bond at the reactive site of the inhibitor is hydrolyzed very slowly by the protease. Although the o v e d sequence of the inhibitors is not conserved, the inhibitors share a common loop structure at their reactive centers.23 The overall structures of the inhibitors are distinct within each family, but the small inhibitors have numerous disulfide bonds that, along with hydrophobic interactions, stabilize the tertiary structure of the proteins and provide a “scaffold” for the loop containing the reactive center. None of the protein modules within TSPlJ: the procollagen, properdin (type l),epidermal growth factor (type 2) and calcium binding (type 3) modules have previously been implicated in inhibition of serine protease.22.23 A leech protein, antistasin, which shares a short sequence homology with part of the properdin modules of TSP,a has recently been identified as a potent inhibitor of Factor Xa and exhibits many functional similarities to other serine protease inhibitors.25 The properdin domain in TSP, however, does not contain the lysine residue identified as the reactive site for Factor Xa in antistasin,25 nor does it contain any other lysine.or arginine residue in an obvious reactive site. Moreover, the homology between antistasin and the properdin modules does not extend to all of the cysteines in the properdin modules, making it unlikely that the properdin modules can adopt an antistasin-like “scaffold.” Owing to the one-to-one stoichiometry of TSP to plasrnin,l5 the most likely candidate for the inhibitory domain of TSP is the procollagen domain, because it is located adjacent to the intermolecular disulfide bridges of TSP, or the bridge region itself. Interestingly, the bridge region, which is thought to be stabilized by the three subunits coming together in an a-helical coiled-coil,26 is different between the two forms of TSP in that five extra residues are inserted between the end of the presumptive coiled-coil and the procollagen repeat to TSP 2.334
SUMMARY Thrombospondin is a large, trimeric glycoprotein secreted by activated platelets and growing cells. Thrombospondin copolymerizes with fibrin during blood coagulation and deposits in extracellular matrix. We tbund that thrombospondin is a slow (rate
68
ANNALS NEW YORK ACADEMY OF SCIENCES
constant approximately 6.3 x lo3 M-1 sec-l), Wt-binding (& < M)inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to degrade fibrinogen, and decreased lysis wnes in fibrin plate assays (Biochmrktty 31: 265469,1992). Thrombospondin also slowly inhibits wkinase plasminogen activator. The lysis zone when urokinase is put on fibrin plates made from whole plasma is less if thrombospondin is present. The stoichiometry of inhibition is approximately one mole plasmin:one mole thrombospondin trimer, a somewhat surprising result considering the trimeric nature of thrombospondin. These results indicate that thrombospondin is an important regulatorof fibrinolysis and degradation of extracellularmatrix, particularly when these processes are initiated by urokinase and even when other inhibitors of fibrinolysis are present. REFERENCES 1. MOSHER,D. F. 1990. Physiology of thrombospondin. Annu. Rev. Med. 41: 85-97. 2. FRAZIER, W. A. 1991. Thrombospondins. Cum. Opinion Cell. Biol. 3: 792-799. 3. BORNSTEIN,P., K. O’RDURYB,K. WIKSTROM,F. W. WOLF, R Urn, P. LI & V. M. DIXIT.1991. A second, expressed thrombospondin gene (Thh2)exists in the mouse genome. J. Biol. Chem. 266: 12821-12824. 4. LAWLER, J., M. DUQUETTE & P. FBRRO.1991. Cloning and sequencing of chicken thrombospondin. J. Biol. Chem. 266: 8039-8043. 5. BORNSTEIN, P., S. DEVABAYALU, P.LI, C. M.DISTECHE & P. FMMSON.1991. A second thrombospondin gene in the mouse is similar in organization to thrombospondin 1 but does not mpond to serum.Proc. Natl. Acad. Sci. USA 88: 8636-8640. 6. LEUNG,L. L. K. & R L. NACHMAN.1982. Complex formation of platelet thrombospondin with fibrinogen. J. Clin. Invest. 70: 542-549. 7. BALE,M. D.,L. G. WESTRICK & D.F. MOSHER.1985. Incorporation of thrombospondin into fibrin clots. J. Biol. Chem. 260: 7502-7508. J. E. & D. F. MOSHER.1985. Localization ofthrombospondin in clots 8. MURPHY-ULLRICH, formed in riru. Blood 66. 1098-1104. R L., L. L. K. LEUNG,P.C. HARPEL& R L. NACHMAN.1984. Complex 9. SILVERSTEIN, 10. 11. 12. 13.
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