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The Plasma Carboxypeptidases and the Regulation of the Plasminogen System
ELSEVIER
BRIEF REVIEWS The Plasma Carboxypeptidases and the Regulation of the Plasminogen System Edward F. Plow, Krishnan Allampallam, Alexander Redlitz
Central to the ~egulation of the plasminogen system, a proteolytic network that mediates degradation of fibn”n and facilitates cell migration, is the binding ofplasminogen to carboxy-terminal lysines. These residues occur either naturallyon plasmin substrates or cell su+aces orare generated as a consequence of partial plasmin degradation. The basic carboxypeptidases of plasma are capable of removing such carboxy-terminal lysines. Carboxypeptidase N, which is constitutively active, suppresses plasminogen binding to cell su~aces; plasma carboxypeptidase B, which must be proteolytically activated, not only suppresses cellular binding of plasminogen but also dampens fibrinolysis. Thus, the plasma carboxypeptidases may constitute an important regulato~ pathway for controlling the activity of the plasminogen system in physiologic, pathophysiologic, and pharmacologic circumstances. (Trends Cardiovasc Med 1997; 7:71-75). 0 1997, Elseviey Science Inc.
Proteolytic networks, such as the complement and the blood coagulation systems, have evolved to provide explosive protection of multicellular organisms against injury and infection. The plasminogen system constitutes another important proteolytic network that plays a central role in a variety of physiological responses. By virtue of its capacity to degrade fibrin, the plasminogen system
EdwardF. Plow,KrishnanAllampallam,and AlexanderRedlitz are at the The Joseph J. Jacobs Centerfor Thrombosisand Vascular Biology,Departmentof MolecularCardiology, The ClevelandClinicFoundation,Cleveland, Ohio44195,USA.AlexanderRedlitzcurrently is also at the Universityof Washington,Departmentof BiologicalStructure,Seattle,WA 98195,USA.
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maintains the patency of vasculature and surrounding tissues (Cohen 1980, Castellino et al. 1988, Vassalliet al. 1991, Takada et al. 1994, Plow et al. 1995, Carmeliet and Cohen 1995). By mediating the degradation of extracellular matrices, the plasminogen system facilitates cell migration in diverse physiological events such as ovulation (Leonardsson et al. 1995) and wound healing (Romer et al. 1996). Numerous and profound pathogenetic consequences, ranging from bleeding to thrombosis, have also been ascribed to excessive or ineffective activity of the plasminogen system. The capacity of tumor cells (Saksela and Rifkin 1988) and microorganisms (Lottenberg et al. 1994) to subvert the proteolytic activity of the plasminogen system for invasion of host tissues extends
the pathophysiological significance of this network. Additionally, the plasminogen system has become a major therapeutic target for the treatment of thrombotic diseases such as myocardial infarction and stroke. With the involvement of the plasminogen system in physiology, pathophysiology, and pharmacology, an understanding of its intricacies is essential. Multiple amplification and suppressive mechanisms are in place to maintain tight control over the proteolytic potential of the plasminogen system (Cohen 1980, Robbins 1991, Takada et al. 1994). Indeed, studies extending over several decades have identified and extensively characterized many of the molecular constituents of the plasminogen system. Nevertheless, recent studies have begun to unravel a previously unappreciated mechanism for the regulation of the plasminogen system. These reports have suggested that the plasma carboxypeptidases may play a significant role in dampening the activity of the plasminogen system, thereby influencing fibrinolysis and cell migration. This article summarizes these recent developments and attempts to place some present and future perspectives upon these findings.
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The Plasminogen System
The central components of the plasminogen system are summarized in Figure 1. Plasminogen, itself, circulates in the blood at a 2 pM concentration. This inactive zymogen is converted to an active serine protease, plasmin, by cleavage of a single peptide bond. This transition is mediated by the plasminogen activators, tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), as well as by bacterial activators such as streptokinase. Plasminogen activation is regulated by the availability of the activators and by inhibitors of these activators, primarily plasminogen activator inhibitor 1 (PAI-1). Plasmin activ-
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Plasminogen Activators
nyl residues (Sottrup-Jensen et al. 1978). Because plasmin cleaves its substrates to PA Inhibitors generate carboxy-terminal lysines, addi/ PleaminOgen_ Plasmin tional plasminogen may be recruited a~ antiplaamin and activated on the degraded substrate / surface. Thus, fibrin or cell surfaces, Fibrin —Fibrin Degradation Products Matrix Pmtains A Degradation Products which have been partially degraded by Pro-metalloprotainase _Metallopro!einase plasmin, can bind more plasminogen, Degradation Products Cell Surface Proteins _ can support more efficient plasminogen activation, and can protect more plasFigure 1. Componentsof the plasminogen min from inactivation than a virgin sursystem. face. Intuitivelythen, agents that remove carboxy-terminal lysines from fibrin or ity also can be directly inhibited, primacell surfaces should suppress plasminorily by az-antiplasmin (Castellino et al. 1988, Robbins 1991, Takada et al. 1994). gen activation and dampen fibrinolysis Plasmin is an endopeptidase, cleaving and pericellular proteolysis. The basic proteins at lysyl or arginyl bonds. Its carboxypeptidases remove carboxy-tersubstrate repertoire is broad. Clearly,fi- minal Iysines and arginines from substrates, which, in turn, suppress the acbrin is its major substrate, but fibrinotivity of the plasminogen system. gen and factor V also are cleaved by plasmin under circumstances of extensive plasminogen activation such as dur● The Plasma Carboxypeptidases ing disseminated intravascular coagulation (DIC). Other notable plasmin The basic carboxypeptidases are exosubstrates are matrix proteins, such as proteinases, which remove lysines and fibronectin (Goldfarb and Liotta 1986), arginines from the carboxy-tennini of laminin (Liotta et al. 1981), and throm- peptides and proteins (Skidgel 1988). On bospondin (Lawler and Slayer 1981). the basis of activity, the plasma basic Plasmin, as well as u-PA, also can acti- carboxypeptidases can be distinguished vate certain metalloproteinases, which, as having constitutive or inducible activin turn, degrade other matrix proteins ity.A single enzyme, carboxypeptidase N (Alexander and Werb 1991). It is the ca- (CpN), accounts for the constitutive acpacity to degrade extracellular matrices, tivity.CpN serves a surveillance function either directly or indirectly, that estab- in the blood, rapidly removing carboxylishes the role of the plasminogen sys- terminal lysines and arginines from peptem in facilitating cell migration. tide effecters (Skidgel 1988). CpN was In addition to the earlier identified ac- first described in the early 1960s as a tivators and inhibitors, surface interac- kinin inactivator (Erdos et al. 1963) and, tions play a central role in regulating the hence, was referred to as kininase I. function of the plasminogen system. Other known CpN substrates include the Plasminogen, whether bound to a fibrin carboxy-terminal residues of C3a, C5a, surface or a cell surface, is more readily and fibrinopeptides A and B. The kinins, activated to plasmin than free plasmino- anaphylotoxins, and fibrinopeptides exgen (Hoylaerts et al. 1982, Plow et al. ert potent and potentially deleterious ef1986), and plasmin bound to a cell or fects, and removal of their carboxy-terfibrin surface is protected from inactiva- mini by CpN is an important regulatory tion by az-antiplasmin (Wiman and Coh- step in limiting or modulating their acen 1978, Plow et al. 1986, Hall et al. tivities (Skidgel 1995). 1991). Surface-activation and surfaceCpN is a large enzyme with a molecuprotection serve to restrict plasmin deg- lar weight of approximately 280 kD. It is radation to fibrin or to the matrix in the a noncovalent tetramer composed of two catalytic (55 kD) and two glycosyimmediate vicinity of the cell surface. Recognition of surface is an intrinsic lated regulatory (83 kD) subunits. As is feature of the plasminogen molecule. characteristic of the carboxypeptidases, Resident within the heavy-chain region zinc is present in the active center of the of plasminogen are five kringles. These enzyme. Removal of the zinc by chelatdisulfide-looped structures of 80–90 ing agents inactivates CpN, whereas reamino acids exhibit lysine-binding func- placement of the zinc with cobalt results tion; they bind carboxy-terminal lysines, in a characteristic enhancement in activas well as certain internal lysyl and argi- ity. CpN has a pH optimum within the 72
neutral range. With synthetic substrates, CpN exhibits a preference for carboxyterminal lysines, particularly with a penultimate alanine, over arginines, although many of its naturally occurring peptide substrates have carboxy-terminal arginines (Skidgel 1988 and 1995, Skidgel et al. 1989). Several independent lines of investigation have led to the identification of an inducible carboxypeptidase activity in blood. CpU was originally defined as the increase in carboxypeptidase activity which arose upon the clotting of blood as serum carboxypeptidase activitywas substantiallyhigher than plasma levels (Hendriks et al. 1989). This unstable (hence CpU) activity was subsequently partially purified and shown to exhibit affinity for plasminogen (Wang et al. 1994). Earlier, Eaton et al. (1991) isolated a plasminogen-binding protein; when its sequence was deduced by cDNA cloning, the mo]ecule showed 40Y0 sequence identityto tissue carboxypeptidases. Plasma carboxypeptidase B (pCPB) was a proenzyme that could be activated by certain serine proteases including thrombin, plasmin, and trypsin. The proenzyme was approximately 60 kD, and the active enzyme was 35 kD. In subsequent studies, the substrate specificity and reactivity with plasminogen of pCPB were characterized in detail (Tanand Eaton 1995).A third route of identification arose from the isolation of a previouslyidentified plasma inhibitor of fibrinolysis(Bajzar and Nesheim 1993). The 58-kD protein, which Bajzar et al. (1995) ultimately isolated, was related to pCPB by sequence analysis and was designated as thrombin activable fibrinolysis inhibitor (TAFI). The relationship between CpU and pCPB remains to be formally proven. It seems likely that a single entity is responsible for the major inducible carboxypeptidase activity of plasma, and a recent publication supports this proposition (Vanhoof et al. 1996). In this review,we shalluse pCPB to designatethe inducible and active carboxypeptidase and pro-pCPB as its precursor form.
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Effects of the Carboxypeptidases on the Plasminogen System Regulation of Fibrinolysis
Newlyexposed carboxy-terminal lysines on partially degraded fibrin catalyze further plasminogen activation and proteolysis of the clot. Removal of these
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nine model of coronary thrombosis. In this model, electric injury to the circumflex coronary artery was used to induce a thrombus composed of both fibrin and platelets. Induction of carboxypeptidase pro-pCPB- depleted plasma activity,presumably mediated by the canine homologue of pCPB, was detected in association with the vascular injury. The level of pCPB activity was low compared with the total potential activity, but the increasewas significant.Afterthe clot was plasma allowed to stabilize, t-PA was administered under a standard therapeutic regir men. Inducible carboxypeptidase levels / remained elevated but did not increase u during t-PA treatment. Interestingly as I I I I shown in Figure 3, a significant linear re10 20 30 40 50 lationship(r= 0.6,p = 0.007) was observed o between the time required for t-PA to reTime (rein) establish blood flow and the level of carFigure 2. Role of plasmacarboxypeptidase B boxypeptidase activity Thus, the higher (pCPB) in fibrinolysis.A whole blood clot, the levelof inducible carboxypeptidaseactrace-labeledwith IZsl.fibrinogen,was SL15- tivity the longer the time for t-PA to inpended in plasma, which was or was not imduce successful reperfusion. This striking munodepleted of pro-pCPB. Clot lysis, mearesult is consistent with a role of pCPB in sured as the release of 1251from the clot was measured over time following addition of tis- suppressingfibrinolysis in vivo (Redlitz et sue-type plasminogen activator (t-PA) (50 WY al 1996). mL). Details are provided in Redlitz et al. (1995).
carboxy-terminal lysines by the carboxypeptidasesshould inhibit the accrual and activation of additional plasminogen and, thereby,inhibit the rate of fibrinolysis. In principle, this suppressive effect could be mediated by both CpN and pCPB. Nevertheless, two independent investigations using different fibrinolytic assays concluded that only pCPB suppressed fibrinolysis (Bajzar et al. 1995, Redlitz et al. 1995).A dramatic illustrationof this effect of pCPB is shown in Figure 2. In this experiment, a whole blood clot, containing tracer 1ZsI-fibrin, was suspended in plasma that was or was not immunodepleted of pro-pCPB. Upon addition of t-PA, the rate of clot lysis in the depleted plasma was markedly enhanced. Dose titration experiments have shown that activation of less than 10% of the pro-pCPB in plasma produces a maximal suppression of fibnnolysis, whereas plasma levels of the constitutivelyactive CpN were without effect (Bajzar et al. 1995, Redlitz et al. 1995). Recently, evidence for induction of carboxypeptidase activity with potential pathogenetic consequences has been obtained in vivo (Redlitz et al. 1996). These data were derived in a well-establishedca-
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Regulation of Plasminogen Binding to Cells Plasminogen binds to a wide variety of cells with high capacity via its lysinebinding sites (Plow et al. 1995). The plasFigure3. Correlationbetween the time to reperfusion following thrombolytic therapy and the levelsof inducible carboxypeptidaseactivity in a canine model of thrombosis. Data are derived from animals with electrically induced thrombosis to the circumflex artery After allowing the thrombus to stabifize, tissue-type plasminogen activator(t-PA)was administered, and the time to restore blood flow was monitored. Inducible carboxypeptidaseactivityis defined as the portion of carboxypeptidase activity detected with a syntheticsubstratethat was inhibitable by the potato carboxypeptidase inhibitor.In humans, this carboxypeptidaseactivity is mediated by plasma carboxypeptidase B (pCPB) but not by carboxypeptidase N (CpN). Detailsare provided in Redlitz et al. (1996), and the data are reprintedwith permission.
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Figure 4. Effect of the carboxypeptidases on plasminogen binding to cells. U937 monocytoid cells were incubated with plasma levels of carboxypeptidase N (CpN) or plasma carboxypeptidase B (pCPB) for 1 hr at 37”C.Afterwashing, lZ51-p1a5minogenbinding was measured.De-
tailsareprovidedin Redlitzet al.(1995).
minogen receptors are heterogeneous; but a number of these binding sites are cell-surface proteins with extracellular carboxy-teminal lysines (Miles et al. 1991). As shown in Figure 4, treatment of U937 cells, a monocytoid cell line that binds plasminogen in a representative fashion, with CpN or pCPB reduced plasminogen binding by approximately 50yo (P1ow et al. 1995). Similar effects were observed with other cell types as well. Sequential treatment of the cells with the two carboxypeptidases was as effective as treatment with each enzyme individually, indicating that the same substrates are ultimately cleaved. Importantly, although carboxypeptidase treatment reduces plasminogen binding by only 50?40,it can completely abolish the kinetic advantage of plasminogen binding to cells (Felez et al. 1996). Thus, a subpopulation of receptors accelerates plasminogen activation on cell surfaces, and this subset is susceptible to regulation by the carboxypeptidases.
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Regulation of the Plasminogen System by the Plasma Carbo~eptidases
The carboxypeptidases complete a regulatory loop for controlling the activity of the plasminogen system (Figure 5). Plasmin or other proteases enhance plasminogen binding and activation on cell or fibrin surfaces, and the carboxypeptidases “downregtdate” this increase. Both plasma carboxypeptidases dampen plasminogen binding to cells, whereas only pCPB was effective in suppressing fibrin degradation. Nevertheless, it would be
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References
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AlexanderCM, Werb Z: 1991. Extracellularmatrix degradation.In Hay ED, ed. Cell Biology of Extracellular Matrix, 2nd ed, New York, Plenum Press, pp 255. Bajzar L, Nesheim M: 1993. The effect of activated protein C in fibrinolysis in cell-free plasma can be attributedspecifically to attenuation of prothrombin activation. J Biol Chem 268:8608–8616.
Blood / Coagulation
Suppression
Figure 5. Integration of the carboxypeptidases into the proteolytic networks of the plasminogen and coagulation systems.
premature
to exclude
entirely an effect
of CpN on fibrinolysis under other assay conditions or in vivo. The capacity of both plasmin and thrombin to activate pro-pCPB broadens and complicates this proteolytic network. Recently have shown that the thrombin/thrombomodulin complex is a particularly efficient activator of pro-pCPB and does not induce an inactivating cleavage (Bajzar et al. 1996a). Thus, there is a direct tie between the carboxypeptidases and the coagulation system, specifically via the protein C pathway. Thisrelationshipappears to account for the long recognized but unexplained profibrinolytic effect of the protein Cpathway (Taylor and Lockhart 1985, Bajzar et al. 1990). Indeed, the recent study by Bajzar et al. (1996b) verifies this linkage, thereby providing an explanation for earlier observations showing that thrombomodulin is a potent inhibitor of fibrinolysis (Gruber et al. 1995) and that thrombin protects fibrin against plasmin degradation (Von Dem Borne et al. 1995). The tight association of pro-pCPB with plasminogen also will undoubtedly be of importance in regulating the delive~ and activity of this carboxypeptidase.
●
ypeptidases may suppress the activity of the plasminogen system at both cell and fibrin surfaces. Nevertheless,the carboxypeptides ultimately constitute a regulatory mechanism and may not be influential under all circumstances. For example, at high plasmin levels, the generation of lysines carboxy-terminal additional should not greatly accelerate an already robust rate of fibnnolysis; hence, a significant effect of the carboxypeptidases may not be discernible. A number of central and immediate questions regarding the interface between the carboxypeptides and the plasminogen system arise. Some obvious examplesare as follows: Are there specific inhibitors or clearance mechanisms that control the function of the carboxypeptidases? Is pro-pCPB activation in vivo controlled solelyby the thrombin–thrombomodulin complex or does plasmin or other pathways induce its function? Are the activitiesof the carboxypeptidases modulated during diseases? Is inhibitionof the carboxypeptidasesan approach to enhance the efficacy of fibrinolytic therapy?Answersto these questions await resolution in order to place the role of the plasma carboxypeptidases in regulating the plasminogen system into a biological perspective.
Concluding Remarks
Although preliminary, the data summarized in this article suggest that the carboxypeptidases represent a significant regulatory loop for control of the plasminogen system. In this capacity,the carbox-
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Acknowledgment
This work is supported by National Institutes of Health grant HL17964.
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clot lysis: in vitro modulation by activated protein C. Thromb Res 37:639-649. Vanhoof G, Wauters J, Schatteman K, et af.: 1996. The gene for human carboxypeptidase U (CPU)—aproposed novel regufatorof plasminogen activation—maps to 13q14.11. Genomics (in press). Vassalli J-D, Sappino A-P, Belin D: 1991. The plasminogen activator/plasminsystem.J Clin Invest 88:1067-1072. Von Dem Borne PA, Meijers JCM, Bouma BN: 1995. Feedback activation of factor XJ by thrombin in plasma resultsin additional formation of thrombin that protects fibrin clots from fibrinolysis. Blood 86:3035-3042. Wang W, Hendriks DF, Scharp4 SS: 1994. Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen. J Biol Chem 269:15,937-15,944. Wiman B, Cohen D: 1978. Molecular mechanism of physiological fibrinolysis. Nature TCM 272:549-550.
Regulation of Cardiac Gene Expression by GATA-4/5/6 Todd Evans Theidentificationof nuclearregulato~ proteinsprovidesgreatpromise for advancingour understandingof the transcn”ptionalcontrol of cardiacgene expression. Three new membem of the GATA family of DNA-binding transcriptionfactors were ~ecentlydiscoveredand designatedGATA-4/5/6. On the basis ofexpressionpattems, the identificationofcandidate cardiac targetgenes and the current understandingof how other GATAfactors function in the hematopoietic system, it appears that these genes are importantfor regulatingprog~amsof cardiac development and terminal differentiation.Indeed, a functional role for GATA-4/5/6 in activating the cardiac differentiationprogram was demonstrated in cell culture and embryonic systems; howeveq recent ~esultsobtained in embryonic stem (ES) cells with a targetedmutation of GATA-4raise new questions regarding specificity of action among the three genes. The future direction of researchin the field is discussed; understandingGATA-4/5/6 function and regulation is likely to provide important insight into the specification and/ordifferentiationof cardiac progenitors, development and morphogenesis of the heart, and regulation of cardiac-specificgene expression. (Trends Cardiovasc Med 1997; 7:75-83). 01997, Ei’.sevierScience Inc.
Todd Evans is at the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine , Bronx, NY 10461, USA.
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BRIEF REVIEWS The Plasma Carboxypeptidases and the Regulation of the Plasminogen System Edward F. Plow, Krishnan Allampallam, Alexander Redlitz
Central to the ~egulation of the plasminogen system, a proteolytic network that mediates degradation of fibn”n and facilitates cell migration, is the binding ofplasminogen to carboxy-terminal lysines. These residues occur either naturallyon plasmin substrates or cell su+aces orare generated as a consequence of partial plasmin degradation. The basic carboxypeptidases of plasma are capable of removing such carboxy-terminal lysines. Carboxypeptidase N, which is constitutively active, suppresses plasminogen binding to cell su~aces; plasma carboxypeptidase B, which must be proteolytically activated, not only suppresses cellular binding of plasminogen but also dampens fibrinolysis. Thus, the plasma carboxypeptidases may constitute an important regulato~ pathway for controlling the activity of the plasminogen system in physiologic, pathophysiologic, and pharmacologic circumstances. (Trends Cardiovasc Med 1997; 7:71-75). 0 1997, Elseviey Science Inc.
Proteolytic networks, such as the complement and the blood coagulation systems, have evolved to provide explosive protection of multicellular organisms against injury and infection. The plasminogen system constitutes another important proteolytic network that plays a central role in a variety of physiological responses. By virtue of its capacity to degrade fibrin, the plasminogen system
EdwardF. Plow,KrishnanAllampallam,and AlexanderRedlitz are at the The Joseph J. Jacobs Centerfor Thrombosisand Vascular Biology,Departmentof MolecularCardiology, The ClevelandClinicFoundation,Cleveland, Ohio44195,USA.AlexanderRedlitzcurrently is also at the Universityof Washington,Departmentof BiologicalStructure,Seattle,WA 98195,USA.
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maintains the patency of vasculature and surrounding tissues (Cohen 1980, Castellino et al. 1988, Vassalliet al. 1991, Takada et al. 1994, Plow et al. 1995, Carmeliet and Cohen 1995). By mediating the degradation of extracellular matrices, the plasminogen system facilitates cell migration in diverse physiological events such as ovulation (Leonardsson et al. 1995) and wound healing (Romer et al. 1996). Numerous and profound pathogenetic consequences, ranging from bleeding to thrombosis, have also been ascribed to excessive or ineffective activity of the plasminogen system. The capacity of tumor cells (Saksela and Rifkin 1988) and microorganisms (Lottenberg et al. 1994) to subvert the proteolytic activity of the plasminogen system for invasion of host tissues extends
the pathophysiological significance of this network. Additionally, the plasminogen system has become a major therapeutic target for the treatment of thrombotic diseases such as myocardial infarction and stroke. With the involvement of the plasminogen system in physiology, pathophysiology, and pharmacology, an understanding of its intricacies is essential. Multiple amplification and suppressive mechanisms are in place to maintain tight control over the proteolytic potential of the plasminogen system (Cohen 1980, Robbins 1991, Takada et al. 1994). Indeed, studies extending over several decades have identified and extensively characterized many of the molecular constituents of the plasminogen system. Nevertheless, recent studies have begun to unravel a previously unappreciated mechanism for the regulation of the plasminogen system. These reports have suggested that the plasma carboxypeptidases may play a significant role in dampening the activity of the plasminogen system, thereby influencing fibrinolysis and cell migration. This article summarizes these recent developments and attempts to place some present and future perspectives upon these findings.
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The Plasminogen System
The central components of the plasminogen system are summarized in Figure 1. Plasminogen, itself, circulates in the blood at a 2 pM concentration. This inactive zymogen is converted to an active serine protease, plasmin, by cleavage of a single peptide bond. This transition is mediated by the plasminogen activators, tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), as well as by bacterial activators such as streptokinase. Plasminogen activation is regulated by the availability of the activators and by inhibitors of these activators, primarily plasminogen activator inhibitor 1 (PAI-1). Plasmin activ-
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Plasminogen Activators
nyl residues (Sottrup-Jensen et al. 1978). Because plasmin cleaves its substrates to PA Inhibitors generate carboxy-terminal lysines, addi/ PleaminOgen_ Plasmin tional plasminogen may be recruited a~ antiplaamin and activated on the degraded substrate / surface. Thus, fibrin or cell surfaces, Fibrin —Fibrin Degradation Products Matrix Pmtains A Degradation Products which have been partially degraded by Pro-metalloprotainase _Metallopro!einase plasmin, can bind more plasminogen, Degradation Products Cell Surface Proteins _ can support more efficient plasminogen activation, and can protect more plasFigure 1. Componentsof the plasminogen min from inactivation than a virgin sursystem. face. Intuitivelythen, agents that remove carboxy-terminal lysines from fibrin or ity also can be directly inhibited, primacell surfaces should suppress plasminorily by az-antiplasmin (Castellino et al. 1988, Robbins 1991, Takada et al. 1994). gen activation and dampen fibrinolysis Plasmin is an endopeptidase, cleaving and pericellular proteolysis. The basic proteins at lysyl or arginyl bonds. Its carboxypeptidases remove carboxy-tersubstrate repertoire is broad. Clearly,fi- minal Iysines and arginines from substrates, which, in turn, suppress the acbrin is its major substrate, but fibrinotivity of the plasminogen system. gen and factor V also are cleaved by plasmin under circumstances of extensive plasminogen activation such as dur● The Plasma Carboxypeptidases ing disseminated intravascular coagulation (DIC). Other notable plasmin The basic carboxypeptidases are exosubstrates are matrix proteins, such as proteinases, which remove lysines and fibronectin (Goldfarb and Liotta 1986), arginines from the carboxy-tennini of laminin (Liotta et al. 1981), and throm- peptides and proteins (Skidgel 1988). On bospondin (Lawler and Slayer 1981). the basis of activity, the plasma basic Plasmin, as well as u-PA, also can acti- carboxypeptidases can be distinguished vate certain metalloproteinases, which, as having constitutive or inducible activin turn, degrade other matrix proteins ity.A single enzyme, carboxypeptidase N (Alexander and Werb 1991). It is the ca- (CpN), accounts for the constitutive acpacity to degrade extracellular matrices, tivity.CpN serves a surveillance function either directly or indirectly, that estab- in the blood, rapidly removing carboxylishes the role of the plasminogen sys- terminal lysines and arginines from peptem in facilitating cell migration. tide effecters (Skidgel 1988). CpN was In addition to the earlier identified ac- first described in the early 1960s as a tivators and inhibitors, surface interac- kinin inactivator (Erdos et al. 1963) and, tions play a central role in regulating the hence, was referred to as kininase I. function of the plasminogen system. Other known CpN substrates include the Plasminogen, whether bound to a fibrin carboxy-terminal residues of C3a, C5a, surface or a cell surface, is more readily and fibrinopeptides A and B. The kinins, activated to plasmin than free plasmino- anaphylotoxins, and fibrinopeptides exgen (Hoylaerts et al. 1982, Plow et al. ert potent and potentially deleterious ef1986), and plasmin bound to a cell or fects, and removal of their carboxy-terfibrin surface is protected from inactiva- mini by CpN is an important regulatory tion by az-antiplasmin (Wiman and Coh- step in limiting or modulating their acen 1978, Plow et al. 1986, Hall et al. tivities (Skidgel 1995). 1991). Surface-activation and surfaceCpN is a large enzyme with a molecuprotection serve to restrict plasmin deg- lar weight of approximately 280 kD. It is radation to fibrin or to the matrix in the a noncovalent tetramer composed of two catalytic (55 kD) and two glycosyimmediate vicinity of the cell surface. Recognition of surface is an intrinsic lated regulatory (83 kD) subunits. As is feature of the plasminogen molecule. characteristic of the carboxypeptidases, Resident within the heavy-chain region zinc is present in the active center of the of plasminogen are five kringles. These enzyme. Removal of the zinc by chelatdisulfide-looped structures of 80–90 ing agents inactivates CpN, whereas reamino acids exhibit lysine-binding func- placement of the zinc with cobalt results tion; they bind carboxy-terminal lysines, in a characteristic enhancement in activas well as certain internal lysyl and argi- ity. CpN has a pH optimum within the 72
neutral range. With synthetic substrates, CpN exhibits a preference for carboxyterminal lysines, particularly with a penultimate alanine, over arginines, although many of its naturally occurring peptide substrates have carboxy-terminal arginines (Skidgel 1988 and 1995, Skidgel et al. 1989). Several independent lines of investigation have led to the identification of an inducible carboxypeptidase activity in blood. CpU was originally defined as the increase in carboxypeptidase activity which arose upon the clotting of blood as serum carboxypeptidase activitywas substantiallyhigher than plasma levels (Hendriks et al. 1989). This unstable (hence CpU) activity was subsequently partially purified and shown to exhibit affinity for plasminogen (Wang et al. 1994). Earlier, Eaton et al. (1991) isolated a plasminogen-binding protein; when its sequence was deduced by cDNA cloning, the mo]ecule showed 40Y0 sequence identityto tissue carboxypeptidases. Plasma carboxypeptidase B (pCPB) was a proenzyme that could be activated by certain serine proteases including thrombin, plasmin, and trypsin. The proenzyme was approximately 60 kD, and the active enzyme was 35 kD. In subsequent studies, the substrate specificity and reactivity with plasminogen of pCPB were characterized in detail (Tanand Eaton 1995).A third route of identification arose from the isolation of a previouslyidentified plasma inhibitor of fibrinolysis(Bajzar and Nesheim 1993). The 58-kD protein, which Bajzar et al. (1995) ultimately isolated, was related to pCPB by sequence analysis and was designated as thrombin activable fibrinolysis inhibitor (TAFI). The relationship between CpU and pCPB remains to be formally proven. It seems likely that a single entity is responsible for the major inducible carboxypeptidase activity of plasma, and a recent publication supports this proposition (Vanhoof et al. 1996). In this review,we shalluse pCPB to designatethe inducible and active carboxypeptidase and pro-pCPB as its precursor form.
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Effects of the Carboxypeptidases on the Plasminogen System Regulation of Fibrinolysis
Newlyexposed carboxy-terminal lysines on partially degraded fibrin catalyze further plasminogen activation and proteolysis of the clot. Removal of these
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nine model of coronary thrombosis. In this model, electric injury to the circumflex coronary artery was used to induce a thrombus composed of both fibrin and platelets. Induction of carboxypeptidase pro-pCPB- depleted plasma activity,presumably mediated by the canine homologue of pCPB, was detected in association with the vascular injury. The level of pCPB activity was low compared with the total potential activity, but the increasewas significant.Afterthe clot was plasma allowed to stabilize, t-PA was administered under a standard therapeutic regir men. Inducible carboxypeptidase levels / remained elevated but did not increase u during t-PA treatment. Interestingly as I I I I shown in Figure 3, a significant linear re10 20 30 40 50 lationship(r= 0.6,p = 0.007) was observed o between the time required for t-PA to reTime (rein) establish blood flow and the level of carFigure 2. Role of plasmacarboxypeptidase B boxypeptidase activity Thus, the higher (pCPB) in fibrinolysis.A whole blood clot, the levelof inducible carboxypeptidaseactrace-labeledwith IZsl.fibrinogen,was SL15- tivity the longer the time for t-PA to inpended in plasma, which was or was not imduce successful reperfusion. This striking munodepleted of pro-pCPB. Clot lysis, mearesult is consistent with a role of pCPB in sured as the release of 1251from the clot was measured over time following addition of tis- suppressingfibrinolysis in vivo (Redlitz et sue-type plasminogen activator (t-PA) (50 WY al 1996). mL). Details are provided in Redlitz et al. (1995).
carboxy-terminal lysines by the carboxypeptidasesshould inhibit the accrual and activation of additional plasminogen and, thereby,inhibit the rate of fibrinolysis. In principle, this suppressive effect could be mediated by both CpN and pCPB. Nevertheless, two independent investigations using different fibrinolytic assays concluded that only pCPB suppressed fibrinolysis (Bajzar et al. 1995, Redlitz et al. 1995).A dramatic illustrationof this effect of pCPB is shown in Figure 2. In this experiment, a whole blood clot, containing tracer 1ZsI-fibrin, was suspended in plasma that was or was not immunodepleted of pro-pCPB. Upon addition of t-PA, the rate of clot lysis in the depleted plasma was markedly enhanced. Dose titration experiments have shown that activation of less than 10% of the pro-pCPB in plasma produces a maximal suppression of fibnnolysis, whereas plasma levels of the constitutivelyactive CpN were without effect (Bajzar et al. 1995, Redlitz et al. 1995). Recently, evidence for induction of carboxypeptidase activity with potential pathogenetic consequences has been obtained in vivo (Redlitz et al. 1996). These data were derived in a well-establishedca-
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Regulation of Plasminogen Binding to Cells Plasminogen binds to a wide variety of cells with high capacity via its lysinebinding sites (Plow et al. 1995). The plasFigure3. Correlationbetween the time to reperfusion following thrombolytic therapy and the levelsof inducible carboxypeptidaseactivity in a canine model of thrombosis. Data are derived from animals with electrically induced thrombosis to the circumflex artery After allowing the thrombus to stabifize, tissue-type plasminogen activator(t-PA)was administered, and the time to restore blood flow was monitored. Inducible carboxypeptidaseactivityis defined as the portion of carboxypeptidase activity detected with a syntheticsubstratethat was inhibitable by the potato carboxypeptidase inhibitor.In humans, this carboxypeptidaseactivity is mediated by plasma carboxypeptidase B (pCPB) but not by carboxypeptidase N (CpN). Detailsare provided in Redlitz et al. (1996), and the data are reprintedwith permission.
50 E =40 * # 30 -* ;
●
*
●
: 20 .-
r.O.W
10 -0
p.o.cG69
-
,~ 0
10
20
30
carboxypeptid3se (Wl)
40
50
Control
Ll CPN
PCPB
Figure 4. Effect of the carboxypeptidases on plasminogen binding to cells. U937 monocytoid cells were incubated with plasma levels of carboxypeptidase N (CpN) or plasma carboxypeptidase B (pCPB) for 1 hr at 37”C.Afterwashing, lZ51-p1a5minogenbinding was measured.De-
tailsareprovidedin Redlitzet al.(1995).
minogen receptors are heterogeneous; but a number of these binding sites are cell-surface proteins with extracellular carboxy-teminal lysines (Miles et al. 1991). As shown in Figure 4, treatment of U937 cells, a monocytoid cell line that binds plasminogen in a representative fashion, with CpN or pCPB reduced plasminogen binding by approximately 50yo (P1ow et al. 1995). Similar effects were observed with other cell types as well. Sequential treatment of the cells with the two carboxypeptidases was as effective as treatment with each enzyme individually, indicating that the same substrates are ultimately cleaved. Importantly, although carboxypeptidase treatment reduces plasminogen binding by only 50?40,it can completely abolish the kinetic advantage of plasminogen binding to cells (Felez et al. 1996). Thus, a subpopulation of receptors accelerates plasminogen activation on cell surfaces, and this subset is susceptible to regulation by the carboxypeptidases.
●
Regulation of the Plasminogen System by the Plasma Carbo~eptidases
The carboxypeptidases complete a regulatory loop for controlling the activity of the plasminogen system (Figure 5). Plasmin or other proteases enhance plasminogen binding and activation on cell or fibrin surfaces, and the carboxypeptidases “downregtdate” this increase. Both plasma carboxypeptidases dampen plasminogen binding to cells, whereas only pCPB was effective in suppressing fibrin degradation. Nevertheless, it would be
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References
Amplification
AlexanderCM, Werb Z: 1991. Extracellularmatrix degradation.In Hay ED, ed. Cell Biology of Extracellular Matrix, 2nd ed, New York, Plenum Press, pp 255. Bajzar L, Nesheim M: 1993. The effect of activated protein C in fibrinolysis in cell-free plasma can be attributedspecifically to attenuation of prothrombin activation. J Biol Chem 268:8608–8616.
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Suppression
Figure 5. Integration of the carboxypeptidases into the proteolytic networks of the plasminogen and coagulation systems.
premature
to exclude
entirely an effect
of CpN on fibrinolysis under other assay conditions or in vivo. The capacity of both plasmin and thrombin to activate pro-pCPB broadens and complicates this proteolytic network. Recently have shown that the thrombin/thrombomodulin complex is a particularly efficient activator of pro-pCPB and does not induce an inactivating cleavage (Bajzar et al. 1996a). Thus, there is a direct tie between the carboxypeptidases and the coagulation system, specifically via the protein C pathway. Thisrelationshipappears to account for the long recognized but unexplained profibrinolytic effect of the protein Cpathway (Taylor and Lockhart 1985, Bajzar et al. 1990). Indeed, the recent study by Bajzar et al. (1996b) verifies this linkage, thereby providing an explanation for earlier observations showing that thrombomodulin is a potent inhibitor of fibrinolysis (Gruber et al. 1995) and that thrombin protects fibrin against plasmin degradation (Von Dem Borne et al. 1995). The tight association of pro-pCPB with plasminogen also will undoubtedly be of importance in regulating the delive~ and activity of this carboxypeptidase.
●
ypeptidases may suppress the activity of the plasminogen system at both cell and fibrin surfaces. Nevertheless,the carboxypeptides ultimately constitute a regulatory mechanism and may not be influential under all circumstances. For example, at high plasmin levels, the generation of lysines carboxy-terminal additional should not greatly accelerate an already robust rate of fibnnolysis; hence, a significant effect of the carboxypeptidases may not be discernible. A number of central and immediate questions regarding the interface between the carboxypeptides and the plasminogen system arise. Some obvious examplesare as follows: Are there specific inhibitors or clearance mechanisms that control the function of the carboxypeptidases? Is pro-pCPB activation in vivo controlled solelyby the thrombin–thrombomodulin complex or does plasmin or other pathways induce its function? Are the activitiesof the carboxypeptidases modulated during diseases? Is inhibitionof the carboxypeptidasesan approach to enhance the efficacy of fibrinolytic therapy?Answersto these questions await resolution in order to place the role of the plasma carboxypeptidases in regulating the plasminogen system into a biological perspective.
Concluding Remarks
Although preliminary, the data summarized in this article suggest that the carboxypeptidases represent a significant regulatory loop for control of the plasminogen system. In this capacity,the carbox-
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Acknowledgment
This work is supported by National Institutes of Health grant HL17964.
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Regulation of Cardiac Gene Expression by GATA-4/5/6 Todd Evans Theidentificationof nuclearregulato~ proteinsprovidesgreatpromise for advancingour understandingof the transcn”ptionalcontrol of cardiacgene expression. Three new membem of the GATA family of DNA-binding transcriptionfactors were ~ecentlydiscoveredand designatedGATA-4/5/6. On the basis ofexpressionpattems, the identificationofcandidate cardiac targetgenes and the current understandingof how other GATAfactors function in the hematopoietic system, it appears that these genes are importantfor regulatingprog~amsof cardiac development and terminal differentiation.Indeed, a functional role for GATA-4/5/6 in activating the cardiac differentiationprogram was demonstrated in cell culture and embryonic systems; howeveq recent ~esultsobtained in embryonic stem (ES) cells with a targetedmutation of GATA-4raise new questions regarding specificity of action among the three genes. The future direction of researchin the field is discussed; understandingGATA-4/5/6 function and regulation is likely to provide important insight into the specification and/ordifferentiationof cardiac progenitors, development and morphogenesis of the heart, and regulation of cardiac-specificgene expression. (Trends Cardiovasc Med 1997; 7:75-83). 01997, Ei’.sevierScience Inc.
Todd Evans is at the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine , Bronx, NY 10461, USA.
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