Clin Biochem, Vol. 23, pp. 197-211, 1990 Printed in Canada. All rights reserved.

0009-9120/90 $3.00 + .00 Copyright ¢ 1990 The Canadian Society of Clinical Chemists.

Biochemical and Biological Aspects of the Plasminogen Activation System MICHAEL MAYER Department of Clinical Biochemistry, Hadassah Medical Center and Hadassah-Hebrew University Medical School, P.O. Box 12000, Jerusalem, IL-91120, Israel Plasminogen activators (PAs) are specific proteolytic enzymes which convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a potent and nonspecific protease which cleaves blood fibrin clots and several other extracellular proteins. In addition to their primary role in the initiation of fibrinolysis, PAs are implicated in a variety of basic biological processes, such as, degradation of the extracellular matrix, tumor invasiveness, tissue remodelling, and cellular differentiation. This review describes recent observations on the biochemical and biophysical characteristics of the different components of the plasminogen activation system. This complex system includes: the proenzymes of tissue type PA (tPA) and urokinase type PA (uPA); the active enzymes tPA, uPA and plasmin; the substrate plasminogen; several natural inhibitors of PA and plasmin activity; and the cellular receptors that bind the proenzymes, enzymes, and inhibitor-enzyme complexes. Through the coordinated interactions of these components, the location, timing, and extent of potent proteolytic activity is controlled. Recent findings on the structure, properties, biological functions, and regulation of the different components of the plasminogen activation cascade are reviewed. Current methods for assay of the amount and activity of the enzymes, inhibitors, and receptors are described. Observations implying specific functions of the system in health and disease, and its potential utilization for diagnosis are examined. Specifically, the potential application of PAs as laboratory markers of neoplasia, as diagnostic tools in diseases of the blood clotting system, their use for monitoring of thrombolytic therapy, and their possible relevance in certain disease states are described.

KEY WORDS: diabetes mellitus; extracellular matrix; fibrin; fibrinogen; fibrinolysis; fibrinolytic agents; neoplasia; plasmin; plasminogen; plasminogen activators; plasminogen activator, inhibitor; plasminogen activator, tissue type; plasminogen activator, urokinase type; receptors; rheumatoid arthritis; thrombolysis; urokinase.

Introduction t lasminogen activators (PAs) are specific serine Fminogen proteases that convert the inactive protein plasto plasmin. Through the generation of the nonspecific and potent protease plasmin, PAs affect numerous important biological processes, including:

Correspondence: Michael Mayer, Department of Clinical Biochemistry, Hadassah Medical Center and Hadassah-Hebrew Medical School, P.O. Box 12000, Jerusalem, IL-91120, Israel. Manuscript received August 23, 1989; revised November 3, 1989; accepted November 9, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, J U N E 1990

fibrinolysis and thrombolysis, cell migration, invasiveness, and metastasis. This review describes the plasminogen activation system and its relevance in h u m a n health and disease.

Components of the plasminogen activation system Figure 1 depicts the major components of the system. The PAs are produced and secreted from cells in the form of proenzymes, which are transformed to the active enzyme by a specific proteolytic cleavage. Two types of active PAs are known: the tissue type (tPA) and the urokinase-like (uPA) enzymes. Both catalyze the specific reaction, converting plasminogen to plasmin by cleavage of a single peptide bond. Both enzymes also form inactive complexes with specific inhibitors; these complexes m a y dissociate to regenerate the enzymatic activity. However, tPA differs from uPA in molecular weight, immunological specificity, and kinetic properties; the enzymes are also encoded by different genes. The details of the plasminogen activation process are reviewed in the following sections. UPA AND ITS PROENZYME

uPA has its major function in tissue-related proteolysis, and it is important in processes that entail dissolution of extracellular matrix and transversion of basement membranes (1-3). It is produced by cells and is present in the extracellular fluid in the form of an inactive single chain proenzyme (pro-uPA) (4). Conversion of pro-uPA to active two-chain uPA by catalytic amounts of plasmin is a crucial regulatory step in plasminogen activation. This conversion provides active uPA and enables an autocatalytic acceleration of uPA formation. By this mechanism, initial traces of plasmin catalyze the production of active PA, which leads to the formation of more plasmin (1-3). Active uPA is a 54 kDa protein molecule composed of two polypeptide chains of 24 kDa and 30 kDa, linked by one disulfide bond. The 24 kDa chain contains a sequence homologous to epidermal growth 197

MAYER PRO-PLASMINOGEN ACTIVATORS

PLASMINOGEN ACTIVATORS uPA

AND t P A

ACTIV[ I

~

OR(S)

ENZYME-INHIBITOR COMPt EXES

I INACTIVE} ,¥ PLASMINOGEN ACTIVATORS

( ACTIVEI

PLASMINOGEN

~) P L A S M I N

:~

=~-~-ANTIPLASMIN

FIBPlN CLOT LYSIS EXTRACELLI.ILAPPROTEOLYCJI$

Figure 1--The plasminogen activation cascade. Schematic presentation of the major steps of the cascade that lead to production ofproteolytic and/or fibrinolytic activity through activation of plasminogen. The broken line indicates conversion of the inhibited complex to active enzyme (185). Some of the reactions take place with the reactants bound to cellular receptors, to macromolecules of the extracellular matrix, or to circulating fibrin. The topography of the reactions is not shown in this scheme.

factor (EGF) and similar to a sequence present in the A chain of tPA (5). Limited proteolysis with trypsin or plasmin in vitro converts the 54 kDa high molecular weight uPA to a smaller molecular weight enzyme (31 kDa) with some loss of activity (6). uPA can be distinguished from tPA by immunological and electrophoretic methods, and by its sensitivity to the protease inhibitor aprotinin. Aprotinin inhibits both the high and low molecular weight forms of uPA in a competitive manner, but it does not inhibit tPA (7). T P A A N D I T S PROENZYME

tPA is a 70 kDa glycoprotein which is primarily active in fibrinolysis and thrombolysis (8). Its concentration in h u m a n plasma is 0.1 nmol/L. The enzyme is synthesized as a single chain molecule; its catalytic activity increases as a result of plasminmediated conversion to a two chain molecule (9). One chain is a '"heavy" 38 kDa polypeptide bearing sites responsible for enzyme regulation; the "light" 198

31 kDa chain contains the active site, similar to that of other serine proteases (10). An important feature of tPA is that its catalytic activity is markedly stimulated when the enzyme binds to certain compounds, such as, fibrin, fibrin(ogen) fragments, polylysine, heparin, or denatured proteins (5,11-14). Binding of these ligands depends on two "kringle" structures and a "finger" domain in the heavy chain (5,15,16). The activation by fibrin involves a sequential reaction of fibrin and plasminogen binding to the enzyme, which leads to formation of a cyclic ternary complex that promotes efficient clot lysis (11). The heavy chain of tPA also contains an EGF-like domain responsible for binding the enzyme to cell surface receptors (15). The domains responsible for interaction of tPA with its substrate plasminogen, with activating ligands such as fibrin, with its specific inhibitors, and with its specific receptor have been defined through the use of deletion m u t a n t s (10,17). PLASMINOGEN

Plasminogen, the physiological substrate for PAs, is a 90 kDa, single chain glycoprotein containing five homologous "kringle" structures. Plasminogen is produced in the liver (18) and in the seminiferous tubules of the testis (19); its normal plasma concentration is 2.2 ~xmol/L. The native form of plasminogen has an N-terminal glutamic acid which can easily be converted to a form with N-terminal lysine through digestion by plasmin (1,5). Plasminogen contains "lysine binding sites" (LBS) which interact specifically with certain amino acids and amino acid analogues (20). Binding of fibrin to plasminogen occurs at these sites and produces marked stimulation of the activation by either tPA or uPA (5,20,21). Plasminogen can also bind to the extracellular matrix in a specific, saturable, and reversible manner (22). Matrix bound plasminogen is a good substrate for activation by tPA or uPA. The plasmin, which is formed on, and consequently bound to, the matrix, is protected from its specific and abundant inhibitor ~2-antiplasmin (22). Therefore, binding seems to have a physiological function in the regulation of plasmin formation and activity. The plasminogen independent cleavage of certain proteins by uPA and tPA suggests that PAs m a y utilize substrates other than plasminogen. The alternative substrates include a 66 kDa matrix protein, fibronectin, and the specific inhibitor PAI-1 (23-25). Supporting a plasminogen independent function for P A s is the finding that phorbol esters induce PA activity and differentiation in HL-60 h u m a n promyelocytic leukemia cells in a system that does not require plasminogen (26). This observation suggests that other protein(s) m a y serve as natural substrate(s) for the induced enzyme. However, the physiological importance of these observations remains to be demonstrated. CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

PLASMINOGEN ACTIVATION SYSTEM

PLASMIN

Plasmin is a trypsin-like endopeptidase that hydrolyzes fibrin and other plasma proteins, as well as many extracellular matrix proteins at susceptible arginine and lysine bonds. It is a two-chain molecule, formed from single chain Glu- or Lys-plasminogen by PAs that cleave a specific Argseo-Valse 1 bond (27). The light and heavy chains of plasmin are linked by two disulfide bonds: the light chain contains the catalytic site which is similar to that of other serine proteases; the heavy chain contains a regulatory domain (20). Plasmin activity is subject to inhibition by binding to its specific inhibitor ~2-antiplasmin (28). Plasmin, like its parent molecule plasminogen, contains LBS that interact with lysine, 6-aminohexanoic acid, tranexamic acid, fibrinogen, fibrin, and related compounds (5). The LBS are located in the protein structure in an area that is distinct and distant from the catalytic site (5,29). Nonetheless, binding of ligands to LBS produces modulatory responses that affect the catalytic activity of the enzyme (30,31). Binding of ligands, such as the amino acid analogue 6-aminohexanoic acid and fibrin to the LBS, also protects plasmin against inhibition by a2-antiplasmin (32,33). This property is responsible for the relative resistance of plasmin, formed on the fibrin clot surface, to inhibition by ~2-antiplasmin. In contrast, plasmin generated in the circulating blood is rapidly neutralized by the inhibitor (8). RECEPTORS Binding of uPA to a specific receptor on the cell surface appears to be important for localization of uPA-catalyzed plasminogen activation (34,35). Binding is rapid, saturable, and with a high affinity of ~ 1 0 - lO M. The bound enzyme is not internalized or rapidly degraded (35,36). Binding of uPA to the receptor does not involve the catalytic site of uPA; it occurs at the EGF domain of uPA and pro-uPA (35). Therefore, bound uPA retains its enzymatic activity (35,36). uPA receptors have been found in monocytes and in several cultured h u m a n cell lines such as, fibroblasts, breast carcinoma, and umbilical vein endothelial cells (34-37). Affinity of binding and concentration of binding sites varies in different cells, subject to regulation by modulators such as tumor promoters, EGF, and dimethylformamide (3538). The receptor has been recently purified and shown to be a single chain, 55-60 kDa, heavily glycosylated protein (37). Single chain pro-uPA binding to the cellular receptors is followed by conversion of the bound enzyme to active, two chain uPA on the surface of the cells (39). Receptor bound uPA can still bind PAI-1 and maintains its susceptibility to inhibition by this inhibitor (40). The surface receptor also binds PA-inhibitor complexes such as uPA-PAI-1 (40).

CLINICALBIOCHEMISTRY,VOLUME23, JUNE 1990

In addition to having receptor sites that bind the enzyme, various cells are also equipped with receptors on the cell surface which bind the substrate, plasminogen (41,42). Binding ofplasminogen is characterized by its low affinity, and seems, at least in part, to involve sites which also bind plasmin (43). Cell bound plasminogen undergoes activation to plasmin by receptor bound uPA, while catalytic amounts of plasmin at these sites serve to produce active uPA from bound pro-uPA. This mechanism is responsible for an autocatalytic process that is subject to regulation by specific inhibitors such as PAL1 and PAI-2 (see below). Cell bound plasmin is refractory to inhibition by its specific inhibiter ~2-antiplasmin (44). Thus, plasminogen activation on receptor sites results in the cells ability to promote pericellular proteolysis. The newly produced proteolytic activity is localized because plasmin generation is limited to the close environment of the cell surface (3,35). Receptors for tPA also exist, and binding of tPA to its specific cellular receptors does not cause loss of the enzymatic activity (45). Simultaneous binding of pro-uPA, uPA, plasminogen and plasmin at the cell surface produces a topographical proximity which is important for the biological function of the plasminogen activation process (3,42,43). The proximity enables efficient autocatalytic activation of plasminogen by uPA, and serves to localize the proteolytic activity that emerges via plasmin formation (1,3,35). Thereby, the cells are armed with a matrix-degrading activity, which is an essential tool for cell migration into solid tissues. Because the receptor bound uPA is distinctly localized at cell-cell and cell-substratum contacts (46,47), the receptors control the position of plasmin activity at focal contact sites. Cell invasiveness and cell migration depend on this focal localization of the components (35). INHIBITORS Emergence of premature and uncontrolled activity of PAs, and activity at inappropriate site(s), is potentially damaging. Time- and site-specific inhibition of the catalytic activity of PAs is, therefore, instrumental in the prevention of aberrant plasminogen activation. Similarly, activity of the newly generated plasmin has to be strictly regulated to prevent uncontrolled proteolysis. It is, therefore, not surprising that potent and specific inhibitors of PAs and plasmin are present in cells, in the circulation, and in other extracellular compartments. The major function of these inhibitors is to regulate the catalytic activities since the balance between enzymes and inhibitors determines net activity. In the circulation, PA inhibitors block premature fibrinolysis (48-50). Plasma levels of the plasminogen activator inhibitors type 1 and type 2 (PAL1 and PAI-2, see below) are elevated in conditions associated with enhanced tendency for thrombus formation. Since

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plasminogen activation is also important in tissue remodelling, neoplasia and invasiveness (see below), the inhibitors may also be implicated in cancer. Several recent studies have demonstrated the ability of natural and synthetic inhibitors of PAs to suppress tumor cell invasion in in vitro experimental models of metastasis (51-53). Various factors might complicate the interaction between inhibitors and the enzymes; therefore, a direct correlation with enzyme/inhibitor ratios and activities is not always observed. For example, binding of tPA to fibrin reduces the ability of PAL1 and PAI-2 to react with the enzyme. In addition, various compounds that affect the PA system do so by independent effects on the enzymes and inhibitors; therefore the final result, in terms of net activity, is not easily predictable (54). In the following section a brief review on specific and potent inhibitors of PAs and plasmin is presented. For more information, refer to recent reviews (48,49). PAI-1

PAI-1 is a 50 kDa single-chain plasma glycoprotein that rapidly and specifically forms equimolar, inactive complexes with one- or two-chain tPA, two-chain uPA, and the anticoagulant protein C, but not with single chain uPA or with plasmin (55,56). PAI-1 has a high second order rate constant for tPA or uPA, and Kd values of 8 × 10-13 M for tPA and 2 × 10- is M for uPA. Therefore, PAI-1 appears to be the physiological inhibitor of PAs. PAL1 has significant homology with members of the serine protease inhibitor (Serpin) family (57). Serpins share structural features, a common functional principal, and are important regulators of the processes of coagulation, inflammation, and complement activation (58). The 1:1 complex formed between PAI-1 and tPA or uPA resists dissociation by denaturing agents such as urea or sodium dodecyl sulphate; however, these agents regenerate activity of the bound enzyme (59). Although PAI-1 is synthesized in an active form, it is rapidly converted to an inactive (latent) variant (60). This inactivation can be partially reversed by sodium dodecyl sulphate, guanidine, and urea (59,60). The physiological relevance of the active and latent forms of the inhibitor remain to be determined. PAI-1 is present in platelets, placenta, serum, and is produced by endothelial cells, monocytes/macrophages, and various normal and neoplastic cells in culture (61,62). Vascular and liver endothelial cells and platelets seem to be the major sources of the inhibitor in the body (50,59,63). The gene for PAI-1 is located on chromosome No. 7 (64). Production of the inhibitor is modulated by a variety of hormones and growth factors (see Table 2). The elevations in PAI-1 protein level as a result of exposure to cytokines, growth factors, and hormones are often preceded by elevations in PAI-1 mRNA level; this

200

indicates transcriptional regulation (54,65,66). PAI-1 activity (and antigen) level in the blood shows an extremely large range. Activities between 0.0-1.3 nmol/L have been found in the plasma of healthy subjects (50,67,68). Activity of PAI-1 in plasma correlates with plasma insulin levels (69) and increases during pregnancy (70). PAI-1 plays an important role in the development of arterial and venous thromboembolism, and it also acts as an acute phase reactant (see below). In plasma, PAI-1 is bound to a protein that was recently identified as vitronectin (71). It also binds to vitronectin in the extracellular cell matrix (72), and is deposited by cultured endothelial cells into the substratum matrix. A large part of PAI-1 in the substratum is associated with the adhesive proteins vitronectin and fibronectin (72,73). PAI-2

PAI-2 is a 46,000 kDa serine protease inhibitor with a higher affinity for uPA than for tPA. The cDNA encoding for PAI-2 in human monocytic cell line U937 has been characterized (74). The gene for PAI-2 is located on chromosome 18q21-23, has two common alleles, and is highly homologous to chicken ovalbumin gene (75). PAI-2 is hardly detectable in nonpregnant plasma, while levels of up to 100 ~g/L (2 ~mol/L) are observed in the third trimester of pregnancy (50,76). Because PAI-2 is found mainly in placental tissue, this suggests a function for the inhibitor in regulation of hemostasis during pregnancy and parturition (77). High levels of the inhibitor may produce microthrombi in the placenta that lead to impaired fetal blood supply and consequently, to retarded intrauterine growth (77). PAI-2 is induced by tumor necrosis factor (TNF), phorbol esters and endotoxin; this suggests its involvement in local inflammatory processes (78,79). Based upon kinetic considerations, PAI-2 is probably more relevant as an inhibitor of uPA than of tPA. It is a serpin that does not inhibit pro-uPA or plasmin. In many cell types, PAI-2 is located intracellulary. The latter observation raises a question concerning the physiological function of PAI-2, since its natural substrates uPA and tPA are mainly extracellular components. Protease nexin

Produced by fibroblasts, heart muscle cells, and kidney epithelial cells, protease nexin is a 51 kDa glycoprotein that inhibits serine proteases such as trypsin, thrombin, plasmin, uPA, single and twochain tPA, but not pro-uPA (80,81). The absence of protease nexin in circulating blood and its relatively unfavorable affinities toward PAs suggest that it does not fulfill a major function in controlling plasminogen activation. The main function of protease nexin appears to be facilitation of endocytosis of proteases. This assumption is based on observations

CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

PLASMINOGEN ACTIVATION SYSTEM

that protease nexin forms covalent complexes with several proteases including uPA and mediates their uptake by cells (48,49).

c~2-antiplasmin cx2-antiplasmin is a 67 kDa single-chain glycoprotein with a plasma concentration of 1 ~Lmol/L. It is an extremely active inhibitor, with a high second order rate constant. By binding to the light chain of plasmin, a 1:1 stoichiometric inactive complex is formed. Ligands such as fibrinogen, lysine, and 6-aminohexanoic acid, prevent interaction between the inhibitor and the enzyme (5,8,32). The inhibitor is synthesized and secreted by liver cells (82); it is the most abundant and active natural inhibitor of plasmin activity. The other plasma proteinase inhibitors do not seem to play a role in the inactivation of blood plasmin (20).

Assays of the components ENZYMES

Plasminogen activation can be measured either by direct assays which follow the action of PAs upon specific substrates, or by coupled, indirect methods. A widely used direct method measures the amidolytic activity of PAs by following the cleavage of chromogenic substrates, such as pyro-glu-gly-argp-nitroanilide, to produce the chromophore p-nitroanilide (83). Another direct assay measures the ability of PAs to convert the single chain of plasminogen to the two peptide chains of plasmin. By reduction of a single disulfide bond, the heavy and light chains can be separated on SDS-PAGE. When the plasminogen is labeled with ~25I, its conversion to plasmin can be quantitated (84). A common disadvantage of the direct assays is that nonspecific proteases might also contribute to the measured activity. Indirect assays of PAs involve a dual-stage reaction. Plasminogen is first converted to plasmin by the assayed enzyme, and the plasmin activity formed is subsequently determined. The advantage of the indirect assays is that only plasminogen-dependent activity is measured, and therefore only net plasminogen activation is determined. A widely used method is the fibrin plate assay that follows generation of plasmin-mediated fibrinolytic activity (85). In this method, PA activity solubilizes fibrin through production of plasmin. Area of lysis on layers of fibrin/ agar plates is measured as an estimate of plasmin activity in a system containing plasminogen. The fibrin plate assay is highly specific and easily quantitated. It utilizes the physiological substrate fibrin and thereby mimicks the fibrinolytic process that occurs in the body (85). When 125I-labeled fibrin is used, fibrinolysis can also be measured quantitatively, with an even higher sensitivity. The disad-

CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

vantage of the fibrinolytic methods is that they are time consuming; the fibrin frequently contains impurities such as proteases and thrombin which contribute to hydrolysis; also, the formed plasmin tends to absorb to fibrin, causing an underestimation of the plasmin level. Insolubility of the fibrin substrate is an additional complication because it results in nonlinear kinetics. Alternative coupled assays follow plasmin formation by its ability to degrade other proteins, such as casein (86) or globin (14). The latter method was useful for studies in different experimental systems (14,26,87-89). Numerous chromogenic substrates are available for the determination of PAs by the plasmin-mediated reaction. Substrate cleavage is followed in a parabolic rate assay (90), or in an end point assay with or without fibrin as a stimulator (91,92). Some of these methods enable assays in plasma without prior precipitation of the euglobulin fraction, while others use the euglobulin fraction (93). Sensitive immunological methods have been devised which utilize specific antibodies for the detection oftPA or uPA antigens, and ELISA methods for use in plasma and other biological fluids (94-96). These reactions provide unmatched specificity and sensitivity. RECEPTOR

The affinity and number of receptors are usually determined by using labeled ligands such as 125Iurokinase, or labeled fragments of the ligands containing the binding site sequence, such a s 125Iamino terminal fragment of urokinase (34,35,37,40). INHIBITORS

In view of the growing interest in the higher risk for thrombosis in individuals displaying increased PAL1 in plasma (see below), reliable analytical methods to measure plasma inhibitors are desired. The inhibitors are often assayed by monitoring their ability to reduce the enzyme activity, measured by the conventional assays of uPA or tPA as described above. For that purpose, standard PA assays are performed in the absence or presence of the inhibitor(s). From the residual activity remaining in the presence of the inhibitor, inhibitory activity can be deduced (92). A simple standard assay for plasma PAI-1 activity follows the inhibition of one chain tPA by serial dilutions of added plasma (97). This assay provides good analytical recovery, has a low detection limit, and is precise. A unique "reversed zymography" system traces PA-inhibitory activity on acrylamide gel electrophoresis. In this assay, lysis of fibrin/agar films is prevented and a lysis-resistant zone appears in regions of the gel where the inhibitor is present. The tested proteins are electrophoresed in polyacrylamide gels containing sodium dodecyl sulphate. The

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gels are subsequently covered with a fibrin overlay containing plasminogen and uPA. The inhibitor(s) appear as zone(s) which inhibit the PA-induced lysis of the fibrin overlay. This technique also provides an estimation of the apparent molecular weight of the inhibitory proteins (98). The inhibitory proteins can also be determined using specific monoclonal and polyclonal antibodies (62,99).

Biological functions FIBRINOLYSIS

The major physiological function of plasminogen activators in the circulation is to provide plasmin that will dissolve blood clots. The ability of PAs to produce plasmin is also the basis for their increasing application in medicine. The use of PAs to treat patients with acute myocardial infarction is based on clinical evidence that occlusive coronary thrombus precipitates most infarctions. Recanalization of the occluded blood vessels can often be achieved by the early administration of the fibrinolytic enzymes. Large clinical trials show improved survival rates after myocardial infarction in patients receiving different types of thrombolytic enzymes, including tPA or uPA (100-104). Thrombolytic therapy with different PAs is also successfully applied in the treatment of deep vein thrombosis, pulmonary embolism, peripheral arterial occlusion and several additional conditions associated with thrombosis (103). The fibrin affinity of the thrombolytic enzyme tPA is an important pharmacological attribute since binding of tPA to plasminogen in the presence of fibrin not only contributes to localization of the catalytic activity to the clot but also increases catalytic efficiency. Thus, fibrin selectivity is important for effective fibrin clot localized thrombolysis without marked activation of systemic plasminogen and without induction of a general hemostatic defect. Due to its fibrin selectivity, tPA appears to have an apparent advantage over uPA or streptokinase in clinical use with cases of localized thrombotic occlusions. It was recently suggested that in physiological fibrinolysis, tPA and pro-urokinase may act in a sequential, complementary, and synergistic manner to maintain the fibrin specifity of plasminogen activation. Such synergistic fibrinolytic effects can potentially be applied as thrombolytic combination therapy with these agents (105). A detailed discussion of the function of plasminogen activation in fibrinolysis, and its importance in clinical and therapeutic aspects of thrombolysis is beyond the scope of this review. This subject has been extensively reviewed (5,8,20,33,103-107). DEGRADATION OF THE EXTRACELLULARMATRIX

uPA plays a major role in tissue destruction and cell migration because it promotes extracellular 202

matrix degradation (1-3,108). This effect is catalyzed by a local, uPA-mediated production of plasmin and probably by additional proteolytic enzymes that are activated by plasmin and/or PAs. Emergence of matrix degrading activity was studied in a variety of tissue and cellular processes, and the invasion of the matrix by neoplastic cells was shown to involve protease induced degradation of the cell substratum (108-110). In a model system of invasion, a requirement for a proteolytic cascade has been observed (53,109). Experimental support for the role of PAs in matrix destruction as a functional stage in invasion also comes from other observations: transformation by oncogenic viruses induces uPA production; uPA activity is involved in tissue involution in many malignant and nonmalignant conditions; and PAs are characteristically localized in areas of tumor invasion (1-3,110). Further support for this is that anticatalytic antibodies against uPA reduce invasiveness and incidence of metastasis (51-53,111), and inhibit the growth pattern and morphological changes that characterize virus induced transformation (112). The finding that plasmin converts procollagenase to active collagenase indicates that in addition to being a direct mediator of matrix cleavage, uPA also recruits additional proteolytic enzymes that can cleave specific matrix proteins (51-53,108,113). Similarly, PA and heparanase were shown to participate in the sequential degradation of the extracellular matrix (114). The distinct pericellular localization of the different components of the plasminogen activation system also supports the role of PAs in cell migration, uPA is found at focal cell-cell and cell-substratum contact sites (46,47,72); whereas, PAI-1 is distributed homogeneously in the matrix under the cells (46), and is present in the growth substratum even before it appears in the culture medium of cells (115). Since cell migration depends on the sequential formation and dissolution of cell-substratum contacts, the specific localization of PAs and plasmin assists in the control of the migration process. The plasmin formed is expected to promote the hydrolysis of bonds between matrix proteins and cells, and to mediate cleavage of adhesive contact proteins. The matrix proteins which appear to be involved in these processes are vitronectin, fibronectin and laminin. In this context, it is interesting that PAI-1 colocalizes with vitronectin in the matrix (46), and that PAI-1 binds to vitronectin (72). Thus, focalized distribution of PAs and their inhibitors in the extracellular space and their specific interaction with matrix components confines and localizes extracellular proteolysis and is .most probably involved in cell movements (3). Net plasminogen activation is a result of an intricate balance between receptors, proenzymes, enzymes, and inhibiters. Although each of these components can be independently and differentially regulated in time and space, together they can direct the activity to specific site(s) and to specific timing, according to requirement. Table 1 lists some important biological CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

PLASMINOGEN ACTIVATION SYSTEM

TABLE 1

Studies Implicating PAs in Biological Processes Other Than Fibrinolysis Organ/Process

Suggested Function

Mammary gland Implantation

Postlactation involution (152) Trophoblast invasion following implantation of fertilized ovum (153) Differentiation, wound re-epithelialization, tissue destruction in psoriasis (154) Gestation (155) Ovulation, oocytematuration (156,157,158) Spermatogenesis (159,160) Angiogenesis (161) Tumor invasion and metastasis (1,2,53,108) Migration, inflammation (1,61,162) Involution post castration (163) Prevention of adhesion (88)

Keratinocytes Amniotic fluid

Ovaries Testes Capillaries Neoplasia Macrophages Prostate Peritoneum

systems which involve plasminogen activation. For more information on these observations see refs. 1-3,110 and the specific citations in Table 1.

Regulation Plasminogen activation is under stringent metabolic control; several sites and mechanisms of regulation have been observed. Among the regulating factors are the specific, high affinity, and fast acting inhibitors PAI-1 and PAI-2. These inhibitors are by themselves subject to regulation at the levels of gene expression, activity, and secretion (33,48,49). Binding to receptors on the cell surface is an additional means of regulation, limiting the activity to specific sites and modifying the mutual interactions of the components and their response to other modulators. Another regulatory mechanism is related to the existence of latent enzyme forms, which are either inactive proenzymes or enzyme-inhibitor complexes. For example, the suppressive effect of dexamethasone on PA activity in cultured cardiac myocytes involves formation of an inactive complex and/or production of an inactive proenzyme (87). Inactive enzyme forms can, under certain conditions, such as exposure to denaturants or proteolytic cleavage, be converted to their active counterparts (1,2,87,88, 106,107,116). The hormones, cytokines, and growth factors listed in Table 2 regulate plasminogen activation by modulating the level of synthesis and/or activity of the components of the system. In addition to regulation by gene expression and through covalent or noncovalent binding to inhibitors and activators, various ligands affect PAs and plasmin activity by inducing conformational changes in the enzymes and/or substrates (20,21,29-32, 106,107,117,118). These conformational changes markedly affect the catalytic activity of the activators and ofplasmin. For example, tPA contains LBS, a flbrin(ogen) binding site and a site that binds with LBS (118). Binding of ligands to these sites allosterically affects the catalytic domain of the enzyme (117,118). CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

Likewise, plasmin undergoes regulation at a site different from the catalytic site by interacting with ~-lactam antibiotics (119). The kinetics of activation of single chain uPA by plasmin, similarly indicate negative cooperativity (120). Susceptibility of plasmin to ~2-antiplasmin is also conformationally controlled by the availability of LBS on the enzyme (32,121). These conformational changes therefore render the enzyme subject to regulatory modulation.

Relevance in clinical biochemistry M A R K E R OF NEOPLASIA

Numerous studies examined the occurence of PAs in neoplasia, their relation to metastasis formation, transformation, and their localization in particular tumors and cell types (1,2,108). Due to the potential role ofproteolytic degradation in neoplasia, researchers sought clear correlations between cell malignancy or spreading, and cell PA activity. Indeed, in certain tumor cell lines, PA activity correlates with the metastatic potential, and PA appears to be an index of the ability of the tumor cells to colonize target organs (122,123). However, these associations remain to be confirmed. A problem in establishing a causal relationship between neoplasia and PA activity is that normal growth processes also involve plasminogen activation (2). Furthermore, conflicting results were obtained in studies on potential correlations between PA activity of tumor cell lines in vitro and their metastatic capacity in vivo (2). Production of PA is probably neither necessary nor sufficient for tumor formation, although plasminogen activation is somehow involved in invasion and metastasis. Recent work supports the involvement of uPA in cancer by demonstrating that antibodies, which inhibit the catalytic activity ofuPA, abort the invasiveness of tumor cells and prevent metastasis (51,53). According to Dano et al. (2), it appears that consistent, but still inconclusive, evidence supports the assumption that uPA production in cancer cells 203

MAYER TABLE 2 Regulation of the PA System by Hormones, Growth Factors, and Cytokines

Component

uPA

Regulator 3',5'-Cyclic adenosine monophosphate (cAMP) (164)

Retinoic acid (165) Phorbol ester (164) Glucocorticoids (148,166,167) Estrogens (168) Tumour necrosis facter-~ (TNF-cx), interleukin-l, lymphotexin (169) tPA

Glucocorticoids (170) Follicular stimulating hormone (FSH), gonadotropinreleasing hormone (GnRH) (157) Phorbol ester (171) cAMP (170)

Receptor

Phorbol ester (34,36,37) Formamide (38) Epidermal growth factor (EGF) (34,36)

PAI-1

Phorbol ester (54,62) Glucocorticoids (62,65,172,173) TNF-~ (174) Thrombin (175) Transforming growth facter-~ (TGF-~) (176) Endotoxin (177,178) Interleukin (179,180,181) Luteinizing hormone (LH) and FSH (157) EGF (182)

PAI-2

TNF, phorbol esters, endotoxin (183) Endotoxin (78,79) Phorbol ester (184) Glucocorticoids (65)

mediates proteolysis of extracellular matrix. However, use of PAs as a clinical marker for neoplasia, invasive growth or metastasis is presently inappropriate. Still, under specific circumstances, the PA system might serve a diagnostic function in malignant diseases. For example, the production of tPA and uPA by leukemic cells can be used to predict prognosis and response to chemotherapy in patients with acute myeloid leukemia (124). Patients whose cells produce only tPA have a lower survival and fail to respond to chemotherapy; uPA producers have a longer survival and a better response to treatment. The production of tPA seems to characterize a more primitive cell, while development of a more specialized, differentiated cell is associated with uPA production. On the basis of this finding, the type of PA released by peripheral blood cells may indicate the leukemic state (125). DIAGNOSIS OF DEFECTS IN CLOT LYSIS

Since excessive fibrinolysis causes bleeding while reduced fibrinolysis causes thrombosis, it is not 204

surprising that maintenance of a proper balance between components of the PA system is crucial for normal clot formation and dissolution. Defciencies in plasma plasminogen, low release of tPA from endothelial cells, abnormal fibrinogen in plasma, increased circulatory levels of PAI-1 and PAI-2, and deficiency of circulating a2-antiplasmin are some of the biochemical abberations of fibrinolysis that have been described. In general, thrombotic patients often exhibit low fibrinolytic activity due to deficient tPA or to elevated PA-inhibitor (126-129). Some defects in clot lysis are directly, and most probably specifically, related to the changes in PA inhibitor activity. For example, in deep vein thrombosis, higher than normal levels of PA inhibitor are found (130-137). An elevated blood PAI-1 level is clearly an independent risk factor for impaired fibrinolytic function in deep vein thrombosis, disseminated intravascular coagulopathy and myocardial infarction (67,130,133,137). By contrast, PAI-1 level is generally decreased in bleeding diatheses. PAI-1 as a risk factor in myocardial infarction is supported by observations that myocardial infarction is associated CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

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ACTIVATION SYSTEM

with increased PA-inhibitory activity and decreased levels of tPA (67,132,133,137), and by high PAI-1 levels found in young survivors of myocardial infarction (138). These observations clearly suggest that the balance between circulatory tPA and PAI-1 may regulate vascular thrombolysis, and that high levels of plasma inhibitors correlate with higher probability of thromboembolism. It is anticipated that the PA-inhibitor level in plasma will be used as a useful marker to evaluate the risk of developing deep vein thrombosis (135). While the relation between PAI-1 and thrombotic diseases is likely to reflect an effect of the inhibitor on clot lysis, other studies suggest that PA-inhibitory activity is nonspecifically increased in a number of disease states. These studies imply that a raised level of the inhibitor is not associated with any specific disease, and may reflect a general response to acute illness (49). This is supported by the increased PAI-1 antigen and PAI-1 activity found in patients with malignant tumors, hepatic insufficiency, after extracorporeal circulation in cardiac bypass surgery, after major surgery, and in severe trauma and septicemia (50,67, 131-133). Laboratory assays of components of the plasminogen activation system in plasma should elucidate the molecular basis of fibrinolytic defects and assist in detecting patients at risk of thromboembolism. It is hoped that further research will indicate therapeutic means to minimize undesired changes in fibrinolytic activity that mediate thrombosis. The potential application of orally-administered stanazolol for enhancement of fibrinolytic activity by its stimulatory effect on endogenous tPA activity is a step toward this goal (139). MONITORING THERAPY WITH THROMBOLYTIC AGENTS

Numerous studies have led to current interest in the clinical use of plasminogen activating agents in thromboembolic conditions, pulmonary embolism, deep vein thrombosis and myocardial infarction (5,103,104). At least five types of active fibrinolytic enzymes are currently being evaluated or used for thrombolytic treatment of acute myocardial infarction and pulmonary embolism. These include: streptokinase-plasminogen, uPA, tPA, single chain uPA, and acylated plasminogen-streptokinase complex. The relative advantages and disadvantages in the therapeutic use of each of these agents are under study (5,140). The increased use of any of these agents for clinical therapy will involve closely monitoring the circulatory level of the components of the plasminogen activation system. Such monitoring is required, since excessive thrombolytic activity is likely to produce the untoward side effects of bleeding, and particularly, cerebral hemorrhage. Routine laboratory tests for clinical follow up have yet to be defined and properly established. Monitoring of the coagulation status and extent of flbrinolysis as routine laboratory procedures have been recommended CLINICAL BIOCHEMISTRY, VOLUME 23, JUNE 1990

during therapy with thrombolytic agents (103,141). Direct determination of plasminogen activation activity in blood samples of patients undergoing fibrinolytic therapy will be an important part of patient monitoring in the future. DIABETES MELLITUS

Vascular complications and disorders of hemostasis represent the most important causes of morbidity and mortality in diabetic patients (142-144). Increased plasma insulin predisposes to atherosclerosis (143). Reduced blood fibrinolytic activity has been identified as a factor that contributes to atherosclerosis (138); variation in plasma insulin concentration, even in the physiological range, modulates the fibrinolytic system at the PA inhibitor level (69). Therefore, the effect of insulin on atherosclerosis could be mediated through its effect on PA inhibitor level. In diabetes mellitus, the normal balance between coagulative and fibrinolytic activity is affected, but no major abnormalities have been reported in levels and activities of the PA system in the plasma of diabetic patients (145). By contrast, abnormally low fibrinolytic activity is produced by vessels from diabetics, and a reduced production of PA in response to venous occlusion occurs in diabetic patients (144). ~2-antiplasmin activity and antigen levels in diabetic patients fall within normal ranges, indicating that this inhibitor is unaffected in this disease (146). Fibrin enhanced plasmin formation in an in vitro plasminogen activation assay was markedly impaired when the plasminogen or the tPA was obtained from patients with uncontrolled diabetes (145); however, the deranged function improved upon normalization of the blood glucose level (147). Therefore, the fibrinolytic defects are secondary to the metabolic consequences of diabetes and are not directly related to the disease (147). Contrasting reports on the implication of PAs in diabetes currently preclude use of these assays in the clinical evaluation of fibrinolysis in diabetic patients. RHEUMATOID ARTHRITIS

Several findings suggest a link between the PA and plasmin activities and the pathogenesis of rheumatoid arthritis (148-150). The PA system is known to be involved in inflammatory processes of different etiologies, and has been found in rheumatoid synovial fluid and tissue. Plasmin can degrade cartilage and activate latent collagenase; anti-inflammatory glucocorticoids that are clinically effective in the treatment of rheumatoid arthritis suppress uPA activity in synovial fibroblasts (148); elevated plasmin levels are found in rheumatoid synovial fluid (149). Cartilage destruction in rheumatoid arthritis involves production of proteases by synovial cells. On the basis of these observations, PA appears to assist in cartilage destruction associated with rheumatoid arthritis. The finding that synovial cell PA 205

MAYER production is stimulated by interleukin-1 (150) furt h e r supports its role as an i m m u n e mechanism in the induction of synovial cell PA. These observations have not yet been applied for diagnostic or therapeutic purposes. Conclusions

The subject of plasminogen activation offers a fascinating area of research because it combines basic science with medically r e l e v a n t and clinical laboratory oriented research. F u t u r e studies in this field should clarify some of the issues discussed. F u t u r e work is likely to examine intriguing new possibilities, such as the apparent association between thrombosis, coronary atherosclerosis, and hyperlipoproteinemia (151). Application of knowledge gained in the area of plasminogen activation will be used for diagnostic and therapeutic purposes; these include: use of components of the system for the diagnosis of cancer, early detection of p r i m a r y tumors and metastasis, and t r e a t m e n t of neoplastic diseases. Similarly, the role of plasminogen activation in cardiovascular and thrombotic conditions will be clarified and applied to diagnosis and therapy.

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