The Plasminogen-Plasmin Jack Henkin,
Patrick Marcotte,
REVIEW of the literature of plasminogen (Pg) and plasmin (Pm) encompasses many fields of research. Studies of physical biochemistry, enzyme kinetics and mechanism, enzyme inhibitors, human genetics and physiological regulation of enzymatic activities are but a few of the relevant topics. This article will focus on studies of the structure and function of Pg and Pm, together with a discussion of their reactions, inhibitors, and activators. The use of Pg activators as therapeutic agents is a major issue for clinical scientists and practicing physicians. This review of the biochemical properties of the proteins has been conceived as an introduction to these enzymes because their use as therapeutic agents is the subject of other articles in this volume of Progressin Cardiovascular Diseases. Previous investigators have reviewed many of the topics covered in this article. In particular, two symposium volumes published in 1988 entitled “Plasminogen: Function, Assay, and Clinical Significance”’ and “Enzymology of Plasminogen Activation”2 are valuable collections of mini-reviews of many subjects. An earlier (1981) review by Castellino3 describes the fundamental work that was carried out in the two decades before 1980 on biochemical characterization of Pg/Pm, fibrinogen/fibrin, and the Pg activators urokinase (UK) ( urinary Pg activator, uPA) and streptokinase (SK). An updated overall review including newer structural and kinetic information was presented by Robbins in 1987. The last decade has expanded the field of Pg activators to include tissue Pg activator (tPA) and prourokinase (pro-uPA, proUK), and the production of these enzymes by recombinant technology. These developments were also reviewed in 1987 by Bachmann.’ The availability and therapeutic importance of thrombolytic agents has resulted in a dramatic increase in research on the enzymatic reactions of fibrinolysis. In order to manage the numerous fields encompassing research on the biochemistry of Pg and Pm, we have subdivided this review into seven major sections. They discuss (1) the common forms, domain organization, and the overall structure and function of the Pg molecule, (2) the reaction of Pm with its major physiologi-
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cal substrate, fibrin (Fn), and with other macromolecular substrates, (3) Pg activators and their mechanism of action, (4) binding of Pg and Pm to cells and macromolecules, (5) inhibitors of Pm, (6) considerations of the assay, stability, and quantitation of Pg and Pm, and (7) characterization of the plasminogen gene and studies of mutant and recombinant forms of the protein. Each of these major topics is further divided into subtopics in which a specific area of research is discussed. In this review, studies that have been published in the last five years (1986-1990) are emphasized. Earlier work is included when it has been deemed necessary for clarity of the description of the topic. The reviews mentioned above are important sources of references to the earlier literature, and they provide the starting point for many of our discussions. Pg STRUCTURE
The isolation of Pg by its specific affinity for lysine-sepharose was described by Deutsch and Mertz in 1970.6 This procedure allowed the isolation of the protein in quantities sufficient for its characterization, and the complete sequence of 790 amino acids was reported by Sottrup-Jensen et al in 1978.7 With a few exceptions (most importantly the addition of one amino acid, IIe 67) the sequence was confirmed on subsequent analysis of the sequence of the gene, whose structure and organization will be detailed in a later section. The major forms of the protein, the organization and functions of the domains, and the conformational properties of the varieties of Pg are discussed below. As shown below, full-size Pg can be divided into a heavy amino-terminal region consisting of five homologous but distinct triple-disulfide bonded domains (kringles) fused to a lighter catalytic C-terminal domain. Activation to two-chain Pm From the Abbott Laboratories, Thrombofytics Venture Discovery Group, Abbott Park, IL. Address reprint requests to Jack Henkin, PhD, Abbott Laboratories, Throtnbolytics Venture Discovery Group, Abbott Park, IL 60064-3500. Copyright 0 1991 by U! B. Saunders Company 0033-0620191/3402-0004$5.00/O
1991:
pp 135164
135
136
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occurs when Pg activators (uPA, tPA, SK-Pg complex) cleave a unique bond, Arg560-Va1561, within a cyclic nonapeptide bridged by the Cys557Cys565 disulfide bond (Fig. 1). Molecular Forms of Human Pg
At least four forms of human Pg are isolable from human plasma based on variation in the N-terminus and the degree of glycosylation. The two major forms in normal plasma having Glul at the N-terminus are collectively called (GluPg), and the other two forms starting with Lys78 or Va179 are called Lys-Pg. Lys-Pg is formed rapidly from Glu-Pg by the catalytic action of Pm.8 Each subgroup is further divided into two forms based on the pattern of glycosylation as
Fig 1.
The primary
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described later. A convenient method to isolate the four major forms of Pg from plasma has recently been reported.’ Purified human Glu-Pg was shown by Brockway and Castellino” to be separable into two forms by gradient elution with l -aminocaproic acid of Pg absorbed to lysine-sepharose. These were designated PLGl and PLG2 because of their order of elution. Hayes and Castellino”-‘3 demonstrated that PLGl contained both N-linked (Asn288) and O-linked (Thr345) polysaccharide, whereas PLG2 contained only the O-linked polysaccharide. Subsequently, Powell and Castellino14 demonstrated that PLG2 contains the same amino acid sequence in the Asn288 region of the molecule, evidence that
structure
of human
Pg.
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SYSTEM
the heterogeneity in glycosylation is due to incomplete posttranslational modification of Pg and not a difference in primary sequence of the two isolated forms. Both forms exhibit microheterogeneity due to varying amounts of sialic acid incorporated into both the N- and O-linked polysaccharide. The sialylation and fucosylation patterns of human, bovine, and porcine plasminogens have been studied using 500 mHz nuclear magnetic resonance (NMR) spectroscopy by Marti et a1.15 Protease-Generated
Forms of Human Pg
In addition to the above forms, several groups have reported the production of lower molecular weight (MW) forms of Pg by limited proteolysis. The first reported is the so-called “mini-Pg” obtained by treatment with porcine pancreatic elastase resulting in cleavage of the amide bond between Va1442 and Va1443’ in a region connecting K4 and K5 kringles. The mini-Pg is thus without K 1 to 4. Moroz has reported formation of mini-Pg-like species after reacting human Pg with human leukocyte elastase,16 but it had an apparent molecular weight of 34,000, considerably smaller than the above mini-Pg (MW 38,000). This mini-Pg-like protein remains to be further characterized. Lower MW forms of human Pg have also been observed in vivo, as Kordich et al reported a mini-Pg-like molecule in septic patients.” Another form, still lower in MW, was obtained when the Pg was treated with human Pm at pH 10.5 resulting in the cleavage of the bond between Arg 530 and Ly~531.l~ This “micro-Pg” without Kl to 5 was hypothesized to have undergone disulfide interchanges among cystines of the residual A chain during incubation at high pH, resulting in an apparent MW of 29,000.” The unique properties of these smaller forms will be discussed following the section on Pg activators. fingle
Structure and Function
The nonprotease, or heavy chain, portion of Pg is comprised of an activation peptide (amino acids 1 through 76), followed by five homologous domains of triple-looped, disulfide-bridged, structures commonly called kringles.” These five domains, each approximately MW 10,000 exhibit a high degree of sequence homology with each other and with similar domains found
137
in prothrombin, tissue Pg activator, urinary Pg activator, factor XII, and perhaps haptoglobin. A structure with substantial homology to kringle 4 of Pg is a repeated unit in apolipoprotein A.*’ Patthy et al” described the homology of the kringle sequences to the gelatin-binding region of fibronectin and have constructed an evolutionary tree of the kringle domains of Pg, prothrombin, tissue-Pg activator, and UK.‘l A statistical evaluation of the alignment of the kringle sequences in six proteins has been reported by Castellino and Beals,** similarly concluding that the Pg kringle 1 and 5 regions are the most closely related sequences among the eleven studied. Both groups concluded that the haptoglobin domain is not a close relative of the structures found in the coagulation proteins. Pg, in its native form, has been shown to contain one high affinity “lysine-binding-site” (LBS): exhibiting a I(d for l -aminocaproic acid (EACA) of 2 p,M, and approximately five sites of lower affinity (Kd about 5 PM). The tight binding site is ascribed to the first kringle domain.z4 Individual kringle domains 1,4, and 5, as well as the larger fragment K1+2+3 have been isolated and studied as described below. Although the quantitative parameters of binding are altered on separation from the native molecule, the isolated domains retain the ability to bind lysine and related molecules and have been shown to be involved in binding of Pg and Pm to Fn, q-antiplasmin, and other macromolecules.25*26Thus the function of the kringles is an important part in the process of fibrinolysis. The following is a brief summary of the work that has been carried out with kringle domains isolated from human plasminogen and studies of their physiological significance. Isolation of human Pg kringles. Through digestion of Pg with porcine elastase, followed by chromatography on lysine-sepharose, the native molecule was separated into three fractions.’ The Pg fractions containing the first three kringles (amino acids 79-353 is the predominant species) and K4 (amino acids 354-439) bind to lysine-sepharose, wherease “mini-Pg” (K5 plus the protease B-chain) does not. It was later found that Kl could be isolated from the larger three-domain fragment after reaction with chymotrypsin,*’ or more efficiently, with staph V8
138
protease% or pepsin.” K5 removed from mini-Pg by sin.3a’31K4 is the most readily studied kringle domain of successful crytallization by reported in 1989.32
HENKIN,
was shown to be reaction with pepisolated and widely human Pg and its Mulichak et al was
Studies of domain stmcture. The isolated kringle domains 1,4, and 5 have been studied by calorimetry,33 fluorometry,2g and extensively by nuclear magnetic resonance.“36V38 The numbering system usually used for isolated kringle domains uses the first cysteine as residue no. 1, rather than the position of the amino acid in the intact protein. The structural data obtained was originally modeled on the X-ray crystal structure of bovine prothrombin kringle 1, which had been determined by Park and Tulinsky in 1986.37 The analysis of the crystals of K4 was reported by Mulichak and Tulinsky’B in 1990, which directly defined the residues involved in forming the lysine binding site. The structure for K4, derived from the NMR and crystallographic data,%%39 showed that aspartic acid 57 and arginine 71 form charge interactions with the amino and carboxylate groups of lysine and hydrophobic interactions between tryptophan 62, tryptophan 72, and phenylalanine 64 with the aliphatic region of the ligand. The structures derived for Kl and K5, primarily from NMR data,28,31,36 are similarly constructed, with a hydrophobic cleft formed by conserved tryptophan and tryosine residues interacting with the ligand. Although mini-Pg (containing K5) is not bound to lysine-sepharose, the isolated kringle domain does bind lysine and its analogues as measured by NMR and calorimetry.36rm Studies on the physiological function of the tingles. In addition to binding Fn and mediating the inhibition by ol-antiPm,26 the kringles of Pg have been impiicated in mediating neutrophil adherence to endothelial cells.41 The K1+2+3 fragment and isolated K4 were effective but mini-Pm was not. Also a lysine analog inhibited the adherence, indicating the importance of the lysine binding sites of the kringIes>l Platelet thrombospondin has been shown to interact with Pg at a specific site, which has been proposed to be within the K5 domain?’ A high-affinity K4 binding protein has been iso-
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lated from plasma and been termed “tetranectin.” It is homologous with asialoglycoprotein receptor and cartilage proteoglycan core protein and has been found to stimulate Pg activation by tissue-type Pg activator.43 However, tetranectin binds 40 times more tightly to lipoprotein (a), which contains multiple repeats of K4-like structures, than to Pg (13 nmol v 500 nmol); therefore, the lipoprotein may be its primary binding species.” Pg Conformation
The conformation of the native Pg molecule has been studied by measurement of its sedimentation velocity,” rotational and thermodynamic properties,46 and most recently by neutron diffraction.47 The molecule exists in a closed conformation, with the domains of the A-chain in close proximity to the protease section. It has a radius of gyration of 39 p and exhibits a sedimentation velocity of 5.7 S.45 Calorimetric studies have shown the presence of 7 cooperative regions in the intact zymogen, 5 from the kringle domains and 2 from the serine protease region. The amino terminal peptide (first 77 amino acids) is not believed to be part of the compact structure.30 The closed structure of Glu-Pg becomes extended upon binding of lysine (or similar molecules such as c-aminocaproic acid or tranexamic acid), with the domains no longer in close contact with each other.47 The conformational change is associated with binding to the weak (Kd = 5 mM for EACA) sites in the kringle domains.” Anions, in particular physiological concentrations of chloride ion, stabilize the closed form of Pg.& The open form exhibits a radius of gyration of 56 A47 and the sedimentation velocity of 4.8 S,” properties similar to those observed for Lys-Pg, in which the amino terminal 77 amino acid residues have been excised through enzymatic cleavage by active Pm from the native zymogen.45 A specific interaction between the N-terminal peptide and the A-chain, which stabilizes the closed form, is lost on removal of the peptide.4g Although Lys-Pg retains lysine-binding sites, its stable conforrnation is the open form and is not significantly altered on binding lysine analogues or anions.” Furthermore, the first four kringle domains of
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Lys-Pg do not exhibit cooperativity in calorimetric studies3’ The change in conformation from the closed to the extended form renders Pg far more readily cleaved to the active enzyme by Pg activators. The kinetics of these processes are discussed in the section of this review on Pg activators. The open conformation also binds more readily to exposed lysine residues on a Fn surface. The major interation between Pg and Fn is through the strong lysine binding site on kringle 1.51,52 Therefore, concentrations of lysine analogues (eg, tranexamic acid, EACA) that will promote the open conformation of Glu-Pg will also prevent its binding to Fn, and hence have an antifibrinolytic effect.s3 Exogenous LysPg, existing in a conformation that is both readily activatable and more favorable for Fn binding, is a potent fibrinolytic agent in combination with Pg activators in in vitro clot lysis models.54 MAJOR
PHYSIOLOGICAL
SUBSTRATES
OF Pm
Attack of Pm on Fg and Fn Fibrinogen (Fg) at 340,000 daltons, the structure of which has been recently reviewed,55,56
and fibrin (Fn), its polymeric form, are primary substrates for the proteolytic activity of Pm in the circulation. Although the exact affinity constants and site occupancy between Fg and Pg or Pm are highly controversial,57 Fg at 3 mg/mL in plasma is sufficiently concentrated to ensure that a significant fraction of circulating Fg molecules carry one or more associated Pg molecules. This may make specific sites on Fg/Fn especially open to attack. Pm readily cleaves Fg and Fn within a number of discreet accessible regions allowing isolation of intact protected domains and thus has played a major role in the elucidation of Fg/Fn structure. An outline of this structure as follows is necessary to understand the sites and sequence of Pm attack. Circulating Fg is comprised of three poiypeptide chains designated Aa, BP, and A, respectively 610, 461, and 410 amino acid residues in length. These chains are intertwined and S-S bonded together. This ensemble is linked via S-S bonds to a second identical one making the 340 kd Fg molecule a dimer of trimers; the intertrimer S-S bonds being near to the N-terFibrinogen
Fig 2. Schematic model of Fg and Fn. Fg consists of three pairs of polypeptide chains, AU, Bg, and y, joined by disulfide bonds to form a symmetric dimeric structure (A). The NH,-terminal regions of all six chains from the central domain (E domain) of the molecule containing fibrinopaptide FPA and FPB sequences that are cleaved by thrombin (Ila) during enzymatic conversion to Fn. Carbohydrate moieties are located at one site on each of the 7 and BP chains. Enzymatic conversion of Fg to Fn ()3) by thrombin cleavage results in release of FPA and FPB. A nonsubstrate (secondary) binding site for thrombin is present in the central domaln of a. &Fn, and depends largely, if not entirely, on the presence of the g15 to 42 sequence. Binding sites for (lla. tPA, fXl1, and %-PI, respectively, are indicated on the Fg of Fn molecule. (Reprinted with permission.“)
D Domain
Fibrin 345
“nl
-I
140
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mini of the Acx and A chains within the so called E-domain. The overall structure is shown in Fig 2. Thrombin sequentially removes a 16 amino acid peptide (FpA) from the N-terminus of the Ao chain, then a 14 amino acid peptide (FpB) from that of the BP chain to form Fn. This exposes new sites on the central E-domain which can associate with two D-domains from the more distal portions of two other Fn molecules, thus initiating a staggered polymerization (see Fig 3). D-domains brought together in this noncovalent polymer are then chemically crosslinked through Gln and Lys side chains catalyzed by factor XIIIa, a transglutaminase. Similar covalent crosslinks are later formed between A-chains of the Fn molecules.” Milhayi et als9 have shown that prolonged exposure to Pm can eventually catalyze the
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hydrolysis of nearly 60 bonds in 340 kd Fg of which only 5 to 6 are cleaved very rapidly. Gaffney and DobosW showed that Acl-chain breakdown preceded that of the BP-chain; X-chain being last to degrade. The earliest cleavage sites are on the exposed polar C-termirial region of AU. One of the first small fragments detected61 consisted of the last 27 amino acids released by hydrolysis of the Lys583Met584 bond. Other attack sites identified in this region include Lys206, Lys230, Arg424, Arg493, and Lys508. Liu et a16’ examined the kinetics of release of small peptides minutes after Pm exposure. Hydrolysis of Lys206Met207 was one of the earliest events essentially removing 2/3 of the distal Ao-chain. A second early attack site at Arg42Ala43 of the BP-chain yields the N-terminal peptide BBl-42, the level of which is often assayed63 as an early measure of Pm action on Fg. Fg and Fn may be
Fibrinogen
a
Thrombin
Intermediate
Plasmin
+
Plasmin
+
Plasmin
Poiymer
* Degradation Complexes
DDIE
YD/DY
YYIDXD
Fig 3. Fn polymerization, cross-linking, and degradation showing the influenca of thrombin, factor Xll a, and plasmin acting in concert on the structure of cross-linked Fn degradation products. After thrombin liberation of the fibrinopeptides, two Fn monomers form a hag-overlap Fn dimer as the Initial step of polymerization. Additional monomers are added to each end by a similar half-overlap process to form an intermediate polymer and then a protoffbril. Factor Xlia catalyzes the formation of cross-links between ‘y chains of contiguous terminal domains. Plasmic degradation of a long two-stranded protofibrii resutts in the series of noncovalently bound complexes (bottom), the smallest of which is DDE The presence of fragments larger than DD {such as DY) attached noncovalently to liberated complementary regions of another Fn strand provides the basis for this scheme of ever-larger cross-linked Fn degradation complexes. {For convenience, the o chain extensions are shown only for the intact fibrinogen molecule). (Rsprinted with permission from the Annual Review of Medicine, Vol37, o 1986 by Annual Reviews Inc.“)
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phosphorylated on the Ada-chains (Ser345, Ser441) and it has recently been shown@ that Fg whose Pi content increased by treatment with protein kinase C from about 1 to about 3.5 moles/mole was attacked two-fold more slowly by Pm. This resistance remained even after enzymatic dephosphorylation. As the cleavages of Fg proceeds, large residual truncated polypeptides are formed (collectively called X-fragment) having masses ranging downward from 337 kd to 246 kd and are designated X,, . . . X,, . . . X,,. These fragments are generally clottable and register as if they were Fg in standard laboratory assays of total plasma Fg. Ferguson65 has pointed out that Fn derived from such truncated Fg may be relatively weak owing to the loss of sites for C-terminal crosslinking. It has been suggestedM that X-fragment can be dynamically incorporated into both newly forming and existing thrombi causing them to be more fragile. The presence of large amounts of X-fragment may be formed during clot-specific thrombolytic therapy with tPA. This has been proposed as an explanation of why fewer bleeding complications have not been observed with tPA as compared with less Fn-specific agents.66 X-fragment accumulates transiently during Pm-catalyzed lysis of Fg, but continues to degrade forming smaller discreet fragments called D, E, and Pl, or a transient fragment Y comprised of a D-E core. Each of these really represents a family of closely related species having variously truncated N and C termini, owing to the presence of clusters of attack sites within accessible regions of polypeptide. Prolonged exposure of Fg to Pm ultimately leads to accumulation of more homogeneous, fully truncated, fragments with such designations as Dl (94 kd), D2, and D3 (76 kd). D-D dimer is formed when crosslinked Fn is the substrate.67 Fn lysis by Pm has been reviewed by Francis and Marder.@ Although the attack sites for Pm available in Fg are also generally accessible, albeit more slowly, in Fn, the covalent crosslinks present in Fn, but not in Fg, lead to a distinctly different pattern of apparent products when these are separated according to size on SDS gels. Thus, unique fragments, such as DD, DY, and YY as well as larger transient fragments,
are characteristic of Fn breakdown. Fg to a much greater extent than Fn is spared from lysis by Pm under normal physiological conditions by the system of circulating Pm inhibitors. These operate efficiently on free Pm but much more slowly on Fn-bound Pm and thus focus Pm action on thrombi. However, in pathological conditions causing plasminemia or during thrombolytic therapy, Pm levels accumulate beyond the capacity of Pm inhibitors and significant Fg lysis also takes place. Higazi and Mayer6’ have reported that Fn acts as a competitive inhibitor of Pm hydrolysis of S-2551, whereas the kinetics of inhibition of the reaction by Fg suggested negative cooperativity. Binding of Fg to a regulatory site in the kringle domains of plasmin was proposed to explain the kinetic observations. Attack of Pm on Other Proteins
Pm is a relatively nonspecific protease and can hydrolyze many proteins in both plasma and extracellular spaces. Both activation and inactivation by plasmic attack has been reported for a number of proteins, although the list of known targets continues to grow and is probably far from complete. In the coagulation pathway factors V, VIII, and vWF are known targets of Pm. Colman7’ reported that, while thrombin cleavages could enhance the activity of factor V, Pm could intercept thrombic intermediates and reduce their subsequent activation. Later it was shown” that factor Va inactivation by Pm is significantly enhanced in the presence of phospholipid (PL) vesicles while factor Xa and prothrombin protect factor Va against Pm attack. In contrast, the attack of Pm on factor V is not dependent on PL7* and leads initially to a rapid increase in procoagulant activity followed by a gradual decline in activity that is PLdependent. This suggests a prothrombotic potential for Pm and may relate to the observed release of FpA observed on thrombolytic treatment with SK or tPA.73 Sakuragawa et al74 have pointed out that plasmin appears in the blood of disseminated intravascular coagulopathy (DIC) patients whereas factors V and VIII are reduced in activity. The latter is activated then subsequently inactivated by Pm.75 Factor VIII circulates in complex with vWF and functions as a cofactor with factor IXa and PL in the
142
HENKIN,
activation of factor X. PL protects factor VIII from Pm.76 Platelets both stimulate the initial activation of this complex by Pm while slowing the subsequent inactivation.77 The complex, even after Pm degradation, supports ristocetininduced platelet aggregation.7s Although vWF can be attacked by Pm, plasmic fragments of vWF have been observed in patients treated with frbrinolytic agents but not in normals.79 Pm can release kinin from HMW-kininogen by cleavage on the N-terminal side of the bradykinin region, although this is probably not normally a major source of kinins compared with kallikrein. However, Pm can directly and indirectly activate prekallikrein’l and thus induce kinin formation. Recently, Lyons et als2 showed that latent transforming growth factor pl can be activated by a single plasmic cleavage in the N-terminal glycopeptide and suggested that Pm may be a significant physiologic activator of this growth factor. Pm can attack protein components of basement membrane as well as activate other proteases within this matrix. Several groups83*84have described a rank order of Pm attack on fibronectin > collagen > laminin. ACTIVATORS
OF Pg
uPA
The fibrinolytic activity of human urine was originally described in the early 1950~~‘~~~and was found to be the property of a glycoprotein that activates Pg to Pm. The enzyme, nameds7 urokinase (UK) or urinary Pg activator (uPA), was shown to be a serine protease of limited substrate specificity. Both high- and low-molecular-weight forms of uPA are prepared commercially, the MW of about 50,000 enzyme is predominant in urinary preparations,s’ whereas a truncated form (MW = 33,000) is isolated from long-term cell culture.89 Lung and kidney are the richest normal sources of uPA,~ and it is also enriched in several tumor cell lines.91 The low-molecular-weight enzyme is comprised of the serine protease region (B-chain) of the protein, with only a short (MW 1,500-2,400) A chain.’ This Pg activator is used clinically for the treatment of pulmonary embolism, acute myocardial infarction, and other thrombotic diseases.92 The zymogen of the UPA~>~~ first termed
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preurokinase93 is known as proUK and has alternatively been named single-chain urokinasetype Pg activator (scupa or pro-uPA). It was first isolated from natural sources,93,96-98then produced through recombinant techno10gy.99-101 The protein consists of 411 amino acids9’ and has a N-terminal domain with homology to the growth factor domain of other proteins, followed by a kringIe domain, homologous to domains on Pg, tissue Pg activator, and other proteins involved in coagulation.” However, the kringle domain of uPA or its zymogen does not contain a lysine-binding site, and it does not confer Fn-binding properties to the enzyme.‘” Specific high-affinity receptors for uPA, which recognize the growth factor domain, have been demonstrated in several cell types.‘“-‘” The focal concentration of UPA on cell surfaces has been postulated as a mechanism by which cells can invade intraceIluIar matrix and may have a role in numerous normal and pathological processes. These possible functions have been reviewed.lM Pro-uPA is rapidly converted to the two-chain enzyme by Pm,% thus these enzymes (uPA and Pm) complementarily activate their zymogens. The investigational use of pro-uPA as a thrombolytic agent has been reported.94s’07 The kinetics of activation of pro-uPA by fulllength Pm have been reported not to follow Michaelis kinetics, whereas addition of e-aminocaproic acid changes the kinetic pattern to the Michaelis form.1o8 With the observation that mini-Pm (lacking kringle domains l-4) activates pro-uPA according to Michaelis kinetics, an interaction between pro-uPA and the kringles of Pm has been proposed’OB as an explanation of the kinetics of the activation reaction. The activation of Pg by uPA is accomplished by specific proteolysis of the Arg560-Va1561 peptide bond of Pg.4 Because two-chain uPA is an active protease, the activation process is readily studied. Evaluation of the interaction of pro-uPA (proUK, scupa) with Pg is more difficult, because both proteins are zymogens and conflicting reports have appeared in the literature concerning the activity of the single-chain pro-UPA. Both uPA and pro-uPA administered intravenously are rapidly removed from the circulation, mainly via hepatic clearance with
PLASMINOGEN-PLASMIN
SYSTEM
dominant half-lives on the order of a few minutes depending on the species tested.“’ Recent studies of the activation of Pg by the urokinase-type Pg activators are dicussed in the next section. Activation of Pg by Urokinase-Type Pg Activator uPA rapidly cleaves Pg to produce two-chain active Pm. It has also been shown to react although slowly, with other proteins, including Fg,“” diphtheria toxin, ‘l’ and itself.“’ The activity of uPA is usually measured using the chromogenic substrate S-2444 (pyro-L-glutamylglycyl-L-lysine-p-nitroanilide).”3 uPA rapidly cleaves the susceptible site of Pg when the latter is in the extended conformation adopted by Lys-Pg but is much less efficient with the intact closed form of Glu-Pg,‘14 which appears mainly to be a K, effect. Effector molecules, such as lysine, E-aminocaproic acid, or tranexamic acid, result in Glu-Pg adopting the extended, more readily activatable, conformation. Physiological concentrations of chloride ion stabilize the closed conformation of Glu-Pg and thereby In comparing the kinetic inhibit activation.“’ constants of activation of plasminogen with uPA determined in different laboratories, the form of Pg used, as well as the exact composition of the media, are important variables to be considered. Studies of the effect of anions, amines, and Fn or Fn-degradation products have been carried out. In general, these molecules act by binding to Pg, the substrate of the reaction, not to the activating enzyme. Urano et ala found that the reaction of uPA and Glu-Pg is inhibited by monovalent anions in the following order of effectiveness: I- > SCN- > Cl- > IO,- > HCOO> F- > OAc-. This inhibition could be reversed by e-aminocaproic acid. Through kinetic analysis, it was shown that chloride ion increased the Km of the activation reaction lo-fold. Watahiki et a1116examined the effect of Fg, Fn, and fibrinogen degradation products (FDPs) on Glu-Pg and Lys-Pg. They reported that fibrin enhances the k,, of the activation reaction of Glu-Pg by uPA, with little effect on K,,,. These same molecules do not influence the kinetics of activation of Lys-Pg by uPA.*16
143
Activation of Pg by the Proenzyme Fom of uPA Pro-uPA (scupa or ProUK) is readily activated by Pm to the two-chain form which complicates evaluation of its properties as a Pg activator. Many attempts have been made to assess the intrinsic activity of the single-chain enzyme both in its reaction with small molecule substrates and Pg. It is clear that the reaction of pro-uPA with the chromogenic substrate S-2444 is very slow (
Patrick Marcotte,
REVIEW of the literature of plasminogen (Pg) and plasmin (Pm) encompasses many fields of research. Studies of physical biochemistry, enzyme kinetics and mechanism, enzyme inhibitors, human genetics and physiological regulation of enzymatic activities are but a few of the relevant topics. This article will focus on studies of the structure and function of Pg and Pm, together with a discussion of their reactions, inhibitors, and activators. The use of Pg activators as therapeutic agents is a major issue for clinical scientists and practicing physicians. This review of the biochemical properties of the proteins has been conceived as an introduction to these enzymes because their use as therapeutic agents is the subject of other articles in this volume of Progressin Cardiovascular Diseases. Previous investigators have reviewed many of the topics covered in this article. In particular, two symposium volumes published in 1988 entitled “Plasminogen: Function, Assay, and Clinical Significance”’ and “Enzymology of Plasminogen Activation”2 are valuable collections of mini-reviews of many subjects. An earlier (1981) review by Castellino3 describes the fundamental work that was carried out in the two decades before 1980 on biochemical characterization of Pg/Pm, fibrinogen/fibrin, and the Pg activators urokinase (UK) ( urinary Pg activator, uPA) and streptokinase (SK). An updated overall review including newer structural and kinetic information was presented by Robbins in 1987. The last decade has expanded the field of Pg activators to include tissue Pg activator (tPA) and prourokinase (pro-uPA, proUK), and the production of these enzymes by recombinant technology. These developments were also reviewed in 1987 by Bachmann.’ The availability and therapeutic importance of thrombolytic agents has resulted in a dramatic increase in research on the enzymatic reactions of fibrinolysis. In order to manage the numerous fields encompassing research on the biochemistry of Pg and Pm, we have subdivided this review into seven major sections. They discuss (1) the common forms, domain organization, and the overall structure and function of the Pg molecule, (2) the reaction of Pm with its major physiologi-
A
Progress
in CardiovascularDiseases,
Vol XXXIV,
No 2 (September/October),
System
and Heechung
Yang
cal substrate, fibrin (Fn), and with other macromolecular substrates, (3) Pg activators and their mechanism of action, (4) binding of Pg and Pm to cells and macromolecules, (5) inhibitors of Pm, (6) considerations of the assay, stability, and quantitation of Pg and Pm, and (7) characterization of the plasminogen gene and studies of mutant and recombinant forms of the protein. Each of these major topics is further divided into subtopics in which a specific area of research is discussed. In this review, studies that have been published in the last five years (1986-1990) are emphasized. Earlier work is included when it has been deemed necessary for clarity of the description of the topic. The reviews mentioned above are important sources of references to the earlier literature, and they provide the starting point for many of our discussions. Pg STRUCTURE
The isolation of Pg by its specific affinity for lysine-sepharose was described by Deutsch and Mertz in 1970.6 This procedure allowed the isolation of the protein in quantities sufficient for its characterization, and the complete sequence of 790 amino acids was reported by Sottrup-Jensen et al in 1978.7 With a few exceptions (most importantly the addition of one amino acid, IIe 67) the sequence was confirmed on subsequent analysis of the sequence of the gene, whose structure and organization will be detailed in a later section. The major forms of the protein, the organization and functions of the domains, and the conformational properties of the varieties of Pg are discussed below. As shown below, full-size Pg can be divided into a heavy amino-terminal region consisting of five homologous but distinct triple-disulfide bonded domains (kringles) fused to a lighter catalytic C-terminal domain. Activation to two-chain Pm From the Abbott Laboratories, Thrombofytics Venture Discovery Group, Abbott Park, IL. Address reprint requests to Jack Henkin, PhD, Abbott Laboratories, Throtnbolytics Venture Discovery Group, Abbott Park, IL 60064-3500. Copyright 0 1991 by U! B. Saunders Company 0033-0620191/3402-0004$5.00/O
1991:
pp 135164
135
136
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occurs when Pg activators (uPA, tPA, SK-Pg complex) cleave a unique bond, Arg560-Va1561, within a cyclic nonapeptide bridged by the Cys557Cys565 disulfide bond (Fig. 1). Molecular Forms of Human Pg
At least four forms of human Pg are isolable from human plasma based on variation in the N-terminus and the degree of glycosylation. The two major forms in normal plasma having Glul at the N-terminus are collectively called (GluPg), and the other two forms starting with Lys78 or Va179 are called Lys-Pg. Lys-Pg is formed rapidly from Glu-Pg by the catalytic action of Pm.8 Each subgroup is further divided into two forms based on the pattern of glycosylation as
Fig 1.
The primary
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described later. A convenient method to isolate the four major forms of Pg from plasma has recently been reported.’ Purified human Glu-Pg was shown by Brockway and Castellino” to be separable into two forms by gradient elution with l -aminocaproic acid of Pg absorbed to lysine-sepharose. These were designated PLGl and PLG2 because of their order of elution. Hayes and Castellino”-‘3 demonstrated that PLGl contained both N-linked (Asn288) and O-linked (Thr345) polysaccharide, whereas PLG2 contained only the O-linked polysaccharide. Subsequently, Powell and Castellino14 demonstrated that PLG2 contains the same amino acid sequence in the Asn288 region of the molecule, evidence that
structure
of human
Pg.
PLASMINOGEN-PLASMIN
SYSTEM
the heterogeneity in glycosylation is due to incomplete posttranslational modification of Pg and not a difference in primary sequence of the two isolated forms. Both forms exhibit microheterogeneity due to varying amounts of sialic acid incorporated into both the N- and O-linked polysaccharide. The sialylation and fucosylation patterns of human, bovine, and porcine plasminogens have been studied using 500 mHz nuclear magnetic resonance (NMR) spectroscopy by Marti et a1.15 Protease-Generated
Forms of Human Pg
In addition to the above forms, several groups have reported the production of lower molecular weight (MW) forms of Pg by limited proteolysis. The first reported is the so-called “mini-Pg” obtained by treatment with porcine pancreatic elastase resulting in cleavage of the amide bond between Va1442 and Va1443’ in a region connecting K4 and K5 kringles. The mini-Pg is thus without K 1 to 4. Moroz has reported formation of mini-Pg-like species after reacting human Pg with human leukocyte elastase,16 but it had an apparent molecular weight of 34,000, considerably smaller than the above mini-Pg (MW 38,000). This mini-Pg-like protein remains to be further characterized. Lower MW forms of human Pg have also been observed in vivo, as Kordich et al reported a mini-Pg-like molecule in septic patients.” Another form, still lower in MW, was obtained when the Pg was treated with human Pm at pH 10.5 resulting in the cleavage of the bond between Arg 530 and Ly~531.l~ This “micro-Pg” without Kl to 5 was hypothesized to have undergone disulfide interchanges among cystines of the residual A chain during incubation at high pH, resulting in an apparent MW of 29,000.” The unique properties of these smaller forms will be discussed following the section on Pg activators. fingle
Structure and Function
The nonprotease, or heavy chain, portion of Pg is comprised of an activation peptide (amino acids 1 through 76), followed by five homologous domains of triple-looped, disulfide-bridged, structures commonly called kringles.” These five domains, each approximately MW 10,000 exhibit a high degree of sequence homology with each other and with similar domains found
137
in prothrombin, tissue Pg activator, urinary Pg activator, factor XII, and perhaps haptoglobin. A structure with substantial homology to kringle 4 of Pg is a repeated unit in apolipoprotein A.*’ Patthy et al” described the homology of the kringle sequences to the gelatin-binding region of fibronectin and have constructed an evolutionary tree of the kringle domains of Pg, prothrombin, tissue-Pg activator, and UK.‘l A statistical evaluation of the alignment of the kringle sequences in six proteins has been reported by Castellino and Beals,** similarly concluding that the Pg kringle 1 and 5 regions are the most closely related sequences among the eleven studied. Both groups concluded that the haptoglobin domain is not a close relative of the structures found in the coagulation proteins. Pg, in its native form, has been shown to contain one high affinity “lysine-binding-site” (LBS): exhibiting a I(d for l -aminocaproic acid (EACA) of 2 p,M, and approximately five sites of lower affinity (Kd about 5 PM). The tight binding site is ascribed to the first kringle domain.z4 Individual kringle domains 1,4, and 5, as well as the larger fragment K1+2+3 have been isolated and studied as described below. Although the quantitative parameters of binding are altered on separation from the native molecule, the isolated domains retain the ability to bind lysine and related molecules and have been shown to be involved in binding of Pg and Pm to Fn, q-antiplasmin, and other macromolecules.25*26Thus the function of the kringles is an important part in the process of fibrinolysis. The following is a brief summary of the work that has been carried out with kringle domains isolated from human plasminogen and studies of their physiological significance. Isolation of human Pg kringles. Through digestion of Pg with porcine elastase, followed by chromatography on lysine-sepharose, the native molecule was separated into three fractions.’ The Pg fractions containing the first three kringles (amino acids 79-353 is the predominant species) and K4 (amino acids 354-439) bind to lysine-sepharose, wherease “mini-Pg” (K5 plus the protease B-chain) does not. It was later found that Kl could be isolated from the larger three-domain fragment after reaction with chymotrypsin,*’ or more efficiently, with staph V8
138
protease% or pepsin.” K5 removed from mini-Pg by sin.3a’31K4 is the most readily studied kringle domain of successful crytallization by reported in 1989.32
HENKIN,
was shown to be reaction with pepisolated and widely human Pg and its Mulichak et al was
Studies of domain stmcture. The isolated kringle domains 1,4, and 5 have been studied by calorimetry,33 fluorometry,2g and extensively by nuclear magnetic resonance.“36V38 The numbering system usually used for isolated kringle domains uses the first cysteine as residue no. 1, rather than the position of the amino acid in the intact protein. The structural data obtained was originally modeled on the X-ray crystal structure of bovine prothrombin kringle 1, which had been determined by Park and Tulinsky in 1986.37 The analysis of the crystals of K4 was reported by Mulichak and Tulinsky’B in 1990, which directly defined the residues involved in forming the lysine binding site. The structure for K4, derived from the NMR and crystallographic data,%%39 showed that aspartic acid 57 and arginine 71 form charge interactions with the amino and carboxylate groups of lysine and hydrophobic interactions between tryptophan 62, tryptophan 72, and phenylalanine 64 with the aliphatic region of the ligand. The structures derived for Kl and K5, primarily from NMR data,28,31,36 are similarly constructed, with a hydrophobic cleft formed by conserved tryptophan and tryosine residues interacting with the ligand. Although mini-Pg (containing K5) is not bound to lysine-sepharose, the isolated kringle domain does bind lysine and its analogues as measured by NMR and calorimetry.36rm Studies on the physiological function of the tingles. In addition to binding Fn and mediating the inhibition by ol-antiPm,26 the kringles of Pg have been impiicated in mediating neutrophil adherence to endothelial cells.41 The K1+2+3 fragment and isolated K4 were effective but mini-Pm was not. Also a lysine analog inhibited the adherence, indicating the importance of the lysine binding sites of the kringIes>l Platelet thrombospondin has been shown to interact with Pg at a specific site, which has been proposed to be within the K5 domain?’ A high-affinity K4 binding protein has been iso-
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lated from plasma and been termed “tetranectin.” It is homologous with asialoglycoprotein receptor and cartilage proteoglycan core protein and has been found to stimulate Pg activation by tissue-type Pg activator.43 However, tetranectin binds 40 times more tightly to lipoprotein (a), which contains multiple repeats of K4-like structures, than to Pg (13 nmol v 500 nmol); therefore, the lipoprotein may be its primary binding species.” Pg Conformation
The conformation of the native Pg molecule has been studied by measurement of its sedimentation velocity,” rotational and thermodynamic properties,46 and most recently by neutron diffraction.47 The molecule exists in a closed conformation, with the domains of the A-chain in close proximity to the protease section. It has a radius of gyration of 39 p and exhibits a sedimentation velocity of 5.7 S.45 Calorimetric studies have shown the presence of 7 cooperative regions in the intact zymogen, 5 from the kringle domains and 2 from the serine protease region. The amino terminal peptide (first 77 amino acids) is not believed to be part of the compact structure.30 The closed structure of Glu-Pg becomes extended upon binding of lysine (or similar molecules such as c-aminocaproic acid or tranexamic acid), with the domains no longer in close contact with each other.47 The conformational change is associated with binding to the weak (Kd = 5 mM for EACA) sites in the kringle domains.” Anions, in particular physiological concentrations of chloride ion, stabilize the closed form of Pg.& The open form exhibits a radius of gyration of 56 A47 and the sedimentation velocity of 4.8 S,” properties similar to those observed for Lys-Pg, in which the amino terminal 77 amino acid residues have been excised through enzymatic cleavage by active Pm from the native zymogen.45 A specific interaction between the N-terminal peptide and the A-chain, which stabilizes the closed form, is lost on removal of the peptide.4g Although Lys-Pg retains lysine-binding sites, its stable conforrnation is the open form and is not significantly altered on binding lysine analogues or anions.” Furthermore, the first four kringle domains of
PLASMINOGEN-PLASMIN
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SYSTEM
Lys-Pg do not exhibit cooperativity in calorimetric studies3’ The change in conformation from the closed to the extended form renders Pg far more readily cleaved to the active enzyme by Pg activators. The kinetics of these processes are discussed in the section of this review on Pg activators. The open conformation also binds more readily to exposed lysine residues on a Fn surface. The major interation between Pg and Fn is through the strong lysine binding site on kringle 1.51,52 Therefore, concentrations of lysine analogues (eg, tranexamic acid, EACA) that will promote the open conformation of Glu-Pg will also prevent its binding to Fn, and hence have an antifibrinolytic effect.s3 Exogenous LysPg, existing in a conformation that is both readily activatable and more favorable for Fn binding, is a potent fibrinolytic agent in combination with Pg activators in in vitro clot lysis models.54 MAJOR
PHYSIOLOGICAL
SUBSTRATES
OF Pm
Attack of Pm on Fg and Fn Fibrinogen (Fg) at 340,000 daltons, the structure of which has been recently reviewed,55,56
and fibrin (Fn), its polymeric form, are primary substrates for the proteolytic activity of Pm in the circulation. Although the exact affinity constants and site occupancy between Fg and Pg or Pm are highly controversial,57 Fg at 3 mg/mL in plasma is sufficiently concentrated to ensure that a significant fraction of circulating Fg molecules carry one or more associated Pg molecules. This may make specific sites on Fg/Fn especially open to attack. Pm readily cleaves Fg and Fn within a number of discreet accessible regions allowing isolation of intact protected domains and thus has played a major role in the elucidation of Fg/Fn structure. An outline of this structure as follows is necessary to understand the sites and sequence of Pm attack. Circulating Fg is comprised of three poiypeptide chains designated Aa, BP, and A, respectively 610, 461, and 410 amino acid residues in length. These chains are intertwined and S-S bonded together. This ensemble is linked via S-S bonds to a second identical one making the 340 kd Fg molecule a dimer of trimers; the intertrimer S-S bonds being near to the N-terFibrinogen
Fig 2. Schematic model of Fg and Fn. Fg consists of three pairs of polypeptide chains, AU, Bg, and y, joined by disulfide bonds to form a symmetric dimeric structure (A). The NH,-terminal regions of all six chains from the central domain (E domain) of the molecule containing fibrinopaptide FPA and FPB sequences that are cleaved by thrombin (Ila) during enzymatic conversion to Fn. Carbohydrate moieties are located at one site on each of the 7 and BP chains. Enzymatic conversion of Fg to Fn ()3) by thrombin cleavage results in release of FPA and FPB. A nonsubstrate (secondary) binding site for thrombin is present in the central domaln of a. &Fn, and depends largely, if not entirely, on the presence of the g15 to 42 sequence. Binding sites for (lla. tPA, fXl1, and %-PI, respectively, are indicated on the Fg of Fn molecule. (Reprinted with permission.“)
D Domain
Fibrin 345
“nl
-I
140
HENKIN,
mini of the Acx and A chains within the so called E-domain. The overall structure is shown in Fig 2. Thrombin sequentially removes a 16 amino acid peptide (FpA) from the N-terminus of the Ao chain, then a 14 amino acid peptide (FpB) from that of the BP chain to form Fn. This exposes new sites on the central E-domain which can associate with two D-domains from the more distal portions of two other Fn molecules, thus initiating a staggered polymerization (see Fig 3). D-domains brought together in this noncovalent polymer are then chemically crosslinked through Gln and Lys side chains catalyzed by factor XIIIa, a transglutaminase. Similar covalent crosslinks are later formed between A-chains of the Fn molecules.” Milhayi et als9 have shown that prolonged exposure to Pm can eventually catalyze the
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hydrolysis of nearly 60 bonds in 340 kd Fg of which only 5 to 6 are cleaved very rapidly. Gaffney and DobosW showed that Acl-chain breakdown preceded that of the BP-chain; X-chain being last to degrade. The earliest cleavage sites are on the exposed polar C-termirial region of AU. One of the first small fragments detected61 consisted of the last 27 amino acids released by hydrolysis of the Lys583Met584 bond. Other attack sites identified in this region include Lys206, Lys230, Arg424, Arg493, and Lys508. Liu et a16’ examined the kinetics of release of small peptides minutes after Pm exposure. Hydrolysis of Lys206Met207 was one of the earliest events essentially removing 2/3 of the distal Ao-chain. A second early attack site at Arg42Ala43 of the BP-chain yields the N-terminal peptide BBl-42, the level of which is often assayed63 as an early measure of Pm action on Fg. Fg and Fn may be
Fibrinogen
a
Thrombin
Intermediate
Plasmin
+
Plasmin
+
Plasmin
Poiymer
* Degradation Complexes
DDIE
YD/DY
YYIDXD
Fig 3. Fn polymerization, cross-linking, and degradation showing the influenca of thrombin, factor Xll a, and plasmin acting in concert on the structure of cross-linked Fn degradation products. After thrombin liberation of the fibrinopeptides, two Fn monomers form a hag-overlap Fn dimer as the Initial step of polymerization. Additional monomers are added to each end by a similar half-overlap process to form an intermediate polymer and then a protoffbril. Factor Xlia catalyzes the formation of cross-links between ‘y chains of contiguous terminal domains. Plasmic degradation of a long two-stranded protofibrii resutts in the series of noncovalently bound complexes (bottom), the smallest of which is DDE The presence of fragments larger than DD {such as DY) attached noncovalently to liberated complementary regions of another Fn strand provides the basis for this scheme of ever-larger cross-linked Fn degradation complexes. {For convenience, the o chain extensions are shown only for the intact fibrinogen molecule). (Rsprinted with permission from the Annual Review of Medicine, Vol37, o 1986 by Annual Reviews Inc.“)
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phosphorylated on the Ada-chains (Ser345, Ser441) and it has recently been shown@ that Fg whose Pi content increased by treatment with protein kinase C from about 1 to about 3.5 moles/mole was attacked two-fold more slowly by Pm. This resistance remained even after enzymatic dephosphorylation. As the cleavages of Fg proceeds, large residual truncated polypeptides are formed (collectively called X-fragment) having masses ranging downward from 337 kd to 246 kd and are designated X,, . . . X,, . . . X,,. These fragments are generally clottable and register as if they were Fg in standard laboratory assays of total plasma Fg. Ferguson65 has pointed out that Fn derived from such truncated Fg may be relatively weak owing to the loss of sites for C-terminal crosslinking. It has been suggestedM that X-fragment can be dynamically incorporated into both newly forming and existing thrombi causing them to be more fragile. The presence of large amounts of X-fragment may be formed during clot-specific thrombolytic therapy with tPA. This has been proposed as an explanation of why fewer bleeding complications have not been observed with tPA as compared with less Fn-specific agents.66 X-fragment accumulates transiently during Pm-catalyzed lysis of Fg, but continues to degrade forming smaller discreet fragments called D, E, and Pl, or a transient fragment Y comprised of a D-E core. Each of these really represents a family of closely related species having variously truncated N and C termini, owing to the presence of clusters of attack sites within accessible regions of polypeptide. Prolonged exposure of Fg to Pm ultimately leads to accumulation of more homogeneous, fully truncated, fragments with such designations as Dl (94 kd), D2, and D3 (76 kd). D-D dimer is formed when crosslinked Fn is the substrate.67 Fn lysis by Pm has been reviewed by Francis and Marder.@ Although the attack sites for Pm available in Fg are also generally accessible, albeit more slowly, in Fn, the covalent crosslinks present in Fn, but not in Fg, lead to a distinctly different pattern of apparent products when these are separated according to size on SDS gels. Thus, unique fragments, such as DD, DY, and YY as well as larger transient fragments,
are characteristic of Fn breakdown. Fg to a much greater extent than Fn is spared from lysis by Pm under normal physiological conditions by the system of circulating Pm inhibitors. These operate efficiently on free Pm but much more slowly on Fn-bound Pm and thus focus Pm action on thrombi. However, in pathological conditions causing plasminemia or during thrombolytic therapy, Pm levels accumulate beyond the capacity of Pm inhibitors and significant Fg lysis also takes place. Higazi and Mayer6’ have reported that Fn acts as a competitive inhibitor of Pm hydrolysis of S-2551, whereas the kinetics of inhibition of the reaction by Fg suggested negative cooperativity. Binding of Fg to a regulatory site in the kringle domains of plasmin was proposed to explain the kinetic observations. Attack of Pm on Other Proteins
Pm is a relatively nonspecific protease and can hydrolyze many proteins in both plasma and extracellular spaces. Both activation and inactivation by plasmic attack has been reported for a number of proteins, although the list of known targets continues to grow and is probably far from complete. In the coagulation pathway factors V, VIII, and vWF are known targets of Pm. Colman7’ reported that, while thrombin cleavages could enhance the activity of factor V, Pm could intercept thrombic intermediates and reduce their subsequent activation. Later it was shown” that factor Va inactivation by Pm is significantly enhanced in the presence of phospholipid (PL) vesicles while factor Xa and prothrombin protect factor Va against Pm attack. In contrast, the attack of Pm on factor V is not dependent on PL7* and leads initially to a rapid increase in procoagulant activity followed by a gradual decline in activity that is PLdependent. This suggests a prothrombotic potential for Pm and may relate to the observed release of FpA observed on thrombolytic treatment with SK or tPA.73 Sakuragawa et al74 have pointed out that plasmin appears in the blood of disseminated intravascular coagulopathy (DIC) patients whereas factors V and VIII are reduced in activity. The latter is activated then subsequently inactivated by Pm.75 Factor VIII circulates in complex with vWF and functions as a cofactor with factor IXa and PL in the
142
HENKIN,
activation of factor X. PL protects factor VIII from Pm.76 Platelets both stimulate the initial activation of this complex by Pm while slowing the subsequent inactivation.77 The complex, even after Pm degradation, supports ristocetininduced platelet aggregation.7s Although vWF can be attacked by Pm, plasmic fragments of vWF have been observed in patients treated with frbrinolytic agents but not in normals.79 Pm can release kinin from HMW-kininogen by cleavage on the N-terminal side of the bradykinin region, although this is probably not normally a major source of kinins compared with kallikrein. However, Pm can directly and indirectly activate prekallikrein’l and thus induce kinin formation. Recently, Lyons et als2 showed that latent transforming growth factor pl can be activated by a single plasmic cleavage in the N-terminal glycopeptide and suggested that Pm may be a significant physiologic activator of this growth factor. Pm can attack protein components of basement membrane as well as activate other proteases within this matrix. Several groups83*84have described a rank order of Pm attack on fibronectin > collagen > laminin. ACTIVATORS
OF Pg
uPA
The fibrinolytic activity of human urine was originally described in the early 1950~~‘~~~and was found to be the property of a glycoprotein that activates Pg to Pm. The enzyme, nameds7 urokinase (UK) or urinary Pg activator (uPA), was shown to be a serine protease of limited substrate specificity. Both high- and low-molecular-weight forms of uPA are prepared commercially, the MW of about 50,000 enzyme is predominant in urinary preparations,s’ whereas a truncated form (MW = 33,000) is isolated from long-term cell culture.89 Lung and kidney are the richest normal sources of uPA,~ and it is also enriched in several tumor cell lines.91 The low-molecular-weight enzyme is comprised of the serine protease region (B-chain) of the protein, with only a short (MW 1,500-2,400) A chain.’ This Pg activator is used clinically for the treatment of pulmonary embolism, acute myocardial infarction, and other thrombotic diseases.92 The zymogen of the UPA~>~~ first termed
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preurokinase93 is known as proUK and has alternatively been named single-chain urokinasetype Pg activator (scupa or pro-uPA). It was first isolated from natural sources,93,96-98then produced through recombinant techno10gy.99-101 The protein consists of 411 amino acids9’ and has a N-terminal domain with homology to the growth factor domain of other proteins, followed by a kringIe domain, homologous to domains on Pg, tissue Pg activator, and other proteins involved in coagulation.” However, the kringle domain of uPA or its zymogen does not contain a lysine-binding site, and it does not confer Fn-binding properties to the enzyme.‘” Specific high-affinity receptors for uPA, which recognize the growth factor domain, have been demonstrated in several cell types.‘“-‘” The focal concentration of UPA on cell surfaces has been postulated as a mechanism by which cells can invade intraceIluIar matrix and may have a role in numerous normal and pathological processes. These possible functions have been reviewed.lM Pro-uPA is rapidly converted to the two-chain enzyme by Pm,% thus these enzymes (uPA and Pm) complementarily activate their zymogens. The investigational use of pro-uPA as a thrombolytic agent has been reported.94s’07 The kinetics of activation of pro-uPA by fulllength Pm have been reported not to follow Michaelis kinetics, whereas addition of e-aminocaproic acid changes the kinetic pattern to the Michaelis form.1o8 With the observation that mini-Pm (lacking kringle domains l-4) activates pro-uPA according to Michaelis kinetics, an interaction between pro-uPA and the kringles of Pm has been proposed’OB as an explanation of the kinetics of the activation reaction. The activation of Pg by uPA is accomplished by specific proteolysis of the Arg560-Va1561 peptide bond of Pg.4 Because two-chain uPA is an active protease, the activation process is readily studied. Evaluation of the interaction of pro-uPA (proUK, scupa) with Pg is more difficult, because both proteins are zymogens and conflicting reports have appeared in the literature concerning the activity of the single-chain pro-UPA. Both uPA and pro-uPA administered intravenously are rapidly removed from the circulation, mainly via hepatic clearance with
PLASMINOGEN-PLASMIN
SYSTEM
dominant half-lives on the order of a few minutes depending on the species tested.“’ Recent studies of the activation of Pg by the urokinase-type Pg activators are dicussed in the next section. Activation of Pg by Urokinase-Type Pg Activator uPA rapidly cleaves Pg to produce two-chain active Pm. It has also been shown to react although slowly, with other proteins, including Fg,“” diphtheria toxin, ‘l’ and itself.“’ The activity of uPA is usually measured using the chromogenic substrate S-2444 (pyro-L-glutamylglycyl-L-lysine-p-nitroanilide).”3 uPA rapidly cleaves the susceptible site of Pg when the latter is in the extended conformation adopted by Lys-Pg but is much less efficient with the intact closed form of Glu-Pg,‘14 which appears mainly to be a K, effect. Effector molecules, such as lysine, E-aminocaproic acid, or tranexamic acid, result in Glu-Pg adopting the extended, more readily activatable, conformation. Physiological concentrations of chloride ion stabilize the closed conformation of Glu-Pg and thereby In comparing the kinetic inhibit activation.“’ constants of activation of plasminogen with uPA determined in different laboratories, the form of Pg used, as well as the exact composition of the media, are important variables to be considered. Studies of the effect of anions, amines, and Fn or Fn-degradation products have been carried out. In general, these molecules act by binding to Pg, the substrate of the reaction, not to the activating enzyme. Urano et ala found that the reaction of uPA and Glu-Pg is inhibited by monovalent anions in the following order of effectiveness: I- > SCN- > Cl- > IO,- > HCOO> F- > OAc-. This inhibition could be reversed by e-aminocaproic acid. Through kinetic analysis, it was shown that chloride ion increased the Km of the activation reaction lo-fold. Watahiki et a1116examined the effect of Fg, Fn, and fibrinogen degradation products (FDPs) on Glu-Pg and Lys-Pg. They reported that fibrin enhances the k,, of the activation reaction of Glu-Pg by uPA, with little effect on K,,,. These same molecules do not influence the kinetics of activation of Lys-Pg by uPA.*16
143
Activation of Pg by the Proenzyme Fom of uPA Pro-uPA (scupa or ProUK) is readily activated by Pm to the two-chain form which complicates evaluation of its properties as a Pg activator. Many attempts have been made to assess the intrinsic activity of the single-chain enzyme both in its reaction with small molecule substrates and Pg. It is clear that the reaction of pro-uPA with the chromogenic substrate S-2444 is very slow (
