The PLG gene is 52.5 kb and is located on chromosome 6q26-q27,¹³ close to two genes for apolipoprotein (a) and for the PLG-related genes A and B.^14,15 Two sequence elements of CTGGGA common to acute-phase reactant genes are at position 76 to 81 and -553 to -558.¹⁶ Three interleukin-6-responsive elements are at positions -830, -518, and +117 in the 5′-flanking region.¹⁷ Two main regions conferring liver specificity of expression to the PLG gene were found in the cis-acting element, AAAAATA, at -2,194 to -2,188 and +48 to +61.¹⁸ Several other putative regulatory transcription elements are present in the 5′-flanking region (reviewed in Ref. 17). On the 3′-flanking region, the location of the consensus polyadenylation sequence AATAAA and of potential CAYTG sequence indicates the presence of three polyadenylation sites.¹⁶

PLG consists of an N-terminal activation peptide, five kringles, and the protease domain with the catalytic triad His603, Asp646, and Ser741¹⁶ (FIGURE 20.3). The kringles are well conserved between species¹⁹ and consist of some 80 residues, the structure being shaped by a characteristic 1-6, 2-4, 3-5 disulfide bond pattern using the conserved Cys residues in positions 1, 22, 51, 63, 75, and 80 (kringle 5 numbering). K2 and K3 are covalently linked by a further disulfide bond.²⁰ Functionally, the kringles give PLG the ability to bind to exposed lysyl residues in fibrin,^21,22 α₂antiplasmin (α₂AP),²³ tetranectin,²⁴ histidine-rich glycoprotein (HRG),²⁵ collagen,²⁶ high molecular weight (HMW) and low molecular weight (LMW) kininogen,²⁷ thrombospondin,²⁸ and cell-surface receptors or binding proteins for PLG (discussed
in more detail below). The affinity of the kringles for lysine is exploited in affinity chromatography.²⁹ The kringles also bind to ω-aminocarboxylic acids, such as 6-aminohexanoic acid (εACA, 6-AHA), p-aminomethyl benzoic acid (p-AMBA), and trans-4-amino-methyl-cyclohexane-carboxylic acid (trans-AMCA, also known as tranexamic acid). These are used experimentally and clinically to inhibit the functional activity of plasmin by competing with the binding of plasmin(ogen) to fibrin and cell surfaces.

Human PLG kringles have different affinities and specificities for ω-amino acids, such as εACA. Kringle 1 exhibits the tightest binding (K_d 9 µM),^30,31 followed by kringle 4^30,32,33 and kringle 5,^32,33 with kringle 2 interacting only weakly and kringle 3 not at all.³⁴ Kringle structures have been solved both in solution and in crystals and show the general feature of a hydrophobic groove with acidic and basic residues that are ideally spaced for docking of zwitterions such as lysine or εACA, the opposite charges of which are approximately 7 Å apart. The lack of binding by kringle 3 is explained by the positive charge at position 57, which in the other kringles is negatively charged.³⁵

Experimentally, kringle 4 can be isolated from PLG by digestion with elastase, typically yielding three major components: a fragment containing kringles 1 to 3, kringle 4, and kringle 5 attached to the protease domain.²⁰ The latter fragment, also called mini-PLG, can be activated to plasmin by PAs,^36,37,38 as can microplasmin, which has only the last 31 amino acid residues of the A chain and a complete B chain.³⁸ Kringle 1 can be obtained from kringles 1 to 3 by further digestion with other enzymes, such as Staphylococcus aureus V8 protease.³⁹ Other fragments of PLG have effects on tumor growth and metastasis; these fragments are known as angiostatins. Their activity depends crucially on their disulfide pattern.⁴⁰

All known PAs cleave the Arg561-Val562 bond in PLG (FIGURE 20.4), generating a two-chain plasmin molecule in which the A and B chains remain attached by two disulfide bonds. PLG exists in different conformations, affected by its N-terminus and also by the presence of lysine analogues. A particularly important consideration is whether PLG is free in solution or bound to a surface, either fibrin or cells, as discussed more fully in the section on “Balance of Plasminogen
Activation and Regulation of Fibrinolysis” At this stage of our discussion, it can be assumed that the binding is primarily dependent on exposed lysine residues, and that the binding to fibrin and to cell receptors for PLG are generally equivalent.

Cleavage of Glu-PLG generates trace amounts of plasmin cleaved at the N-terminus at one or several of the following bonds: Arg68-Met69, Lys77-Lys78, or Lys78-Val79.^41,42 The final product obtained is termed Lys-plasmin (FIGURE 20.4). These two conformations differ in several features, including efficiency of activation and binding to fibrin. Small-angle neutron scattering revealed that Glu-PLG has a more closed form than Lys-PLG,⁴³ making it less readily activated. In the closed form, the five kringle domains and the catalytic domain interact to produce an ellipsoid shape. The open form of Lys-PLG, in contrast, has increased flexibility, which facilitates the binding of PAs some 10-fold.^44,45,46 Binding of εACA to Glu-PLG elicits a similar, but probably not identical change in conformation.⁴³ Lys 50 of Glu-PLG binds to kringle lysine binding site (LBS),^47,48 making them less available for binding to C-terminal lysine in fibrin, PLG receptors, and other proteins, so that Lys-PLG binds with higher affinity to fibrin than does Glu-PLG. The susceptibility of Glu-PLG to activation is dependent on additional factors, such as the presence of anions, which in the human plasma are mainly chloride ions, and of the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺. These ions counteract the effect of εACA and stabilize PLG in the Glu-form.^46,49

The PLG molecule is subject to considerable variation in the normal population. In addition to the limited proteolysis and activation processes already discussed, the two important sources of variation are glycosylation and genetic polymorphism.

The gene for tPA is located on bands p12-p11 on chromosome 8⁹⁷; it contains four exons⁹⁸ and comprises 32.7 kb. The mature protein exists in two forms, the N-terminus being Gly or Ser in Bowes melanoma cell tPA,⁹⁹ but predominantly Ser in recombinant tPA,¹⁰⁰ and the numbering in this chapter is based on Ser at position one. tPA expression in cultured cells can be stimulated through multiple intracellular signaling pathways. Vasoactive substances, such as thrombin and histamine, increase tPA synthesis in human umbilical vein endothelial cell (HUVEC), acting through their G-coupled receptors and the protein kinase C pathway.^101,102,103 Steroid hormones^104,105 and retinoids can increase synthesis of tPA.^106,107 The expression of tPA has been reviewed¹⁰⁸ and will not be covered in detail here. It is important to note that not all endothelial cells synthesize tPA in vivo and that expression is restricted to small vessels.^109,110

The structure of tPA (FIGURE 20.1) shows a finger and EGF domain, and two kringle domains in the A chain, whereas the protease domain resides in the B chain. The finger domain extends from residues 6 to 43. It contains a binding site for fibrin, as mutants lacking kringle domains are still capable of binding fibrin.¹¹¹ Consistent with this, a degraded form of tPA that had lost the N-terminal 12 kDa bound less well to fibrin than wild-type tPA.¹¹² The binding of the finger domain is independent of LBS, in that it cannot be blocked by εACA.¹¹³ Structurally, the finger domain is very similar to the seventh type 1 repeat of human fibronectin.¹¹⁴

The EGF domain (residues 44 to 92) is structurally similar to other EGF structures, as shown by nuclear magnetic resonance (NMR).¹¹⁵ There is some evidence from deletion mutagenesis that the EGF domain binds to the mannose receptor and is involved in tPA clearance.¹¹⁶ Kringle 2 has affinity for lysine, ω-amino acids, and fibrin, whereas no function has yet been ascribed to kringle 1.¹¹¹ The affinity for the binding of εACA (a model for C-terminal lysine residues) and of N-acetyllysine methyl ester (a model for intrachain lysine residues) is approximately equal, suggesting that unlike PLG, tPA does not have a preference for C-terminal lysine residues binding.¹¹³ Intact tPA and a variant consisting only of kringle 2 and the protease domains were found to bind to the cyanogen bromide (CNBr) fibrinogen fragment FCB-2, which also binds PLG and acts as a stimulator of tPA-catalyzed PLG activation.¹¹¹ In both cases, binding was completely inhibited by εACA, pointing to the involvement of LBS in this interaction.¹¹¹

The structure-function relations among kringle 2 and ω-amino acids and lysine have been studied in detail, using NMR,¹¹⁷ microcalorimetry,¹¹⁸ crystallography,¹¹⁹ and site-directed mutagenesis.^118,120,121 The crystal structure of kringle 2 resembles that of PLG kringle 4; there are, however, differences in the lysine-binding pocket. The core of kringle 2 is formed by a hydrophobic cluster of three tryptophan residues in positions 25, 63, and 74, surrounded by aromatic and hydrophobic side chains that form, at the surface of the kringle, a hydrophobic grove. Ligand binding appears to rely mostly on the integrity of Trp63 and Trp74, and an aromatic residue at position 76, which is normally Tyr.¹²² Mutation of the critical amino acids Lys33, Asp55, Asp57, or Trp72 markedly diminished binding to lysine-Sepharose, εACA, or both.¹²³

The protease domain of tPA has the typical catalytic triad His322, Asp371, and Ser478. The 2.3-Å crystal structure of the protease domain revealed strong structural similarity with other trypsin-like serine proteases, thrombin in particular.¹²⁴ The active site cleft is shaped and narrowed by four surface loops. The loop around Arg299 exhibits five additional residues, Arg298-Arg-Ser-Pro-Gly302, compared with chymotrypsin. It projects out of the molecular surface as a β-hairpin and is of fundamental importance for the interaction with PAI-1.³ The 60-loop around Arg327 is similar to but shorter than the corresponding loop in thrombin. Further loops are found around Ser381 and Gly465. The fully solvent-exposed hydrophobic region, comprising amino acids 420 to 423 of tPA, which forms a surface loop near one edge of the active site of tPA, is an important secondary site for the interaction of tPA with PLG in the absence of fibrin.

tPA is secreted as a single-chain molecule (sctPA) that is converted to the two-chain form (tctPA) by plasmin by cleavage at Arg275-Ile276. Unlike most serine proteases the single-chain form is not a zymogen but a protease that, in the presence of fibrin, is nearly as active as the two-chain form.¹²⁵ Mutagenesis studies have revealed that tPA lacks a specific zymogen triad Asp194, His40, and Ser32 (chymotrypsin numbering).^3,126 The constructed double mutant Phe305His and Ala292Ser was more zymogenic.¹²⁶

The major source of tPA in the circulation under basal conditions is thought to be the endothelial cell, from which it is constitutively released.¹²⁷ A secondary pool of tPA is present in specialized storage vesicles and is released in response to specific extracellular stimuli.^{128,129,130,131} High concentrations of tPA are contained within these storage vesicles that occur in various cells, such as endothelial, neuroendocrine, and adrenal chromaffin cells.^129,131 In rats, the tissue stores of tPA were calculated to be sufficient to maintain a steady-state plasma level of tPA for 2 days in the absence of protein synthesis.¹³² Compounds that triggered release within a few minutes include bradykinin, histamine, eledoisin, acetylcholine, β-adrenergic agents, platelet-activating factor, endothelin, calcium ionophore A-23187, and acidosis.^133,134 These compounds induce calcium influx into the endothelial cell and activate G-protein-coupled receptors.¹³⁵ Some interventions that cause elevated tPA act through decreased clearance, including exercise and α-adrenergic agents.¹³⁶ In all situations in which epinephrine levels are increased, such as stress, anxiety, and exercise, tPA antigen levels increase.^96,137

Acute release of tPA is established from early studies on venous occlusion.^138,139,140 Young patients with a history of recurrent venous thromboembolism exhibited an abnormal response to venous occlusion and this could be attributed either to subnormal tPA release or, more often, to elevated PAI-1.^95,139,140 In isolated cases with severe forms of von Willebrand disease, a complete lack of response to venous stasis and the infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) was found.¹⁴¹ These patients may have a functional or structural defect in their endothelial cells. It should be noted, however, that release of von Willebrand factor (vWF) and tPA occur by different mechanisms.¹⁴² Lower PA activity is found in leg veins than in proximal veins and may be one etiologic factor for development of deep venous thrombosis. DDAVP was widely used in the 1980s to study the release potential for tPA in patients with idiopathic or recurrent thrombosis.^143,144,145 Comparison of the effects of DDAVP and sodium nitroprusside showed that nitroprusside produced a greater increase of forearm blood flow but no increase of tPA; however DDAVP stimulated tPA release from the vascular bed.¹⁴⁶ The increase of circulating tPA levels after intra-arterial infusion of acetylcholine and methacholine was shown to be mediated by muscarinic receptors.^146,147,148 Bradykinin and substance P both induced tPA release after being infused into the forearm of volunteers,^149,150 and the releasable pool of tPA, rather than the availability of PAI-1 to inhibit it, was the key factor.¹⁵¹

tPA is unusually specific among the serine proteases. Its major substrate is PLG, in which it cleaves the Arg561-Val562 bond but is a relatively inefficient activator of PLG in the absence of its cofactor, fibrin.¹⁵² Binding of sctPA and tPA to fibrin is roughly comparable.¹⁵³ The K_d for binding of tPA to fibrin clots in the absence of PLG ranges from 140 to 400 nM.^125,153,154 In the presence of PLG, the affinity of tPA to fibrin increases approximately 20-fold (K_d 20 nM).¹⁵⁵ These observations are explained by formation of a ternary complex between tPA, PLG, and fibrin or binding of tPA to PLG, which subsequently, upon binding to fibrin, takes on an open conformation. The isolated A chain of tPA has been shown to bind to Glu- and mini-PLG with a K_d of 100 nM.¹⁵⁶

There is a wide range of kinetic parameters reported for the activation of PLG by tPA due to many potential experimental variables, such as different protein preparations, substrate
concentrations, and nature of the fibrin stimulator. In the absence of fibrin, K_M values for the activation of Glu-PLG by tPA range from 9 µM to slightly more than 100 µM.^125,157,158 In general, K_M is three to four times lower with tPA than with sctPA for the activation of Glu-PLG in the absence of fibrin. In the presence of fibrin, this difference is less apparent with K_M values typically two orders of magnitude smaller, with only moderate increases of k_cat. Values for K_M in the presence of fibrin range from 0.16 to 1.1 µM PLG, and k_cat values from 0.1 to 1.1 per second.^125,158 Several authors found nonlinear enzyme kinetics,^156,157,159 and there appear to be two phases in the activation of Glu-PLG by tPA in the presence of fibrin, with an initial K_M of 1.05 µM and k_cat of 0.15 per second. Later in the process, as new high-affinity binding sites for PLG and tPA are exposed in partially digested fibrin,^{160,161,162,163,164} K_M decreases to 0.07 µM, whereas the k_cat is unchanged.¹⁵⁹ The key message is that PLG is not readily activated by tPA except in the presence of fibrin, because it is only in this situation that the K_M for the reaction is consistent with the circulating PLG concentration of 2 µM.

Distinct sites in the fibrin molecule have been identified as accelerating PLG activation; notably, they are not available for binding in fibrinogen but become exposed in fibrin.¹⁶⁵ The D-region of fibrin binds both PLG and tPA in a region that encompasses the residues Aα148-160. Both these associations are of low affinity and in the circulation the Aα148-160 site would bind only PLG, which is present in much higher concentrations than tPA. Another binding in the D-region encompasses γ311-336 and γ337-379, which are linked by a disulfide bond.¹⁶⁶ Antibodies have revealed that there is a tPA binding site that includes γ312-324.¹⁶⁷ Higher affinity sites for binding tPA and PLG are in the C-terminus of the α chain, within the region Aα392-610.¹⁶⁸ The conformational changes that occur on fibrinogen cleavage and fibrin assembly¹⁶⁹ reveal new sites for binding of tPA and PLG, and for enhancement of PLG activation.

Mutations have been engineered in tPA to change its characteristics, with the aim of making it an even more effective therapeutic agent. The most striking of these is the series of mutations to make tPA resistant to PAI-1. Madison et al.¹⁷⁰ identified the importance of interactions between the positively charged tPA sequence 298 to 302 and the negatively charged PAI-1 sequence 350 to 355. Mutagenesis of Arg298, 299, and 304 resulted in a mutant that was inhibited 120,000 times less rapidly by PAI-1 than wild-type tPA.¹⁷¹ These observations led, in part, to the development of the new tPA mutant tenecteplase (TNK-tPA), in which the sequence 296 to 299 (Lys-His-Arg-Arg) is replaced by four alanines.¹⁷² This variant also exhibits slower clearance, by virtue of its changed glycosylation, and its enhancement of PLG activation is more selective for fibrin over fibrinogen and the fibrin degradation product DD/E.¹⁷³

The human uPA gene is located on chromosome 10q24 and is 6.4 kb in length.¹⁸⁰ It contains 11 exons, and the intron-exon organization of the uPA gene closely resembles the tPA gene. The regulation of its expression has been reviewed in detail elsewhere¹⁰⁸ and is not included here. uPA is produced as a 54-kDa, single-chain glycoprotein, 411 residues long (Table 20.1) and containing three domains: an EGF domain, a kringle, and a protease domain (FIGURE 20.1). The EGF domain interacts with urokinase plasminogen activator receptor (uPAR) found on many cells, as discussed in subsequent text. The kringle domain has no affinity for fibrin but has been reported to stabilize the interaction of uPA with uPAR.¹⁸¹ The protease domain contains the catalytic triad, His204, Asp255, and Ser356, and has approximately 40% sequence identity with tPA.⁹⁸ uPA is normally glycosylated at Asn302, whereas a covalently attached single monosaccharide, fucose, to Thr18,¹⁸² appears to affect the mitogenicity of uPA.¹⁸³ There are two phosphorylation sites on Ser138 and Ser303. The phosphorylated form diminishes uPA’s interaction with cells and PAI-1.¹⁸⁴

Activation of single-chain urinary plasminogen activator (scuPA, also called proUK) to the active enzyme, uPA (UK), is by cleavage of Lys158-Ile159.¹⁸⁵ Many enzymes can cleave this bond but the most important to hemostasis are plasmin,^186,187 factor XIIa, and kallikrein.¹⁸⁸ In PLG-deficient mice activation of scuPA is ascribed to kallikrein.¹⁸⁹ Cleavage close to the activation site (Lys158-Ile159) generates an inactive form of uPA. Thrombin cleaves Arg156-Phe157 in scuPA^188,190,191 and the inactive product can be subsequently activated through release of the N-terminal dipeptide of its B chain by cathepsin C or, albeit slowly, by plasmin.^185,192 Granulocyte elastase and cathepsin G cleave the Ile159-Ile160 peptide bond to yield an inactive product.¹⁹³ uPA has two active forms: HMW-UK and LMW-UK.¹⁹⁴ These are, respectively, full-length active uPA and a processed form, cleaved at Lys135-Lys136 to yield the amino-terminal fragment (ATF), which interacts with uPAR, leaving 21 residues of the A chain and an intact B chain.

The question of whether scuPA is a true zymogen was debated for several years, a controversy that was fuelled by the problem of trace plasmin or uPA contamination, with reciprocal activation of scuPA and PLG.¹⁹⁵ The issue has been resolved by studies on mutant forms of the proteins and by examining scuPA in the absence of PLG. For instance, the Lys158Glu mutant of scuPA, which cannot be cleaved by plasmin into uPA, still converted PLG to plasmin.¹⁹⁶ The consensus is that scuPA has approximately 0.5% of the activity of uPA.^{197,198,199,200} Therefore, scuPA lies on the spectrum between PLG, a true zymogen, and tPA, which is almost fully active in its single-chain form. Lys300 and Asp355 are key structural elements of scuPA’s enzymatic
activity. Lys300, situated in the flexible loop region 297 to 313, forms a weak interaction with Asp355 and pulls the adjacent active site Ser356 into the position found in the fully active protease. Site-directed mutagenesis of Lys300 to Ala²⁰¹ or of Asp355 to Asn²⁰² resulted in a 40-fold and 270-fold reduction in activity, respectively, compared to wild-type scuPA. Altering the flexibility of the 297-313 loop changes the intrinsic catalytic activity of scuPA.²⁰³

A cellular receptor for uPA (uPAR; CD87) was first isolated on human monocytes²⁰⁴ but is expressed on several cell types. The gene for uPAR (gene code PLAUR) is on chromosome 19 and consists of seven exons and six introns extending over 23 kb.^205,206 Regulation of its expression has been reviewed¹⁰⁸ and is not included here. The 1.4-kb mRNA encodes a signal peptide of 22 and a protein of 313 residues, with a glycosylphosphatidyl inositol (GPI) anchor (FIGURE 20.5). The N-terminus of the receptor consists of three homologous domains; region 1 interacts with uPA and regions 2 and 3 enhance binding.²⁰⁷