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Molecular Basis of Fibrinolysis
Fibrinolysis is the process by which the insoluble protein fibrin is converted to a defined set of soluble degradation products. It occurs in both intravascular and extravascular locations and is essential to human health and survival. Modern molecular biologic techniques have identified the major fibrinolytic genes, the mechanisms regulating their expression, and the consequences of their deficiency or overexpression in genetically engineered mice. In many cases these experiments have yielded surprising and instructive data that have revealed physiologic and pathophysiologic roles for the fibrinolytic system that are much more complex than originally thought. Furthermore, in the current genomic age, molecular analysis of human syndromes has identified specific mutations that result in either thrombosis secondary to fibrinolytic deficiency or hemorrhage secondary to fibrinolytic excess. In this chapter the molecular basis of fibrinolysis and its physiologic roles in human health and disease are reviewed.
Basic Concepts of Fibrinolysis
In response to vascular injury and activation of the coagulation cascade, thrombin induces polymerization of the soluble plasma protein fibrinogen, thereby producing an insoluble, cross-linked end product called fibrin (see Chapter 27 ). Once the flow of blood has been stemmed and vascular integrity restored, fibrin, which is found in both intravascular and extravascular sites, is cleared by a process known as fibrinolysis ( Fig. 28-1, A ). Normally, the fibrinolytic process is tightly regulated by a series of activators, inhibitors, cofactors, and receptors. In the presence of fibrin, which serves as a cofactor for its own destruction, tissue plasminogen activator (tPA) is released from endothelial cells and possibly other cell types and interacts with the circulating zymogen plasminogen. Plasminogen, tPA, and fibrin form a ternary complex that accelerates the catalytic efficiency of plasmin generation by approximately 500-fold. Urokinase is also an efficient plasminogen activator (uPA), but its action is only minimally enhanced by fibrin. The action of plasmin on fibrin generates soluble fibrin degradation products, many of which have their own unique biologic properties.
The dynamic regulation of plasmin generation is complex. On the surface of a fibrin-containing thrombus, tPA and plasmin are protected from their major circulating inhibitors plasminogen activator inhibitor 1 (PAI-1) and α 2 -antiplasmin (α 2 -AP), respectively. On release into the circulation, however, plasmin and tPA are rapidly neutralized by these inhibitors and cleared by the liver. Because uPA and the nonenzymatic plasminogen activator streptokinase do not use fibrin as a cofactor, they function well in the fluid phase. Plasminogen may also be activated, albeit rather inefficiently, by proteases of the contact system such as kallikrein, factor XIa, and factor XIIa (see Chapter 27 ).
Cell surfaces represent protected environments in which plasmin can be generated without the risk of neutralization by fluid-phase inhibitors ( Fig. 28-1, B ). Endothelial cells, platelets, monocytes, macrophages, and some tumor cells all express protein receptor sites for plasminogen, tPA, or uPA. The broad substrate specificity of plasmin observed in vitro may relate to its generation in nonvascular sites through fibrin-independent mechanisms. Plasmin may thus play an important role in extravascular events such as the modification of growth and differentiation factors, processing of matrix proteins, and activation of procoagulant molecules.
Components of the Fibrinolytic System
Plasminogen
Synthesized primarily in the liver, plasminogen is a single-chain proenzyme with a molecular weight (M r ) of approximately 92,000 that circulates in plasma at a concentration of approximately 1.5 µmol/L ( Table 28-1 ). The plasma half-life of plasminogen in humans is approximately 2 days. Its 791 amino acids are cross-linked by 24 disulfide bridges, 16 of which yield five homologous triple-loop structures called kringles ( Fig. 28-2 ). The first (K1) and fourth (K4) of these 80–amino acid structures with an M r of 10,000 impart high- and low-affinity lysine binding, respectively. The lysine-binding domains of plasminogen appear to mediate its specific interactions with fibrin, cell surface receptors, and other proteins, including its circulating inhibitor α 2 -AP.
TABLE 28-1
Fibrinolytic Proteins
| PROTEASES | ||||
| Property | Plasminogen | tPA | uPA | |
| Molecular mass (D) | 92,000 | 72,000 | 54,000 | |
| Amino acids | 791 | 527 | 411 | |
| Chromosome | 6 | 8 | 10 | |
| Site of synthesis | Liver | Endothelium | Endothelium | |
| Neuronal cells | Neuronal cells | Kidney | ||
| Glial cells | Glial cells | |||
| Plasma concentration | ||||
| nmol/L | 1500 | 0.075 | 0.150 | |
| µg/mL | 140 | 0.005 | 0.008 | |
| Plasma half-life | 48 hr | 5 min | 8 min | |
| N -Glycosylation (%) | 2 | 13 | 7 | |
| Form 1 | — | N117, N184, N448 | N302 | |
| Form 2 | N288 | N117, N448 | — | |
| O -Glycosylation | ||||
| α-Fucose | — | T61 | T18 | |
| Complex | T345 | — | — | |
| Two-chain cleavage site | R560-V561 | R275-I276 | K158-I159 | |
| Heavy chain domains | ||||
| Finger | No | Yes | No | |
| Growth factor | No | Yes | Yes | |
| Kringles (no.) | 5 | 2 | 1 | |
| Light-chain catalytic triad | H602, D645, S740 | H322, D371, S478 | H204, D255, S356 | |
| MAJOR SERPIN INHIBITORS | ||||
| Property | α 2 -AP | PAI-1 | PAI-2 | |
| Molecular mass (D) | 70,000 | 52,000 | 60,000 (glycosylated) | |
| 47,000 (nonglycosylated) | ||||
| Amino acids | 452 | 402 | 393 | |
| Chromosome | 18 | 7 | 18 | |
| Sites of synthesis | Kidney, liver | ECs | Placenta | |
| Monocytes/Macrophage | Monocytes/Macrophage | |||
| Hepatocytes | Tumor cells | |||
| Adipocytes | ||||
| Plasma concentration | ||||
| nmol/L | 900 | 0.1–0.4 | ND | |
| µg/mL | 50 | 0.02 | ND | |
| Serpin reactive site | R364-M365 | R346-M347 | R358-T359 | |
| Specificity | Plasmin | uPA = tPA | uPA > tPA | |
| NONSERPIN INHIBITORS | ||||
| Property | TAFI | α 2 -MG | ||
| Molecular mass (D) | 45,000 | 725,000 (monomer ~180,000) | ||
| Amino acids | 423 | 1451 | ||
| Chromosome | 13 | 12 | ||
| Sites of synthesis | Liver | Liver, Macrophage | ||
| Endothelium | ||||
| Fibroblasts | ||||
| Plasma concentration | ||||
| nmol/L | ~75 | ~2000–5000 | ||
| µg/mL | 4 | ~1450–3625 | ||
| Activators | Plasmin >> thrombin | — | ||
| Specificity | C-terminal K and R | Broad spectrum | ||
| SOME PROPOSED ACTIVATION RECEPTORS | ||||
| Property | uPAR | Annexin A2 Complex (A2-p11) 2 | PlgR- KT | |
| A2 | S100A10 (p11) | |||
| Molecular mass (D) | 55-60,000 | 36,000 | 11,000 | 17,000 |
| Amino acids | 313 | 339 | 97 | 147 |
| Chromosome | 19 | 15 | 1 | 9 |
| Source | ECs | ECs | M | Monocytes |
| Monocytes | ||||
| Macrophage | Macrophage | |||
| Fibroblasts | Early myeloid cells | |||
| Tumor cells | Tumor cells | |||
| Ligand(s) | uPA | tPA, Plg | Plg, tPA | Plg |
α 2 -AP, α 2 -Antiplasmin; α 2 -MG, α 2 -macroglobulin; EC, endothelial cell; LRP, low-density lipoprotein receptor-like protein; PAI, plasminogen activator inhibitor; ND, not determined; Plg, plasminogen; TAFI, thrombin activatable fibrinolysis inhibitor; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor.
Posttranslational modification of plasminogen results in two glycosylation variants (forms 1 and 2) (see Table 28-1 ). An O -linked oligosaccharide on Thr345 is common to both forms. Only form 2, however, contains an N -linked oligosaccharide on Asn288. The carbohydrate portion of plasminogen appears to regulate its affinity for cellular receptors and may also specify its physiologic degradation pathway.
Activation of plasminogen results from cleavage of a single Arg-Val peptide bond at position 560 to 561, which produces the active protease plasmin (see Table 28-1 ). Plasmin contains a typical serine protease catalytic triad but exhibits broad substrate specificity in comparison to other proteases of this class. The circulating form of plasminogen, amino-terminal glutamic acid plasminogen (Glu-Plg), is readily converted by limited proteolysis to several modified forms known collectively as lysine plasminogen (Lys-Plg). Hydrolysis of the Lys77-Lys78 peptide bond results in an altered conformation that more readily binds fibrin, displays twofold to threefold higher avidity for cellular receptors, and is activated 10 to 20 times more rapidly than Glu-Plg is. Lys-Plg does not normally circulate in plasma but has been identified on cell surfaces.
Spanning 52.5 kilobases (kb) of DNA on chromosome 6q26-27, the plasminogen gene consists of 19 exons and directs expression of a 2.7-kb messenger RNA (mRNA) (see Fig. 28-2 ). Plasminogen gene activity is stimulated by the acute phase mediator interleukin-6, both in vitro and in vivo. The gene is closely linked and structurally related to that of apolipoprotein(a), an apoprotein associated with the highly atherogenic low-density lipoprotein–like particle lipoprotein(a) and more distantly related to other kringle-containing proteins such as tPA, uPA, hepatocyte growth factor, and macrophage-stimulating protein. The significance of the latter two proteins to the fibrinolytic system remains to be determined.
Plasminogen Activators
Tissue Plasminogen Activator
One of the two major endogenous plasminogen activators, tPA, is a 527–amino acid glycoprotein with an M r of approximately 72,000 (see Table 28-1 ). tPA contains five structural domains, including a fibronectin-like “finger,” an epidermal growth factor–like domain, two kringle structures homologous to those of plasminogen, and a serine protease domain (see Fig. 28-2 ). Cleavage of the Arg275-Ile276 peptide bond by plasmin converts tPA to a disulfide-linked, two-chain form. Although single-chain tPA is less active than two-chain tPA in the fluid phase, both forms demonstrate equivalent activity when bound to fibrin.
The two glycosylation forms of tPA are distinguishable by the presence (type 1) or absence (type 2) of a complex N -linked oligosaccharide moiety on Asn184 (see Table 28-1 ). Both types, however, contain a high-mannose carbohydrate on Asn117, a complex oligosaccharide on Asn448, and an O -linked α-fucose residue on Thr61. The carbohydrate moieties of tPA may modulate its functional activity, regulate its binding to cell surface receptors, and specify degradation pathways.
Located on chromosome 8p12-q11.2, the gene for human tPA is encoded by 14 exons spanning a total of 36.6 kb. Most of the structural domains of tPA are encoded by one or two exons, and the organization of these exons is similar across related domains of tPA and the other fibrinolytic proteases (see Fig. 28-2 ). This observation suggests that the tPA gene arose by an evolutionary process called “exon shuffling,” whereby functionally related genes are generated through rearrangement of exons encoding autonomous domains. Consistent with this hypothesis, various functions of tPA can be localized to specific domains. For example, deletion of the fibronectin-like finger or kringle 2, but not kringle 1, results in a tPA resistant to the cofactor activity of fibrin, whereas catalytic activity in the absence of fibrin remains intact.
The proximal promoter of the human tPA gene contains binding sequences for potentially important transcriptional factors, including AP1, NF1, SP1, and AP2, as well as a potential cyclic adenosine monophosphate (cAMP)-responsive element. In vitro, many agents have been shown to exert small effects on the expression of tPA mRNA, but relatively few enhance tPA synthesis without augmenting PAI-1 synthesis as well. Agents that regulate tPA gene expression independently of PAI-1 include arterial shear stress, thrombin, endotoxin, histamine, butyrate, retinoids, and dexamethasone. Forskolin, which increases intracellular cAMP levels, has been reported to decrease the synthesis of both tPA and PAI-1.
tPA is synthesized in the endothelial cell and stored within Rab3D-negative granules that are distinct from classical Weibel-Palade bodies. Its release is governed by a variety of stimuli such as shear stress, butyrate, thrombin, histamine, bradykinin, epinephrine, acetylcholine, arginine vasopressin, gonadotropins, exercise, and venous occlusion. Its circulating half-life is exceedingly short (≈5 minutes). By itself, tPA is a poor activator of plasminogen. However, in the presence of fibrin, the catalytic efficiency of tPA-dependent plasmin generation ( k cat / K m ) increases by at least 2 orders of magnitude because of a dramatic increase in affinity (decreased Michaelis constant [ K m ]) between tPA and its substrate plasminogen in the presence of fibrin. Although it is expressed by extravascular cells, tPA appears to represent the major intravascular activator of plasminogen.
Urokinase
The second endogenous plasminogen activator, single-chain uPA or prourokinase, is a glycoprotein with an M r of approximately 54,000 and consists of 411 amino acids (see Table 28-1 ). uPA contains an epidermal growth factor–like domain and a single plasminogen-like kringle and possesses a classic catalytic triad within its serine protease domain (see Fig. 28-2 ). Cleavage of the Lys158-Ile159 peptide bond by plasmin or kallikrein converts single-chain uPA to a disulfide-linked two-chain derivative. Located on chromosome 10, the human uPA gene is encoded by 11 exons spanning 6.4 kb and expressed by activated endothelial cells, macrophages, renal epithelial cells, and some tumor cells. As noted earlier, its intron-exon structure is closely related to that of the tPA gene.
uPA may be induced during neoplastic transformation, possibly through a mechanism involving the transcription factors AP1 and AP2. Other agents that appear to induce expression of uPA in vitro include hormones, growth factors, and cAMP. Inflammatory cytokines such as interleukin-1 and lipopolysaccharide induce only small increments in uPA expression, whereas tumor necrosis factor and transforming growth factor β (TGF-β) have a more dramatic (5- to 30-fold) effect.
Two-chain uPA occurs in both high-molecular-weight (M r of 54,000) and low-molecular-weight (M r of 33,000) forms that differ by the presence or absence, respectively, of a 135-residue amino-terminal fragment released by plasmin cleavage between Lys135 and Lys136. Although both forms are capable of activating plasminogen, only the high-molecular-weight form binds to the uPA receptor (uPAR). uPA has much lower affinity for fibrin than tPA does and is an effective plasminogen activator both in the presence and in the absence of fibrin. The extent to which prourokinase possesses intrinsic plasminogen-activating capacity is an area of controversy.
Accessory Plasminogen Activators
Under certain conditions, proteases traditionally classified within the intrinsic arm of the coagulation cascade (see Chapter 26 ) have been shown to be capable of activating plasminogen directly. Such proteases include kallikrein, factor XIa, and factor XIIa. Normally, however, they account for no more than 15% of the total plasmin-generating activity in plasma.
Inhibitors of Fibrinolysis
Plasmin Inhibitors
The action of plasmin is negatively modulated by a family of ser ine p rotease in hibitors called serpins (see Table 28-1 ). All serpins share a common mechanism of action by forming an irreversible complex with the active-site serine of the target protease after proteolytic cleavage of the inhibitor by the target protease. Within such a complex, both protease and inhibitor lose their activity.
A single-chain glycoprotein with an M r of approximately 70,000, α 2 -AP circulates in plasma at relatively high concentrations (≈0.9 µmol/L) and has a plasma half-life of 2.4 days (see Table 28-1 ). This serpin contains about 13% carbohydrate by mass and consists of 452 amino acids with two disulfide bridges. In humans the gene is located on chromosome 18 and contains 10 exons distributed over 16 kb of DNA. The promoter region of the α 2 -AP gene contains a hepatitis B–like enhancer element that directs tissue-specific expression in the liver. α 2 -AP is also a constituent of platelet alpha granules. Plasmin released into flowing blood or in the vicinity of a platelet-rich thrombus is immediately neutralized on forming an irreversible 1 : 1 stoichiometric, lysine binding site–dependent complex with α 2 -AP. Interaction with plasmin is accompanied by cleavage of the Arg364-Met365 peptide bond, and the resulting covalent complexes are cleared in the liver.
Several other proteins inhibit the activity of fibrinolytic serine proteases (see Table 28-1 ). α 2 -Macroglobulin (α 2 -MG) is a tetrameric protein with an M r of 725,000 that is synthesized by the liver, endothelial cells, macrophages, and fibroblasts and is found in platelet alpha granules. The gene for α 2 -MG, which consists of 36 exons distributed over 48 kb of DNA on chromosome 12, directs the expression of a 1451–amino acid polypeptide. As a generic inhibitor of all four classes of proteases (serine, cysteine, aspartyl, and metallo), α 2 -MG is a nonserpin that inhibits plasmin with approximately 10% of the efficiency exhibited by α 2 -AP by forming a noncovalent complex. C1 esterase inhibitor can also serve as an inhibitor of tPA in plasma. Protease nexin may function as a noncirculating cell surface inhibitor of trypsin, thrombin, factor Xa, urokinase, or plasmin and result in protease-inhibitor complexes that are endocytosed via a specific nexin receptor.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that acts as a potent inhibitor of fibrinolysis (see Table 28-1 ). Identical to the previously cloned carboxypeptidase B and the previously isolated carboxypeptidase U, this single-chain polypeptide with an M r of 45,000 circulates in plasma at concentrations of about 75 nmol/L and undergoes limited proteolysis in the presence of plasmin or thrombin, which leads to its activation. Carboxypeptidase B–like molecules remove carboxyl-terminal lysine or arginine residues from fibrin and other proteins, thereby reducing binding of plasminogen to fibrin and cell surfaces and limiting plasmin generation. The potentially antifibrinolytic effect of thrombin appears to be mediated through its ability to activate TAFI in the presence of thrombomodulin. Anticoagulation by inhibition of factor XI also appears to have an antifibrinolytic effect in vivo by downregulation of thrombin-mediated activation of TAFI. In a system of purified components, TAFI has been shown to reduce tPA-induced fibrinolysis half-maximally at approximately 1 nmol/L, which is well below its concentration in plasma.
Plasminogen Activator Inhibitors
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