Plasminogen activation: biochemistry, physiology, and therapeutics

The mammalian serine protease zymogen, plasminogen, can be converted into the active enzyme plasmin by vertebrate plasminogen activators urokinase (uPA), tissue plasminogen activator (tPA), factor XII-dependent components, or by bacterial streptokinase. The biochemical properties of the major components of the system, plasminogen/plasmin, plasminogen activators, and inhibitors of the plasminogen activators, are reviewed. The plasmin system has been implicated in a variety of physiological and pathological processes such as fibrinolysis, tissue remodeling, cell migration, inflammation, and tumor invasion and metastasis. A defective plasminogen activator/inhibitor system also has been linked to some thromboembolic complications. Recent studies of the mechanism of fibrinolysis in human plasma suggest that tPA may be the primary initiator and that overall fibrinolytic activity is strongly regulated at the tPA level. A simple model for the initiation and regulation of plasma fibrinolysis based on these studies has been formulated. The plasminogen activators have been used for thrombolytic therapy. Three new thrombolytic agents--tPA, pro-uPA, and acylated streptokinase-plasminogen complex--have been found to possess better properties over their predecessors, urokinase and streptokinase. Further improvements of these molecules using genetic and protein engineering tactics are being pursued.     

Bacterial plasminogen activators and receptors

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.     

Nonfibrinolytic functions of plasminogen

Although the roles of plasminogen and plasmin in mediating blood clot dissolution are well known, the availability of mice deficient for components of the fibrinolytic system has allowed direct approaches to be made toward elucidating the role of these proteins in other diverse physiological and pathophysiological processes. A number of these studies have identified plasminogen as playing an important role in inflammation and other cell migratory processes. With the identification of receptors for plasminogen on a number of pathogens, and the ability to activate plasminogen through either endogenous production of plasminogen activators or utilization of host activators, mice deficient for components of the fibrinolytic system offer a unique approach toward further elucidating the importance of this system in pathogen infection and dissemination.     

Cyclic AMP phosphodiesterase inhibitors depress production of plasminogen activator by Chinese hamster ovary cells.

Oncogenic transformation in a limited number of cell systems has been shown by others to be associated with an increased production of extracellular proteolytic activators that convert the plasma proenzyme, plasminogen, to the active protease, plasmin. In the present study, two cyclic AMP phosphodiesterase inhibitors (theophylline, papaverine) markedly depressed the production of intracellular and extracellular plasminogen activator by Chinese hamster ovary cells of the CHO-Kl line in serum-free medium. Prostaglandin E₁ had a moderately similar effect on the production of only extracellular plasminogen activator. The ability to control experimentally the level of production of plasminogen activator should be of value in elucidating the possible biological role of the proteolytic action of plasmin on the surface of CHO cells, and the cell surface alterations which accompany oncogenic transformation.     

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system

Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1–3, K1–4, and K1-4.5 (0–2 μM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 μM) had no effect, while 2 μM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.     

Protease nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.     

Bacterial metastasis: the host plasminogen system in bacterial invasion

Several pathogenic bacterial species intervene with the mammalian proteolytic plasminogen-plasmin system. Recent developments have been made in understanding the structure and the virulence-associated functions of bacterial plasminogen receptors and activators, in particular by using plasminogen-deficient or transgenic gain-of-function mice. Bacteria can affect the regulation of the plasminogen system by degrading circulating plasmin inhibitors and by influencing the expression levels of mammalian plasminogen activators and activation inhibitors. Interaction with the plasminogen system promotes damage of extracellular matrices as well as bacterial spread and organ invasion during infection, suggesting common mechanisms in migration of eukaryotic and prokaryotic cells.     

Plasminogen Activators in Vascular Remodeling and Angiogenesis

This review considers cellular and molecular mechanisms of the involvement of plasminogen activators in extracellular proteolysis and cell migration and proliferation. The role of plasminogen activators in vascular remodeling in atherosclerosis, restenosis, and angiogenesis is discussed.     

The human plasma fibrinolytic system: Regulation and control

Summary The factors involved in the regulation and control of the human plasma fibrinolytic system at the cellular level are unknown at this time. The physiological regulation of plasmin formation in plasma depends primarily on the nature of the circulating zymogen, plasminogen, the physiological activators formed both in the blood and in the vascular endothelium, and the specific plasmin inhibitors found both in plasma and in certain of the cellular elements of the blood. The biosynthesis of the zymogen must be under genetic control, and the activators are probably released, after thrombus and clot formation, from components involved in the surface-mediated initiation of the coagulation system, and from the vascular endothelium. Activation of plasminogen can occur both in the fluid phase surrounding the thrombus and probably at thrombus surfaces, involving both the fibrin clot and the platelet membrane. The plasmin inhibitors act to control the system in order to prevent proteolytic degradation of important physiological trace proteins of the coagulation, complement and kallikrein-kinin systems by the enzyme.     

Cellular mechanisms regulating non-haemostatic plasmin generation

A variety of proteases have the potential to degrade the extracellular matrix (ECM), thereby influencing the behaviour of cells by removing physical barriers to cell migration, altering cell-ECM interactions or releasing ECM-associated growth factors. The plasminogen activation system of serine proteases is particularly implicated in this pericellular proteolysis and is involved in pathologies ranging from cancer invasion and metastasis to fibroproliferative vascular disorders and neurodegeneration. A central mechanism for regulating plasmin generation is through the binding of the two plasminogen activators to specific cellular receptors: urokinase-type plasminogen activator to the glycolipid-anchored membrane protein uPAR, and tissue plasminogen activator to a type-II transmembrane protein recently identified on vascular smooth muscle cells. These binary complexes interact with membrane-associated plasminogen to form higher order activation complexes that greatly reduce the K(m) for plasminogen activation and, in some cases, protect the proteases from their cognate serpin inhibitors. Various other proteins that are involved in cell adhesion and migration also interact with these complexes, modulating the activity of this efficient and spatially restricted proteolytic system. Recent observations demonstrate that certain forms of the prion protein can stimulate tissue plasminogen activator-catalysed plasminogen activation, which raises the possibility that these proteases may also have a role in the pathogenesis of the transmissible spongiform encephalopathies.     

Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots

Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM epsilon-aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen-depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.     

Molecular mechanisms of plasminogen activation: bacterial cofactors provide clues

Plasminogen activation is a key event in the fibrinolytic system that results in the dissolution of blood clots, and also promotes cell migration and tissue remodelling. The recent structure determinations of microplasmin in complex with the bacterial plasminogen activators staphylokinase and streptokinase have provided novel insights into the molecular mechanisms of plasminogen activation and cofactor function. These bacterial proteins are cofactor molecules that contribute to exosite formation and enhance the substrate presentation to the enzyme. At the same time, they modulate the specificity of plasmin towards substrates and inhibitors, making a 'specificity switch' possible.     

The Receptor for Urokinase-Type Plasminogen Activator of a Human Keratinocyte Line (HaCaT)

It is assumed that plasmin participates in pericellular proteolysis in the epidermis. Plasmin is generated by keratinocyte-associated plasminogen activators from the proenzyme plasminogen; plasminogen activation can proceed at the keratinocyte surface. The resultant plasmin interferes with cell to matrix adhesion and does possibly contribute to keratinocyte migration during reepithelialization. Here we describe the receptor for urokinase-type plasminogen activator (uPA-R) in the human keratinocyte cell line HaCaT, which serves to direct plasminogen activation to the cell surface; we relate the receptor to the uPA-R previously described in human myclo-/monocytes. Binding of uPA to the receptor accelerated plasminogen activation by a factor of ≈10, compared to uPA in solution. Receptor-bound uPA was susceptible to inhibition by the plasminogen activator inhibitors 1 and 2. uPA and uPA-R antigen, as well as uPA activity, were localized to the leading front of expanding sheets of HaCaT cells. Exposure of HaCaT cells to plasminogen was followed by detachment of the cells. Detachment was prevented by an anti-catalytic anti-uPA antibody, by the plasmin-specific inhibitor aprotinin, and by the lysine analogue tranexamic acid, the latter of which prevents plasmin(ogen) binding to the cell surface. Our findings support the hypothesis that uPA-mediated plasminogen activation is characteristic of mobile rather than sessile keratinocytes. Moreover, the uPA-R seems to focalize plasminogen activation to the surface of cells at the site of keratinocyte migration.     

Fibrinolysis and Proliferative Endarteritis: Two Related Processes in Chronic Infections? The Model of the Blood-Borne Pathogen Dirofilaria immitis

The interaction between blood-borne pathogens and fibrinolysis is one of the most important mechanisms that mediate invasion and the establishment of infectious agents in their hosts. However, overproduction of plasmin (final product of the route) has been related in other contexts to proliferation and migration of the arterial wall cells and degradation of the extracellular matrix. We have recently identified fibrinolysis-activating antigens from Dirofilaria immitis, a blood-borne parasite whose key pathological event (proliferative endarteritis) is produced by similar mechanisms to those indicated above. The objective of this work is to study how two of this antigens [actin (ACT) and fructose-bisphosphate aldolase (FBAL)] highly conserved in pathogens, activate fibrinolysis and to establish a relationship between this activation and the development of proliferative endarteritis during cardiopulmonary dirofilariasis. We demonstrate that both proteins bind plasminogen, enhance plasmin generation, stimulate the expression of the fibrinolytic activators tPA and uPA in endothelial cell cultures and are located on the surface of the worm in contact with the host’s blood. ELISA, western blot and immunofluorescence techniques were employed for this purpose. Additionally, the implication of lysine residues in this interaction was analyzed by bioinformatics. The involvement of plasmin generated by the ACT/FBAL and plasminogen binding in cell proliferation and migration, and degradation of the extracellular matrix were shown in an “in vitro” model of endothelial and smooth muscle cells in culture. The obtained results indicate that ACT and FBAL from D. immitis activate fibrinolysis, which could be used by the parasite like a survival mechanism to avoid the clot formation. However, long-term overproduction of plasmin can trigger pathological events similar to those described in the emergence of proliferative endarteritis. Due to the high degree of evolutionary conservation of these antigens, similar processes may occur in other blood-borne pathogens.     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Key Role for the Urokinase Plasminogen Activator (uPA) in Invasive Group A Streptococcal Infection

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.     

Tissue-type plasminogen activator gene is on chromosome 8

Tissue plasminogen activator is one of the two plasminogen activators, both serine proteases, that catalyze the conversion of inactive plasminogen to plasmin, which then degrades the fibrin network of blood clots. By combining somatic cell genetics, in situ hybridization, and Southern blot hybridization, we localized the human tissue plasminogen activator gene to the pericentromeric region of chromosome 8.     

Antiprotease inactivation by Salmonella enterica released from infected macrophages

The mammalian serine protease plasmin, which has an important role in extracellular matrix degradation during cell migration, is regulated by the plasma antiprotease alpha(2)-antiplasmin (alpha(2)AP). The surface protease PgtE of Salmonella enterica serovar Typhimurium proteolytically inactivated alpha(2)AP. PgtE also activates the plasma zymogen plasminogen to plasmin, and bacteria expressing PgtE promoted degradation of extracellular matrix laminin in the presence of plasminogen and alpha(2)AP. alpha(2)AP inactivation was detected with the rough derivative of S. enterica 14028, but not with the smooth wild-type strain, suggesting that the O-antigen of lipopolysaccharide prevented contact of PgtE with the substrate molecule. After growth of S. enterica 14028 in murine J774A.1 macrophage-like cells, the infected cell lysate as well as bacteria from isolated Salmonella-containing vacuoles (SCVs) cleaved alpha(2)AP. Bacteria from SCVs produced an elevated level of PgtE and had a reduced O-antigen chain length. The lysate from S. enterica 14028-infected macrophages promoted formation of plasmin in the presence of alpha(2)AP, whereas plasmin formation by lysates from uninfected macrophages, or from macrophages infected with the pgtE-negative derivative of 14028, was inhibited by alpha(2)AP. Salmonella disseminates in the host within macrophages, which utilize plasmin for migration through tissue barriers. The results suggest that intracellular enhancement of PgtE activity in Salmonella may promote macrophage-associated proteolysis and cellular migration by altering the balance between host plasmin and alpha(2)AP.     

Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes

The binding of urokinase-type plasminogen activators (u-PA) to receptors on various cell types has been proposed to be an important feature of many cellular processes requiring extracellular proteolysis. We have investigated the effect of single-chain u-PA binding to the monocyte-like cell line U937 on plasminogen activation. A 16-fold acceleration of the activation of plasminogen was observed at optimal concentrations of single-chain u-PA. This potentiation was abolished by the addition of either 6-aminohexanoic acid or the amino-terminal fragment of u-PA, thus demonstrating the requirement for specific binding of both single-chain u-PA and plasminogen to the cells. The mechanism of the enhancement of plasmin generation appears to be due primarily to an increase in the rate of feedback activation of single-chain u-PA to the more active two-chain u-PA by cell-bound plasmin, initially generated by single-chain u-PA. This increased activity of the plasminogen activation system in the presence of U937 cells provides a mechanism whereby u-PAs may exert their influence in a variety of cell-associated proteolytic events.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.