Fibrinolytic system of cultured endothelial cells: regulation by plasminogen activator inhibitor

Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.     

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.     

Development of research into the physiology and pathophysiology of the fibrinolysis system

A modern data review on the importance of fibrinolysis system is given. A considerable success has been scored during the study of molecular parameters of fibrinolysis system: the plasminogen, plasmin, its inhibitors, plasminogen activators and the mechanism of activation system have been characterized. The entrance of A, K, C, P and PP vitamins has been established to be necessary for the normal functioning of the fibrinolysis system; the dependence of the blood fibrinolytic activity upon the initial plasminogen content and concentration of its activators in blood has been revealed. The plasminogen activator depletion in tissues has been shown to be one of the reasons of some pathological states development, especially at cardiovascular diseases. The increase of fibrinolysis level by the active fibrinolytic ferment injection in blood has a medical effect at thrombosis. The ferment fibrinolysin received in the laboratory is successfully used in clinical practice. Some other activators of fibrinolytic system: tricholysine and longolytin from the culture of saprophyte fungi, plasminogen activator from the pig heart and the cells culture of the calf kidney have been received and are being studied.     

Structure and function of plasminogen/plasmin system

The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: degradation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in various functions performed by plasmin thus formed.     

Serine proteases of the fibrinolysis pathway are not involved in lethal hepatitis and fibrinogen breakdown induced by tumor necrosis factor

Tumor necrosis factor (TNF) plays a key role in several types of fulminant and acute hepatitis, and induces massive apoptosis and necrosis of hepatocytes. Our previous studies described the central role played by several matrix metalloproteinases (MMPs) and one or more unknown serine proteases. The aim of this study was to investigate the involvement of serine proteases of the fibrinolytic pathway, known to be activators of several MMPs, in TNF-induced hepatitis and fibrinogen (FG) breakdown. Experiments were performed in a model of TNF-induced hepatitis, consisting of administration of TNF in combination with D-(+)-galactosamine (GalN) to mice deficient in urokinase-type plasminogen (PG) activator (u-PA), tissue-type PG activator (t-PA) or PG. Lethality, transaminase release, increased plasma clotting time and FG levels were measured. In PA- and PG-deficient mice, TNF/GalN still induced hepatitis, as well as increased clotting time and FG breakdown. MMP-9 activation still occurred in the liver despite the lack of plasmin. The data suggest that the serine proteases involved in TNF-induced lethal hepatitis are no constituents of the fibrinolytic cascade.     

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)     

Development of new thrombolytic agents using recombinant DNA technology.

The increasing incidence of thromboembolic diseases has sustained the search for new agents able to stimulate the natural fibrinolytic system. The first generation of antithrombotic agents include bacterial streptokinase and human urine urokinase. Because these molecules lack specificity for the fibrin clot, important efforts have been made to produce, using recombinant DNA technology, agents presenting higher fibrin clot selectivity such as t-PA (tissue-type plasminogen activator) and scu-PA (single chain urokinase-type plasminogen activator). In parallel, several laboratories are presently attempting to create mutants and hybrids plasminogen activators displaying improved thrombolytic properties with respect to the natural molecules. In this paper, we describe briefly the mechanisms of fibrinolysis and the role of the different natural thrombolytic agents. In addition, we review the possibilities of genetic engineering for the production of natural and novel plasminogen activators.     

Recent advances in understanding endogenous fibrinolysis: implications for molecular-based treatment of vascular disorders

The fate of a forming thrombus is determined through the delicate balance between the coagulation cascade, favouring clot formation, and the fibrinolytic system, favouring clot lysis. These processes occur simultaneously, and enhancement of fibrinolysis has been shown to reduce occlusive thrombus formation in animal models. This review examines the roles of the major fibrinolytic factors involved in clot lysis. The regulation of plasmin activity by plasminogen activators, alpha-2-antiplasmin, plasminogen activator inhibitor 1, and thrombin-activatable fibrinolysis inhibitor, and their effects on thrombus formation in vivo are discussed. Since alterations in fibrinolytic capacity appear to affect thrombus formation in animal models, there is considerable interest in the pharmacological manipulation of fibrinolysis.     

The multifaceted role of plasminogen in inflammation

A fine-tuned activation and deactivation of proteases and their inhibitors are involved in the execution of the inflammatory response. The zymogen/proenzyme plasminogen is converted to the serine protease plasmin, a key fibrinolytic factor by plasminogen activators including tissue-type plasminogen activator (tPA). Plasmin is part of an intricate protease network controlling proteins of initial hemostasis/coagulation, fibrinolytic and complement system. Activation of these protease cascades is required to mount a proper inflammatory response. Although best known for its ability to dissolve clots and cleave fibrin, recent studies point to the importance of fibrin-independent functions of plasmin during acute inflammation and inflammation resolution. In this review, we provide an up-to-date overview of the current knowledge of the enzymatic and cytokine-like effects of tPA and describe the role of tPA and plasminogen receptors in the regulation of the inflammatory response with emphasis on the cytokine storm syndrome such as observed during coronavirus disease 2019 or macrophage activation syndrome. We discuss tPA as a modulator of Toll like receptor signaling, plasmin as an activator of NFkB signaling, and summarize recent studies on the role of plasminogen receptors as controllers of the macrophage conversion into the M2 type and as mediators of efferocytosis during inflammation resolution.     

Role of plasminogen activators in peritoneal adhesion formation

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.     

Mechanism of fibrinolysis and thrombolytic therapy

The thrombolytic treatment with plasminogen activators, such as physiological tissue-type plasminogen activator (t-PA), suffers from a number of significant limitations. There is a resistance to reperfusion and acute coronary reocclusion. The peculiarity of t-PA and one-chain urokinase treatment is their using in very high doses. Thus the process of thrombolytic therapy is proceeding with a deviation from the fibrinolytic mechanism, which is needs of a little quantity of tissue-type plasminogen activator and provides the physiologic thrombolysis without systemic complication. The estimation of this disaccordance suggests, the possible reasons of these complications.     

Effect of physical exercise on fibrinolytic properties of erythrocytes

The fibrinolytic properties of blood and erythrocytes were studied before and after physical exercise in male volunteers. Their fibrinolytic responses were of two distinct types. In type 1 response, fibrinolytic activities of blood and erythrocytes increased; the plasminogen activator and active plasmin contents in erythrocytes also increased, whereas the profibrinolysin content correspondingly decreased. In addition, physical exercise increased the erythrocyte adsorption properties for plasma activators of fibrinolysis. Type 2 response was characterized by a decrease in the fibrinolytic activity of blood; neither fibrinolytic activity nor adsorption properties of erythrocytes increased. The type of blood and erythrocyte response to muscular activity was determined by the pre-exercise level of red blood cell fibrinolytic activity. It was low in type 1 response due to a lesser content of plasmin activators and greater content of antiplasmin. In type 2 response, the initially high lytic capacity is connected with a greater reserve of activators and lesser reserve of inhibitors of the fibrinolytic system. A conclusion was made that individual differences in fibrinolytic responses to physical exercise were largely accounted for by the properties of erythrocytes.     

Biological control of tissue plasminogen activator-mediated fibrinolysis

Fibrinolysis, the body's ability to degrade fibrin, is an integrated part of hemostasis. Overactivity in the fibrinolytic system causes bleeding and underactivity causes thrombosis. Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), alpha 2-antiplasmin (alpha 2-AP) and plasminogen are definitely involved in fibrinolysis because: (1) these components can be assigned a fibrinolytic role in purified systems, i.e. in vitro, and (2) abnormal structural variants and abnormal levels of these components give rise to bleeding or to thrombosis. The biological control of tPA-mediated fibrinolysis is both cellular and humoral. The cellular regulation compasses synthesis of tPA and PAI-1 and release/uptake of these components. The humoral regulation involves: (1) the reaction between tPA and PAI-1; (2) the fibrin-stimulated plasminogen activation; (3) the reaction between plasmin and alpha 2-AP and (4) plasmin degradation of fibrin. The highly developed biological control of tPA-mediated fibrinolysis is indicative of its physiological importance.     

Fibrinolytic activity of human peripheral blood granulocytes

The fibrinolytic activity of human peripheral blood leukocytes was studied by plating the cells on 125I-fibrin coated dishes. The separation of the three major leukocyte types allowed to demonstrate that most of the activity was produced by granulocytes. The rate of fibrinolysin was found to be linear with incubation time and cell number in the range of 1-4 X 10(5) cells/ml. Since little activity was found in absence of exogenous plasminogen, it was concluded that the cell fibrinolytic activity depended mostly upon the release of plasminogen activator. Plasmatic and granulocytic activators obtained from the same amount of blood were found to be of similar level suggesting a possible clinical implication of the cellular activity in the thrombolytic system.     

Role of the fibrinolytic and matrix metalloproteinase systems in development of adipose tissue

Obesity is a common disorder and related diseases such as diabetes, atherosclerosis, hypertension, cardiovascular disease and cancer are a major cause of mortality and morbidity in Western-type societies. Development of obesity is associated with extensive modifications in adipose tissue involving adipogenesis, angiogenesis and extracellular matrix proteolysis. The fibrinolytic (plasminogen/plasmin) and matrix metalloproteinase (MMP) systems cooperate in these processes. A nutritionally induced obesity model in transgenic mice has been used extensively to study the role of the fibrinolytic and MMP systems in the development of obesity. These studies support a role of both systems in adipogenesis and obesity; the role of specific members of these families, however, remains to be determined.     

Plasminogen activators: general aspects and recent developments

Considerable interest in plasminogen activators as human thrombolytic drugs has stimulated rapid biotechnologic progresses. These enzymes have been classified in two immunochemically distinct groups: "urokinase-like" activators or u-PA which do not interact with fibrin and "tissue activator-like" activators or t-PA which interact with fibrin. Plasminogen activators are widely distributed in normal and malignant tissues and they are implicated in various physiological and pathological processes. They maintain the functional integrity of the vascular system and their presence may be of importance in tissue remodeling and cell migration. Urokinase and streptokinase are used in human thrombolytic therapy. However, the properties displayed by t-PA suggest that this enzyme may be a superior fibrinolytic agent. The primary structures of urokinase and t-PA are known; both enzymes have been synthesized by DNA technology. In order to produce t-PA in large quantities by gene cloning, intensive studies are conducted by pharmaceutical industries. Clinical trials using t-PA for dissolving thrombi in coronary heart disease, strokes and pulmonary embolism are in progress. This review presents the molecular and structural properties of plasminogen activators, as well as related physiological, pathological and therapeutic aspects.     

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.     

Hormonal regulation of plasminogen activator in rat hepatoma cells

Plasminogen activators are membrane-associated, arginine-specific serine proteases which convert the inactive plasma zymogen plasminogen to plasmin, an active, broad-spectrum serine protease. Plasmin, the major fibrinolytic enzyme in blood, also participates in a number of physiologic functions involving protein processing and tissue remodelling, and may play an important role in tumor invasion and metastasis. In HTC rat hepatoma cells in tissue culture, glucocorticoids rapidly decrease plasminogen activator (PA) activity. We have shown that this decrease is mediated by induction of a soluble inhibitor of PA activity rather than modulation of the amount of PA. The hormonally-induced inhibitor is a cellular product which specifically inhibits PA but not plasmin. We have isolated variant lines of HTC cells which are selectively resistant to the glucocorticoid inhibition of PA but retain other glucocorticoid responses. These variants lack the hormonally-induced inhibitor; PA from these variants is fully sensitive to inhibition by inhibitor from steroid-treated wild-type cells. Cyclic nucleotides dramatically stimulate PA activity in HTC cells in a time- and concentration-dependent manner. Paradoxically, glucocorticoids further enhance this stimulation. Thus glucocorticoids exert two separate and opposite effects on PA activity. The availability of glucocorticoid-resistant variant cell lines, together with the unique regulatory interactions of steroids and cyclic nucleotides, make HTC cells a useful experimental system in which to study the multihormonal regulation of plasminogen activator.     

Role of the pleiotropic effects of plasminogen deficiency in infection experiments with plasminogen-deficient mice

Plasminogen-deficient mice hold great promise as tools for analyzing the contribution of plasminogen activators produced by infectious agents to pathogenesis. However, the pathology caused by congenital plasminogen deficiency complicates the interpretation of infection experiments conducted with these animals. This pathology, the most prominent features of which are poor weight gain, wasting after about 60 days of age, and shortened lifespan, results from the inability of the mice to clear small fibrin thrombi. This article describes strategies for distinguishing the contribution of this pathology from the direct effects of depriving infectious agents of plasminogen. These strategies depend on the use of mouse genotypes in which the correlation of plasminogen deficiency with fibrin-dependent pathology is broken. Mice with plasminogen activator deficiencies are unable to generate plasmin and develop pathologies identical to those seen in plasminogen-deficient mice. However, unlike plasminogen-deficient mice, they do make plasminogen available to the infectious agent. Fibrinogen-deficient mice also deficient for plasminogen do not develop the pathology typical of plasminogen deficiency. These mice allow examination of plasminogen deficiency in the absence of fibrin-dependent pathology. Use of fibrinogen-deficient mice is complicated by the possibility that fibrin may be the key substrate of plasmin generated by the infectious agent.