The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

Mechanisms of plasminogen activation by mammalian plasminogen activators

Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg560-Val561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH2-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system.     

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.     

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)     

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159).

The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for 'Lys-plasminogen' (human plasminogen with N-terminal lysine) and 'Glu-plasminogen' (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation.     

A spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-tPA) for high-fibrin-affinity tissue plasminogen activators

A new spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-tPA), specific for the quantitation of tissue plasminogen activators, is described. The method is based on (1) the high-affinity binding (Kp = 1.4 +/- 2 nM) of tPA to a solid-phase fibrin network constructed by thrombin proteolysis of fibrinogen covalently coupled to polyglutaraldehyde-activated polyvinyl chloride microtiter plates, and (2) the subsequent development of PA activity by the fibrin-tPA complex and its measurement with a coupled assay using a chromogenic substrate highly selective for plasmin. Conditions were chosen such that the rate of para-nitroaniline release from the substrate is directly proportional to the concentration of tPA. The support is able to isolate tPA from the bulk of proteins present in any biological fluid allowing the assay to specifically detect tPA activity (range: 0.01 to 50 IU/ml) even in the presence of other activators, proteases, and inhibitors. Since the assay is done in a well-defined reaction mixture (the fibrin-tPA complex, plasminogen, and the synthetic substrate), kinetics studies using pure or crude tPA can be performed. Standard curves (rate measurement and endpoint methods) were made using the international standard (preparation 83/517) for tPA.     

Spectrophotometric method to quantify and discriminate urokinase and tissue-type plasminogen activators.

Plasminogen activator and urokinase are often used as biological markers of cell activation. However, the methods currently used are cumbersome, make no discrimination between tissue-type plasminogen activator and urokinase, and do not allow expression of the results of the overall reaction in International Units. The one-step method described in this paper lacks these drawbacks. Moreover, we propose use of H-D-Val-Phe-Lys-4-nitroanilide as substrate which has a lower Km than the standard H-D-Val-Leu-Lys-4-nitroanilide which is commercially available. Low concentrations of sodium dodecyl sulfate in the reaction mixture dramatically and preferentially accelerate the reaction catalyzed by tissue-type plasminogen activators. Identical results are obtained under kinetic or fixed-time assay conditions using either a photometer or 96-well plate reader. The corresponding formulae are provided.     

Electrochemical assay of plasminogen activators in plasma using polyion-sensitive membrane electrode detection

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these "clot-busting" drugs.     

Plasminogen activators and their potential in therapy

Plasminogen activators (PAs) are proteases that convert plasminogen to plasmin. Plasmin, in turn, is a protease that can lyse a fibrin clot and, therefore, PAs have a primary role in fibrinolysis. Two PAs, urokinase (UK) and streptokinase (SK), have been available for therapeutic use for years. Unfortunately, both can cause systemic fibrinogenolysis and other side effects which have limited their use. Interest has focused on a different enzyme, tissue plasminogen activator (t-PA), which will cause specific clot lysis without systemic problems. The gene for t-PA has been cloned and many biotechnology firms are preparing to produce t-PA for therapeutic use. The properties and potential for therapy of t-PA are reviewed and compared to new forms of other activators, such as pro-urokinase. How the interactions of PAs and inhibitors may affect the use of PAs is also discussed.     

Tissue plasminogen activator mRNA in murine tissues

The urokinase-type and tissue-type plasminogen activators are the two enzymes found in mammals, which specifically convert the zymogen plasminogen to plasmin. Using cDNA probes, we have assayed for the presence of the two types of plasminogen activator mRNAs in murine tissues. We demonstrate that tissue-type plasminogen activator mRNA can be detected in a wide variety of tissues. In contrast, the accumulation of urokinase-type plasminogen activator mRNA is observed in only a few of the tissues analyzed. Using an S1 nuclease assay, we demonstrate that the tPA mRNA detected contains the complete sequences encoding the non-protease finger, growth-factor and kringle domains.     

Highly potent fibrinolytic serine protease from Streptomyces

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.     

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.     

The potential improvement of thrombolytic therapy by targeting with bispecific monoclonal antibodies: Why they are used and how they are made

The generation of the proteolytic enzyme plasmin from its inactive precursor plasminogen, mediated by so called plasminogen activators, is the essential step in thrombolytic therapy. Plasmin is responsible for the degradation of the insoluble fibrin, the major component of a thrombus, to soluble fibrin degradation products. So far, the use of the more recently developed thrombolytic agents single-chain urokinase-type plasminogen activator (scu-PA) and tissue-type plasminogen activator (t-PA) were disappointing, mainly due to some of their negative propertiesin vivo, i.e., rapid inhibition and/or hepatic clearance. Besides some background information on the haemostatic balance; t-PA and scu-PA structure; and mechanisms of action, we here review some reported attempts to improve on these agents for thrombolytic therapy following various strategies. One of the more potential strategies, antibody-targeted thrombolytic therapy using bispecific monoclonal antibodies, is discussed somewhat more extensively, as are the several procedures that can befollowed for bispecific antibody preparation.     

The X-ray crystal structure of full-length human plasminogen

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.     

Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation

The extracellular serine protease, plasmin, is activated from its precursor, plasminogen (Plg), by the urokinase-type and tissue-type Plg activators (uPA and tPA respectively). One of the main plasmin substrates, fibrin, is formed from fibrinogen via thrombin activity. We have previously shown that mice deficient for Plg are strikingly less able to support a litter during lactation compared to wild type mice. Here we suggest a mechanism responsible for this lactation defect. Reduced epithelial content and increased apoptosis are observed in Plg-deficient mammary glands at lactation day 7. Immunofluorescence analysis reveals the presence of fibrin(ogen) in the stroma surrounding mammary alveoli and adipocytes and identifies fibrin(ogen) as a component of breast milk in both wild type and Plg-deficient mice. Furthermore, a large accumulation of fibrin(ogen) together with apoptotic epithelial cells is observed in the lactating mammary alveoli and ducts of some Plg-deficient mice. This suggests that fibrin plays a key role in the malfunction of mammary glands in the absence of Plg, possibly through blockade of mammary ducts inducing milk stasis, inhibiting milk expulsion and thereby inducing premature apoptosis and involution.     

Monolithic peptidyl sorbents for comparison of affinity properties of plasminogen activators

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein's denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA-EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.     

A colorimetric assay for the simultaneous measurement of plasminogen activators and plasminogen activator inhibitors in serum-free conditioned media from cultured cells

The coupled photometric assay for plasminogen activator reported by Coleman and Green (1981) Methods in Enzymology (Lorand, L., Ed.), Vol. 80, pp. 408-414, Academic Press, San Diego, CA) has been adapted for use with 96-well plates and an automatic microplates spectrophotometer. The assay allows the discrimination between tissue-type and urokinase-type plasminogen activators in cell culture-conditioned media. It provides a level of detection of these enzymes in the range 10(-17) to 10(-13) mol (determined using purified human plasminogen activators), uses no radioisotopes, and is faster and more economical than similar assays using specific peptide substrates for plasminogen activators. Levels of free plasminogen activator inhibitor activity can be simultaneously measured on the same samples by a simple adaptation of the assay. This method allows an easy treatment of the data by interfacing with a computer and should thus be useful when large numbers of samples are assayed.     

Serum amyloid A enhances plasminogen activation: implication for a role in colon cancer

We have recently reported that the acute phase protein serum amyloid A (SAA), is locally and differentially expressed in neoplastic tissues of human colon. In the present study, we demonstrate that SAA enhances the plasminogen activation (PA)-activity of HT-29 colon cancer cell line. Cell-associated PA-activity was measured following the plasminogen-dependent ability of the cells to cleave the chromogenic substrate S-2251. The SAA-enhanced PA-activity was inhibited by anti-SAA antibodies. These antibodies also decreased the basal PA-activity of HT-29 cells and neutralized their cytokines (Interleukin-1β + Interleukin-6)—enhanced PA-activity. Using specific chromogenic substrates and the fibrin clot-lysis assay, we found that SAA enhances also the PA-activity mediated by purified urokinase- and tissue-type plasminogen activators. Together, the data indicate that SAA enhances plasminogen activation and suggest its possible role in plasmin(ogen)-mediated colon cancer progression.     

Activated thrombin-activatable fibrinolysis inhibitor reduces the ability of high molecular weight fibrin degradation products to protect plasmin from antiplasmin

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B-like plasma enzyme that can slow clot lysis by removing lysine residues exposed on fibrin as it is cleaved by plasmin. Previously, it was shown that fibrin treated with TAFIa is less able to promote plasminogen activation by tissue-type plasminogen activator. In this study, the effect of TAFIa modification of a fibrin surface on the rate of plasmin inhibition by antiplasmin was studied using high molecular weight fibrin degradation products (HMw-FDPs) as a soluble model for intact plasmin-modified fibrin. To quantify the inhibition, a novel end point assay was employed where plasmin, antiplasmin, and cofactors were mixed in the presence of a chromogenic substrate and the end point in the substrate hydrolysis reaction was used to measure the second order rate constant of inhibition. When HMw-FDPs were titrated in the presence of plasmin and antiplasmin, the rate constant for inhibition decreased by 16-fold at saturation (9.6 x 10(6) m(-1) s(-1) to 0.59 x 10(6) m(-1) s(-1)). When HMw-FDPs were pretreated with TAFIa, nearly two-thirds of the protective effect was lost. When 730 nm HMw-FDPs were treated for 20 min with TAFIa, the rate constant for plasmin inhibition was increased 3-fold from 1.9 x 10(6) m(-1) s(-1) to 6.2 x 10(6) m(-1) s(-1). Therefore, a novel mechanism was identified whereby TAFIa can modulate plasmin levels by increasing the susceptibility of plasmin to inhibition by antiplasmin.     

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.