Is the streptokinase responsible for the endothelial injury and the platelet activation during fibrinolytic therapy?
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Complement Activation↗
Biomedical subjects
R Machovich
Publications and source records attributed to R Machovich.
Inhibition of plasmin activity by sulfated polyvinylalcohol-acrylate copolymers.
The effect of four sulfated polyvinylalcohol-acrylate copolymers and heparin on plasminogen activation and on plasmin activity is studied. The molecules differing in charge (proportion of negatively charged units 40.5%-73.5% of the total) and in size (5600 Da-8800 Da) accelerate plasminogen activation by 2- up to 4-fold at a 7-fold molar excess of the polyvinylacrylates over plasminogen. They, however, exert a concentration and charge-dependent effect on plasmin: both the amidolytic (half-maximal effect at a 1.33-3.66 molar excess of the polyvinylacrylates) and fibrinolytic (half-maximal effect at 1.23-1.72 molar excess of the polyvinylacrylates) activities of plasmin are inhibited. In contrast, heparin (a similarly carboxylated and sulfated polymer) and polyvinylacrylates with a low number of sulfate groups (30% sulfated monomers) at concentrations up to 2.2 microM do not affect plasminogen activation and plasmin activity in a milieu of physiological ionic strength. Experiments with plasmin derivatives lacking N-terminal peptides of different length (des-kringle(1-4) and des-kringle(1-5) plasmin) show identical changes in the protease activities, precluding involvement of the kringle-domain in the interaction with the polyvinylacrylates. Fluorescence studies evidence the charge-dependent binding of the polyvinylacrylates to plasmin, but not to plasminogen. Thus, through non-covalent interaction with the protease-domain of plasmin the polyvinylacrylates inhibit fibrinolysis. Since these sulfated copolymers inhibit both thrombin [4] and plasmin activity, they may be a useful therapeutic tool in situations when both the blood coagulation and the fibrinolytic system are activated (such as intravascular coagulation and fibrinolysis, ICF).
Acrylic Resins↗
Modulation of plasminogen activation and plasmin activity by methylglyoxal modification of the zymogen.
The effect of methylglyoxal on the plasminogen-plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH(2) side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations: the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus.
Animals↗
Streptokinase does not activate the complement system.
Streptokinase is an extensively used thrombolytic agent. However, different preparations cause severe hypotension during therapy, partially related to the complement cascade activation. In four ischaemic stroke patients treated with Streptase, an increased level of soluble terminal complement complex (SC5b-9) was measured. In the sera of normal subjects, the increase in SC5b-9 induced by Streptase, Kabikinase and Calbiochem streptokinases was highly significant (P < 0.005). Sigma streptokinase did not activate the complement system. Sigma streptokinase analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a homogeneous band. The other three preparations were contaminated with albumin and other proteins. Based on our in vivo and in vitro data, we conclude that complement activation is related to contamination of different streptokinase products rather than the streptokinase itself.
Acute Disease↗
Identification of thrombin receptors in rat brain capillary endothelial cells.
Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.
Animals↗
Facilitation of plasminogen activation by denatured prothrombin.
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Aminocaproates↗
Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors.
Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).
Animals↗
Flow rate-modulated dissolution of fibrin with clot-embedded and circulating proteases.
The efficiency of plasmin, miniplasmin, and neutrophil leukocyte elastase in fibrin digestion is well characterized in static systems. Since in vivo the components of the fibrinolytic system are permanently exposed to flow, we have developed two in vitro models and studied the effect of shear forces on fibrin dissolution with these proteases. Cylindrical nonocclusive fibrin clots are perfused at various flow rates through their preformed axial channel, and dissolution of fibrin is followed by measuring the absorbance of degradation products released into the circulating fluid phase. In one experimental setting, fibrin surface is degraded with enzymes applied in the recirculating fluid phase; in another setting, clots containing gel-embedded proteases are perfused with enzyme-free buffer. As shear rate at fibrin surface is changed from 25 to 500 s(-1), the rate of product release by recirculated enzymes increases 2.8-, 2.9-, and 4-fold for plasmin, miniplasmin, and porcine pancreatic elastase, respectively. Buffer-perfused fibrin containing gel-embedded plasmin or miniplasmin is disintegrated by shear forces at a relatively early stage of dissolution, and this disassembly is related to the formation of fragment Y (150 kDa) and fragment D (100 kDa) fibrin degradation products. Fibrin clots degraded by incorporated polymorphonuclear leukocyte elastase, which yields different degradation products, do not disassemble abruptly, even at the highest shear rate (500 s(-1)). Our results suggest that fibrin surface degradation is accelerated with increasing shear rate and that plasmin or miniplasmin embedded in the clot promotes the release of particular clot remnants into the circulating phase, whereas polymorphonuclear leukocyte elastase does not.
Animals↗
Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4.
The inflammatory mediators, cytokines and complement proteins are believed to regulate the sequential events during the development of lesions secondary to ischaemia and reperfusion. The endothelial cell monolayer of the brain microvasculature is the critical interface between the blood-borne mediators and brain tissue. The involvement of these cells in complement production and regulation has not been well documented. In the present study, expression of complement proteins (C1 inhibitor, factor H, factor B, C4) by cultured endothelial cells obtained from human brain microvessels has been characterized. Interferon gamma upregulates the production of all the complement factors studied. Serine proteases, plasmin and miniplasmin induce the expression of C4, decrease the level of ELISA detectable C1 inhibitor, and do not affect the production of factors H and B. These data indicate that complement proteins are expressed locally by the brain microvessels, and may modulate the inflammatory responses of brain tissue.
Brain↗
Prostaglandin-independent stimulation of interleukin-6 production by fibrinogen degradation product D in perfused murine liver.
Bacterial endotoxin (LPS) and fibrinogen degradation product D (FDP-D) are both potent stimulators of interleukin-6 (IL-6) production in liver, however, there are differences in their metabolic effects. The aim of the present study was to compare the role of prostaglandins in the enhancement of IL-6 production by LPS or FDP-D in perfused mouse livers. Indomethacin inhibited the effect of LPS significantly but was ineffective in the case of FDP-D. Accordingly, production of prostaglandins D2 and E2 was not elevated following the addition of FDP-D, while their formation was increased several fold by LPS. At the same time interleukin-1 (IL-1) production in perfused liver rose markedly upon the addition of FDP-D. It is suggested that prostaglandins are not involved in the effects of FDP-D on the liver. The stimulatory effect of FDP-P on IL-6 production might be the consequence of elevated IL-1 levels.
Animals↗
Perturbation of the integrity of the blood-brain barrier by fibrinolytic enzymes.
The action of fibrinolytic enzymes (plasmin, miniplasmin, neutrophil leukocyte elastase) on the blood-brain barrier is investigated. The binding and the effects of the fibrinolytic enzymes are studied in the first subcultivation of human brain capillary endothelial cells. 125I-labeled plasmin, miniplasmin and neutrophil leukocyte elastase bind to confluent monolayers of cultured endothelial cells with dissociation constants of 1 x 10(-8) mol/l, 4.8 x 10(-7) mol/l and 1.8 x 10(-8) mol/l, respectively, and the number of binding sites varies between 2.3 x 10(5) and 7.5 x 10(6) per cell. Following treatment of the cultured cells with purified and active-site titrated proteases, the changes in morphology of individual cells are analyzed with computerized morphometry. At low concentrations (in nanomolar range) all studied fibrinolytic proteases induce reduction of the cell area; the minimal size is achieved in 20-80 min after the application of an enzyme and the effect is completely reversed in 15 min after its removal. A possible in-vivo consequence of these in-vitro findings is studied in an organ-perfusion model: rat hemisphere is perfused with a protease solution followed by a circulating phase-borne tracer (horse-radish peroxidase). In perfused rat hemisphere, the fibrinolytic enzymes open the blood-brain barrier to the circulation-borne tracer. These results support the concept that fibrinolytic enzymes interact with the brain microvascular endothelium and thus affect the integrity of the blood-brain barrier through active cell contraction.
Blood-Brain Barrier↗
[Alternative ways of fibrinolysis].
This study surveys our knowledge on the molecular mechanisms of fibrinolysis; the plasmin activity and the formation of the enzyme (plasminogen activation and its regulation). The author tries to indicate the cause of clinical problems arising from the limitations of our present knowledge. On the basis of these and the most recent data (not available yet in handbooks), he attempts to call the attention of physicians to molecular and cellular reactions, which may modify our concept on the fibrinolysis.
Fibrinolysis↗
Denatured proteins as cofactors for plasminogen activation.
Activation of covalently intact plasminogen by tissue-type plasminogen activator (tPA) is facilitated by a majority of proteins subjected to denaturing conditions. Except for heat-denatured apoferritin, the denatured proteins examined require partial proteolysis by plasmin for cofactor activity. The same proteins in their native state are resistant to proteolysis with plasmin and develop no activity. Denatured preparations of apoferritin, antithrombin, alpha1-protease inhibitor, alpha2-macroglobulin, and albumin also accelerate des(1-77)-plasminogen activation by tPA. The rate enhancements are comparable with that of the fibrin(ogen) fragments on a w/w basis. The cofactor activities are inhibited by 6-aminohexanoate and inactivated by pepsin. Analysis of heat-denatured apoferritin and albumin preparations by ultracentrifugation and gel chromatography indicates that cofactor is associated predominately with aggregates, which have binding capacity for both tPA and zymogen. Heat-denatured albumin pretreated with plasmin decreases K(M) and increases k(cat) for both intact plasminogen and des(1-77)-plasminogen activation by tPA, yielding catalytic efficiencies in excess of 8 x 10(3) M(-1) s(-1) and 2 x 10(4) M(-1) s(-1), respectively. Because of enhanced plasmin-catalyzed proteolysis of plasminogen to des(1-77)-plasminogen, activation by urokinase-type plasminogen activator is also facilitated by denatured proteins; activation of des(1-77)-plasminogen is not affected. It is concluded that denatured proteins serve as both cofactors and substrates in the fibrinolytic system, and that enhancement of plasminogen activation by denatured proteins is mechanistically indistinguishable from that observed with fibrin.
Animals↗
Functional evaluation of the structural features of proteases and their substrate in fibrin surface degradation.
A new model has been introduced to characterize the action of a fluid phase enzyme on a solid phase substrate. This approach is applied to evaluate the kinetics of fibrin dissolution with several proteases. The model predicts the rate constants for the formation and dissociation of the protease-fibrin complex, the apparent order of the association reaction between the enzyme and the substrate, as well as a global catalytic constant (kcat) for the dissolution process. These kinetic parameters show a strong dependence on the nature of the applied protease and on the structure of the polymerized substrate. The kinetic data for trypsin, PMN-elastase, and three plasminogen-derived proteases with identical catalytic domain, but with a varied N-terminal structure, are compared. The absence of kringle5 in des-kringle1-5-plasmin (microplasmin) is related to a markedly lower kcat (0.008 s-1) compared with plasmin and des-kringle1-4plasmin (miniplasmin) (0.039 s-1). The essentially identical kinetic parameters for miniplasmin and plasmin with the exception of kdiss, which is higher for miniplasmin (81.8 s-1 versus 57.6 s-1), suggest that the first four kringle domains are needed to retain the enzyme in the enzyme-fibrin complex. Trypsin, a protease of similar primary specificity to plasmin, but with a different catalytic domain, shows basically the same kcat as plasmin, but its affinity to fibrin is markedly lower compared with plasmin and even microplasmin. The latter suggests that in addition to the kringle domains, the structure of the catalytic domain in plasmin also contributes to its specificity for fibrin. The thinner and extensively branched fibers of fibrin are more efficiently dissolved than the fibers with greater diameter and lower number of branching points. When the polymer is stabilized through covalent cross-linking, the kcat for plasmin and miniplasmin is 2-4-fold higher than on non-cross-linked fibrin, but the decrease in the association rate constant for the formation of enzyme-substrate complex explains the relative proteolytic resistance of the cross-linked fibrin. Thus, the functional evaluation of the discrete steps of the fibrinolytic process reveals new aspects of the interactions between proteases and their polymer substrate.
Aminocaproic Acid↗
Myosin as cofactor and substrate in fibrinolysis.
Myosin accelerates plasminogen activation by tissue-type plasminogen activator (tPA), and is degraded extensively by plasmin. Myosin binds both tPA and plasminogen, and enhances activation of des1-77-plasminogen by tPA but not by urokinase-type plasminogen activator (uPA). Myosin decreases K(M) and increases k(cat) for des1-77-plasminogen activation by tPA, to yield catalytic efficiencies in excess of 8000 M-1 s-1. The effect of myosin is attributed to its C-terminal portion, the myosin rod. With a K(M) of 3 microM, myosin is a high-affinity substrate for plasmin. The findings indicate that myosin is a cofactor for plasminogen activation and a substrate for plasmin.
Animals↗
Kinetic parameters for plasminogen activation by tissue type plasminogen activator.
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Fibrinolysin↗
Human brain microvessel endothelial cell culture as a model system to study vascular factors of ischemic brain.
Cerebral ischemia is caused by reduced blood supply at the microcirculatory level. In the microvessels, the main elements of the reperfusion injury following brain ischemia are the transformation of endothelial cell-surface from anticoagulant to procoagulant property, leukocyte adhesion, sludge or clot formation. There is a paucity of information on how hemostatic factors, cytokines, lipoprotein(a) (Lp(a)) and endothelin-1 (ET-1), being responsible for ischemic/reperfusion injury, interact with human brain microvessel endothelium (HBEC). There are no data furthermore about the expression of complement proteins of HBEC influenced by cytokines or fibrinolytic factors. Previously we established optimal conditions for culturing HBEC. Cell contraction induced by thrombin, plasmin, miniplasmin was recorded. The reassembly of F-actin was observed after thrombin treatment. ICAM-1 upregulation was measured following TNF-alpha, IL-1-alpha and thrombin incubation. Plasmin and miniplasmin downregulated the ICAM-1 in our cell culture system. Lp(a) modulated the thromboresistant cell-surface by reduction of t-PA and u-PA, but PAI-1 remained unchanged. Lp(a) modulated the ET-1 production by early increasing and late decreasing, in a bimodal manner. The increased secretion of ET-1 by cytokines (TNF-alpha, IL-1-alpha) was reduced in the presence of Lp(a). Gradual increase of complement proteins (factor H, factor B, C4) was induced by cytokines. Plasmin and miniplasmin augmented a rapid increase of C4. Some factors of complex relationship between regulators and modulators of endothelial adhesion molecules have been demonstrated in a human cell culture system prepared from brain microvessel endothelium. A unified concept of sequential events of ischemia/reperfusion in the brain has not yet developed.
Biological Factors↗
Quantitative comparison of fibrin degradation with plasmin, miniplasmin, neurophil leukocyte elastase and cathepsin G.
The relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using spectrophotometric kinetic analysis of the competition between fibrin and synthetic peptide substrates for the proteases, turbidimetric assay for fibrin dissolution and gel-filtration of the partially degraded fibrin. When the substrate is fibrin polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 x 10(5) M-1.s-1 for plasmin, 1.9 x 10(5) M-1.s-1 for miniplasmin, 5.0 x 10(4) M-1.s-1 for PMN-elastase and 2.2 x 10(3) M-1.s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6 for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive cross-linking of fibrin decreases the catalytic action of all studied proteases, but no change in their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at physiological concentration) is also cross-linked to fibrin, the most efficient fibrinolytic enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity lysine binding site in the N-terminal kringle domain of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to newly exposed lysine residues. These results provide a quantitative basis for the evaluation of fibrinolytic efficiency and support the concept of synergistic fibrinolysis.
Amino Acid Sequence↗