Basic mechanisms and regulation of fibrinolysis.

Fibrinolysis appears in many diverse physiological situations, and the components of the system are well established, along with mechanistic details for the individual reactions and some high-resolution structures. Key questions in understanding the regulation of fibrinolysis surround mechanisms of initiation and propagation, the localization of fibrinolysis reactions to the fibrin clot, and the influence of fibrin structure and clot composition on thrombolysis. This review covers these key areas with a focus on recent developments on fibrin structure and binding, the effects of a variety of cell types, the consequences of histones and DNA released by neutrophils, and the influence of flow. A complete understanding of the regulation of fibrinolysis will come from the building of detailed mathematical models. Suitable models are at an early stage of development, but may improve as model clots increase in complexity to incorporate the components and interactions listed above.

Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components.

BACKGROUND: Proteases expressed in atherosclerotic plaque lesions generate collagen fragments, release glycosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and expose extracellular matrix (ECM) proteins (e.g. decorin) at sites of fibrin formation. OBJECTIVE: Here we address the effect of these vessel wall components on the lysis of fibrin by the tissue plasminogen activator (tPA)/plasminogen system and on the mechanical stability of clots. METHODS AND RESULTS: MMP-8-digested collagen fragments, isolated CS, DS, glycosylated decorin and its core protein were used to prepare mixed matrices with fibrin (additives present at a 50-fold lower mass concentration than fibrinogen). Scanning electron microscopy (SEM) showed that the presence of ECM components resulted in a coarse fibrin structure, most pronounced for glycosylated decorin causing an increase in the median fiber diameter from 85 to 187 nm. Rheological measurements indicated that these structural alterations were coupled to decreased shear resistance (1.8-fold lower shear stress needed for gel/fluid transition of the clots containing glycosylated decorin) and rigidity (reduction of the storage modulus from 54.3 to 33.2 Pa). The lytic susceptibility of the modified fibrin structures was increased. The time to 50% lysis by plasmin was reduced approximately 2-fold for all investigated ECM components (apart from the core protein of decorin which produced a moderate reduction of the lysis time by 25%), whereas fibrin-dependent plasminogen activation by tPA was inhibited by up to 30%. CONCLUSION: ECM components compromise the chemical and mechanical stability of fibrin as a result of changes in its ultrastructure.

Hindered dissolution of fibrin formed under mechanical stress.

BACKGROUND: Recent data indicate that stretching forces cause a dramatic decrease in clot volume accompanied by gross conformational changes of fibrin structure. OBJECTIVE: The present study attempts to characterize the lytic susceptibility of fibrin exposed to mechanical stress as a model for fibrin structures observed in vivo. METHODS AND RESULTS: The relevance of stretched fibrin models was substantiated by scanning electron microscopic (SEM) evaluation of human thrombi removed during surgery, where surface fibrin fibers were observed to be oriented in the direction of shear forces, whereas interior fibers formed a random spatial meshwork. These structural variations were modeled in vitro with fibrin exposed to adjustable mechanical stress. After two- and three-fold longitudinal stretching (2 × S, 3 × S) the median fiber diameter and pore area in SEM images of fibrin decreased two- to three-fold. Application of tissue plasminogen activator (tPA) to the surface of model clots, which contained plasminogen, resulted in plasmin generation which was measured in the fluid phase. After 30-min activation 12.6 ± 0.46 pmol mm(-2) plasmin was released from the non-stretched clot (NS), 5.5 ± 1.11 pmol mm(-2) from 2 × S and 2.3 ± 0.36 pmol mm(-2) from 3 × S clot and this hampered plasmin generation was accompanied by decreased release of fibrin degradation products from stretched fibrins. Confocal microscopic images showed that a green fluorescent protein-fusion variant of tPA accumulated in the superficial layer of NS, but not in stretched fibrin. CONCLUSION: Mechanical stress confers proteolytic resistance to fibrin, which is a result of impaired plasminogen activation coupled to lower plasmin sensitivity of the denser fibrin network.

Neutrophil granulocyte-dependent proteolysis enhances platelet adhesion to the arterial wall under high-shear flow.

SUMMARY BACKGROUND: Under high shear stress platelets adhere preferentially to the adventitia layer of the arterial vessel wall in a von Willebrand factor (VWF)-dependent manner. OBJECTIVE: The present study was undertaken in an attempt to characterize the structural background of the relative thromboresistance of the media and the impact of neutrophil leukocyte-derived proteases (matrix metalloproteinases, neutrophil elastase) on platelet adhesion in this layer of the arteries. METHODS AND RESULTS: Platelet adhesion to cross-sections of the human iliac artery was monitored by indirect immunofluorescent detection of GpIIb/IIIa antigen. Exposure of the vessel wall to activated neutrophils or neutrophil-derived proteases increased platelet adhesion to the media about tenfold over the control level at 3350 s(-1) surface shear rate. In parallel with this enhanced thrombogenicity morphological changes in the media were evidenced by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The fine proteoglycan meshwork seen with Cupromeronic Blue enhancement of the SEM images was removed by the proteolytic treatment and the typical collagen fiber structure was exposed on the AFM images of the media. CONCLUSION: Through their proteases activated neutrophils degrade proteoglycans, unmask VWF binding sites and thus abolish the thromboresistance of the media in human arteries.

Proteolytic resistance conferred to fibrinogen by von Willebrand factor.

The formation of platelet-rich thrombi under high shear rates requires both fibrinogen and von Willebrand factor (VWF) as molecular adhesives between platelets. We attempted to describe the role of VWF as a potential substrate and modulator of the fibrinolytic system using binding assays, as well as kinetic measurements on the cleavage of fibrin(ogen) and a synthetic plasmin substrate (Spectrozyme-PL). The similar dissociation constants for the binding of plasminogen, plasmin, and active site-blocked plasmin onto immobilised VWF suggest that the primary binding site in plasmin(ogen) is not the active site. The progressive loss of clottability and generation of degradation products during fibrinogen digestion with plasmin were delayed in the presence of VWF at physiological concentrations, while VWF cleavage was not detectable. Determination of kinetic parameters for fibrinogen degradation by plasmin, miniplasmin and microplasmin showed that VWF did not modify the Km, whereas kcat values decreased with increasing VWF concentrations following the kinetic model of non-competitive inhibition. Inhibitory constants calculated for VWF were in the range of its physiological plasma concentration (5.4 mg/ml, 5.7 mg/ml and 10.0 mg/ml for plasmin, miniplasmin and microplasmin, respectively) and their values suggested a modulating role of the kringle 5 domain in the interaction between VWF and (mini)plasmin. VWF had no effect on the amidolytic activity of plasmin on Spectrozyme-PL, or on fibrin dissolution by (mini)plasmin. Our data suggest that VWF, while a poor plasmin substrate relative to fibrinogen, protects fibrinogen against degradation by plasmin preserving its clottability in plasma and its adhesive role in platelet-rich thrombi.

Fibrinolysis in a lipid environment: modulation through release of free fatty acids.

BACKGROUND: Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, but the structure of clots in vivo includes additional constituents (proteins, phospholipids) that modulate their solubilization. OBJECTIVE: We examined the presence of free fatty acids in thrombi and their effects on distinct stages of fibrinolysis (plasminogen activation, plasmin activity). METHODS AND RESULTS: Using the fluorescent probe acrylodated intestinal fatty acid-binding protein, variable quantities (up to millimolar concentrations) of free fatty acids were demonstrated in surgically removed human thrombi. Oleic acid at relevant concentrations reversibly inhibits more than 90% of the amidolytic activity of plasmin on a synthetic substrate (Spectrozyme PL), but only partially inhibits its fibrinolytic activity measured using turbidimetry. Chromogenic assays detecting the generated plasmin activity show that plasminogen activation by tissue-type plasminogen activator (t-PA) is completely blocked by oleic acid in the fluid phase, but is accelerated on a fibrin matrix. A recombinant derivative of t-PA (reteplase) develops higher fibrin specificity in the presence of oleic acid, because both the inhibition of plasminogen activation in free solution and its enhancement on fibrin template are stronger than with wild-type t-PA. CONCLUSION: Through the stimulation of plasminogen activation on a fibrin template and the inhibition of plasminogen activators and plasmin in the fluid phase, free fatty acids confine the action of fibrinolytic proteases to the site of clotting, where they partially oppose the thrombolytic barrier function of phospholipids.

Fibrinolysis at the fluid-solid interface of thrombi.

Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, a protease generated by plasminogen activators from its inactive precursor, plasminogen. Typically plasminogen activation occurs on the surface of the clot, where fibrin behaves as a cofactor in this process, and plasmin also initiates its proteolytic action at the fluid-solid interface. Although the basic reactions of the plasminogen/plasmin system in fluid phase are well characterized in terms of classical enzymology, they cannot explain completely the interfacial fibrinolytic events. Recently new methods have been introduced for quantitative evaluation of plasminogen activation on gel-phase fibrin and heterogenous-phase proteolysis, an overview of the new methodology is presented. Following formation of an interfacial lytic zone, fibrin dissolution proceeds through propagation of this zone to the core of the clot, which depends on diffusion and permeation phenomena affected by the composition of thrombi. Phospholipids (originating from platelets) form a diffusion barrier to the thrombolytic agents and also bind some of them; structural cellular proteins (namely myosin) interact with the fibrin fibers masking their cofactor and plasmin-cleavage sites. The contribution of these recent findings to our understanding of the limitations of current thrombolytic therapy is discussed. Finally, attention is focused on the termination of thrombus-associated proteolytic action in an environment abundant in proteinase inhibitors. Thus, combining together the interfacial events in the initiation, progress and termination of thrombolysis, a concept for modeling the thrombus as a temporary fibrinolytic compartment is presented.

Fluorescence studies on native and bound to trifluraline soy bean Lb"a" in the enhanced N2 fixation.

The differences in the tryptophan (Trp) fluorescence of native (control) Lb"a" and experimental substance isolated from nodules of the Williams' soy beans variety treated with trifluraline at a concentration of 2.1 x 10(-10) M have been studied. A positively charged environment has been proved for the tryptophans of the native Lb"a" and a negative one for the tryptophans of the experimental Lb"a". The difference in the tryptophan emission spectra at lambdaex = 280 and 300 nm may be assigned to conformational alterations occurring in the experimental Lb"a". This is also confirmed by the greater energy transfer from tyrosine to tryptophan in the experimental Lb"a"--30% compared to the 10% in the native Lb"a". The value of the constant of acrylamide quenching (Ksv = 2.77 M(-1)) shows that the tryptophans are buried more deeply in the experimental Lb"a" than in the native Lb"a" (Ksv = 4 M(-1)). They are substantially lower than Ksv of the standard compound N-Ac-Trp-NH2 (16.30 M(-1)). The activation energy (Ea) of the thermal quenching of tryptophan fluorescence is higher for the experimental Lb"a" (37 kJ mol(-1)) as compared to the standard compound N-Ac-Trp-NH2 (24 kJ mol(-1)) and the native Lb "a" (32 kJ mol(-1)). The dissociation constant of the complex of trifluraline with Lb "a" (6.32 x 10(-11) M) has been determined as well as the stoichiometric ratio trifluraline/Lb"a" (1:1). The estimated nitrogenase activity (microM/gfrw h) and the total Lb (mg/gfrw) for trifluraline are higher as compared to those for the control.

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).

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.

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.

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.

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.

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).

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.

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.

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.