Cleavage of surfactant-incorporating fibrin by different fibrinolytic agents. Kinetics of lysis and rescue of surface activity

Incorporation of surfactant into polymerizing fibrin causes loss of surface activity and marked retardation of clot lysis by plasmin (Günther and colleagues, Am. J. Physiol. 1994;267:L618-L624). We compared the efficacy of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), activated anisoylated streptokinase-plasminogen activator complex (APSAC), and plasmin to dissolve surfactant-incorporating fibrin. Alveofact was employed as a natural surfactant source, and plasminogen was coincorporated into the fibrin matrix at a physiologic ratio to fibrin. Fibrinolysis was quantified by the release of tracer from (125)I-labeled fibrin, and the pattern of split products was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, we investigated the fibrinolysis-related restoration of surface activity by measurement in the pulsating bubble surfactometer. Concentrations of all fibrinolytic agents were chosen to effect approximately 40% lysis of clot material in the absence of surfactant (control). When incorporated into the fibrin matrix, but not when admixed after clot formation, surfactant inhibited the cleavage of fibrin by all fibrinolytic agents in a dose-dependent manner. Interestingly, t-PA and u-PA were significantly less inhibited than was plasmin or APSAC. The pattern of arising fibrin scission products was identical for all fibrinolytic approaches and was independent of surfactant incorporation. Adsorption and minimum surface tension-lowering properties of Alveofact were almost completely lost upon incorporation into fibrin, but surface activity was fully restored upon sustained clot lysis with all fibrinolytic agents. We conclude that the fibrinolytic capacity of all agents investigated is markedly inhibited by surfactant incorporation in fibrin, but this inhibition is significantly less pronounced in the agents employing preincorporated plasminogen (t-PA and u-PA), as compared with plasmin and APSAC. The plasminogen activators may thus proffer to "rescue" pulmonary surfactant function by induction of fibrinolysis in the alveolar compartment.     

A comparison of the rates of clot lysis in a plasma milieu induced by tissue plasminogen activator (t-PA) and rec-pro-urokinase: Evidence that t-PA has a more restricted mode of action

Tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) were previously found to have distinct and complementary mechanisms of action in a plasma milieu. The t-PA activated primarily plasminogen bound to intact fibrin (internal lysine-bound), whereas pro-UK activated plasminogen bound to partially degraded fibrin (C-terminal lysine-bound). These observations therefore suggested that each of these activators was restricted in its fibrin-dependent lytic effect. At least this is true at activator concentrations where activation of plasminogen is strictly fibrin dependent; at some high concentration all plasminogen is activated including free plasminogen in the ambient plasma. However, since some pro-UK is converted to UK at the plasmin rich fibrin surface during the course of lysis and since UK is a non-selective plasminogen activator, the pro-UK/UK system should not be similarly restricted. The present study was therefore designed to examine whether this is the case by determining the highest rates of lysis achievable at the threshold of specificity for t-PA and for pro-UK/UK respectively. Maximal rates of clot lysis by t-PA (0.1–5 μg/ml) and by rec-pro-UK (0.5–5 μg/ml) were determined and correlated with the degree of preservation of fibrinogen. The highest rate of clot lysis induced by a fibrin-specific (<10% fibrinogen degradation) dose of rec-pro-UK was 80%/h. A similar rate could not be induced by t-PA without causing >80% fibrinogen degradation. The highest fibrin-specific rate of lysis achievable with t-PA was only 42%/h. When a small amount of t-PA was added at intervals during clot lysis induced by rec-pro-UK, the t-PA significantly foreshortened the lag phase, but it did not enhance the maximal rate of clot lysis. By contrast, when rec-pro-UK was added during the course of clot lysis induced by a large, but fibrin-specific amount of t-PA, rec-pro-UK accelerated the rate of lysis thereby demonstrating the presence of some fibrin-bound plasminogen not activated by t-PA. The study indicates that in a plasma milieu t-PA is more restricted in its action and activates only about half the fibrin-bound plasminogen which is activated by rec-pro-UK/UK.     

Tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) is a serine protease with a molecular weight of about 70,000. It activates plasminogen to plasmin by cleavage of the Arg 560-Val 561 peptide bond. Kinetic analysis showed that the activation obeys Michaelis-Menten kinetics and that the presence of fibrin strikingly enhances the activation rate. The directed action of plasmin towards fibrin in vivo can be explained by the low Michaelis constant in the presence of fibrin (0.16 microM) which allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin (65 microM) prevents efficient activation in plasma. Plasmin formed on the fibrin surface is protected from rapid inactivation by alpha 2-antiplasmin. Studies on the thrombolytic properties of t-PA (purified from melanoma cell cultures or obtained by recombinant DNA technology) in various animal models and in selected patients revealed that t-PA is a specific thrombolytic agent which induces thrombolysis without causing extensive systemic activation of the fibrinolytic system.     

Characterisation of rt-PA I276G,a recombinant human tissue-type plasminogen activator mutant with altered plasmin cleavage site

rt-PA I276G, a mutant of recombinant human tissue-type plasminogen activator (rt-PA) with altered plasmin cleavage site, was obtained by site-directed mutagenesis of Ile²⁷⁶ to Gly. rt-PA I276G, purified to homogeneity from the conditioned medium of transfected Chinese hamster ovary cells, was obtained as a single chain molecule, which was quantitatively converted to a two chain moiety by cleavage of the Lys²⁷⁷-Gly²⁷⁸ peptide bond with plasmin. The specific activities on fibrin films of the single chain and two chain forms of rt-PA I276G were 8900 and 15000 IU/mg respectively, as compared to 210000 and 130000 IU/mg respectively for the single chain and two chain forms of wild-type rt-PA obtained in the same expression system. The amidolytic activity of the rt-PA I276G moieties was 3- to 5-fold lower and their catalytic efficiency for plasminogen activation 20- to 50-fold lower than those of the wild-type rt-PA moieties. Both single chain and two chain rt-PA I276G induced concentration-dependent lysis of a ¹²⁵I-fibrin labelled plasma clot submersed in human plasma; equi-effective concentrations (causing 50% clot lysis in 2 h) were 0.55 and 1.40 μg/ml respectively, as compared to 0.36 and 0.60 μg/ml for single chain or two chain wild-type rt-PA respectively. Continuous infusion over 60 min of single chain rt-PA 1276G or wild-type rt-PA in hamsters with a pulmonary embolus, revealed an approximately 2-fold lower thrombolytic potency (clot lysis versus dose) for the mutant, but a comparable specific thrombolytic activity (clot lysis versus steady state plasma antigen level). It is concluded that replacement of Ile²⁷⁶ by Gly in single chain rt-PA significantly reduces the intrinsic enzymatic activity in purified systems. In a plasma milieu in the presence of fibrin the fibrinolytic potential of the mutant is, however, only 2-fold lower than that of wild-type rt-PA obtained in the same expression system.     

A study of the activation of fibrin-bound plasminogen by tissue-type plasminogen activator,single chain urokinase and sequential combinations of the activators

The efficacy of tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) to activate fibrin-bound plasminogen was determined. With the use of a solid-phase model of intact and plasmin-degraded fibrin, the initial rate, Vi (fmol of plasmin/min), of plasmin generation by t-PA, scu-PA and two chain u-PA (tcu-PA) and the ratio, R (mol of plasmin generated/mol of activator) were determined as indicators of the efficiency of plasminogen activation. [Glu)t-plasminogen bound to intact fibrin was most efficiently activated by t-PA (Vi=0.843 and R=4.08), while scu-PA and tcu-PA produced markedly lower activation coefficients (Vi=0.011 and 0.023, and R=0.13 and 0.50 respectively). In contrast, when [Glu]¹-plasminogen was bound to the degraded surface, it became activatable with equivalent efficiencies by both t-PA and scu-PA (R=4.95 and 4.72, respectively). No evidence of Lys-plasminogen formation was observed. Rather, the selective activation of plasminogen bound to degraded fibrin by scu-PA is consistent with a particular interaction of Glu-plasminogen with carboxy-terminal lysine residues on the degraded surface. The consequences of this interaction of scu-PA with plasminogen bound to degraded fibrin on sequential activations of plasminogen by both t-PA and scu-PA were further explored. While the activation by scu-PA and then t-PA produced only an additive effect on plasmin generation, the opposite combination induced a greater than additive affect; the concentration of activators needed to produce 50% of plasmin generation was 70pM for the scu-PA/t-PA combination; in contrast, the amount required to produce a similar degree of activation with the opposite combination (t-PA/scu-PA) was only 17 pM. These results indicate that the potentiation of the scu-PA activity requires both the previous generation of plasmin by t-PA, and plasminogen bound to carboxy-terminal lysine residues of the degraded fibrin surface as a substrate.     

A zymogenic tissue plasminogen activator variant: The Phe305→ His mutation suppresses fibrin(ogen) stimulated plasminogen activation by one chain t-PA

Tissue plasminogen activator (t-PA) is a unique serine protease because it does not require proteolytic activation to exhibit full enzymatic activity. In the presence of physiological stimulators related to fibrinogen and fibrin, the one chain form of the enzyme has equivalent activity to the two chain form against the physiological substrate, plasminogen. This report describes a Phe 305→His (F305H) variant of t-PA which displays kinetics more typical of a serine protease zymogen. Plasminogen activation by F305H t-PA in the presence of a fibrin-like stimulator was shown to exhibit non-linear kinetics, presumably due to the ability of plasmin in the reaction mixture to convert the one chain F305H t-PA to the more active two chain form. Comparison of the apparent kinetic constants of two chain F305H t-PA and a single chain variant (R275E/F305H), indicated that the two chain form of the enzyme had approximately 3-fold higher activity than the one chain form. The increase in activity was due primarily to an increase in the k_cat of the reaction. Formation of a hydrogen bond between His 305 and Asp 477 in F305H t-PA, analogous to the His 40-Asp 194 hydrogen bond in chymotrypsinogen, is postulated to account for the partially zymogenic characteristics of this variant.     

The activation of plasminogen by tissue plasminogen activator is not enhanced by different heparin species as assessed in vitro with a solid-phase fibrin method

The aim of this study was to evaluate the effect of several heparin species (standard heparin, heparin of low molecular weight: IC 831422, heparin of high affinity for antithrombin III: IC 831435, and heparin of low affinity for antithrombin III: IC 831436) on the different steps of the fibrinolytic mechanism i.e., interaction of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor-1 (PAI-1), binding of t-PA and plasmin(ogen) to fibrin and activation of plasminogen on the fibrin surface, in the presence of plasma proteins and factors that modulate fibrinolysis. Fibrinolytic and plasmin amidolytic activities were measured in the presence and absence of heparin. The spectrophotometric assays were performed in the presence and absence of solid-phase fibrin using selective chromogenic substrates for plasmin and saturating concentrations of plasminogen.The overall fibrinolytic activity, the amidolytic activity of mixtures of t-PA or urokinase with plasminogen in the absence of fibrin, the binding of t-PA and plasmin(ogen) to fibrin and the t-PA/PAI-1 interaction were not modified by heparin. In contrast, the activation of plasminogen by fibrin-bound t-PA decreased as a function of the concentration of heparin. Although this effect was not significant at concentrations usually used in routine heparin therapy (0.1 to 1 iu/ml) it might have some relevance when heparin is injected in bolus doses.In conclusion, by using a solid-phase fibrin method which allows the analysis of t-PA/PAI-1 balance, data were obtained indicating that the heparin species tested here neither competed with fibrin for the binding of t-PA, nor potentiated the activation of plasminogen at the fibrin surface.     

Albumin concentration influences fibrinolytic activity in plasma and purified systems

Delayed clot lysis was observed in 9 patients with nephrotic syndrome. This delay, assessed by fibrin degradation product release, was corrected by supplementing patients' plasma with purified human albumin to reach the normal albumin concentration of 45g/1. The effect of albumin on clot lysis was further assessed in the presence of albumin-depleted plasma and in a purified system containing tissue-type plasminogen activator (t-PA), plasminogen, fibrin monomers and increasing albumin concentrations. An increase in plasminogen uptake was observed when clots were prepared in the presence of such concentrations. We conclude that low albumin levels may alter clot structure and reduce spontaneous thrombolysis, thereby increasing the risk of thrombosis.     

Fibrin-specific thrombolytic agents

The mammalian fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme plasmin, which will degrade fibrin. Plasminogen activation is mediated by plasminogen activators which are classified as either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, however via entirely different mechanisms. 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 activity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic efficiency of the enzyme. scu-PA has a high affinity for plasminogen but, however, does not activate plasminogen in plasma in the absence of a fibrin clot, due to competitive inhibition. Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition, but this does not seem to occur via specific binding of scu-PA to fibrin. 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 thrombolysis and relative fibrinogen sparing effect of t-PA was recently confirmed in several multicenter clinical trials in patients with acute myocardial infarction. Specific thrombolysis by scu-PA has also been demonstrated in animal models of pulmonary embolism, venous thrombosis and coronary artery thrombosis.     

Importance of the structure of the clot in thrombolysis

Activation of plasminogen by tissue-type plasminogen activator (tpA) is potentiated by fibrin. We have demonstrated the role of fibrin polymerization in the potentiating effect of tpA-induced fibrinolysis. Therefore a pathogenic mechanism of thrombotic disorder may be related to an abnormal fibrin polymerization: the abnormal clot being less accessible to fibrinolysis than normal one. This defective lysis may be due to a defective enhancement by the abnormal fibrin of plasminogen activation by tpA, as demonstrated for fibrinogen Dusard, a congenital dysfibrinogenemia associated with a very severe thrombotic disorder. In some other cases, a decrease in the availability of the plasmin cleavage sites in fibrin clot may be involved. On the contrary, some antithrombotic drugs such as pentosane polysulfate in modifying clot structure allow a better degradation of fibrin clot by fibrinolytic enzymes. It is speculated that this enhanced fibrinolysis could explain, almost in part, the antithrombotic action of these drugs.     

Diversity in catalytic properties of single chain and two chain tissue-type plasminogen activator

Like many other serine proteases, human tissue-type plasminogen activator (t-PA) is synthesised and released from cells in a single chain (sc-) form. This may later be converted proteolytically to a two chain (tc-) form, in which the chains are held together by a disulfide bridge. With most serine proteases, the sc-form is a genuine zymogen with an activity several orders of magnitude smaller than that of the tc-form. But in the case of t-PA, the relative activity of the two forms has been a controversial issue. We review here old and new investigations of the problem, emphasizing recent experiments with genetically engineered t-PA mutants that cannot be cleaved to the tc-form. Provided steps are taken to prevent plasmin-catalysed conversion of sc- to tc-t-PA during the assays, the results consistently point to a plasminogen activation activity of sc-t-PA that is at least one order of magnitude smaller than that of tc-t-PA. Fibrin stimulates the activity of sc-t-PA strongly and to a higher extent than tc-t-PA, resulting in equal activities of the two forms in the presence of fibrin. On the other hand, there is no doubt that sc-t-PA as such possesses a distinct activity even in the absence of fibrin. It reacts with synthetic substrates, with reactive site reagents and with its natural inhibitors. These properties have important implications as to the way t-PA may fulfil its physiological function in fibrinolysis: sc-t-PA is not likely to circulate as a reservoir of potential plasminogen activation activity. Rather, plasmin generation and subsequent fibrinolysis is likely to be initiated by release of sc-t-PA from the endothelium and to depend on the presence of polymerised fibrin as a cofactor. Other mechanisms of t-PA activation may be involved in non-fibrinolytic functions of the activator.     

The role of urokinase generation during clot lysis by pro-urokinase in a plasma milieu

The conversion of single chain urokinase-type plasminogen activator (scu-PA) to two chain urokinase-type plasminogen activator (tcu-PA) during lysis of normal and tissue-type plasminogen activator (t-PA) pre-treated clots by scu-PA was examined using a combined ELISA assay system which selectively measures either scu-PA or tcu-PA in plasma. At fibrin-specific doses, conversion did not exceed 20% against normal clots. Conversion was found to be dose-dependent and correlated with fibrinolysic efficiency. Against t-PA pre-treated clots the efficacy of lysis by scu-PA, as measured by the length of the lag-phase and maximum rate of lysis, was increased and was accompanied by 30% conversion of scu-PA to tcu-PA. An infusion of tcu-PA equivalent to that generated from scu-PA in the above experiments induced 70% less fibrinolysis and 25% more fibrinogenolysis. These findings indicate that local activation of scu-PA on the fibrin surface induces far more lysis and less non-specific effects than systemic conversion to tcu-PA, and that local activation is well correlated with scu-PA lytic efficiency.     

Thrombolytic effect of pro-urokinase in vitro

The thrombolytic mechanism of pro-urokinase (Pro-UK) was investigated. Pro-UK is of a single-chain form, and is converted to UK with plasmin-Sepharose. The [3H]DFP incorporation was strongly enhanced by plasmin treatment, which induces conformational changes as observed from the circular dichroism. A relatively higher affinity of Pro-UK for fibrin than that of UK was found by adsorption on fibrin-Sepharose and also by incorporation into fibrin during fibrinogen-fibrin conversion. Pro-UK produced effective thrombolysis without degrading fibrinogen. The presence of t-PA in the thrombus promoted thrombolysis effectively. Further, coexistence of t-PA and Pro-UK in the plasma markedly enhanced the thrombolysis. The adsorption of Pro-UK, plasminogen and t-PA on fibrin-Sepharose induced rapid activation of Pro-UK to UK as well as rapid production of plasmin on the fibrin surface. These results suggest that a trace amount of t-PA causes marked enhancement of Pro-UK induced thrombolysis.     

Construction,expression and biochemical characterisation of a novel triskringle plasminogen activator gene

A DNA sequence coding for the second kringle of tissue-type plasminogen activator (t-PA) was synthesised chemically and inserted just before the double-kringle region of the t-PA gene. This tris-kringle PA or hybrid F gene was expressed in a BPV-based expression vector and the expression product was purified to homogeneity. The specific activity of the protein on fibrin-agar plate was about 250 000 iu/mg compared to ∼ 500 000 iu/mg for rt-PA. Analysis of the protein by SDS-PAGE showed that it was present mainly in the single chain form and displayed doublet bands of Mr 68 000 and 73 000 Da, probably due to differential glycosylation. Although hybrid F showed tighter binding to lysine-Sepharose, its binding to a fibrin clot was comparable to that of t-PA. Hybrid F and t-PA were found to have comparable amidolytic activities toward a synthetic chromogenic substrate, S-2288, suggesting that the extra kringle in hybrid F did not affect its catalytic function. However, hybrid F exhibited considerably lower activity in fibrin-dependent plasminogen activation characterised by higher Km and lower kcat values than the native t-PA. This shows that addition of an extra kringle in t-PA somehow interferes in the ternary complex formation with fibrin and plasminogen and thereby adversely affects its fibrin-stimulated activity.     

Plasminogen activators in bovine milk during mastitis,an inflammatory disease

We have investigated the content of plasminogen activators in bovine milk during mastitic inflammation induced by Staphylococcus aureus. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with fibrin agarose zymography and a coupled peptidyl anilide plasminogen activation assay of samples of whey prepared by acidification, we found that the level of tisue-type plasminogen activator (t-PA) in milk was increased immediately after infection and remained elevated during an experimental period of 42 days. The maximal increase was 10 to 20-fold. By zymography, we also demonstrated a strong increase in urokinase-type plasminogen activator (u-PA) associated with the bovine cells in the milk. By ligand blotting, we demonstrated an increase in the level of the urokinase-receptor (u-PAR) on the milk cells during inflammation. Plasma kallikrein was also detected as a plasminogen dependent proteolytic activity by zymography of whey samples. When analyzed in the presence of the t-PA in the milk, the plasma kallikrein lysis zone was strongly increased in mastitic whey, but when analyzed after separation from t-PA, its level was unaffected by mastitis; this could be ascribed to a t-PA dependent stimulation of plasma prekallikrein. These results suggest an important role for plasminogen activators in the inflammatory response during bovine mastitis. Using an enzyme-linked immunosorbent assay we measured the plasminogen/plasmin level during the inflammation, but found a less than 2-fold increase during the experimental period.     

A variant of tissue plasminogen activator (t-PA) comprised of the Kringle 2 and the protease domain shows a significant difference in the in vitro rate of plasmin formation as compared to the recombinant human t-PA from transformed chinese hamster ovary cells

BM 06.022 is a tissue plasminogen activator (t-PA) variant comprised of the kringle 2 and the protease domain. In vivo data from animal studies provided evidence that BM 06.022 is a potent and fibrin specific fibrinolytic agent. In this study we present a detailed analysis of the in vitro rate of plasmin formation by BM 06.022. The activation of plasminogen by BM 06.022 was reduced as compared to CHO-t-PA when using CNBr-fragments of fibrinogen as a stimulator. However, a more detailed analysis revealed that the rate of plasmin formation by t-PA and BM 06.022 in the presence of CNBr-fragments of fibrinogen is dependent on the exact concentration of the stimulator because the activity-stimulator curves of both enzymes are non-parallel. In contrast, in the presence of fibrin-monomer and its plasmin derived degradation products the course of the activity-stimulator curve of BM 06.022 and t-PA was parallel. Furthermore, the concentrations of these stimulators which allowed 50% and maximal stimulation were very similar for BM 06.022 and CHO-t-PA. The rate of plasmin formation by BM 06.022 in the presence of fibrin-monomer and fibrin degradation products was lower by a factor of 2.0 and 4.3, respectively, as compared to CHO-t-PA. Furthermore, BM 06.022 and t-PA were not or only marginally stimulated by fibrinogen indicating that the systemic activation of fibrinolytic agents as it may occur during the therapy of the myocardial infarction may be due to a stimulation by fibrin degradation products rather than due to a stimulation by fibrinogen.     

The sequential,complementary and synergistic activation of fibrin-bound plasminogen by tissue plasminogen activator and pro-urokinase

Tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) are the two principal activators of the fibrinolytic system. They share a relative fibrin specificity related to their selective activation of fibrin-bound plasminogen, but achieve this by very different mechanisms. Some of the properties of pro-UK, which contrast with those of t-PA, are reviewed. These include zymogenicity and stability in plasma, lack of significant fibrin affinity and requirement for a certain conformational change in Glu-plasminogen. This conformation appears to be induced principally when Glu-plasminogen is bound to carboxyterminal lysine residues which interact with the high affinity lysine binding sites. These residues become available on fibrin only after degradation has been initiated. By contrast, in a plasma milieu, fibrin-bound t-PA was found to activate plasminogen on intact fibrin. This plasminogen binding involves internal lysine residues which interact with the weak lysine binding site on Kringle 5. The two activators are therefore sequential and complementary in their activation of fibrin-bound plasminogen. This complementariness helps to explain their synergistic fibrinolytic effects when combined. The findings also explain the lag phase characteristic of pro-UK but not t-PA-induced clot lysis. They also help account for the unusually high dose requirements for t-PA and pro-UK in therapeutic thrombolysis when each is used alone. A rationale for combination therapy is provided which may mimic the natural design for fibrinolysis.     

Quantification of a specific inhibitor (PAI-1) of tissue-type plasminogen activator (t-PA) in the plasma

In human plasma, the activation of plasminogen by tissue plasminogen activator (t-PA) is a fibrin localized process which allows the specific dissolution of thrombi. Most of the t-PA circulates as a complex with its inhibitor, PAI-1, which thereby regulates its activity. In the present work the authors have studied the kinetics of inhibition of t-PA by PAI-1 and have developed an assay for its specific detection. The assay is performed in microtitration plates containing a solid-phase fibrin network, as follows: the source of inhibitor is mixed with solutions containing increasing amounts of t-PA, then the residual t-PA is separated by means of a solid-phase fibrin support and detected with a coupled reaction using a plasmin selective chromogenic substrate. The change in absorbance is measured in a microtiter plate reader and converted to t-PA activity by reference to a standard curve. The residual t-PA activity is inversely proportional to the concentration of PAI-1. The quantitation of PAI-1 is based on the variation of the dissociation constant of the fibrin/t-PA interaction obtained in the presence of the inhibitor. Since other serine-protease inhibitors do not interfere with the assay, the method is specific for PAI-1 and can be safely used in other biological fluids.     

Lysine-derivatized polyurethane as a clot lysing surface: conversion of adsorbed plasminogen to plasmin and clot lysis in vitro

Polyurethane surfaces to which lysine residues are immobilized by photochemical methods are proposed as a basis for clot lysing surfaces. The lysines are attached in such a way that the epsilon-amino and carboxyl groups are free. We showed previously that these surfaces, when placed in contact with plasma, adsorb only plasminogen and virtually no other proteins (McClung et al., J. Biomed. Mater. Res. 49 (2000) 409). In this communication, data based on a chromogenic substrate assay are presented showing that plasminogen adsorbed to these surfaces is readily converted to plasmin in the presence of tissue-plasminogen activator (t-PA). Moreover, the rate of activation on the surface is considerably greater than in solution. Experiments demonstrating the ability of these surfaces to dissolve fibrin clots are also reported. Surfaces exposed to plasma and then to t-PA were placed in citrated plasma. On recalcification, clotting was initiated, but the incipient clots were soon dissolved. On control surfaces (no lysine or lysine in which the epsilon-amino groups were not available) coagulation continued until a stable clot was formed. Similar observations were made when the plasma/t-PA exposed surfaces were placed in a pure fibrinogen solution and thrombin was added.     

The effect of poloxamer 188 on the rate of in vitro thrombolysis mediated by t-PA and streptokinase

Poloxamer 188 is a nonionic block copolymer surfactant which is undergoing clinical trials as a haemorheologic agent. A simulated thrombus preparation was designed to evaluate its effects on fibrinolysis and thrombolysis under flow conditions similar to those which might be encountered within an occlusive coronary thrombus. ¹²⁵I labelled human fibrinogen (20 μCi/ml) was mixed with platelet-poor citrated plasma, recalcified, and allowed to clot for 1 h at 37°C in a tuberculin syringe filled with small glass beads. This produced a fibrin clot permeated with microscopic channels simulating those of a recently formed thrombus. After washing the plasma bead clots for 30 min with normal saline, heparinised blood containing tissue plasminogen activator (t-PA) or streptokinase, with and without 0.1% poloxamer 188, was added to the columns and rates of flow and elution of 12 ¹²⁵-fibrin degradation products were measured. Poloxamer 188 alone had no ability to lyse fibrin. However, it increased the rate at which t-PA or streptokinase caused lysis of simulated thrombi by as much as 4-fold. This accelerated lysis was associated with up to a 20-fold increase in flow through the thrombus and with elution of larger fragments of fibrin, but not with increased plasmin activity as assessed with a colourimetric substrate. Consequently, poloxamer 188 appeared to accelerate thrombolysis without directly affecting any of the components of the fibrinolytic system. We propose that the ability of poloxamer 188 to modulate thrombolysis is due to its haemorheologic properties.