A miniaturized fibrinolytic assay for plasminogen activators.

This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.     

The fibrinolytic action of the synthetic agent S-1623 in a purified system of fibrin and plasminogen

The effects of the synthetic fibrinolytic agent α(isobutyl-4 cyclohexene-1 yl) propionic acid (S-1623) were investigated in a purified system. Bovine fibrin clots were incubated at 37°C with varying concentrations of S-1623 (0–10 mM) and a constant concentration of human plasminogen (1 μM). Two methods were used to measure lysis, e.g. visual observation of the clots and SDS-polyacrylamide gel electrophoresis. Plasmin formed during incubation was measured quantitatively by a spectrophotometric method using a chromogenic substrate. After 24 hours of incubation, a partial or total degradation of the clots was observed visually for S-1623 concentrations between 3 and 8 mM, with maximum degradation occuring at 4–5 mM. Analysis of the samples by acrylamide gel electrophoresis revealed total fibrinolysis at a concentration of 4–5 mM, partial fibrinolysis at 3 and 6–7 mM, and no fibrinolysis at other concentrations (0–2 mM and 8–10 mM). Proteolysis of plasminogen also occured, with maximum effect at 4–5 mM, and resulted in the production of fragments smaller than the intact plasmin molecule. The amidolytic activity coincided with the lytic concentrations of S-1623, with maximum activity occuring at 4–5 mM. No activity was observed in the absence of fibrin, plasminogen, or S-1623. Amidolytic activity was also generated, though to a lesser extent, when the fibrin substrate was replaced by fibrinogen. These results seem to show that the agent S-1623 is capable of generating plasmin-like activity in this system.     

Influence of detergents on the measurement of the fibrinolytic activity of plasminogen activators

The influence of detergents on the apparent activity of tissue plasminogen activator and of urokinase was investigated. The nonionic detergents Tween 80 and Triton X-100 and the zwitterionic detergent Zwitter-gent 314 caused a marked increase in the apparent activity of tissue plasminogen activator and, to a lesser degree, of urokinase in the fibrin plate assay, particularly at higher activator concentrations. These detergents did not enhance the plasma clot lysis in the presence of urokinase or tissue plasminogen activator. The anionic detergent sodium dodecyl sulfate had a small inhibitory action on the fibrin plate activity of urokinase and strongly inhibited the fibrin plate activity of tissue plasminogen activator. A systematic study into the mechanism of the stimulatory action of nonionic and zwitter-ionic detergents is presented.     

The fibrinolytic action of flufenamate and its influence on plasminogen binding to fibrin

The effects of the synthetic fibrinolytic agent flufenamate were investigated in a purified system made up of bovine fibrin and human plasminogen. The lysis of the fibrin clots was observed after a 12-hour incubation for flufenamate concentrations ranging from 0.25 to 3 mM. Below 0.25 mM and above 3 mM, no lysis occurred, even after a longer incubation. An increase in plasminogen adsorption on the fibrin clot was also observed in the presence of flufenamate. The amount of plasminogen bound was estimated using 125I labelled Glu-plasminogen or Lys-plasminogen, in the presence of a protease inhibitor to avoid the fibrinolysis induced at lytic concentrations of flufenamate. Maximum binding was observed with both types of plasminogen at a flufenamate concentration of 2.5 mM. The binding, relatively fast at the start of incubation, slowed down progressively, but a real plateau was not reached, even after a 6-day incubation. Plasminogen binding was not saturable, suggesting a non-specific binding type. Although Lys-plasminogen binding was always greater than that of Glu-plasminogen, the effect of flufenamate was more pronounced on Glu-plasminogen binding.     

The comparison of solid phase and fibrin plate methods for the measurement of plasminogen activators

A rapid and highly sensitive solid phase assay was compared with the fibrin plate method for the measurement of urokinase, streptokinase and the plasminogen activators in human euglobulin fractions. The solid phase assay was run using glu - or lys - plasminogen, and significant differences were observed in the activation of the plasminogens by urokinase and streptokinase. Plasminogen activator levels in euglobulin fractions were also measureable. Very good agreement was obtained between the fibrin plate and solid phase methods in all cases.     

Study on the mechanism of action of heparin and related substances on the fibrinolytic system: Relationsship between plasminogen activators and heparin

It is now generally well accepted that heparin and related substances increase the fibrinolytic activity . The stimulation of the amidolytic, plasminogenolytic and fibrinogenolytic activity of tissue plasminogen activator and urinary plasminogen activator through heparin was investigated . A concentration-dependent stimulation of the plasminogenolytic and fibrinogenolytic activity of both urinary and tissue-type plasminogen activators was observed in the presence of heparin. No heparin dependence was observed in the amidolytic assay. Heparin stimulates the plasminogenolytic activity of tissue plasminogen activator in the same manner as fibrin. Both activators form complexes with heparin; the heparin-binding-site seems to be identical or related with the fibrin-binding-site of tissue plasminogen activator. The physiological role of these interactions is discussed.     

A fibrin specific monoclonal antibody which interferes with the fibrinolytic effect of tissue plasminogen activator

Monoclonal antibodies to human fibrin have been prepared from stable hybridomas, obtained by fusion of a mouse myeloma cell line (NS-1) and spleen cells of Balb/c mice immunized with a suspension of human fibrin. One cell line, DG1, producing a monoclonal antibody of the IgG1, kappa subclass, reacted specifically with human fibrin (KD = 1.2 nM). Western blotting analysis indicates that DG1 crossreacts with the fibrin fragment D-dimer. Using both a chromogenic and an 125I-fibrin release assay it was illustrated that in the presence of the fibrin specific antibody the t-PA mediated generation of plasmin was significantly inhibited. An animal model system, developed to monitor thrombosis and induced reactive fibrinolysis, was used to investigate the interference of plasminogen activation, by the antibody, in vivo. This fibrin specific antibody prolonged the onset of reactive fibrinolysis in a dose dependent manner.     

Increased euglobulin fibrinolytic potential in women on oral contraceptives low in oestrogen--levels of extrinsic and intrinsic plasminogen activators, prekallikrein, factor XII, and C1-inactivator

Components of the fibrinolytic system were studied in samples of plasma from 15 normal, young women and from 11 women taking oral contraceptives containing 30 micrograms ethinyl oestradiol and 150 micrograms levo-norgestrel. Fibrinolytic activity of euglobulins precipitated at pH 5.9 was higher than normal in the hormone group, with significant fluctuations related to the cycle. Normal women showed only minor fluctuations. The concentration of C1-inactivator was lower in euglobulins of the hormone group. However, the difference in fibrinolytic activity was retained, when C1-inactivator was inactivated with sodium flufenamate. Fluctuations of the extrinsic (tissue-type) plasminogen activator (t-PA) activity parallelled those of the euglobulin activity. The intrinsic plasminogen activator activity (dextran sulphate precipitated euglobulin) was significantly increased in the hormone group and the cyclic pattern differed from that of the normal group. The increased activity was factor XII-dependent. Plasma prekallikrein did not differ. The factor XII level was increased in the hormone group but this could not explain the increased intrinsic fibrinolytic activity, suggesting an increase in the quantity of an additional factor XII-dependent proactivator.     

A comparative ex vivo study of plasminogen activators and proteases for fibrinolytic activity and side effects in rabbits

Urokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose were administered to monitor potential toxicity revealing that Brinase, trypsin and SN 687 were very toxic at this concentration. Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo. The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.     

Interfering factors in the assay of plasminogen activators by the fibrin plate method. Occurrence of different inhibitors against tissue plasminogen activator and urokinase.

The assay of plasminogen activator activities on fibrin plates was re-evaluated with special reference to fibrinolysis inhibitors present in samples and in fibrin plates. The nature, action and stability of inhibiting material were studied in tissue with considerable differences in activator and inhibitor contents: human lung, liver and placenta. Extracts were tested for inhibitory capacity against purified human uterine tissue plasminogen activator, urokinase and plasmin of fibrin plates prepared from different grades of fibrinogen and fibrin. The tissue extracts inhibited fibrinolysis on fibrin plates to varying degrees, dependent on the sample medium, the type of fibrin plate and the kind of plasminogen activator. The influence of inhibitors in the sample and in the fibrin plate was partly abolished by the presence of 2 M KSCN in the sample. The procedure for preparing the samples as described by Astrup and Albrechtsen did not completely eliminate the inhibitory action against the added plasminogen activators. Comparison of urokinase inhibition with tissue activator inhibition by the tissue extracts as to the degree of denaturation in the Astrup and Albrechtsen procedure showed that they have much in common. Nevertheless, some differences were found which indicated the possible existence of separate urokinase and tissue activator inhibitors or of different inhibition mechanisms for these plasminogen activators.     

Effect of oxidants on proteases of the fibrinolytic system: possible role for methionine residues in the interaction between tissue type plasminogen activator and fibrin.

Evidence has recently been presented that activated macrophages (M phi) express both urinary (u-PA) and tissue type (t-PA) plasminogen activator. Major cell products of M phi and polymorphonuclear neutrophils (PMN) are reactive oxidants of the HOCl/chloramine type. Since PMN and M phi are involved in inflammatory and fibrinolytic processes, we were interested in the interaction of u-PA, t-PA, and plasmin with oxidants of the leukocyte type. The enzymes were treated with chloramine-T, which at pH 8.5 is a selective oxidant for methionine residues. Oxidation by chloramine-T of t-PA abolishes about 40% of both stimulation susceptibility of t-PA by fibrinogen degradation products (FDP) and affinity of t-PA to FDP. However, the plasminogenolytic and amidolytic activity of unstimulated t-PA as well as the plasminogenolytic activity of u-PA and the amidolytic activity of plasmin are not impaired. Identification of the amino acid residues in the t-PA responsible for the interaction with fibrin might be of great importance in order to understand the mechanism of the clot- selectivity of t-PA. The present study gives evidence that fibrin specificity of t-PA partly depends on chloramine oxidizable amino acids, presumably methionine residues. Hence, experimental data on the interaction between t-PA and fibrin, using oxidized and labelled t-PA should be interpreted with caution. It may be suggested that oxidants of the leukocyte type might regulate t-PA activity and selectivity for fibrin.     

A comparison of the effects of cimetidine and epsilon-aminocaproic acid on fibrinolysis induced by activators of plasminogen and on fibrin formation

A comparison of the effects of cimetidine and 6-aminohexanoic acid (EACA) on activator-induced fibrinolysis showed, that cimetidine does not have any antifibrinolytic effect, when applied in the concentration range used in the treatment of haemorrhagic gastritis or hypertrophic protein-losing gastritis. Concentrations of cimetidine above the therapeutic level impaired fibrin formation.     

Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator

The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.     

Changes of the fibrinolytic system of animals induced by injection of tissue plasminogen activator

The changes in the fibrinolytic system of rats induced by repeated injections of tissue plasminogen activator (TPA) have been studied. The euglobulin fraction fibrinolytic activity increases 10 min after the first injection of TPA. Repeated injections of TPA for one or more days do not stimulate the fibrinolytic activity or TPA accumulation in the blood. Apparently in the absence of fibrin clots in the blood flow of healthy animals TPA is incapable of activating plasminogen conversion into plasmin. TPA, in its turn, is rapidly bound to the antiactivator and is excreted from the organism.     

Fibrinolysis in normal subjects--comparison between plasminogen activator inhibitor and other components of the fibrinolytic system

We analyzed fibrinolytic parameters in 20 healthy men and 20 healthy women, aged from 25 to 59, before and after 10 and 20 min venous occlusion. The 10 min post-occlusion fibrinolytic activity measured directly in diluted unfractionated plasma by a highly sensitive 125I-fibrin plate assay correlated well with the activity of euglobulins determined by the classical fibrin plate assay (r = 0.729), but pre-stasis activities determined with these two methods did not correlate (r = 0.084). The enhancement of fibrinolytic activity after venous occlusion was mainly due to an increase of t-PA in the occluded vessels (4-fold increase t-PA antigen after 10 min and 8-fold after 20 min venous occlusion). Plasminogen activator inhibitor (PAI) activity and plasminogen activator inhibitor 1 (PAI-1)1 antigen levels at rest showed considerable dispersion ranging from 1.9 to 12.4 U/ml, respectively 6.9 to 77 ng/ml. A significant increase of PAI-1 antigen levels was observed after 10 and 20 min venous occlusion. At rest no correlation was found between PAI activity or PAI-1 antigen levels and the fibrinolytic activity measured by 125I-FPA. However, a high level of PAI-1 at rest was associated with a high prestasis antigen level of t-PA and a low fibrinolytic response after 10 min of venous stasis. Since the fibrinolytic response inversely correlated with PAI activity at rest, we conclude that its degree depends mainly on the presence of free PAI.     

Delayed fibrin elimination from the lungs of burned rats with endogenous inhibition of the fibrinolytic system

The elimination of fibrin from the lungs of burned rats with endogenous inhibition of the fibrinolytic system was studied after intravenous injection of thrombin. The fibrin content of the lungs was quantified, using labelled fibrinogen and albumin. It was found that in burned rats the elimination of fibrin from the lungs was delayed. The lungs of these rats showed micro-atelectases which were more numerous than in rats without fibrinolysis inhibition.