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Background Many proteins bind to fibrin during clot formation in plasma. We previously identified by mass spectrometry the most abundant proteins that noncovalently bind to fibrin clots. Several of these proteins (e.g., apolipoprotein J/clusterin, haptoglobin, α2-macroglobulin, α1-antitrypsin) can act as extracellular chaperones. Objective We hypothesize that clot-binding proteins may interact with fibrin as chaperones. The goal of this study is to test this hypothesis and to investigate the origin of the cross-β or amyloid structures in fibrin clots, which are associated with protein unfolding. Methods and Results A thioflavin T assay was used to detect cross-β structures. A steadily increasing amount was measured in the fibrinogen fraction of plasma during heat stress, a standard treatment to induce unfolding of proteins. Heat-stressed plasma was clotted and clot-bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results showed that the amounts of the clot-bound proteins were related to the duration of the heat stress. This indicates that cross-β structures in unfolded fibrin(ogen) are involved in clot binding of the proteins, which supports our chaperone hypothesis. A contributing role of fibrin formation itself was studied by clotting purified fibrinogen with thrombin in the presence of thioflavin T. The fluorescence intensity increased in time in the presence of thrombin, but did not increase in its absence. This provides evidence for the generation of cross-β structures during fibrin formation. Conclusion Fibrin clots generated in plasma are decorated with extracellular chaperones. The binding of these chaperones involves cross-β structures originating both from unfolded fibrinogen and from fibrin formation.

SummaryHypofibrinolysis is a risk factor for venous and arterial thrombosis, and can be assessed by using a turbidimetric tPA-induced clot lysis time (CLT) assay. Biological variation in clot lysis time may affect the interpretation and usefulness of CLT as a risk factor for thrombosis. Sufficient information about assay variation and biological variation in CLT is not yet available. Thus, this study aimed to determine the analytical, within-subject and between-subject variation in CLT. We collected blood samples from 40 healthy individuals throughout a period of one year (average 11.8 visits) and determined the CLT of each plasma sample in duplicate. The mean (± SD) CLT was 83.8 (± 11.1) minutes. The coefficients of variation for total variation, analytical variation, within-subject variation and between-subject variation were 13.4%, 2.6%, 8.2% and 10.2%, respectively. One measurement can estimate the CLT that does not deviate more than 20% from its true value. The contribution of analytical variation to the within-subject variation was 5.0%, the index of individuality was 0.84 and the reference change value was 23.8%. The CLT was longer in the morning compared to the afternoon and was slightly longer in older individuals (> 40 years) compared to younger (≤40 years) individuals. There was no seasonal variation in CLT and no association with air pollution. CLT correlated weakly with fibrinogen, C-reactive protein, prothrombin time and thrombin generation. This study provides insight into the biological variation of CLT, which can be used in future studies testing CLT as a potential risk factor for thrombosis.

SummaryThe role of ethanol metabolism in possible haemostatic cardioprotective effects has not yet been determined. To this end, we investigated the effect of a moderate dose of ethanol (35 g) and its metabolism, on haemostatic variables over 14 hours (h). Eighteen Caucasian males participated in a placebo-controlled, randomised, cross-over study. Blood was collected prior to alcohol consumption, and at 10 time points for 14 h. Blood ethanol peaked at 1 h and was cleared after 8 h following ethanol consumption, significantly increasing plasma acetate (p=0.0028). Ethanol did not influence the coagulation factors significantly. PAI-1act increased (p

SummaryIn this study, the in-vitro fibrinolytic efficacy of Tenecteplase, Amediplase and scu-PA was investigated in different external lysis models by measuring the lysis of human plasma clots after the addition of the plasminogen activators (PAs) to the surrounding plasma. The effect ofTAFI was examined for each PA by neutralizing TAFI a with potato carboxypeptidase inhibitor (PCI). The lytic efficacy of Amediplase was lower than that of Tenecteplase at low PA concentrations but slightly higher at therapeutic concentrations. The activity of scu-PA was clearly lower than that of either Tenecteplase or Amediplase. The TAFI system inhibited external clot lysis mediated by all the PAs when thrombomodulin was present in the model. In the therapeutic range (5–10 µg/ml) however, the TAFIa effect was negligible for both Amediplase and Tenecteplase. At lower PA concentrations the effect of TAFI on Amediplase was slightly stronger than that on Tenecteplase. Under static conditions the lysis rates were lower than with stirring. The role of TAFI was similar under both conditions. In conclusion, at therapeutic concentrations Amediplase was slightly more active than Tenecteplase and scu-PA under all conditions used. Therefore, Amediplase might possibly be a more potent thrombolytic agent at these concentrations and increase the efficacy of thrombolysis. The potential of TAFI for inhibiting thrombolytic therapy is probably low. However in conditions where the local PA concentrations are sub-optimal TAFI might affect the lysis rate.

SummaryIn order to study the role of the asialoglycoprotein receptor (ASGPr) in the rapid plasma clearance of urokinase-type plasminogen activator (u-PA), a microtiter plate binding assay was developed using ASGPr purified from rat liver extracts. Urinary two-chain u-PA bound to immobilized ASGPr in a saturable manner with an EC50 of 0.2 µM. Binding was inhibited by rabbit antibodies against the ASGPr. In line with the known carbohydrate specificity of the ASGPr, GalNAc proved to be the most effective inhibitor from a series of monosaccharides, followed by Gal and Fuc, whereas GlcNAc was ineffective. The N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element, but with a GalNAc-GlcNAc element which is partially sulfated. Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30% of the total) appeared to bind to the ASGPr. From different u-PA preparations used for thrombolytic therapy only urinary u-PA and u-PA produced by kidney cell cultures strongly bound to the ASGPr, whereas (recombinant) u-PA expressed in mouse myeloma cells, Chinese hamster ovary cells or E. coli scarcely bound to the receptor. It is concluded that u-PA bearing non-sulfated GalNAc-GlcNAc elements is specifically recognized by the ASGPr present on liver cells.

SummaryFibrinolysis initially generates channels in an occluding thombus which results in blood flow through the thrombus. Since the impact of flow along the surface of a thrombus on thrombolysis has not been investigated in detail, we studied in vitro how such a flow affects lysis. Compacted and noncompacted plasma clots were used as model thrombi. With compacted clots, fibrin-specific lysis induced by alteplase in the outer plasma was accelerated about 2-fold by strong flow (arterial shear rate). Non-fibrin-specific lysis induced either by a high concentration of alteplase or by streptokinase was slow, was accompanied by rapid depletion of plasminogen in the outer plasma, and was only slightly accelerated by flow. With noncompacted clots, similar acceleration factors were documented, when mild flow (venous shear rate) was applied. Strong flow further accelerated fibrin-specific lysis, up to 10-fold as compared to lysis without flow, but paradoxically retarded non-fibrin-specific lysis. The data suggest that flow accelerates lysis by enhancing transport of plasminogen from the outer plasma to the surface of the clot. Both opposite effects of the strong flow were mediated by forceful intrusion of the outer plasma into the noncompacted clot due to flow irregularities. In the case of non-fibrin-specific lysis this resulted in the replacement of the plasminogen-containing milieu by plasminogen-depleted outer plasma in certain areas of the clot turning them into virtually unlysable fragments. This flow-enforced “plasminogen steal” may contribute to the relatively high percentage of incomplete thrombolysis (TIMI-2 grade flow) documented in a number of trials for non-fibrin-specific thrombolytic agents. In the case of fibrin-specific lysis, the effect of flow on the speed of fibrinolysis is always beneficial.

SummaryIn a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed.Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot.In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

SummaryDextran is used during surgery as a prophylactic agent to prevent deep venous thrombosis. Recently it has been shown that dextran increases t-PA plasma concentrations in patients. As dextran is a potential ligand for the mannose receptor, we studied whether this glucose-polymer would be able to inhibit mannose receptor-mediated clearance of t-PA.In this report we show that dextran 40 and dextran 70 were able to inhibit t-PA binding to the isolated human mannose receptor (IC5014 and 4 mg/ml, respectively). Both glucose-polymers inhibited mannose receptor-mediated t-PA degradation by human monocyte-derived macrophages in vitro (IC50 7 and 2 mg/ml, respectively). The α2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP)-mediated t-PA degradation by the macrophages was not affected by dextran. During and after a 45 min infusion of dextran 70 (Macrodex) in rats, in plasma endogenous t-PA concentrations increased to 162 ± 33% and 122 ± 35% respectively. The plasma clearance of a bolus injection of exogenous t-PA was decreased by 33 ± 9% in the same rats.We conclude that dextran inhibits mannose receptor-mediated t-PA clearance. The inhibition of t-PA clearance during dextran infusion results in increased endogenous t-PA plasma concentrations. Increased t-PA concentrations present during thrombus formation are known to increase thrombus lysability. Thus the inhibition of t-PA clearance can contribute to the antithrombotic effect of dextran.

To study the role of the mannose receptor in cellular uptake and degradation of tissue-type plasminogen activator (t-PA), a set of five monoclonal antibodies (Moab) was generated against the mannose receptor isolated from human placental tissue. All Moab specifically recognised the 175 kDa mannose receptor in a crude placenta extract, as shown in Western blot analysis. By use of immunohistochemistry, we showed that in human placenta only the Hofbauer cells (fetal macrophages) express the mannose receptor. Epitope competition experiments indicated that the Moab bound to at least two different epitopes on the receptor molecule. Moab 14-3, 14-5, and 15-2, which are directed against one of these epitopes, strongly inhibited the interaction between the purified mannose receptor and t-PA. These Moab also inhibited mannose receptor-mediated degradation of t-PA by cultured human macrophages. The low density lipoprotein receptor-related protein (LRP) mediated t-PA degradation was not affected by the Moab. It is concluded that the Moab are useful for studying the expression of the human mannose receptor in Western blot and in immunohistochemistry, and for studying the interactions between the human mannose receptor and the mannose-containing ligand t-PA. Copyright © 1997 Schattauer Verlag. Chemicals/CAS: Antibodies, Monoclonal; Epitopes; Lectins, C-Type; mannose receptor; Mannose-Binding Lectins; Receptors, Cell Surface; Tissue Plasminogen Activator, EC 3.4.21.68

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a two-chain form (tcu-PA/T), which is virtually inactive in plasminogen activator assays. Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA) for the assessment of tcu-PA/T in human body fluids. In this BIA, urokinase antigen was immuno-immobilized in microtiter plates and treated with cathepsin C, a specific activator of tcu-PA/T, after which plasminogen activator activity was measured. The occurrence of tcu-PA/T was examined in the plasma of 27 healthy individuals and of 17 sepsis patients, and in the synovial fluid of 16 rheumatoid arthritis patients. In addition, the concentration of urokinase antigen and scu-PA were measured in all three groups. In the plasma of the healthy individuals no measurable amounts of tcu-PA/T could be found (< detection limit of 0.2 ng/ml). In the plasma of almost all sepsis patients tcu-PA/T could be detected (median value 0.4 ng/ml). The amount of tcu-PA/T was 12% of the amount of scu-PA and accounted for about 9% of urokinase antigen. In the synovial fluid of all rheumatoid arthritis patients tcu-PA/T could be measured (median value 5.4 ng/ml) at a concentration which was twofold higher than the concentration found for scu-PA. In this group tcu-PA/T contributed to about 47% of the urokinase antigen. From these data we conclude that inactivation of scu-PA by thrombin can take place in vivo under pathological conditions which involve the production of large amounts of thrombin. This way thrombin may regulate fibrinolysis and extracellular proteolysis. The BIA for tcu-PA/T can be of use for further research on the physiological role of tcu-PA/T. Chemicals/CAS: Thrombin, EC 3.4.21.5; Urinary Plasminogen Activator, EC 3.4.21.73

SummaryThe recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding charactaristics to freshly isolated rat parenchymal liver cells.The binding of 125I-HMW-scu-PA at 4° C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding.Recombinant scu-PA also bound with high affinity (Kd= 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37° C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems. The binding of rscu-PA was decreased for 91%, while that of HMW-scu-PA showed a decrease of 51%.It is suggested that native HMW-scu-PA is bound and degraded by the rat parenchymal liver cells via a lectin-like recognition site, while non-glycosylated recombinant scu-PA is bound and degraded by rat parenchymal liver cells via the low density lipoprotein receptor-related protein (LRP). The differences in recognition system for native and recombinant proteins by liver cells suggest that the glycosylation of recombinant proteins, as obtained in mammalian expression systems, can be important for their physiological fate and their pharmacological application.

Fibrinogen γ’ and variation in fibrinogen gamma genes in the etiology of portal vein thrombosis

BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against C1-inactivator, α2-antiplasmin and α1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of α2-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by C1-inactivator, α2-antiplasmin and α1-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form. Chemicals/CAS: alpha 1 antitrypsin, 9041-92-3; alteplase, 105857-23-6; fibrinogen, 9001-32-5; plasmin, 9001-90-5, 9004-09-5; reteplase, 133652-38-7; tissue plasminogen activator, 105913-11-9; proteinase inhibitor, 37205-61-1; BM 06.022; Fibrinolytic Agents; Protease Inhibitors; Recombinant Proteins; Tissue Plasminogen Activator, EC 3.4.21.68

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

SummaryThrombolytic therapy successfully reopens obstructed blood vessels in the majority of cases. However, it is not known why a substantial amount of thrombi are resistant to lysis by a fibrinolytic agent. In vitro studies have demonstrated that tissue-type plasminogen activator (t-PA) and plasminogen incorporated in the clot (during formation) increase lysibility. To test whether lysibility of in vivo formed human thrombi is related to their composition, we studied 25 venous thrombi obtained at autopsy and 21 arterial thrombi obtained during embolectomy.Plasminogen activator inhibitor-1 (PAI-1) antigen was measured in a phosphate-buffered saline (PBS) extract of each thrombus; t-PA antigen and plasminogen antigen were determined in a 6 M urea extract of the thrombus, representing bound proteins. Lysibility was measured as weight reduction during 8 h of incubation in PBS containing streptokinase (SK) 100 U/ml, corrected for spontaneous lysis, reflected by weight loss in PBS without SK. In addition, lysibility in SK was compared with lysibility in urokinase (UK) 100 U/ml and in t-PA 200 U/ml.Spontaneous lysis amounted to 29 ± 5% (mean ± SEM) and 33 ± 5% in venous and arterial thrombi, respectively, and inversely correlated with the PAI-1 content of thrombi (r = —0.43, p We conclude that spontaneous lysis of thrombi in saline is dependent on PAI-1 content and that susceptibility of thrombibi to lysis by SK ex vivo is dependent on the plasminogen content

It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and alpha 2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 micrograms/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and alpha 2-antiplasmin consumption were most pronounced at 37 degrees C, were less pronounced at 25 degrees C, but surprisingly, did not further decrease at 10 degrees C, 0 degrees C or -8 degrees C. In contrast, when plasma was supplemented with 160 IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and alpha 2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10 degrees C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogen, alpha 2-antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0 degrees C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples. In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 micrograms/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0 degrees C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy.

SummaryTAFIa was shown to attenuate fibrinolysis. In our in vitro study, we investigated how the inhibitory effect of TAFIa depended on the type and concentration of the plasminogen activator (PA). We measured PA-mediated lysis times of plasma clots under conditions of maximal TAFI activation by thrombin-thrombomodulin in the absence and presence of potato carboxypeptidase inhibitor. Seven different PAs were compared comprising both tPA-related (tPA, TNK-tPA, DSPA), bacterial PA-related (staphylokinase and APSAC) and urokinase-related (tcu-PA and k2tu-PA) PAs. The lysis times and the retardation factor were plotted against the PA concentration. The retardation factor plots were bell-shaped. At low PA concentrations, the retardation factor was low, probably due to the limited stability of TAFIa. At intermediate PA concentrations the retardation factor was maximal (3-6 depending on the PA), with TNK-tPA, APSAC and DSPA exhibiting the strongest effect. At high PA concentrations, the retardation factor was again low, possibly due to inactivation of TAFIa by plasmin or to a complete conversion of glu-plasminogen into lys-plasminogen. Using individual plasmas with a reduced plasmin inhibitor activity (plasmin inhibitor Enschede) the bell-shaped curve of the retardation factor shifted towards lower tPA and DSPA concentrations, but the height did not decrease. In conclusion, TAFIa delays the lysis of plasma clots mediated by all the plasminogen activators tested. This delay is dependent on the type and concentration of the plasminogen activator, but not on the fibrin specificity of the plasminogen activator. Furthermore, plasmin inhibitor does not play a significant role in the inhibition of plasma clot lysis by TAFI.

In order to study the role of the asialoglycoprotein receptor (ASGPr) in the rapid plasma clearance of urokinase-type plasminogen activator (u-PA), a microtiter plate binding assay was developed using ASGPr purified from rat liver extracts. Urinary two-chain u-PA bound to immobilized ASGPr in a saturable manner with an EC50 of 0.2 microM. Binding was inhibited by rabbit antibodies against the ASGPr. In line with the known carbohydrate specificity of the ASGPr, GalNAc proved to be the most effective inhibitor from a series of monosaccharides, followed by Gal and Fuc, whereas GlcNAc was ineffective. The N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element, but with a GalNAc-GlcNAc element which is partially sulfated. Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30% of the total) appeared to bind to the ASGPr. From different u-PA preparations used for thrombolytic therapy only urinary u-PA and u-PA produced by kidney cell cultures strongly bound to the ASGPr, whereas (recombinant) u-PA expressed in mouse myeloma cells, Chinese hamster ovary cells or E. coli scarcely bound to the receptor. It is concluded that u-PA bearing non-sulfated GalNAc-GlcNAc elements is specifically recognized by the ASGPr present on liver cells.

In a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed. Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot. In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

Dextran is used during surgery as a prophylactic agent to prevent deep venous thrombosis. Recently it has been shown that dextran increases t-PA plasma concentrations in patients. As dextran is a potential ligand for the mannose receptor, we studied whether this glucose-polymer would be able to inhibit mannose receptor-mediated clearance of t-PA. In this report we show that dextran 40 and dextran 70 were able to inhibit t-PA binding to the isolated human mannose receptor (IC50 14 and 4 mg/ml, respectively). Both glucose-polymers inhibited mannose receptor-mediated t-PA degradation by human monocyte-derived macrophages in vitro (IC50 7 and 2 mg/ml, respectively). The alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP)-mediated t-PA degradation by the macrophages was not affected by dextran. During and after a 45 min infusion of dextran 70 (Macrodex) in rats, in plasma endogenous t-PA concentrations increased to 162 +/- 33% and 122 +/- 35% respectively. The plasma clearance of a bolus injection of exogenous t-PA was decreased by 33 +/- 9% in the same rats. We conclude that dextran inhibits mannose receptor-mediated t-PA clearance. The inhibition of t-PA clearance during dextran infusion results in increased endogenous t-PA plasma concentrations. Increased t-PA concentrations present during thrombus formation are known to increase thrombus lysability. Thus the inhibition of t-PA clearance can contribute to the antithrombotic effect of dextran.

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta 125-rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta 125-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a two-chain form (tcu-PA/T), which is virtually inactive in plasminogen activator assays. Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA) for the assessment of tcu-PA/T in human body fluids. In this BIA, urokinase antigen was immuno-immobilized in microtiter plates and treated with cathepsin C, a specific activator of tcu-PA/T, after which plasminogen activator activity was measured. The occurrence of tcu-PA/T was examined in the plasma of 27 healthy individuals and of 17 sepsis patients, and in the synovial fluid of 16 rheumatoid arthritis patients. In addition, the concentration of urokinase antigen and scu-PA were measured in all three groups. In the plasma of the healthy individuals no measurable amounts of tcu-PA/T could be found(detection limit of 0.2 ng/ml). In the plasma of almost all sepsis patients tcu-PA/T could be detected (median value 0.4 ng/ml). The amount of tcu-PA/T was 12% of the amount of scu-PA and accounted for about 9% of urokinase antigen. In the synovial fluid of all rheumatoid arthritis patients tcu-PA/T could be measured (median value 5.4 ng/ml) at a concentration which was twofold higher than the concentration found for scu-PA. In this group tcu-PA/T contributed to about 47% of the urokinase antigen. From these data we conclude that inactivation of scu-PA by thrombin can take place in vivo under pathological conditions which involve the production of large amounts of thrombin. This way thrombin may regulate fibrinolysis and extracellular proteolysis. The BIA for tcu-PA/T can be of use for further research on the physiological role of tcu-PA/T.

The present study was undertaken in order to determine the recognition site for tissue-type plasminogen activator-plasminogen activator inhibitor type 1 [t-PA-PAI-1] complexes in rat liver in vivo and in vitro. After intravenous injection into rats t-PA-PAI-1 complexes were rapidly removed from the plasma and the liver took up 80% of the injected dose. Within the liver parenchymal and endothelial liver cells contributed mainly to the uptake of t-PA-PAI-1, and were responsible for 62% and 24% of the liver uptake, respectively. The interaction of t-PA-PAI-1 with isolated rat parenchymal liver cells was of high affinity (Kd 17 nM). A well-known antagonist of the alpha 2-macroglobulin receptor (alpha 2MR/low-density lipoprotein receptor-related protein (LRP), GST-39kDa protein (GST-39kDaP) efficiently inhibited the binding (IC50 0.7 nM) of t-PA-PAI-1 to rat parenchymal liver cells. The interaction of t-PA-PAI-1 with LRP on rat parenchymal liver cells was not Ca2(+)-dependent and is most probably mediated by a specific determinant on PAI-1, since an anti-PAI-1 monoclonal antibody inhibited the binding of t-PA-PAI-1, where as free t-PA did not. The binding of t-PA-PAI-1 to rat hepatocytes could not be inhibited by a complex of plasmin and alpha 2-antiplasmin nor by various other ligands of LRP like beta-VLDL and lactoferrin. Binding of t-PA-PAI-1 to rat parenchymal liver cells was followed by internalization and subsequent degradation in the lysosomal compartment. It is concluded that parenchymal and endothelial liver cells mediate the removal of t-PA-PAI-1 complexes from the circulation. LRP on rat parenchymal liver cells is responsible for the uptake and degradation of t-PA-PAI-1 and may therefore be important for the regulation of the t-PA levels in the circulation.

The recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding characteristics to freshly isolated rat parenchymal liver cells. The binding of 125I-HMW-scu-PA at 4 degrees C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding. Recombinant scu-PA also bound with high affinity (Kd = 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37 degrees C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems.(ABSTRACT TRUNCATED AT 250 WORDS)

BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 microgram/ml to 38 min at 10 micrograms/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of alpha 2-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 micrograms/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparin-agarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

The fibrinolytic activity of low molecular weight (LMW) single-chain urokinase-type plasminogen activator (scu-PA) lacking the epidermal growth factor domain and the kringle domain was compared with the activity of high molecular weight (HMW) scu-PA. LMW scu-PA was 1-5 times less active than HMW scu-PA in a fibrin plate method, in a purified fibrin clot lysis assay and in a plasma clot lysis assay. Time course experiments in a chromogenic plasminogen activator assay suggested that LMW scu-PA was less sensitive to activation by plasmin than HMW scu-PA. This was confirmed in a scu-PA activation test, which showed that at a concentration of 40 IU/ml LMW scu-PA required a three-fold higher plasmin concentration for 50% activation in 20 min than did HMW scu-PA. Kinetic experiments in the presence of 0.1 M NaCl showed non-standard Michaelis-Menten kinetics for the activation by plasmin of both HMW and LMW scu-PA. In contrast, standard kinetics was observed at 0.15 M NaCl, showing a 2.6-fold lower catalytic efficiency for LMW scu-PA than for HMW scu-PA. It is concluded that the plasmin activation of LMW scu-PA is about three times slower than the activation of HMW scu-PA. This explains, at least partially, the lower fibrinolytic activity of LMW scu-PA in comparison with HMW scu-PA.

It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and alpha 2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 micrograms/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and alpha 2-antiplasmin consumption were most pronounced at 37 degrees C, were less pronounced at 25 degrees C, but surprisingly, did not further decrease at 10 degrees C, 0 degrees C or -8 degrees C. In contrast, when plasma was supplemented with 160 IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and alpha 2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10 degrees C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogen, alpha 2-antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0 degrees C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples. In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 micrograms/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0 degrees C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy.

The Inactivation of Single-chain Urokinase-type Plasminogen Activator by Thrombin May Provide an Additional Explanation for the Antifibrinolytic Effect of Factor XI