Migration of the activation peptide of thrombin-activatable fibrinolysis inhibitor (TAFI) during SDS-polyacrylamide gel electrophoresis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which upon activation is capable of delaying fibrinolysis. We investigated the migration and detection of the activation peptide of TAFI during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Purified TAFI before and after activation by thrombin/thrombomodulin was electrophoresed on 4-20% polyacrylamide gels and stained with Coomassie blue as well as Western blotting. Before activation, Coomassie blue staining resulted in one main band of TAFI. After activation, a sharp band corresponding to TAFIa was observed. No distinct activation peptide was detected, in agreement with the literature. Western blotting using a polyclonal anti-TAFI antibody, on the other hand, showed one additional broad band with an Mr of about 33 000 after TAFI activation. N-terminal sequence analysis confirmed that this band represented the activation peptide of TAFI. In addition, we tested the reactivity of two anti-TAFI monoclonal antibodies (MA-T3D8 and MA-T18A8) towards TAFI before and after activation by Western blotting. Both monoclonal antibodies recognized TAFI. After activation of TAFI, MA-T3D8 reacted with TAFIa, while MA-T18A8 reacted with the activation peptide. We identify the 33 000 band as the activation peptide of TAFI and exemplify the use of this information for the characterization of monoclonal antibodies against TAFI.

Characterization of the binding of urokinase-type plasminogen activator to the asialoglycoprotein receptor.

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.

Role of carbohydrate and protein in the binding of tissue-type plasminogen activator to the human mannose receptor.

The 175-kDa mannose receptor is one of the receptors that mediates the clearance of tissue-type plasminogen activator (t-PA). The affinity of t-PA for the mannose receptor is much higher than the affinity of other high-mannose-type oligosaccharide-containing glycoproteins. In order to find an explanation for this high affinity, we studied the biochemical interaction of various forms of t-PA with the isolated human mannose receptor in several in vitro binding assays. t-PA showed a high affinity (Ki = 0.2 nM) for the mannose receptor and the interaction could be fully inhibited by mannan or polyclonal antibodies against the mannose receptor. The interaction was not affected by non-glycosylated t-PA. The high affinity differed slightly between t-PAs synthesized by various cell types (range Ki 0.2-0.7 nM) and between various glycoforms of t-PA. No statistically significant difference in affinity between t-PA and t-PA complexed to inhibitors was observed. In contrast to intact t-PA, a trypsin digest of t-PA had a low affinity (Ki = 0.5 microM) for the mannose receptor. Both intact and trypsin digests of the high-mannose-type oligosaccharide-containing glycoproteins ribonuclease B and ovalbumin had a low affinity (Ki 0.5-1.5 microM) for the mannose receptor. We conclude that neither protein-protein interactions, nor the complex-type oligosaccharides and the fucose residue on t-PA contribute significantly to the high-affinity binding of t-PA. We suggest that the conformation of the high-mannose-type oligosaccharide on t-PA is influenced by the protein moiety of t-PA in such a way that the oligosaccharide has a high affinity for the mannose receptor.

Inhibition of mannose receptor-mediated clearance of tissue-type plasminogen activator (t-PA) by dextran: a new explanation for its antithrombotic effect.

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.

Cluster mannosides can inhibit mannose receptor-mediated tissue-type plasminogen activator degradation by both rat and human cells.

Recently, we developed a series of cluster mannosides that were able to inhibit tissue-type plasminogen activator (t-PA) binding to the isolated mannose receptor. The mannoside with the highest affinity was able to inhibit t-PA clearance by the liver in the rat. To test whether these mannosides would also be efficient inhibitors in humans, we studied the expression of the mannose receptor in the human liver and determined the efficacy of the mannosides to inhibit mannose receptor-mediated t-PA degradation by both rat and human cells. Immunohistochemistry indicates that, like the rat, human liver endothelial cells and human Kupffer cells do express the mannose receptor. The mannosides do inhibit mannose receptor-mediated t-PA binding, association, and degradation by isolated rat liver endothelial cells and t-PA association and degradation by cultured human macrophages at similar concentrations. The cluster mannoside with six mannose residues connected with a backbone of five lysine groups (M6L5) was, like unlabeled t-PA, able to inhibit 125I-t-PA degradation in the nmol/L range, while the mannoside M5L4 inhibited 125I-t-PA degradation in the micromol/L range. The concentrations of mannoside necessary to inhibit 125I-t-PA degradation in vitro were comparable with the concentrations necessary to inhibit mannose receptor-mediated 125I-t-PA clearance in vivo. We conclude that there is no species difference between rat and humans with respect to the distribution of the mannose receptor in the liver and the affinity of the cluster mannosides, establishing the relevance of the inhibition of mannose receptor-mediated t-PA clearance by M6L5 as observed in the rat, for the human situation.

Antagonists of the mannose receptor and the LDL receptor-related protein dramatically delay the clearance of tissue plasminogen activator.

BACKGROUND: Clinical application of tissue plasminogen activator (TPA) as a fibrinolytic agent is complicated by its rapid clearance from the bloodstream, which is caused by TPA liver uptake. The mannose receptor on endothelial liver cells and the LDL receptor-related protein (LRP) on parenchymal liver cells were reported to contribute to liver uptake. METHODS AND RESULTS: In this study, we addressed whether TPA clearance can be delayed by inhibiting receptor-mediated endocytosis of TPA. A series of cluster mannosides was synthesized, and their affinity for the mannose receptor was determined. A cluster mannoside carrying six mannose groups (M6L5) displayed a subnanomolar affinity for the mannose receptor (Ki = 0.41 +/- 0.09 nmol/L). Preinjection of M6L5 (1.2 mg/kg) reduced the clearance of 125I-TPA in rats by 60% because of specific inhibition of the endothelial cell uptake. The low toxicity of M6L5, combined with its accessible synthesis and high specificity for the mannose receptor, makes it a promising agent to improve the pharmacokinetics of TPA. Blockade of LRP by 39-kD receptor-associated protein (GST-RAP) also inhibited TPA clearance by 60%. Finally, combined preinjection of M6L5 and GST-RAP almost completely abolished reduced liver uptake of TPA and delayed its clearance by a factor of 10. CONCLUSIONS: It can be concluded that (1) the mannose receptor and LRP appear to be the sole major receptors responsible for TPA clearance and (2) therapeutic levels of TPA can be maintained for a prolonged time span by coadministration of the aforementioned receptor antagonists.

In vitro stability of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma.

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)

Isolation and characterization of the mannose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator.

Various studies have shown that mannose receptors rapidly eliminate glycoproteins and microorganisms bearing high mannose-type carbohydrate chains from the blood circulation. The purpose of this study was to characterize the mannose receptor in the liver, which in vivo is involved in the rapid clearance of tissue-type plasminogen activator from the circulation. Human liver membranes were solubilized in Triton X-100, and the solution was applied to a tissue-type plasminogen activator Sepharose column. Bound proteins were eluted with ethylenediaminetetraacetate (10 mmol/L). A second, similar purification step rendered a single liver protein of 175,000 daltons. A combination of ligand blotting and a chromogenic assay for tissue-type plasminogen activator demonstrated that the identified liver protein is a mannose receptor because it bound tissue-type plasminogen activator, this tissue-type plasminogen activator binding being fully inhibited by 0.2 mol/L D-mannose. Western-blot analysis revealed that the isolated liver protein is immunologically identical to the human mannose receptor from placenta. Treatment of the liver protein and the placenta mannose receptor with trypsin yielded the same pattern of proteolytic degradation products as identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the physiologically relevant mannose receptor for tissue-type plasminogen activator clearance isolated from human liver is immunologically and structurally similar to or identical with the human mannose receptor isolated from placenta.

Binding of tissue-type plasminogen activator by the mannose receptor.

Previous studies have shown that tissue-type plasminogen activator (t-PA) in blood is cleared by the liver partially through a mannose-specific uptake system. The present study was undertaken to investigate, in a purified system, whether t-PA is recognized by the mannose receptor which is expressed on macrophages and liver sinusoidal cells. The mannose receptor was isolated and purified from bovine alveolar macrophages and migrated as a single protein band at Mr 175,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Ligand blotting revealed that this protein specifically bound t-PA. The t-PA-receptor interaction was further characterized in a binding assay, which showed saturable binding with an apparent dissociation constant of 1 nM. t-PA binding required calcium ions and was negligible in the presence of EDTA or at acid pH. Mannose-albumin was an effective inhibitor, whereas galactose-albumin did not have a significant effect. From a series of monosaccharides tested, D-mannose and L-fucose were the most potent inhibitors, N-acetyl-D-glucosamine was a moderate inhibitor, whereas D-galactose and N-acetyl-D-galactosamine were ineffective. t-PA, deglycosylated by endoglycosidase H, did not interact with the receptor. It is concluded that the mannose receptor specifically binds t-PA, probably through its high mannose-type oligosaccharide.

Plasminogen activation at low temperatures in plasma samples containing therapeutic concentrations of tissue-type plasminogen activator or other thrombolytic agents.

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.

Pharmacokinetics of antigen and activity of recombinant tissue-type plasminogen activator after infusion in healthy volunteers.

Pharmacokinetics of recombinant tissue plasminogen activator (rt-PA, large-scale process) were determined based on antigen and activity after infusion of 0.25 mg/kg in 8 healthy volunteers. Plasma antigen was measured using enzyme-linked immunosorbent assay (ELISA) with and without treatment of blood at collection with D-Phe-Pro-Arg-CH2Cl (P PACK); activity was quantified in acidified plasma both on fibrin plates and in a chromogenic assay. Highest rt-PA concentrations were measured in ELISA with P PACK-treated samples, yielding the following pharmacokinetic parameters (2-compartment model, mean +/- S.D.): Cmax = 973 +/- 133 ng/ml, CL = 687 ml/min, dominant half-life t1/2 alpha = 3.3 +/- 0.4 min, t1/2 beta = 26 +/- 12 min, V1 = 3.9 +/- 0.6 l and Vss = 7.2 +/- 1.0 l. The other assays yielded lower rt-PA concentrations, which affected clearance and volume parameters but not t1/2 beta and t1/2 beta. Linear regressions of the fibrin plate and chromogenic assay results vs. ELISA yielded excellent correlations (R greater than 0.96, n = 55-57) but slopes of 0.76 and 0.64, respectively. This indicates that about 25-35% of rt-PA antigen in thawed plasma samples are not detected in activity assays, due at least partially to in vitro binding of rt-PA by proteinase inhibitors.

The inability of propranolol and aspirin to inhibit the response of fibrinolytic activity and factor VIII-antigen to infusion of DDAVP.

We tested the response of the fibrinolytic activity and factor VIII-antigen levels to infusion of DDAVP in healthy volunteers and we studied the influence of propranolol and aspirin on this response. After DDAVP, 0.4 microgram/kg in 10 min i.v., the fibrinolytic activity of redissolved euglobulins rose from 179 to 452 mm2 (lysis area of fibrin plates); after pretreatment with propranolol, 320 mg per day during 7 days, DDAVP induced a similar rise (from 166 to 471 mm2) and after pretreatment with a single dose of aspirin, 600 mg, ingested 5 hr before the DDAVP infusion, the lysis area increased from 159 to 455 mm2. Factor VIII-antigen level increased within 60 min after DDAVP from 104 to 208%; after pretreatment with propranolol from 111 to 230% and after a single dose of aspirin, DDAVP induced a rise from 107 to 206%. From these data we conclude that neither baseline levels nor the release of plasminogen activator or factor VIII after DDAVP infusion are influenced by beta-blockade or by interference with prostaglandin synthesis.

The use of desmopressin acetate (DDAVP) as a test of the fibrinolytic capacity of patients--analysis of responders and non-responders.

Intravenous infusion of desmopressin (DDAVP, 0.4 micrograms/kg b.w. in 12') causes an increase in the level of extrinsic plasminogen activator, measured in plasma euglobulin fractions with added C1-inactivator on fibrin plates. A poor response or no response at all was elicited in two out of 21 patients with spontaneous thrombosis, 18/38 with hyperlipoproteinaemia and 10/14 with terminal renal insufficiency requiring haemodialysis. Haemodilution during the first 30' after starting the DDAVP-infusion occurred both in responders and in non-responders; so did haemodynamic reactions: increase in heart rate, drop in diastolic blood pressure, facial flushing. The rise of fibrinolytic activity was shown not to be associated with decreased hepatic blood flow. Normal factor VIII-rises in "non-responders" indicate the responsiveness of the receptive organs, including the hypothalamus, to DDAVP. Despite a normal baseline level of fibrinolytic activity in the blood, as occurs for instance in terminal renal insufficiency, the vascular endothelium may be refractory to stimulation. In some patients especially in type IV hyperlipoproteinaemia, a selective defect of the release of plasminogen activator is postulated. In subjects with low fibrinolytic activity at rest, as observed in spontaneous thromboembolism and in hypertriglyceridaemia, the failure to release plasminogen activator upon stimulation with DDAVP might be a consequence of an impairment of synthesis as well.

Response to fibrinolytic activity and factor VIII-related antigen to stimulation with desmopressin in hyperlipoproteinemia.

Impairment of fibrinolysis is supposed to contribute to CVD. In 38 hyperlipoproteinemic patients, known to be at risk for early CVD, fibrinolytic activity was measured before and after stimulation with DDAVP. A negative correlation was found between serum triglyceride levels and fibrinolytic activity, both before and after DDAVP. A subnormal activity was invariably found when serum triglyceride concentration was above 8 mmol/L. The defect can be attributed to low levels of extrinsic plasminogen activator. High cholesterol levels were not associated with impairment of fibrinolysis. Fibrinolytic activity and response to DDAVP were lowest in those patients with hypertriglyceridemia who also had a tendency to develop hyperchylomicronemia. (type V/IV). The low fibrinolytic activity in this type of hyperlipoproteinemia cannot be explained by obesity. Factor VIII was higher than normal in most patients with hyperlipoproteinemia; the level increased after stimulation with DDAVP in every patient. This imbalance between coagulation and fibrinolysis might increase the risk of CVD.