(Circulation. 1997;95:46-52.)
© 1997 American Heart Association, Inc.
Articles |
Antagonists of the Mannose Receptor and the LDL Receptor–Related Protein Dramatically Delay the Clearance of Tissue Plasminogen Activator
the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden (E.A.L.B., M.v.T., H.V., M.K.B., T.J.C.v.B., J.K.), and Gaubius Laboratory, TNO Prevention and Health (M.M.B.-B., D.C.R.), Leiden, The Netherlands.
Correspondence to Dr Ir E.A.L. Biessen, Division of Biopharmaceutics, LACDR, University of Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands.
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Abstract |
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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.
Key Words: plasminogen activators • thrombolysis • cluster mannoside • GST-RAP
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Introduction |
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Tissue plasminogen activator is a serine protease that plays a central role in the fibrinolytic system.1 2 TPA converts plasminogen to plasmin, which degrades blood clot–associated fibrin. The fibrin-specific thrombolytic TPA has proved to be a potent drug in several clinical trials.3 4 5 Despite its widespread clinical application, the thrombolytic efficacy of TPA is complicated by its rapid clearance from the circulation, and large doses of TPA must be administered.6 7 8 9 10 The short plasma half-life of TPA (ranging from 1 minute in rats to about 6 minutes in humans) results from a rapid liver uptake of TPA.6 7 8 9 10 11 In vivo studies on TPA have indicated that at least two different hepatic uptake mechanisms are involved in the clearance of TPA from the circulation, because both parenchymal and endothelial liver cells contribute to the liver uptake of TPA.7 11 12 13
The characteristics of the TPA uptake sites on parenchymal and endothelial liver cells differ markedly.7 12 14 Uptake by endothelial liver cells is mediated by the mannose receptor, which recognizes the mannose-rich oligosaccharide chain at Asn117 of TPA.7 11 12 13 The receptor involved in parenchymal liver cell uptake is not unequivocally identified to date.7 12 13 15 16 17 In vitro binding studies revealed that TPA may interact with LRP, the asialoglycoprotein receptor,11 and a novel carbohydrate recognition system.7 12 13 15 16 17 Warshawsky et al16 showed that an established LRP antagonist, GST-RAP, reduced the in vivo clearance of TPA. Major efforts have been undertaken to construct TPA variants with prolonged plasma half-lives.18 19 20 21 22 23 24 25 26 To circumvent endothelial cell uptake of TPA via the mannose receptor, deglycosylated TPA variants were developed, and the clearance of these variants was significantly reduced.18 19 Alternatively, deletion of the finger and epidermal growth factor domains also resulted in a significant increase of the plasma half-life,21 22 23 24 25 26 whereas blockade of the active site of TPA (protease domain) only marginally affected the plasma half-life.12 26 However, the benefit in overall thrombolytic activity of these variants was often too low to justify further development as a thrombolytic drug.
Therefore, we pursued an alternative approach to improve the pharmacokinetics of TPA. We investigated whether the in vivo half-life of wild-type TPA can be prolonged by blockade of its clearance. We devised and synthesized a series of high-affinity ligands for the mannose receptor. Combination of the developed mannose receptor antagonist with an LRP antagonist reduced the liver uptake of TPA strongly and prolonged the plasma half-life of TPA 10-fold.
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Materials
BSA (fraction V), collagenase (types I and IV), and iodogen were purchased from Sigma Chemical Co [125I]NaI (carrier free) and streptavidin–alkaline phosphatase conjugate were from Amersham. Pronase and DNase I were from Boehringer Mannheim GmbH. Nycodenz was from Nycomed Pharma AS (Oslo, Norway). HEPES was from Merck. Recombinant TPA was from Boehringer Ingelheim GmbH. All other chemicals were of analytic grade. The synthesis of M6L5 is described in detail elsewhere.27
Production and Isolation of GST-RAP
A plasmid (pGEX) encoding for a fusion protein (GST-RAP) of GST and the 39-kD protein or receptor-associated protein (RAP), which was transformed in Escherichia coli (DH5
), was a generous gift of Dr J. Herz (Dallas, Tex). GST-RAP was produced exactly as described.28 The potency of GST-RAP to displace trypsin-activated 125I-2M binding from its receptor was essentially equal to values described in the literature (IC50, 1 nmol/L).
Isolation of Human Mannose Receptor
Human mannose receptor was isolated from human placenta after solubilization with Triton X-100 and subsequently purified by affinity chromatography over mannosylated albumin-sepharose according to Otter et al.29
Biotinylation and Radiolabeling of TPA
TPA was dialyzed against 0.1 mol/L NaHCO3 (pH 8.5) and reacted with N-hydroxysuccinimide–activated biotin (Zymed Laboratories Inc) at a ratio of 1 mol TPA to 200 mol N-hydroxysuccinimide–activated biotin at room temperature for 3 hours. After reaction, the modified protein was dialyzed against 20 mmol/L Tris buffer, pH 7.4, containing 0.01% Tween-80 (vol/vol).
Recombinant TPA was iodinated by the iodogen method as described, and a specific radioactivity of 3500 to 5000 cpm/ng protein was obtained.7
Mannose Receptor Binding Assay
Displacement studies of the binding of biotinylated TPA to isolated human mannose receptor were performed according to the procedure of Otter et al.29 Plates were coated with 100 µL solubilized receptor in loading buffer (pH 7.4) containing 0.02 mol/L Tris-HCl, 5 mmol/L CaCl2, and 0.15 mol/L NaCl at 4°C overnight. Loading buffer supplemented with 0.5% Tween 80 and 0.1% BSA (125 µL) was added for 30 minutes at room temperature to minimize aspecific binding of ligand to the wells. The receptor-coated wells were preincubated with the indicated amounts of competitor for 30 minutes at room temperature. Biotinylated TPA (1.5 nmol/L) was added and incubated for 2 hours at room temperature. Streptavidin–alkaline phosphatase conjugate was added subsequently, and the wells were incubated for 1 hour at room temperature. Next, p-nitrophenolphosphate was added, the wells were incubated for 4 hours at 25°C, and finally the absorption at 405 nm was monitored with a microplate reader. Wells were washed three times with 0.5% Tween-80 in loading buffer supplemented with 0.5% Tween and 0.1% BSA after each step of the procedure. Uncoated wells were used as a control for aspecific binding of biotinylated TPA to uncoated wells.
In Vivo Plasma Clearance and Organ Uptake
Twelve-week-old male Wistar rats (225 to 275 g) were anesthetized by injection with 20 mg pentobarbital IP. The abdomen was opened, radiolabeled TPA (600 µg/kg body wt) was injected via the vena penis, and at the indicated times, blood samples (0.3 mL) were taken with heparinized syringes from the vena cava and liver lobules were tied off. The liver uptake of the injected compound was corrected for the radioactivity in plasma in the liver at the time of sampling.7
Cell Isolation Procedures
For determination of the contributions of different liver cell types to total liver uptake, rats were anesthetized and injected with 125I-labeled TPA via the vena penis. After 10 minutes, the vena porta was cannulated and a liver perfusion at low temperature (<8°C) was started with Hanks' buffer (supplemented with 10 mmol/L HEPES). Parenchymal liver cells, endothelial liver cells, and Kupffer cells were isolated exactly as described.7 The contributions of the various liver cell types to total liver uptake were calculated as described.7 As found for a number of substrates, no loss of cell-bound label and/or formation of acid-soluble radioactivity occurred during the low-temperature cell isolation procedure, leading to a quantitative recovery of radioactivity associated with the isolated liver cells compared with the total liver association. This was checked for each individual liver cell isolation by comparison of the calculated liver association (from the relative contributions of the various cell types) and the determined total liver association.
Toxicity Studies
Rats (Wistar, male, 250 g) were anesthetized with ether, and PBS (500 µL) or M6L5 (6.0 mg/kg) in 500 µL PBS was injected in the vena penis. At 2 and 24 hours after injection, blood samples (600 µL) were taken. Serum levels of alanine aminotransferase, aspartate aminotransferase, and 
-glutamyl transferase were determined enzymatically with Boehringer Mannheim SYS-3 BM/Hitachi 747 enzyme kits. Kinetic determination of lactate dehydrogenase activity in serum was determined on an SYS-3 BM/Hitachi 747 with the Boehringer Mannheim LDH kit. After 24 hours, rats were killed and liver, spleen, and kidney were excised, weighed, and analyzed histologically.
Data Analysis
The displacement binding data were analyzed according to a single-site model with a computerized nonlinear fitting program (Prism, GraphPad Software) to calculate the IC50 values.30 The Ki was calculated from the corresponding IC50 by the Cheng-Prussoff equation [Ki=IC50/(1+Ligand/Kd)] and assuming the Kd of TPA to be 1.0 nmol/L. Pharmacokinetic studies of TPA clearance were analyzed according to a two-phase exponential decay model using the same program. Clearance (Cl) was calculated from the area under curve (AUC) of the plasma decay and the injected dose of TPA according to the equation Cl=Dose/AUC. The significance of differences between means was tested by unpaired two-way Student's t test. Significance of the differences in TPA clearance between control and treated rats was analyzed by one-way ANOVA with a Student-Newman-Keuls multiple-comparison post hoc test (Instat, GraphPad software).
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Mannose Receptor Binding Studies
A series of cluster mannosides on a base of an oligolysine backbone was synthesized27 (for chemical structure see Fig 1
). The cluster mannosides contain an increasing number of mannose residues, and their affinity for the isolated human mannose receptor was tested (Fig 2). All cluster mannosides completely inhibited the binding of biotinylated TPA to the mannose receptor, and the potency to compete for the binding of TPA increased dramatically with increasing mannose valency. From the inhibition curves, the inhibition constants were calculated. It was found that the inhibition constant of M6L5 (0.41±0.09 nmol/L), which showed the highest affinity for the mannose receptor, was almost 107-fold lower than that of mannose (4.0±0.6 mmol/L).
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), M2L (
), M3L2 (
), M4L3 (
), M5L4 (
), and M6L5 (
). Binding of biotinylated TPA is expressed as percentage of the control binding of biotinylated TPA (without displacer).
Effect of M6L5 on the Plasma Clearance and Liver Uptake of TPA
Since TPA is in part cleared from plasma via the liver mannose receptor, we determined the effect of the high-affinity ligand for the mannose receptor, M6L5, on TPA clearance. In control rats, 125I-TPA (600 µg/kg) was rapidly cleared from the bloodstream (t1/2, 1.1±0.1 minutes; Fig 3) because of a rapid uptake of TPA by the liver, and a maximum of 86±1.5% of the injected dose was recovered in the liver. Injection of M6L5 1 minute before 125I-TPA resulted in a significant and dose-dependent reduction in TPA clearance. At a dose of 0.12 mg M6L5/kg, the rate of TPA clearance was reduced by 48% (1.9±0.1 and 3.5±0.2 mL/min for 0.12 mg M6L5/kg and controls, respectively; Fig 4), whereas 1.2 mg M6L5/kg inhibited the clearance for 59% (1.46±0.07 mL/min). Concomitantly, the liver uptake of TPA was delayed, and the maximal liver uptake was reduced to 73±1% and 62.5±1.0% of the injected dose after preinjection of 0.12 and 1.2 mg M6L5/kg, respectively.
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Effect of GST-RAP on the Plasma Clearance and Liver Uptake of 125I-TPA
To address the involvement of LRP in TPA clearance, we studied the effect of an established antagonist of LRP,30 GST-RAP, on the clearance of TPA in the rat. Fig 3
shows that preinjection of GST-RAP (40 mg/kg) strongly affected the pharmacokinetics of 125I-TPA (600 µg/kg). At 10 minutes after injection, 21±1% of the injected dose still resided in the circulation, and the clearance was reduced significantly, by 63% (1.30±0.03 and 3.5±0.2 mL/min for GST-RAP–treated and control, respectively; Fig 4). GST-RAP pretreatment led to a delay in liver uptake of 125I-TPA, and maximal liver uptake was reduced to 60±2% of the injected dose.
Effect of M6L5 and GST-RAP on the Hepatocellular Distribution of TPA
To determine whether the receptor antagonists M6L5 and GST-RAP indeed blocked uptake of 125I-TPA via the corresponding receptors, we studied their effects on the uptake of 125I-TPA in the various liver cell types (Table 1). As described before,7 parenchymal and endothelial liver cells appeared to be the major cell types responsible for liver uptake of 125I-TPA in control rats; 55±1.5% of total liver uptake of 125I-TPA was recovered in parenchymal liver cells, and 40±2% was recovered in endothelial cells. Preinjection of M6L5 (1.2 mg/kg) caused a significant shift in the liver cell distribution profile. Parenchymal liver cell uptake increased significantly, to 71±3%, while at the same time the relative contribution of endothelial cells to 125I-TPA uptake decreased to 19.5±1% of the total liver uptake. The increase of the relative contribution of parenchymal liver cells was not caused by enhanced uptake per milligram of cell protein. The specific parenchymal liver cell uptake of 125I-TPA was not influenced by preinjection of M6L5, in contrast to the specific endothelial cell uptake, which was reduced by 72% (124±5% and 430±40% of injected dosex103/mg cell protein for M6L5-treated rats and for controls, respectively).
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By contrast, GST-RAP treatment reduced parenchymal cell uptake by 65% (7.6% versus 21±4% of injected dosex103/mg cell protein for GST-RAP–treated and control rats, respectively). Concomitantly, specific endothelial cell uptake was increased by 44% to 619% of injected dosex103/mg cell protein on GST-RAP treatment. Apparently, TPA uptake is partly compensated by an increased uptake by mannose receptor in case the LRP-mediated pathway is blocked. Both GST-RAP and M6L5 preinjection did not significantly affect Kupffer cell uptake of TPA.
Effect of Combined Treatment With M6L5 and GST-RAP on the Plasma Clearance and Liver Uptake of 125I-TPA
These findings demonstrate that although GST-RAP and M6L5 both affect TPA clearance, blockade of either receptor system is not sufficient to prevent clearance of TPA. Therefore, we treated rats with both M6L5 (1.2 mg/kg) and GST-RAP (40 mg/kg) and determined that the plasma clearance of 125I-TPA (600 µg/kg) was almost completely blocked (Fig 5). At 10 minutes after injection, 70±7% of the injected dose is still recovered in the plasma, which is significantly more than in untreated controls (8±0.4%), in M6L5-treated rats (21±3%), or in GST-RAP–treated rats (21±1%). The TPA clearance is reduced almost 10-fold, from 3.5±0.2 mL/min for the control rats to 0.42±0.05 mL/min for the combined treatment (Fig 4). Moreover, liver uptake of 125I-TPA was almost completely abolished after preinjection with GST-RAP and M6L5. Only 18.7±0.8% of the injected dose, at maximum, was recovered in the liver, compared with 86±1.5% for controls.
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To exclude the possibility that the observed effect of combined treatment with GST-RAP plus M6L5 on TPA clearance resulted from an aspecific effect of GST-RAP and/or M6L5 on hepatic blood flow or receptor-mediated endocytosis in general, we also tested the effect of combined treatment on the in vivo kinetics of 125I-ASOR, which is an established substrate for the asialoglycoprotein receptor. No effect of combined treatment was observed on liver uptake or plasma clearance of 125I-ASOR. The plasma half-life of ASOR was 0.53 minute in treated and 0.51 minute in untreated rats (data not shown).
Toxicity of M6L5
To validate the potential of M6L5 as a therapeutic additive in thrombolytic therapy, we assessed the acute toxicity of M6L5 (Table 2). Even at doses (6 mg/kg) 5 to 50 times higher than doses used in this study, M6L5 was essentially nontoxic after single bolus injection. Liver, spleen, and kidney weights remained unaffected, and serum parameters for systemic (lactate dehydrogenase) and liver toxicity (alanine aminotransferase, aspartate aminotransferase, and -glutamyl transferase) at 2 hours and at 24 hours after injection were essentially unaltered. Histological analysis of liver did not show any signs of toxicity. We may therefore assume that the toxicity of M6L5 is very low.
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Discussion |
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The therapeutic effectiveness of the highly potent thrombolytic agent TPA is reduced by its rapid elimination from the bloodstream, which results from an efficient liver uptake. A first approach to improve the pharmacokinetics of TPA has been the construction of TPA mutants that lack those domains responsible for hepatic uptake.18 19 20 21 22 23 24 25 26 31 32 33 We used another, still unexplored, approach and developed a highly specific antagonist for the mannose receptor, which is responsible for 40% of the liver uptake of TPA.
In search of high-affinity mannose receptor ligands, we synthesized a series of cluster mannosides that contained two (M2L) to six (M6L5) mannose residues per cluster molecule. The cluster mannoside that carried two mannose groups (M2L; Ki, 16 µmol/L) already displayed a 250-fold higher affinity than -D-mannose (Ki, 4.0 mmol/L). The most potent mannoside, M6L5, had an affinity for the mannose receptor of 0.41 nmol/L, which is substantially higher than that of ovalbumin (Ki, 290 nmol/L14 ) or mannosylated BSA (Ki, 2.2 nmol/L34 ) and quite similar to that of TPA (Ki, 0.6 nmol/L14 ). Previously developed synthetic mannosides—mostly branched oligosaccharides—possessed affinities only in the low micromolar range,35 which is >1000-fold lower than the affinity of M6L5. The subnanomolar affinity of M6L5, in combination with its accessible synthesis, makes M6L5 a promising compound to inhibit mannose receptor–mediated uptake of TPA.
In vivo, M6L5 significantly and dose-dependently inhibited the clearance of 125I-TPA (injected at a therapeutic dose of 600 µg/kg) by up to 59%. The reduction in liver uptake of TPA by M6L5 treatment resulted from a specific inhibition of TPA uptake by endothelial liver cells. This corresponds well with earlier studies showing that the clearance of deglycosylated TPA mutants was retarded by a factor of 3 compared with unmodified TPA.18 30 35 Blockade of the plasma clearance of TPA could be reduced 2.6-fold on blockade of the mannose receptor by high doses of mannan (20 mg/kg) or ovalbumin (80 mg/kg).7 11 These data illustrate that M6L5 is 15- to 70-fold more effective than ovalbumin and mannan in the in vivo blockade of TPA clearance via the mannose receptor. Toxicity studies showed that M6L5 is tolerated well at doses 5- to 50-fold higher than the doses that were needed to inhibit TPA clearance. No signs of acute systemic or liver toxicity were observed after single injection of 6.0 mg M6L5/kg. Moreover, M6L5 is probably far less immunogenic than mannan or ovalbumin. It can therefore be concluded that the high affinity and specificity of M6L5 for the mannose receptor, together with its low toxicity, makes it a valuable therapeutic to improve the pharmacokinetics of TPA.
To establish the involvement of LRP in TPA clearance, we quantified the effect of preinjection of GST-RAP (40 mg/kg) on TPA clearance. GST-RAP, a widely used antagonist of LRP,28 appeared to increase the plasma half-life of 125I-TPA 2.7-fold. Warshawsky et al16 showed a similar effect of GST-RAP on TPA clearance. In an extension of their study, we show that GST-RAP pretreatment delayed and reduced liver uptake of TPA significantly, by 30%, and the data on the liver cell distribution of TPA show that GST-RAP specifically reduced the uptake by parenchymal liver cells.
The effect of GST-RAP on liver uptake was comparable to that of M6L5. Apparently, neither antagonist can fully block the plasma clearance of TPA or TPA uptake by the liver. The simultaneous blockade of LRP and the mannose receptor by preinjection of GST-RAP and M6L5 almost completely abolished liver uptake and at the same time reduced TPA clearance 10-fold. Combined treatment did not affect clearance and liver uptake of another fast-clearing glycoprotein, ASOR, excluding the theory that the blockade of TPA clearance results from aspecific effects of the combined treatment on hepatic blood flow or receptor-mediated endocytosis. The effect of the combined treatment on the clearance of TPA matches very well with the kinetics of TPA reported in rats preinjected with an excess of unlabeled TPA (20 mg/kg).7 16 In these studies, half of the injected dose of TPA was still present in the circulation at 30 minutes after injection.7 16 Prevention of the liver uptake of TPA by hepatectomy also resulted in a 10-fold decreased clearance.6 9 10 Apparently, TPA clearance is prolonged by a factor of 10 by prevention of its liver uptake. Recently, Narita et al36 reported that the plasma half-life of TPA (10 µg/kg) in RAP-overexpressing mice was enhanced to 20 minutes after preinjection of 150 mg ovalbumin/kg body wt. Although this suggested that the mannose receptor and LRP are the sole contributors to liver uptake of TPA, that was not conclusively established. First, ovalbumin blocks not only mannose receptors but also asialoglycoprotein receptors, which was also suggested to be involved in TPA clearance.11 Second, RAP is a chaperone protein involved in intracellular trafficking of proteins, suggesting that systemic RAP overexpression in mice may also affect other endocytotic pathways that are important for TPA clearance. Most importantly, Narita et al used tracer doses of TPA (10 µg/kg, which is 60-fold lower than therapeutic doses). At therapeutic doses of 600 µg/kg, alternative TPA uptake pathways may contribute to TPA liver uptake. This study therefore provides additional information that the mannose receptor and LRP are indeed the only major contributors to the liver uptake and rapid clearance of TPA.
In conclusion, we now show that therapeutic levels of plasma TPA can be maintained for a prolonged time span by blockade of both LRP and the mannose receptor–mediated liver uptake of TPA (Fig 6). The rather unexplored approach to improve the clinical effectiveness of TPA by means of receptor blockade involves the combined application of the mannose receptor ligands used in this study and TPA-specific LRP antagonists. As a result, lower doses of costly TPA will suffice for thrombolytic therapy, and TPA pharmacokinetics will be greatly improved, leading to fewer unwanted side effects. Blockade of LRP-mediated uptake of TPA by GST-RAP requires rather high doses, which qualifies its potential in thrombolytic therapy. However, more specific and potent LRP antagonists may be developed by combinatorial immunoglobulin repertoire cloning,37 or recently described truncated RAP mutants38 may be applied for this purpose. Compared with application of new slow-clearing TPA variants, application of one of the above antagonists in thrombolytic therapy offers the advantage that it may improve the thrombolytic activity of wild-type TPA, an acknowledged and successful fibrinolytic agent.
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Acknowledgments |
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This study was supported by grants M93.001 and 90.294 from the Dutch Heart Foundation.
Received March 18, 1996; revision received August 7, 1996; accepted August 13, 1996.
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References |
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