Marrie Barrett-Bergshoeff

Lysine-based Cluster Mannosides That Inhibit Ligand Binding to the Human Mannose Receptor at Nanomolar Concentration

In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysine-based oligomannosides containing two (M2L) to six (M6L5) terminal α-D-mannose groups that are connected with the backbone by flexible elongated spacers (16 Å). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissue-type plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18-23 μM to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional α-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M3L2 to M6L5 displayed negative cooperative inhibition of ligand binding to the mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M6L5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M6L5 not only is a powerful tool to study the mechanism of ligand binding by the mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques.

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.

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

Summary.  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.

Clot penetration and fibrin binding of amediplase, a chimeric plasminogen activator (K2 tu-PA)

Amediplase (K(2) tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase. The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 micro g/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase. We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeation-driven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.

Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot lysis models. Sensitivity to the inhibitory action of thrombin activatable fibrinolysis inhibitor (TAFI)

In 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 of TAFI was examined for each PA by neutralising TAFIa 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 mug/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.

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 µmol/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.

The effect of 40 kHz ultrasound on tissue plasminogen activator‐induced clot lysis in three in vitro models

In previous work from the same laboratory, high‐frequency ultrasound (US) (3 MHz) was shown to promote in vitro fibrinolysis through enhanced supply of plasminogen to the clot surface. The application of high‐frequency US is limited in vivo due to tissue heating. Low‐frequency US, however, has less tissue heating and improved penetration. Internal plasma clot lysis and external lysis with compacted and non‐compacted plasma clots were used to determine the magnitude of the effect of low‐frequency US (40 kHz; 0.5 W/cm2) on tissue plasminogen activator‐induced lysis and to elucidate the mechanisms behind the effect. Ultrasound enhanced lysis in all three models, with the largest effects (4‐fold) in the external lysis model with compacted plasminogen‐poor clots. The acceleration effect of ultrasound in this model decreased with increasing t‐PA—and decreasing plasminogen concentrations. Ultrasound had a much smaller effect in this model when compacted plasminogen‐rich clots were used. In the external lysis, no...