In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysinebased oligomannosides containing two (M 2 L) to six (M 6 L 5 ) terminal ␣-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 tissuetype plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18 -23 M to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional ␣-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M 3 L 2 to M 6 L 5 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 M 6 L 5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M 6 L 5 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.The mannose receptor is a 175-kDa membrane-associated protein that is localized on sinusoidal liver cells, peripheral and bone marrow macrophages, and dendritic cells (1-4). It recognizes and internalizes mannosylated polysaccharides from pathological microorganisms (5), tumor cells (6), and yeast cells (7) and glycoproteins like type-I procollagen (8), tissue-type plasminogen activator (9), or various lysosomal enzymes (10). As such, the mannose receptor participates in the nonimmune host-defense system. In addition, the macrophage receptor is implicated in major histocompatability complex-mediated antigen presentation by dendritic cells (11).The cDNA of the mannose receptor has been sequenced by Taylor et al. (12) and codes for five types of domains (13): an N-terminal cysteine-rich domain, a transmembrane domain, a fibronectin type II-like domain, a domain composed of eight strongly homologous repeats (the so-called carbohydrate recognition domains or CRDs)1 and a C-terminal cytoplasmic tail. Taylor and Drickamer (13, 14) have established that the CRDs are involved in ligand binding. Recent structure-function studies of recombinant truncated forms of the mannose receptor provided new insight into the mechanism of ligand binding by the mannose receptor (13,14). On basis of these results, it was proposed that CRD4 is the only CRD to display a monosaccharide specificity characteristic for the mannose receptor (15). CRD4 and CRD5 appear to be required for high affinity binding of high mannose-type glycoproteins and mannosylated bovine serum albumin (BSA). B...
Abstract:Recently we developed mouse monoclonal antibodies (inAb) against the isolated human 175-kDa mannose receptor.
SummaryIntravenous infusion of desmopressin (DDAVP, 0.4 μg/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.
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.
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