Yeast expression and phagemid display of the human urokinase plasminogen activator epidermal growth factor-like domain

The human urokinase plasminogen activator (uPA) epidermal growthfactor-like domain (residues 1-48) and a variant with a C-terminalepitope tag have been secreted from recombinant yeast Purifiedhuman uPA 1-48 and uPA l-48glu compete for binding to the humanuPA receptor with Kji of 180 and 400 pM respectively, in anin vitro assay using an immobilized recombinant uPA receptor.A synthetic gene encoding human uPA 1-48 with an N-terminalepitope tag was inserted into a phagemid expression vector asa fusion with residues 249-406 of the M13 pill protein withan intervening amber codon (TAG). Phagemid production led toinfectious particles which were selectively bound and elutedfrom both epitope tag antibody and urokinase receptor. Sequentialbinding to this antibody and receptor demonstrated a substantialenrichment, where up to 10% of the infectious particles werethen retained on urokinase receptor-coated plates. A PCR strategywas used to convert previously described peptide bacteriophageligands for the urokinase receptor to phagemid display. Theyields of these peptide phagemids and the uPA 1-48 phagemidshowed a correlation with peptide affinity, in contrast to whenthe peptides are multivalently displayed on a bacteriophage.     

Moscatilin Inhibits Metastatic Behavior of Human Hepatocellular Carcinoma Cells: A Crucial Role of uPA Suppression via Akt/NF-κB-Dependent Pathway

Hepatocellular carcinoma (HCC) frequently shows early invasion into blood vessels as well as intrahepatic metastasis. Innovations of novel small-molecule agents to block HCC invasion and subsequent metastasis are urgently needed. Moscatilin is a bibenzyl derivative extracted from the stems of a traditional Chinese medicine, orchid Dendrobium loddigesii. Although moscatilin has been reported to suppress tumor angiogenesis and growth, the anti-metastatic property of moscatilin has not been elucidated. The present results revealed that moscatilin inhibited metastatic behavior of HCC cells without cytotoxic fashion in highly invasive human HCC cell lines. Furthermore, moscatilin significantly suppressed the activity of urokinase plasminogen activator (uPA), but not matrix metalloproteinase (MMP)-2 and MMP-9. Interestingly, moscatilin-suppressed uPA activity was through down-regulation the protein level of uPA, and did not impair the uPA receptor and uPA inhibitory molecule (PAI-1) expressions. Meanwhile, the mRNA expression of uPA was inhibited via moscatilin in a concentration-dependent manner. In addition, the expression of phosphorylated Akt, rather than ERK1/2, was inhibited by moscatilin treatment. The expression of phosphor-IκBα, and -p65, as well as κB-luciferase activity were also repressed after moscatilin treatment. Transfection of constitutively active Akt (Myr-Akt) obviously restored the moscatilin-inhibited the activation of NF-κB and uPA, and cancer invasion in HCC cells. Taken together, these results suggest that moscatilin impedes HCC invasion and uPA expression through the Akt/NF-κB signaling pathway. Moscatilin might serve as a potential anti-metastatic agent against the disease progression of human HCC.     

Prevention and Treatment of Thrombosis Associated With Long-Term Hemodialysis Catheters

Soft, cuffed, central vein hemodialysis catheters are used in about 20% of chronic hemodialysis patients in the United States, because long-term arteriovenous blood access cannot be maintained in an aging patient population with a large proportion of diabetics. The most frequent complication of these catheters is thrombosis. The treatment of catheter-related thrombosis is difficult and expensive; thus the emphasis should be on prevention. The preferred material for a long-term catheter is silicone rubber, since it is the least thombogenic. Anticoagulation should be more vigorous during “catheter dialysis” than during “fistula dialysis.” Heparin is the least expensive and most convenient anticoagulant, suitable for over 99% of chronic dialysis patients. The dose of heparin for sufficient anticoagulation depends on many factors, varies widely, and should be established for each patient based on activated clotting time (ACT). ACT should be kept over 270 sec throughout dialysis. Recently we introduced a method of locking catheter lumina with a predetermined amount of heparin; this heparin is not discarded before the next dialysis, but serves as a loading dose. This saves a number of connections/ disconnections and decreases dialysis-associated blood losses. To prevent catheter thrombosis, over 60% of patients require warfarin in sufficient doses to keep the international normalized ratio (INR) between 1.5 and 2.5. The most common catheter-related thrombus is a periluminal fibrin sleeve. Locking the catheter with urokinase to dissolve the clot is of little value, because the bulk of the thrombus is outside the catheter. We have found a high-dose (250 000 U or more) intradialytic urokinase infusion through the venous chamber to be a very efficient and convenient method for dissolving clots. Cumulative success of up to three infusions is over 99%. This obviates the need of catheter stripping or replacement, which is more cumbersome and expensive.     

Mutations affecting the activity of urokinase-type plasminogen activator

Mutagenesis throughout the single-chain urokinase-type plasminogenactivator (scu-PA) cDNA molecule, followed by expression ofthe mutant genes and secretion of the resulting mutant proteinsfrom yeast, has been used to determine the amino acid residuesimportant for activity of scu-PA molecules. Twelve out of 13colonies secreting variant scu-PA molecules with decreased abilityto form a zone of fibrinolysis had mutant genes with a singlecodon alteration in the serine protease encoding domain (B-chain).Many of these changes are of highly conserved residues in theserine proteases and are consequently of considerable interest.A model three-dimensional structure of the protease domain ofurokinase was used to explain the basis for the effects of thesedown mutations. The model showed that the strongest down mutationsresult from either interference of the mutated side chain withsubstrate binding at the active site or the introduction ofbulky or charged groups at structurally sensitive internal positionsin the molecule. Attempts to find second site revertants offive down mutants, altered either at the plasmin activationsite or near the serine at the active site, only resulted insame-site revertants, with the original or closely related aminoacids restored.     

Successful Long-Term Central Venous Access; Daily Home Hemodialysis Blood Access

Successful long-term central venous access is a complex subject. The concept of “long term” implies that continued surveillance will be required. This also requires the catheter to be placed, initially, in its best configuration. To achieve long-term performance and durability, a thorough understanding of all aspects related to the catheter, catheter placement, and catheter maintenance is essential.     

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a K_D of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a K_D of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.