scholarly journals Hdxms Reveals the Role of Dynamics on the Serine Protease Activity of the Urokinase-Type Plasminogen Activator (UPA)

2021 ◽  
Vol 120 (3) ◽  
pp. 100a
Author(s):  
Constanza Torres-Paris ◽  
Lufan Xiao ◽  
Harriet Song ◽  
Elizabeth A. Komives
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Protease Activity ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Serine Protease Activity

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

1997 ◽  
Vol 77 (04) ◽  
pp. 710-717 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
J Kar Kruijt ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Liver Uptake ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Urokinase-type plasminogen activator receptor is associated with the development of adipose tissue

2010 ◽  
Vol 104 (12) ◽  
pp. 1124-1132 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Eri Kawashita ◽  
Kiyotaka Okada ◽  
Hidetaka Suga ◽  
Shigeru Ueshima ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasminogen Activator ◽  
Adipocyte Differentiation ◽  
Cellular Adhesion ◽  
Wild Type ◽  
Tissue Mass ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Plasminogen Activator Receptor

SummaryUrokinase-type plasminogen activator receptor (uPAR) plays a role in cellular responses which include cellular adhesion, differentiation, proliferation and migration. The aim of this study was to clarify the role of uPAR on the development of adipose tissue. To clarify the role of uPAR on adipogenesis, we examined the effect of uPAR overexpression and uPAR deficiency on the adipocyte differentiation. Adipocyte differentiation was induced by incubation of 3T3-L1 cells with differentiation media containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthin. uPAR overexpression by transfection of uPAR expression vector induced adipocyte differentiation. In addition, we examined the difference in adipocyte differentiation of mesenchymal stem cells from wild-type mice and uPAR knockout (uPAR-/-) mice. The uPAR deficiency attenuated differentiation media-induced adipocyte differentiation. Moreover, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway attenuated uPAR overexpression-induced adipocyte differentiation, and uPAR overexpression induced the activation of Akt. We also found that an increase of the adipose tissue mass in uPAR-/- mice was less than that observed in wild-type mice. The present results suggest that uPAR plays a pivotal role in the development of adipose tissue through PI3K/Akt pathway.

Influence of t-PA and u-PA on Adipose Tissue Development in a Murine Model of Diet-Induced Obesity

2002 ◽  
Vol 87 (02) ◽  
pp. 306-310 ◽  
Author(s):  
P.E. Morange ◽  
D. Bastelica ◽  
M.F. Bonzi ◽  
B. Van Hoef ◽  
D. Collen ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasminogen Activator ◽  
The Body ◽  
Tissue Type ◽  
Tissue Development ◽  
Diet Induced Obesity ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Adipose Tissue Development

SummaryTo investigate the potential role of tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) in development of adipose tissue, we have used a nutritionally induced obesity model in t-PA (t-PA−/−) and u-PA (u-PA−/−) deficient mice. Five week old male wild-type (WT), t-PA−/− or u-PA−/− mice (n = 9 to 16) were fed a high fat diet (HFD, 42% fat). After 16 weeks of HFD, the body weight of t-PA−/− mice was significantly higher than that of WT mice (48 ± 1.1 g vs. 39 ± 2.2 g, p = 0.004). The total weight of the isolated subcutaneous (sc) fat deposit was higher in t-PA−/− than in WT mice (2.4 ± 0.22 g vs. 1.2 ± 0.29 g, p = 0.002), accompanied with higher adipocyte diameters (80 ± 1.7 µm vs. 61 ± 4.7 µm, p < 0.01). These differences were not observed in the intra-abdominal fat deposit. The number of stroma cells in both adipose tissue territories was increased in t-PA−/− as compared to WT mice (2.0 ± 0.13 vs. 1.5 ± 0.10 p = 0.02 and 3.0 ± 0.17 vs 1.6 ± 0.17, p = 0.0001, stroma cells/ adipocytes in sc and intra-abdominal tissue, respectively), partly as a result of an increased number of endothelial cells (192 ± 9 vs. 154 ± 18 p = 0.06 and 108 ± 13 vs. 69 ± 8 p = 0.04 CD31 stained/adipocyte area). In contrast the weight gain and adipose tissue development in u-PA−/− mice was not different from that in WT mice. These data suggest that t-PA but not u-PA plays a role in adipose tissue development.

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