Faculty Opinions recommendation of Development of serine protease inhibitors displaying a multicentered short (<2.3 A) hydrogen bond binding mode: inhibitors of urokinase-type plasminogen activator and factor Xa.

Author(s):  
David Matthews
1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen

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.


2002 ◽  
Vol 124 (39) ◽  
pp. 11657-11668 ◽  
Author(s):  
Bradley A. Katz ◽  
Jeffrey R. Spencer ◽  
Kyle Elrod ◽  
Christine Luong ◽  
Richard L. Mackman ◽  
...  

PLoS ONE ◽  
2011 ◽  
Vol 6 (4) ◽  
pp. e19302 ◽  
Author(s):  
Joakim E. Swedberg ◽  
Simon J. de Veer ◽  
Kei C. Sit ◽  
Cyril F. Reboul ◽  
Ashley M. Buckle ◽  
...  

2003 ◽  
Vol 328 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Ewa Zeslawska ◽  
Uwe Jacob ◽  
Andrea Schweinitz ◽  
Gary Coombs ◽  
Wolfram Bode ◽  
...  

2003 ◽  
Vol 329 (1) ◽  
pp. 93-120 ◽  
Author(s):  
Bradley A. Katz ◽  
Kyle Elrod ◽  
Erik Verner ◽  
Richard L. Mackman ◽  
Christine Luong ◽  
...  

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