A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir

2015 ◽  
Vol 28 (9) ◽  
pp. 521-527
Author(s):  
Balaji Somasundaram ◽  
Conan J. Fee ◽  
Rayleen Fredericks ◽  
Andrew J. A. Watson ◽  
Antony J. Fairbanks ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Antiviral Drugs ◽  
Wild Type ◽  
Neuraminidase Inhibition

Surface plasmon resonance measurement of binding and dissociation of wild-type and mutant streptavidin on mixed biotin-containing alkylthiolate monolayers

1999 ◽  
Vol 54 (1-2) ◽  
pp. 137-144 ◽  
Author(s):  
Linda S Jung ◽  
Kjell E Nelson ◽  
Charles T Campbell ◽  
Patrick S Stayton ◽  
Sinclair S Yee ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Wild Type ◽  
Resonance Measurement

Lys 42/43/44 and Arg 12 of thrombin-activable fibrinolysis inhibitor comprise a thrombomodulin exosite essential for its antifibrinolytic potential

2017 ◽  
Vol 117 (08) ◽  
pp. 1509-1517 ◽  
Author(s):  
Chengliang Wu ◽  
Alan Stafford ◽  
James Fredenburgh ◽  
Jeffrey Weitz ◽  
Ann Gils ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Catalytic Efficiency ◽  
Clot Lysis ◽  
Wild Type ◽  
Activation Kinetics ◽  
Resonance Data ◽  
Fibrinolysis Inhibitor ◽  
Rate Of Activation

SummaryThe thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K1/2) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay at physiologic levels of TAFI. Similar trends in the antifibrinolytic activity of the TAFI variants were observed when plasma clots were formed using HUVECs as the source of TM. When thrombin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1-fold compared with wild-type TAFI, suggesting that these mutations only impacted activation kinetics in the presence of TM. Surface plasmon resonance data suggest that mutation of the four residues abrogates TM binding with or without thrombin. Therefore, Lys 42, Lys 43, Lys 44 and Arg 12 are critical for the interaction of TAFI with the thrombin-TM complex, which modulates its antifibrinolytic potential.

A novel gene mutation in the 60s loop of human coagulation factor VII – inhibition of interdomain crosstalk

2004 ◽  
Vol 91 (01) ◽  
pp. 28-37 ◽  
Author(s):  
Anita Kavlie ◽  
Merete Wiiger ◽  
Mette Husbyn ◽  
Helge Stormorken ◽  
Hans Prydz
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Chinese Hamster Ovary Cells ◽  
Peptide Sequence ◽  
Factor Vii ◽  
Mutant Protein ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Mammalian Expression

SummaryA novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively.The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon resonance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177−206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC50 value of 0.5 μM in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent Ki of 7 μM.

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