A novel assay for factor XIII based on cross-linking of synthetic peptides: analysis of different substrates

Abstract

A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.

Quantification of coagulation factor XIII activity by a thio-NADH based assay using factor XIII immuno-depleted plasma as a diluent for calibration

“…FXIII therapy is recommended for patients with plasma FXIII activity levels below 5%, but abnormal bleeding can also occur in patients with plasma FXIII activity at approximately 30% (7). However, many are microtiter plate based assays, and are therefore not compatible with the routine work flow in the hemostasis clinical laboratory, which demands highly automated methods for application on routine coagulation analyzers (9)(10)(11)(12)(13). However, many are microtiter plate based assays, and are therefore not compatible with the routine work flow in the hemostasis clinical laboratory, which demands highly automated methods for application on routine coagulation analyzers (9)(10)(11)(12)(13).…”

Fully automated immunoassay for quantitative determination of FXIII

“…Several methods for quantification of either FXIII plasma activity or antigen levels are available for clinical diagnosis (18). Many of them are microtitre plate (MTP) based (19)(20)(21)(22). In certain settings that demand highly automated methods for application on routine, random access, high throughput coagulation analyzers or point-of-care instruments other assay formats are desirable.…”