Abstract
The recent availability of a sensitive chromogenic method approach for determination of FXIa activity has been explored for designing sensitive methods for FXIIa and kallikrein, both using FXa formation as the read-out. For both enzymes the assay range 1–10 nmol/L provides a resolution of about 0.8 absorbance units with a total assay time of about 20 min. For studies on activation kinetics, subsampling and extensive dilution can be performed in MES–bovine serum albumin (BSA) buffer pH 5.7 for quenching of enzyme activity and with ensuing determination of FXa generation in a chromogenic FXIa method. Optionally, suitable inhibitors such as aprotinin and/or corn trypsin inhibitor may be included. The stability of FXIa, FXIIa, and kallikrein in MES–BSA buffer was shown to be at least 5 h on ice. In conclusion, the use of a sensitive chromogenic FXIa method either per se or in combination with MES–BSA buffer pH 5.7 are new and potentially valuable tools for the study of contact factor enzymes and their inhibitors. So far, dose–response studies of FXIIa and kallikrein have been limited to purified systems, and hence more data are required to learn whether these new methods might or might not be applicable to the determination of FXIIa and kallikrein activities in plasma.
Highlights
The contact activation pathway, involving factor XII (FXII), prekallikrein (PK), high molecular weight kininogen (HK), and factor XI (FXI) displays complex interactions and is involved in both coagulation and inflammation [for reviews, see Ref. [1, 2]]
The recent availability of a sensitive chromogenic method approach for the determination of FXIa activity has been explored for designing sensitive methods for FXIIa and kallikrein, both using FXa formation as the read-out
For studies on activation kinetics, subsampling and extensive dilution can be performed in MES–BSA buffer pH 5.7 for quenching of enzyme activity, optionally including aprotinin and/or CTI, and with ensuing determination of FXa generation in the chromogenic FXIa method
Summary
The contact activation pathway, involving factor XII (FXII), prekallikrein (PK), high molecular weight kininogen (HK), and factor XI (FXI) displays complex interactions and is involved in both coagulation and inflammation [for reviews, see Ref. [1, 2]]. In vivo as well as for in vitro studies on plasma, contact activation is surface-bound, i.e., contact factors assemble on a negatively charged surface with the initial step being binding and autoactivation of FXII. PK and FXI bind to the surface through HK. In vitro, this is utilized in any APTT-based method, and the negatively charged surface is provided as a constituent in the APTT reagent, typically as some variant of silica or as ellagic acid. Studies performed more than 40 years ago demonstrated that surface-bound FXII and FXI are much more effectively activated than in solution and that HK is a cofactor to such activation, not a mandatory requirement [14,15,16]
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