Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5510084 | Clinical Biochemistry | 2016 | 7 Pages |
â¢New global method for measuring kallikrein generation potential in plasmaâ¢Fluorogenic substrate Boc-LKR-AMC was specific for kallikrein activity.â¢Signal was externally calibrated with α2M-kallikrein complexes.â¢Method detects abnormalities of the contact activation system.
ObjectivesThe physiological role of the contact system remains inconclusive. No obvious clinical complications have been observed for factor XII (FXII), prekallikrein (PK), or high molecular weight kininogen deficiencies even though the contact system in vitro is associated with coagulation, fibrinolysis, and inflammation. A global generation assay measuring the initial phase of the contact system could be a valuable tool for studies of its physiological role.Design and methodsWe investigated whether such a method could be developed using the principle of the Calibrated Automated Thrombin generation method as a template.ResultsA suitable kallikrein specific fluorogenic substrate was identified (KM = 0.91 mM, kcat = 19 sâ 1), and kallikrein generation could be measured in undiluted plasma when silica was added as activator. Disturbing effects, including substrate depletion and the inner-filter effect, however, affected the signal. These problems were corrected for by external calibration with α2-macroglobulin-kallikrein complexes. Selectivity studies of the substrate, experiments with FXII and PK depleted plasmas, and plasma with high or low complement C1-esterase inhibitor activity indicated that the obtained and calibrated signal predominantly was related to FXII-dependent kallikrein activity.ConclusionsThe findings described show that establishment of a kallikrein generation method is possible. Potentially, this setup could be used for clinical studies of the contact system.