D Biltoft1, J J Sidelmann2, L F Olsen3, Y Palarasah2, J Gram2. 1. Unit for Thrombosis Research, Department of Public Health, University of Southern Denmark, Esbjerg, Denmark; Department of Clinical Biochemistry, Hospital of South West Denmark, Esbjerg, Denmark. Electronic address: dbiltoft@health.sdu.dk. 2. Unit for Thrombosis Research, Department of Public Health, University of Southern Denmark, Esbjerg, Denmark; Department of Clinical Biochemistry, Hospital of South West Denmark, Esbjerg, Denmark. 3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Abstract
OBJECTIVES: The 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 METHODS: We investigated whether such a method could be developed using the principle of the Calibrated Automated Thrombin generation method as a template. RESULTS: A suitable kallikrein specific fluorogenic substrate was identified (KM=0.91mM, kcat=19s-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. CONCLUSIONS: The 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.
OBJECTIVES: The 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 METHODS: We investigated whether such a method could be developed using the principle of the Calibrated Automated Thrombin generation method as a template. RESULTS: A suitable kallikrein specific fluorogenic substrate was identified (KM=0.91mM, kcat=19s-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. CONCLUSIONS: The 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.