Calibrated kallikrein generation in human plasma

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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.

Original languageEnglish
JournalClinical Biochemistry
Volume49
Issue number15
Pages (from-to)1188-1194
ISSN0009-9120
DOIs
Publication statusPublished - 29. Jun 2016

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Plasma (human)
Kallikreins
Factor XII
Prekallikrein
Tissue Kallikreins
Complement C1 Inhibitor Protein
Plasmas
High Molecular Weight Kininogens
Fibrinolysis
Complement C1
Fluorescent Dyes
Macroglobulins
Substrates
Coagulation
Thrombin
Silicon Dioxide
Assays
Calibration
Experiments

Cite this

@article{589b2eb192314d9b8af2c41ccf47902c,
title = "Calibrated kallikrein generation in human plasma",
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.",
author = "D Biltoft and Sidelmann, {J J} and Olsen, {L F} and Y Palarasah and J Gram",
note = "Copyright {\circledC} 2016. Published by Elsevier Inc.",
year = "2016",
month = "6",
day = "29",
doi = "10.1016/j.clinbiochem.2016.06.011",
language = "English",
volume = "49",
pages = "1188--1194",
journal = "Clinical Biochemistry",
issn = "0009-9120",
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}

Calibrated kallikrein generation in human plasma. / Biltoft, D; Sidelmann, J J; Olsen, L F; Palarasah, Y; Gram, J.

In: Clinical Biochemistry, Vol. 49, No. 15, 29.06.2016, p. 1188-1194.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Calibrated kallikrein generation in human plasma

AU - Biltoft, D

AU - Sidelmann, J J

AU - Olsen, L F

AU - Palarasah, Y

AU - Gram, J

N1 - Copyright © 2016. Published by Elsevier Inc.

PY - 2016/6/29

Y1 - 2016/6/29

N2 - 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.

AB - 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.

U2 - 10.1016/j.clinbiochem.2016.06.011

DO - 10.1016/j.clinbiochem.2016.06.011

M3 - Journal article

VL - 49

SP - 1188

EP - 1194

JO - Clinical Biochemistry

JF - Clinical Biochemistry

SN - 0009-9120

IS - 15

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