Choline inhibition of prothrombin activation

Richard D. Leach; Sally A. Dewind; Charles W. Slattery; E. Clifford Herrmann

doi:10.1016/0049-3848(91)90368-7

Choline inhibition of prothrombin activation

Richard D. Leach, Sally A. Dewind, Charles W. Slattery, E. Clifford Herrmann

Biochemistry and Microbiology

Research output: Contribution to journal › Article › peer-review

Abstract

A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A₄₀₀ of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely >0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent K_i and K_i values (mM): 22±3 and 48±7 without factor Va, 25±4 and 41±4 with factor Va.

Original language	English
Pages (from-to)	635-648
Number of pages	14
Journal	Thrombosis Research
Volume	62
Issue number	6
DOIs	https://doi.org/10.1016/0049-3848(91)90368-7
State	Published - Jun 15 1991

ASJC Scopus Subject Areas

Hematology

Keywords

anticoagulant
choline
chromogenic peptide substrate
enzyme inhibitor
factor Va
prothrombinase

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title = "Choline inhibition of prothrombin activation",

abstract = "A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely >0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki values (mM): 22±3 and 48±7 without factor Va, 25±4 and 41±4 with factor Va.",

keywords = "anticoagulant, choline, chromogenic peptide substrate, enzyme inhibitor, factor Va, prothrombinase",

author = "Leach, {Richard D.} and Dewind, {Sally A.} and Slattery, {Charles W.} and Herrmann, {E. Clifford}",

note = "A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-spe...",

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AU - Leach, Richard D.

AU - Dewind, Sally A.

AU - Slattery, Charles W.

AU - Herrmann, E. Clifford

N1 - A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-spe...

PY - 1991/6/15

Y1 - 1991/6/15

N2 - A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely >0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki values (mM): 22±3 and 48±7 without factor Va, 25±4 and 41±4 with factor Va.

AB - A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely >0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki values (mM): 22±3 and 48±7 without factor Va, 25±4 and 41±4 with factor Va.

KW - anticoagulant

KW - choline

KW - chromogenic peptide substrate

KW - enzyme inhibitor

KW - factor Va

KW - prothrombinase

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JF - Thrombosis Research

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Choline inhibition of prothrombin activation

Abstract

ASJC Scopus Subject Areas

Keywords

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