Optimization of a mechanized amidolytic factor-X assay; influence of reaction conditions and reagents

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 27; no. 6; p. 918
• Main Authors: van Wijk, E M, Kahlé, L H, Wiebosch, M, ten Cate, J W
• Format: Journal Article
• Language: English
• Published: England 01-06-1981
• Subjects: Anticoagulants - therapeutic use; Colorimetry - methods; Factor X - analysis; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Reference Values; Viper Venoms
• ISSN: 0009-9147
• DOI: 10.1093/clinchem/27.6.918

We describe the effect of pH and NaCl concentration on the activation of factor X in plasma by Russell's viper venom and on the amidolytic activity of the activated enzyme towards factor Xa-sensitive chromogenic substrates. An increasing NaCl concentration results in a decrease in the activation rate of factor X by Russell's viper venom, whereas in this step no pH effect is observed. Increasing NaCl concentrations decrease the Km and Vmax for both the factor Xa-sensitive chromogenic substrates, S 2222 and S 2337. Km values were lowest between pH 7.8 and 8.6; Vmax increased with increasing pH. Comparison of NaCl and KCl in the activation step as well as in the amidolytic step shows that the observed effect is specific for Na+, not just an effect of ionic strength. No difference was detected between activation by the crude venom or its purified factor X-activating enzyme. Also, values with the substrates S 2222 or S 2337 were the same in the mechanized amidolytic factor X assay.