MEASUREMENT OF FACTOR XIII ACTIVITY IN HUMAN PLATELET HOMOGENATE BY A NEW UV-KINETIC METHOD

Factor XIII (FXIIl) of blood coagulation is a zymogen which is converted into an active transglutaminase during the clotting process. Earlier methods used for its determination are cumbersome, laborious, and not suitable for routine laboratory measurements.Most recently we have designed a new simple UV-kinetic assay for the determination of FXIII in the plasma (Muszbek et al., Clin. Chem., 3JL, 35, 1985). The assay is performed on def:j.b-rinated plasma in which FXIII is activated by thrombin and Ca²⁺. Acetylateddephosphorylated (AD)β-casein and ethylamine are used as substrates and the ammonia released during thereaction is continuously monitored by a NADPH dependent indicator reaction at 340 nm. As the enzymatically active a subunit of FXIII is also present in platelets and monocytes/macrophages we attempted to adapt the above method for the measurement of cellular FXIII activity. Experiments were carried out on Lubrol extract of washed sonicated platelets. It was found that the small amount of fibrinogen present in platelets does not need to be removed and in the blank hirudin used for preventing activation of plasma FXIII should be replaced by EGTA. The concentration of substrates and activators were optimized. The methodwas found linear at least up-to 40 U/l enzyme activity. It had a good reproducibility (optimal conditionvariance was less than 3%) and correlated well with the most commonly used fluorescent amine (dansylcadaverine) incorporation assay. The method was adapted to a centrifugal fast analyser (Baker, Centrifichem). In addition to congenital FXIII deficiency the determination of FXIII in platelets by this new methodmight have a diagnostic importance in haemopoietic diseases with diminished or accelerated platelet production.