Thrombin clotting activity measurement and standardization with lyophilized plasma

Abstract

Thrombin-induced clotting times were inversely proportional to fibrinogen concentrations within the range of 98 to 2900 nmol/L; these reciprocal velocity measurements had units of seconds per clot. By analogy with classical enzyme kinetics, we defined rectangluar hyperbolic parameters where K(m(clot)) ranged from 0.14 to 0.56 μmol/L and k(clot) from 0.020 to 0.075 clot per second. Specificity ratios (k(clot/K(m(clot)) showed that reconstituted lyophilized human plasma is as good, if not better, a source of clottable fibrinogen as the purified protein itself. These ratios also showed that the presence of >98% of the autoproteolytic forms (β- and γ-thrombins) of the human enzyme did not interfere with clotting activity attributable to α-thrombin. Contrary to simple enzyme kinetics, velocities (reciprocal clotting times) were rectangular hyperbolically related to clotting activities. Clotting times between 5 and 90 s/clot correlated (r >0.999) with reciprocal α-thrombin concentrations, permitting standardization with lyophilized plasma (100 U.S. 'NIH' thrombin-clotting units/L corresponded to ~21 s/clot with these plasma). Lyophilized plasma re-examined after 15 months displayed no change in clottability.