Clinical Chemistry, Vol 32, 320-324, Copyright © 1986 by American Association for Clinical Chemistry

Thrombin clotting activity measurement and standardization with lyophilized plasma

JW Fenton 2d, CS Wideman and BL Evatt

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 rectangular hyperbolic parameters where Km(clot) ranged from 0.14 to 0.56 mumol/L and kclot from 0.020 to 0.075 clot per second. Specificity ratios (kclot/Km(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 greater than 98% of the autoproteolytic forms (beta- and gamma- thrombins) of the human enzyme did not interfere with clotting activity attributable to alpha-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 greater than 0.999) with reciprocal alpha-thrombin concentrations, permitting standardization with lyophilized plasma (1000 U.S. "NIH" thrombin-clotting units/L corresponded to approximately 21 s/clot with these plasma). Lyophilized plasma re- examined after 15 months displayed no change in clottability.