Some Effects of Factor VII on Rates of Thrombin Production and one Stage Pi’s

Attempts to automate a chromogenic one stage PT assay required hastening the onset of thrombin production without altering the rate of production. Assays were performed by combining 0.25ml chromogenic substrate S-2238 (2mM), 0.40ml tissue thromboplastin,0.050ml plasma, and 0.050ml diluted serum (40%v/v) in 1.35ml TRIS buffer (pH=8-5,I=0.15). Sera were prepared by using the supernatant of a) whole blood, b) recalcified plasma, c) recalc fied plasma and tissue thromboplastin, d)recalcified plasma and partial thromboplastin (cephalin and ellagic acid) and e) thrombin clotted plasma. The results indicated that S-2238, a potent thrombin inhibitor, delayed the onset and altered the rate of thrombin; production. Small amounts of thrombin shortened the initial generation of thrombin without altering its rate. Sera from a), c) and d) but not b) and e) resulted in immediate thrombin production at most plasma concentrations without affecting the rate, had no residual prothrombin, and had similar residual S-2238 activity not reduced by hirudin or At III-heparin. In particular, scrum b) had ~80% plasma Factor X which could be adsorbed without affecting thrombin production, had ~40% Factor V and ~300% Factor VII. These results suggest that when Factor VII activity is greater than ~7% there is an effect on the time but not on the rate of thrombin production via positive thrombin feedback scheme. Thus, the standard PT can measure the time of thrombin appearance, and/or the rate of thrombin production, and/or fibrin polymerization.