Some Effects of Purified Autoprothrombin C in Blood Clotting

Abstract

SummaryPurified autoprothrombin C was added to rabbit blood or recalcified plasma in order to measure the exact influence on clotting time. At a concentration of 10 u of autoprothrombin C per ml blood there was clotting in 13 sec. At that concentration recalcified plasma clotted in 7 to 8 sec. In the acceleration of the whole blood clotting time with autoprothrombin C, the effect was greater in glass tubes than in silicone coated tubes. The difference was probably due to lipids from platelets. With autoprothrombin C at a concentration of 10 u/ml reaction mixture, the prothrombin time and partial thromboplastin time of plasma was brought down to 3 to 4 sec. The partial thromboplastin time supplied the most sensitive conditions for detecting autoprothrombin C activity. Less than 2 × 10−5 micrograms of autoprothrombin C were detected. Rabbits that were treated with Coumadin had their prothrombin concentration reduced to 9 u/ml plasma, as measured by two-stage analysis. Such plasma yielded twice as much thrombin when purified autoprothrombin C and purified Ac-globulin were used in the assay to determine the thrombin potential. Even when the prothrombin concentration was brought to low levels with Coumadin a partial thromboplastin time or prothrombin time of 7 sec was easily obtained by using autoprothrombin C as procoagulant. The important effect of Coumadin or related drugs is the reduction of prothrombin concentration. This involves a lowering of the autoprothrombin C potential as well as the thrombin potential of plasma, and also the amount of inhibitor that can be obtained from prothrombin.Normal blood was found to contain prethrombin in small amounts as well as prothrombin. The synthesis of prothrombin was stopped with Coumadin and it is likely that the residual prothrombin was in part utilized by degradation to prethrombin, inhibitor and autoprothrombin III. At the height of the anticoagulant effect the prethrombin concentration was higher than the prothrombin concentration. With the resumption of prothrombin synthesis, when vitamin K was given, it may be that prethrombin, inhibitor, and autoprothrombin III was first produced in the liver and some of the latter entered the blood stream to compensate for substrate deficiency. Free autoprothrombin III would account for the short prothrombin time observed before prothrombin concentration rose.

Two-Stage Procedure for the Quantitative Determination of Autoprothrombin III Concentration and Some Applications

“…During those 3 hrs the liver must have been synthesizing the prethrombin and autoprothrombin III portion of the molecule and these appeared in the blood, partly as separate entities and partly in the usual combination found in prothrombin. The disparity between the concentration of the 2 subunits must have been even higher than indicated from our experiments because Miiller-Berghaus and Seegers (8) found that the prethrombin assay shows an even higher thrombin precursor titre than the prothrombin assay when vitamin K 1 is given under conditions similar to those in our experiment. Most likely prethrombin and autoprothrombin III are always synthesized independently and then incorporated uniformly in the prothrombin molecule.…”

“…In one study the reason for the discrepancy was largely clarified. The observed effects were ascribed to the combined effect of some prethrombin not detected by the two-stage analysis and some free autoprothrombin III (8).…”

“…Approaches to the quantitative measurement of autoprothrombin C activity were discussed in previous papers from this laboratory (2,7,8). A small amount of the enzyme shortens the partial thromboplastin time of plasma, and if the conditions are standardized this fact can be the basis for quantitative measurements.…”

“…The latter are expressed in unit numbers and the prothrombin time is based on another scale because it is just a number from the stop watch. It takes only a small amount of autoprothrombin C to change tremendously the values read directly from the watch (8), and in the prothrombin time the thromboplastin certainly converts any autoprothrombin III rapidly to autoprothrombin C. Hence, the first appearance of autoprothrombin III, after there was practically none, is reflected in the prothrombin time. From the work with rabbits by Miiller-Berghaus and Seegers (8), we know that the prethrombin assay shows an early response after vitamin K administration.…”