SummaryI have considered the accuracy of the “enzyme cascade” sequence or “waterfall sequence” for blood clotting proposed recently. My remarks are not intended to be exhaustive, but rather representative of a perspective. I regard these presentations as being at the speculative level of science, created with a wild use of the imagination. Some of the clotting factor precursors that are supposed to be converted in succession to active enzymes may not exist and certainly are not known to be enzymes. I find it against my sense of order to believe that six proteolytical enzymes are needed to get thrombin. That would require six special sites in proteins where each enzyme does its work. Each enzyme would not split the other five sites if located as single or multiple frequencies in the five other substrates. While creating hypothetical enzymes and assigning Roman numerals to them the well documented function of the best known enzyme; namely, thrombin is disregarded. The capacity of prothrombin to form derivatives is disregarded. This immediately places the superstructure on a false base. The sequence is definitely not in accurate order. There is no elaborate mobilization of chemical events on the periphery of prothrombin which leads to doing something to prothrombin. The second known enzyme, autoprothrombin C, is included in the plasma-platelet system (intrinsic) whereas its main, if not exclusive place, is in the conversion of prothrombin to thrombin when tissue-plasma-platelet mechanisms are in operation. What is meant by t he term prothrombin is not clarified, and the way in which the required phospholipid originates is not accounted for. If there is a factor XII it is not the exclusive way to create conditions that will initiate prothrombin activation in the plasma-platelet system. Most of the steps postulated could only be accurate if it were possible to deny important conclusions stated in papers which are not at all considered. Evidently refutation of facts in these papers was not possible and a parochial kind of authority was promoted1 ) on the basis of “consistent with most of the current investigations”. I like to recal11 ) the words of Henry David Thoreau: “Any man more right than his neighbor, constitutes a majority of one.” The advances in prothrombin chemistry are sufficiently substantial for the formulation of a conceptual theme consistent with principles of enzymology, and as I have indicated above, can also serve as a clarification of the proposed cascade or waterfall diagrams.

Autoprothrombin I, was obtained by activating prothrombin preparations with autoprothrombin C. Autoprothrombin I, was obtained by activating prothrombin with the use of calcium ions and purified platelet factor 3. Both of these prothrombin derivatives were found to be inactivated by plasma and serum. Autoprothrombin I, was studied most extensively and was found to be inactivated by purified antithrombin. When antithrombin was first combined to capacity with purified thrombin the power to neutralize autoprothrombin I was almost eliminated. Autoprothrombin I1 was not destroyed by antithrombin and was found in serum as the main procoagulant. Most likely it is not in plasma.Autoprothrombin I,, autoprothrombin I,, autoprothrombi11 C, and thrombin all have reactive groups that combine with antithrombin. The activity in 1 n~g of purified autoprothrombin I, was neutralized by 50 ml plasma. One milligram of autoprothrombin C was neutralized by 5 ml plasma, and 7 ml plasma was sufficient to neutralize 1 mg of thrombin. Antithrombin IV is regarded as a kinetic effect and not as a substance. From a review of the literature it is concluded that there is no adequate evidence for appreciable quantities of factor VII in serum, or that it occurs in plasma independent of prothrombin.

SummaryPurified prethrombin was converted to thrombin in an activating system consisting of purified autoprothrombin C, purified Ac-globulin, lipid and calcium chloride. With the concentration of calcium and pH fixed, the effect of varying the other 3 pro-coagulants was studied.Conditions influencing the conversion of prethrombin to thrombin were determined. Autoprothrombin C is the enzyme, but with it alone activation was very slow. Best results were obtained with the simultaneous presence of autoprothrombin C, Ac-globulin and lipid. Reducing any one of the three toward zero concentration decreased the rate and yield of thrombin generation. In association with rapid thrombin generation Ac-globulin and autoprothrombin C were represented in approximately a 1:1 molar ratio. In that combination of Ac-globulin and autoprothrombin C the Michaelis constant for autoprothrombin C with prethrombin as a substrate was 3.14 × 10-6M.

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.

SummaryThe procoagulant or P fraction obtained from human urine was analyzed to determine how it functions in the activation of purified prothrombin. Prothrombin converts to thrombin in the presence of P fraction, Ac-globulin, calcium chloride and either whole platelets or platelet factor 3. The yield of thrombin can be made to depend upon the concentration of P fraction. This conversion of prothrombin to thrombin can be partially or completely inhibited by adding various concentrations of soybean trypsin inhibitor introduced into the activation mixture.When purified prothrombin was activated with platelet factor 3 and calcium chloride, the prothrombin activity, as measured by the two-stage assay, decreased. When it was at the lowest possible level, P fraction was added. Quite quickly prothrombin activity began to reappear, and soon 100% of the original prothrombin activity was accounted for as either prothrombin-R or thrombin.