CHROMATOGRAPHY OF BLOOD-CLOTTING FACTORS AND SERUM PROTEINS ON COLUMNS OF DIATOMACEOUS EARTH

1. In batch adsorptions with prothrombin solutions, hyflo was the weakest adsorbent, standard super-cel intermediate, and filter-cel strongest. Of these three grades of diatomaceous earth, hyflo has the smallest surface area per gram and filter-cel the largest. In parallel breakthrough experiments, a column of standard super-cel had a capacity almost six times that of a hyflo column. 2. After partial removal of impurities by diatomaceous earth, prothrombin preparations contained less thrombokinase, were more stable, and displayed less tendency to form thrombin "spontaneously." Thrombokinase (or its precursor) was removed from a preparation of prothrombin by passage through a filter cake of standard super-cel. The specific activity of the prothrombin was increased; and 62 per cent of the activity was recovered. 3. Prothrombin was adsorbed from an ammonium sulfate solution at pH 5.26 by columns of hyflo or standard super-cel. When eluted by phosphate solutions, the protein moved down the columns more readily at higher pH and higher concentration of phosphate salts, within the pH range 5.0 to 6.6, and within the phosphate range 0.1 to 1.0 M. 4. Thrombin was adsorbed on a column of standard super-cel at pH 5.11. As successive eluents passed through the column, the thrombin emerged between two bands of impurities. The specific activity of the thrombin was raised; and 83 per cent of the activity was recovered. 5. With a column of standard super-cel, and with a series of eluents within the pH range 5.1 to 6.3, total serum proteins were separated into four major bands. About 94 per cent of the protein was recovered

PREPARATION OF THROMBOKINASE FROM BOVINE PLASMA

Thrombokinase is prepared from bovine plasma by a procedure involving: treatment with diatomaceous silica, adsorption on barium sulfate, flowing elution with two successive phosphate buffers, ammonium sulfate fractionation, "spontaneous" activation in concentrated solution, and isoelectric precipitation. The yield of nitrogen is 0.002 per cent, corresponding to 1.2 mg. protein per liter of plasma. When diluted back to the volume of parent plasma, and complemented by calcium plus cephalin, the product causes appreciable activation of prothrombin in 1 minute. Thus, the quantity of thrombokinase obtainable is compatible with a physiologic role. In the more complex system used for routine assay, thrombokinase can be supplied by crude plasma at a dilution of 1/500. In parallel tests, the product appears to be more active than its parent plasma, although it contains only 0.002 per cent of the nitrogen. However, the thrombokinase of the product has been activated, whereas the thrombokinase of the plasma is probably in an inactive precursor state. When diluted back to the volume of parent plasma, to a concentration of 0.2 microgram nitrogen per ml., thrombokinase can slowly activate prothrombin in the presence of oxalate, and without the addition of accessory factors. Activation of prothrombin in the presence of oxalate is faster with higher concentrations of thrombokinase

PURIFICATION OF THROMBIN

1. Under certain conditions crude prothrombin changes to thrombin without the addition of extraneous activators and in the absence of ionic calcium. 2. Thrombin is soluble in 0.45 saturated ammonium sulfate, whereas crude prothrombin is not. 3. Partially purified thrombin is comparatively stable in concentrated ammonium sulfate solutions at pH 5.2. 4. A method based on the above facts yields a thrombin preparation the specific activity of which is 100 to 175 times the potential specific activity of whole plasma

Thrombokinase of the Blood as Trypsin-Like Enzyme

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity

EFFECT OF BLOOD THROMBOKINASE, AS INFLUENCED BY SOY BEAN TRYPSIN INHIBITOR; ULTRACENTRIFUGATION, AND ACCESSORY FACTORS

1. Crystallized soy bean trypsin inhibitor, at a concentration of 100 µg./ml., suppressed the production of thrombin from a mixture of prothrombin and blood thrombokinase. The experiment was performed in the presence of 0.011 M oxalate, in order to minimize the possibility of participation by accessory factors which require ionic calcium. The results are in accord with the view that thrombokinase is a trypsin-like enzyme. 2. When a solution of blood thrombokinase was centrifuged at 85,000 g for 120 minutes, almost all the activity remained in the supernate. This supernate activated the supernate from a prothrombin solution which had been similarly centrifuged. The activation of prothrombin by thrombokinase can proceed in the absence of material completely sedimentable in 120 minutes at 85,000 g. 3. An "accelerator" reagent was prepared by treating bovine serum with barium carbonate, and then passing the serum through a column of diatomaceous earth. This "accelerator" was used together with prothrombin, blood thrombokinase, Howell's cephalin, and calcium chloride to compose a five-reagent thrombin-producing system. In this system, no thrombin was produced without thrombokinase. On the other hand, thrombin was produced from prothrombin and thrombokinase, even when all the other reagents were omitted. When calcium was omitted, thrombokinase was able to function; but cephalin and the "accelerator" reagent were ineffective. 4. Quantitative tests indicated that the "accelerator" reagent exerted an effect distinct from those of thrombokinase and cephalin. However, it is not certain whether the "accelerator" reagent functioned as an accessory factor, as a potential source of more thrombokinase, or both. In the experiments reported, thrombokinase was primary to, or necessary for, the effect of "accelerator." 5. The effectiveness of thrombokinase was multiplied a hundred times or more, when complemented by calcium, cephalin, and "accelerator" reagent. Ionic calcium was a necessary component of this complementing system. This may help to explain why removal of calcium ions keeps blood fluid, even though thrombokinase, by itself, is little influenced either by calcium ions or by oxalate

THREE-STAGE ANALYSIS OF BLOOD COAGULATION

1. Blood-clotting mechanism has been analyzed by a procedure which devotes a separate experimental step to each of the three primary reactions: 1. Prothrombokinase → thrombokinase 2. Prothrombin → thrombin 3. Fibrinogen → fibrin 2. Activation of prothrombin by thrombokinase followed the course of a unimolecular reaction, and the concentration of thrombokinase determined the initial rate. By this relation thrombokinase was measured, and the activation of its precursor was charted. 3. When the activation of prothrombokinase was plotted against time, the experimental points fell close to the theoretical curve for a simple autocatalytic reaction. Moreover, the process was accelerated by seeding with a small amount of crude thrombokinase. It was concluded that the activation of prothrombokinase involves an autocatalytic or chain reaction. 4. The three-stage procedure made possible the separate estimation of the power to activate prothrombin, on one hand, and the capacity to accelerate the transformation of prothrombokinase on the other. Drastic losses of both activities occurred when crude thrombokinase solutions were heated at 60°C., or adsorbed with barium sulfate. 5. The concentration of calcium was important for the normal progress of prothrombin activation, and also for the transformation of prothrombokinase

Outstanding Characteristics of Thrombokinase Isolated from Bovine Plasma

Thrombokinase has been isolated from bovine plasma by a procedure which begins with the highly purified product of a previously described method, chromatographs it on DEAE-cellulose, and then fractionates it by continuous flow electrophoresis, yielding 0.2 mg per liter of oxalated plasma. The electrophoretic fraction has shown a single boundary in the ultracentrifuge; and its esterase activity on toluenesulfonylarginine methyl ester has been about the same as that of thrombokinase previously isolated by repeated electrophoretic fractionations. Thrombokinase is a euglobulin with minimum solubility near pH 5.0. It is most stable within the pH range 7.5 to 9.5; but there is also a peak in the stability curve near pH 1.8. A few micrograms of thrombokinase per milliliter can activate prothrombin in the presence of EDTA. A few thousandths of a microgram causes rapid production of thrombin in the system: prothrombin, thrombokinase, calcium chloride, phosphatide, "accelerator." But, thrombokinase has less than 1/175 the proteolytic activity of crystallized trypsin