Search

The enzyme thrombin is sensitive to diisopropylfluorophosphate and phenylmethanesulfonyl flouride, but not to p-toluenesulfonyl-phenylalanyl-chloromethylketone. It is relatively resistant to -SS- reducing agents. Under appropriate conditions the enzyme activity regenerates after reduction. Autoprothrombin C activity is easily destroyed 3y -SS- reducing agents, but resistant to the other three reagents cited above.

1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner.

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.

A concentrate of antithrombin was made from bovine plasma. When examined with an analytical ultracentrifuge it was found to contain a main component and a small amount of more rapidly sedimentating material. For the main component [Formula: see text] was 4.01 S. The antithrombin concentrate destroyed the activity of purified autoprothrombin C, and most likely antithrombin and antiautoprothrombin C are one and the same substance. The antithrombin preparation was limited in its capacity to destroy autoprothrombin C activity, and equilibrium conditions were reached in 3 to 4 hours. At equilibrium the quantity of autoprothrombin C destroyed was proportional to the antithrombin added to an "excess" of autoprothrombin C. The last traces of the latter activity could only be neutralized with difficulty. Under optimum conditions 1 ml of plasma can neutralize the autoprothrombin C that might generate from 27 ml of plasma. About 0.83 ml of oxalated bovine plasma can neutralize the activity in 1 mg of purified autoprothrombin C.

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.

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.

SummaryReduction of thrombin yields in an enzymic (thrombokinase)-activated 2-stage clotting system is achieved in vitro by mM additions of organic phosphates (ATP, ADP, AMP). This is investigated in relation to pH and Ca++ concentration, optima for which are defined in control systems. The evidence indicates that the major inhibition must be related to some unknown calcium complex, which is a yield-determining factor in thrombin generation, even without the organic phosphates. ATP-ase degrades ATP and largely removes its inhibitory action.

SummaryThrombokinase, purified from 738 liters of bovine plasma, was subjected to repeated fractionations by continuous flow paper electrophoresis. This increased the specific activity of kinase and led to a 3rd electrophoretic run which placed almost all the protein in a single peak. Kinase activity was closely accompanied by TAMe esterase activity through successive stages of electrophoretic fractionation. The kinase fractions of the 3rd run showed esterase activity of the order of 337 TAMe units/mg protein. The results are in accord with the view that TAMe is a substrate for thrombokinase and that thrombokinase is a trypsin-like enzyme.

In every prothrombin activation two different enzymatic reactions can be distinguished. The first reaction is the prothrombin disappearance and the second one is the thrombin formation. Autoprothrombin C catalyzes both reactions and thrombin only the first one. The inhibition of thrombin with heparin allows a more complete activation of the thrombin. The two reactions can be independently catalyzed by different substances. Heparin by itself can provide the environment for activation.

SummaryHighly purified thrombokinase, derived from bovine plasma, activated chymo-trypsinogen. Chymotrypsin was assayed by the hydrolysis of BTEE, which was not split by thrombokinase under the conditions used. Activation of chymotrypsinogen may have been due to thrombokinase, per se, or to an impurity, since a large quantity of thrombokinase was required. However, phosphatide accelerated the process, provided that ionic calcium was included. Without thrombokinase, phosphatide plus ionic calcium failed to activate chymotrypsinogen.Much of the preparative work was done by William S. Bartek and Harriet F. Brown. We are indebted to Nadia Oulianoff for thrombokinase assays.

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.

SummaryThrombokinase prepared from bovine plasma was further purified by continuous flow paper electrophoresis. Thrombokinase was assayed by its capacity to activate prothrombin in the presence of oxalate, and also by production of thrombin in a system containing prothrombin, cephalin, calcium and bovine “barium carbonate serum.” Curves of these 2 assays were similar and formed a peak ahead of the thrombin peak. TAMe esterase activity appeared in 2 peaks which corresponded respectively to thrombin and thrombokinase peaks.