Although the blood-clotting system presents increasing complexities, it is still to be hoped that there is a simple basic mechanism' at its core. If this basic mechanism were first apprehended, one could then begin to superimpose the complexities.The facts at hand are consistent with the following concept of the basic mechanism:Kinase
Ever since Morawitz published his classical review in 1905, it has been the custom to divide the blood-clotting process into two phases, or stages: (1) Prothrombin is converted to thrombin in the presence of calcium ions and certain factors derived from platelets and tissues. (2) Under the influence of thrombin, soluble fibrinogen is transformed to the insoluble fibrin.This formulation was put forth as tentative, and it was generally appreciated that a two-stage theory could not give an adequate exphnation of the mechanism. Several attempts were made to construct a three-stage framework on which further detail might be built. Morawitz (1904) suggested that prothrombin was first acted upon by thrombokinase, then by calcium. In somewhat similar fashion, Howell (1935) believed that cephalin freed prothrombin by removing heparin from it; then the prothrombin was activated directly by calcium. The 1940 schema of W~klisch postulated the conversion of prothrombin I to prothrombin II. Bordet (1919) had proposed that prothrombin must be "unmasked" in order to be readily convertible to thrombin; and his experiments had shown with particular clarity that some timeconsuming reaction did, in fact, precede the development of thrombin. However, his data did not prove the assumption basic to all these three-stage theories, namely that the preliminary reaction was concerned with some preparatory change in the status of prothrombin.Meanwhile another idea had been growing. Mellanby (1909) interpreted some of his data as indicating that serum contained an inactive form of thrombokinase. Closer to the point was the demonstration by Dale and Walpole (1916) that fowl plasma contained an inactive form of thrombokinase, from which active thrombokinase could be released by treating the plasma with chloroform or trypsin. The concept of a plasma precursor of thrombokinase was embodied in a three-stage theory of blood coagulation by Lenggentiager in 1936; and the proposed mechanism was explicitly diagrammed by him in 1940, in principle, as follows:1. Prothrombokinase --* thrombokinase in the presence of calcium. 2. Prothrombin --, thrombin in the presence of calcium and 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.Thrombokinase was first isolated from bovine plasma by a series of three electrophoretic fractionations (1). Later, it was reported (2, 3) that chromatography on DEAE-cellulose offered a less arduous means for attaining a comparable specific activity. However, it has been found that the chromatographic preparation is not homogeneous electrophoretically. Now, one chromatographic step and one electrophoretic step have been incorporated into the procedure for isolation of thrombokinase.Thrombokinase so prepared, has about the same esterase activity on toluenesulfonylarginine methyl ester (TAMe) as thrombokinase prepared by the more laborious procedure; and it sediments in the ultracentrifuge with a single, grossly symmetrical boundary. A few of its outstanding enzymatic and physicochemical properties are herein delineated.
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.
The clot-promoting effect of platelet suspensions is attributed to the platelets themselves, rather than to accompanying blood components. The mode of this platelet action still remains to be clarified. It is often stated that platelets plus calcium ions activate prothrombin. But i m t is obvious that the facts are not that simple; for platelets, calcisum and prothrombin coexist in the circulating blood. I t seem that at least one other * Aided by a grant from the Fluid Research t This work was done during the tenure of a Fund
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