Human and bovine antithrombin, purified by affinity chromatography on heparin‐agarose, have been characterized with regard to chemical composition, size, shape and conformation. Both preparations were found to contain several active components of identical or similar size but different electrical charge. Amino acid and carbohydrate analyses revealed striking similarities between human and bovine antithrombin, while immunological analyses failed to demonstrate any cross‐reactivity. The molecular weights were determined by sedimentation equilibrium to be 58000 for human and 56000 for bovine antithrombin. The small molecular weight difference suggested by these values was verified by several empirical methods of molecular weight estimation. Hydrodynamic measurements indicated that the two proteins have similar molecular shapes, both of which are slightly more extended than that of typical globular proteins. The internal folding of the two polypeptide chains is also similar, as evidenced by the identity of the far‐ultraviolet circular dichroism spectra. Specifically, these analyses suggested a low α‐helix content of both proteins. In conclusion, the marked structural similarity of human and bovine antithrombin indicates that the two proteins may also exhibit extensive functional similarities in the binding of heparin and the inhibition of various coagulation factors.

The interaction between antithrombin and two forms of heparin, differing in their affinity for the matrix-linked protein, has been studied by fluorescence. The binding of the high-affinity heparin fraction to antithrombin leads to activation of the inhibitor, allowing it to react more rapidly with a number of serine proteases of the coagulation cascade. The interaction with the low-affinity heparin fraction, however, has considerably less influence on this inhibition rate. The binding of either fraction to antithrombin was found to result in an increase of the tryptophan fluorescence of the protein. This increase was much larger for high-affinity heparin than for low-affinity heparin, suggesting a different mode of binding of the two fractions to the protein. The fluorescence enhancement caused by high-affinity heparin is consistent with a conformational change of antithrombin related to its activation. Only the fluorescence enhancement observed on the binding of high-affinity heparin was of a sufficient magnitude to allow quantitative studies. These showed high-affinity heparin to bind to antithrombin with a stoichiometry of about one and with a binding constant at physiological ionic strength of about 8 x lo7 M-l. At higher ionic strengths, however, the affinity decreased markedly.

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The accelerating effect of heparin on the reaction between purified human antithrombin and thrombin has been investigated by measuring the amount of thrombin inactivated during a short incubation of the enzyme with the inhibitor in the presence and absence of non-stoichiometric amounts of heparin. It was demonstrated that one molecule of heparin was able to promote the binding of a large number of antithrombin molecules to thrombin. Thus heparin may affect the rate of the inactivation of thrombin by antithrombin in a catalytic manner.The potent anticoagulant activity of heparin has been well known since the discovery of this polysaccharide by McLean in 1916 [l]. Recently several lines of evidence have suggested that at least part of the anticoagulant activity is due to the interaction of heparin with antithrombin 111, a plasma inhibitor capable of inactiviting thrombin and also a number of other serine proteases, notably several of the coagulation cascade [2 -71. It has thus been established that heparin binds to antithrombin, and that the addition of heparin to a mixture of antithrombin and thrombin (or certain other activated coagulation factors, such as factors VII, IX,, X, and XI,) results in a dramatic acceleration of the slow rate at which the inhibitor normally inactivates these enzymes. This may be due to a conformational change of antithrombin induced by its interaction with the polysaccharide [4]. Thus antithrombin is identical to the heparin cofactor, a plasma factor postulated by earlier workers in the field to be essential for heparin anticoagulant activity [S, 91. However, heparin has been shown to bind not only to antithrombin but also to thrombin [10,11], and the possibility exists that the rate of the reaction between thrombin and the inhibitor is influenced also by a direct interaction of heparin with the enzyme. The aim of this communication is to demonstrate that the accelerating effect of heparin in a system in vitvo, in which purified antithrombin and thrombin are used, is exerted by amounts of polysaccharide far below those of either of the two proteins present. This indicates that heparin under these conditions may function as a catalyst in the inactivation process. Such a catalytic role of heparin has been suggested previously [12,13], but on the basis of -~ E~y r n c . Thrombin (EC 3.4 21 5)

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