Abstract
The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.
Highlights
MethodsCovalent Heparin-Antithrombin III Complexes (AT-H,AT-hlG,and AT-h,,i-The coupling of H and of the heparin fragments (h16 and h12)to antithrombin 111 was performed as previously described [22, 23]
The mechanism of the heparin-promoted reactionof Antithrombin I11 is a plasmaprotein thrombin with antithrombin I11 was investigated by that can inhibit several serine proteinases, including thromusingcovalent complexes of antithrombin I11 with either high-affinity heparin ( M, = 15,000) or heparin bin, factors IXa, Xa, XIa, XIIa, and plasmi(n1).Antithrombin I11 is distinguished from other protease inhibitorsby the fragments having an averageof 16 and 12 monosaccharide units (Mr= 4,300 and 3,200)
The inhibition rateof the enzyme is saturable bin with antithrombin I11by binding of heparin to theenzyme
Summary
Covalent Heparin-Antithrombin III Complexes (AT-H,AT-hlG,and AT-h,,i-The coupling of H and of the heparin fragments (h16 and h12)to antithrombin 111 was performed as previously described [22, 23]. Complex was purified further by gel filtration on Ultrogel AcA 44 in Kinetic Analysis of the Inactivation of Thrombin by Covalent Hep-. T o remove residual free heparin (about lo%), the preparations, rene tube (8.3 mm inner diameter) was added buffer diluent and dissolved in 0.05 M NaCl, 0.1 M Tris-HC1, pH 7.6, were applied to antithrombin 111 reagent (AT-H, AT-h16,or AT-h,,) to a finalvolume columns of concanavalin A-Sepharose equilibratedin thesame buffer. About 10%of the heparin but no antithrombin 111 appeared in the 1000rpm, and thrombin (5~ lw)as added with a positive displacement breakthrough. With [thrombin] = 1 nM, a rate of 80 mA/min was radioelectrophoresis and no free antithrombin 111 as determined by obtained. Antithrombin 111 concentration determined mixture before thrombin was added.
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