The molecular-weight-dependence of the anti-coagulant activity of heparin

Abstract

It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.

Binding to antithrombin of heparin fractions with different molecular weights

“…A similar molecular-weight-dependence of the anticoagulant activity of high-affinity heparins as that observed in the present work has been shown earlier (Laurent et al, 1978;Andersson et al, 1979). However, these investigators measured the average activity of the whole broad high-affinity peak from affinity chromatography and did not quantitatively characterize the binding of the fractions to antithrombin.…”

“…2, fractions V-IV, peak NA), an observation also made by Laurent et al (1978). When rechromatographed, this nonadsorbed heparin fraction was eluted at its original position, which shows that its appearance was not due to overloading of the column.…”

“…The different molecular-weight fractions of heparin were further fractionated according to their affinity for antithrombin on a column containing matrix-linked antithrombin (Hook et al, 1976;Nordenman & Bj6rk, 1978a;Laurent et al, 1978). The fractions behaved differently on this column.…”

“…Stoicheiometries (expressed as mol of heparin bound/mol of antithrombin) and binding constants were determined by computer-fitting the data from these titrations to a theoretical equation (Krause et al, 1974;Nordenman & Bjork, 1978a). Anticoagulant activities were determined by a whole-blood clotting assay (British Pharmacopoeia, 1968), or by a spectrophotometric assay based on the ability of heparin to accelerate the reaction between purified bovine antithrombin and thrombin (Bjork & Nordenman, 1976;Laurent et al, 1978;. In the latter procedure the amounts of thrombin inactivated at several sample concentrations were related to the inactivation produced by the IIIrd International Heparin Standard under the same conditions.…”

A historical perspective of the biophysics of the thrombin–heparin system: an example of nonspecific binding and the consequent parking problem in action

“…For thrombin, this inactivation of blood clotting entails the formation of a ternary complex in which enzyme and antithrombin are both bound to heparin (Olson and Shore 1981;Olson 1988). Physicochemical characterization of the interaction between antithrombin and the polysaccharide proved to be relatively straightforward in that the reaction involves binding of the enzyme to a unique pentasaccharide sequence of heparin (Rosenberg and Damus 1973;Laurent et al 1978;Olson 1988). On the other hand, the initial enthusiasm for quantitative characterization of the corresponding interaction between thrombin and heparin (Nesheim et al 1986;Evington et al 1986) seems to have been quashed by the realization (Olson et al 1991) that heparin does not possess a specific binding site for thrombin, which merely binds electrostatically to the highly charged polyanion.…”