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Kinetic analysis of the heparin-enhanced plasmin–antithrombin III reaction. Apparent catalytic role of heparin

Authors
  • Raymund Machovich
  • Pál I. Bauer
  • Péter Arányi
  • Éva Kecskés
  • Kálmán G. Büki
  • István Horváth
Publication Date
Dec 01, 1981
Source
PMC
Keywords
Disciplines
  • Biology
License
Unknown

Abstract

Inactivation of plasmin by a 3–4-fold molar excess of antithrombin III follows pseudo-first-order kinetics and the apparent rate constants are proportional to the concentration of the inhibitor. Heparin accelerates the inactivation reaction without changing its pseudo-first-order character, and the apparent rate constants are also proportional to the concentration of the polysaccharide. Heparin results in a minimum 20-fold rate enhancement of the reaction between plasmin and antithrombin III when the concentrations of heparin and plasmin are approx. 0.5μm and 1μm respectively. Heparin at a molar concentration well below that of plasmin still accelerates the reaction: one molecule of the polysaccharide is able to facilitate the inactivation of about 100 molecules of plasmin. Heparin must bind to plasmin to accelerate the plasmin–antithrombin III reaction, since the modification of four to five lysine residues of the enzyme inhibits the rate-enhancement effect of heparin and the dissociation of heparin–plasmin complex decreases the inactivation rate of plasmin. Increasing the concentration of antithrombin III, at a constant amount of heparin, results in increase of the inactivation rate. By contrast, the effect of increasing the amount of plasmin in the presence of constant amount of heparin and antithrombin III is such that higher plasmin-to-heparin ratios are associated with lower rates of inactivation. It seems, therefore, that to obtain `optimal' conditions for fast enzyme inactivation, the amount of heparin should be matched to plasmin rather than to antithrombin III. Arrhenius plots of the plasmin–antithrombin III reaction are linear both in the absence and presence of heparin, at concentrations of 1 or 2μg/ml, over a range of 26K. Under these experimental conditions, heparin increases activation entropy. The findings show that heparin seems to fulfil some criteria that are characteristic for biological catalysis: binding, reaction-rate enhancement (increasing activation entropy), recycling of heparin (effectiveness of non-stoichiometric amounts of the polysaccharide) and specificity.

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