Abstract
Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.
