Antithrombin III Affinity for Thrombin, Trypsin and α-Chymotrypsin

The relative affinity of human AT III for thrombin, trypsin and α-chymotrypsin was evaluated tn reactions proceeding in the molar excess of inhibitor, to reflect more closely natural conditions occurring in plasma. Residual AT III was titrated as total factor Xa-binding capacity independent of the velocity of inhibition. The decrease in binding properties of purified AT III reacting with thrombin or trypsin was consistent with formation of enzyme-inhibitor complexes at 1:1 molar ratio and was associated with a partial obliteration of antigenic determinants. Purified α₁-trypsin inhibitor competed with AT III but not for thrombin. α-chymotrypsin caused an excessive loss ot AT III binding properties; once the enzyme-inhibitor ratio exceeded 1:5 proteolysis of AT III occurred with formation of multiple protein fragments evident in gel filtration in fibrinogen-depleted human plasma where the overall binding capacity for both trypsin and thrombin exceeded that of AT III, a marked difference was seen in the rate of AT III utilization by each of these enzymes. Every μM of trypsin gradually added utilized on the average only 2% of AT III capacity, whereas the amount of thrombin capable of binding 1 μM of purified AT III obliterated of AT III capacity in plasma. Neutralization of thrombin proceeded after AT III was totally depleted. These results contribute to the notion that AT III is the primary inhibitor of thrombin with an unique affinitv for this enzyme in blood.