Interaction of Soybean Trypsin Inhibitor with Thrombin and Its Effect on Prothrombin Activation

Abstract

Abstract Because of its importance to an understanding of the mechanisms involved in the conversion of prothrombin to thrombin, the inhibitory properties of soybean trypsin inhibitor on this system were investigated. Kinetic studies showed that the inhibitor participates in a bimolecular reversible reaction with one of the components of the prothrombin-thrombin conversion system and to have an association constant dependent upon the rate of prothrombin conversion. With the use of three different systems for the activation of prothrombin, soybean trypsin inhibitor was found to block the conversion of the zymogen at two stages in the activation sequence. The first stage occurs during the initial peptide bond-breaking step which produces a polymorphic prothrombin intermediate plus a peptide fragment. The second point of inhibition occurs when this intermediate is split into smaller molecules, one of which has thrombin activity. Indirect evidence suggests that the proteolytic activity of thrombin is responsible for these initial events and that some of the bonds normally broken at these two stages are similar to bonds which have been implicated with the reactive site of soybean trypsin inhibitor. By the use of gel filtration columns equilibrated with buffers containing soybean trypsin inhibitor, it is possible to show that human or bovine thrombin (mol wt approximately 30,000 g) combines with soybean trypsin inhibitor (mol wt approximately 20,000 g) to form a mole for mole dissociable enzyme-inhibitor complex (mol wt approximately 50,000 g). This was confirmed by ultracentrifuge analysis. The complex thus formed is enzymatically active against fibrinogen (Arg-Gly bonds) and synthetic ester substrates but its formation is not affected when these catalytic properties are inhibited by sulfonation or alkylation of the residues at the active site of thrombin. Because of this, it is suggested that the mechanism of inhibition may be associated with alterations of secondary binding sites on the enzyme or with steric effects. From other experiments and for reasons which are discussed, these results cannot be explained on the basis of Factor X contamination of the thrombin preparations used. Although these studies do not dispute the possible interaction of soybean trypsin inhibitor with preparations having Factor X activity, they do not support the view that the inhibitor combines with intermediates or derivatives of the prothrombin-thrombin transition. Rather, these studies indicate that soybean trypsin inhibitor interacts with thrombin to block its specific proteolytic capacity to initiate prothrombin activation.