Importance of the P2 Glycine of Antithrombin in Target Proteinase Specificity, Heparin Activation, and the Efficiency of Proteinase Trapping as Revealed by a P2 Gly → Pro Mutation

Yung-Jen Chuang, Peter G. W. Gettins, Steven T. Olson
1999 Journal of Biological Chemistry  
A sequence-specific heparin pentasaccharide activates the serpin, antithrombin, to inhibit factor Xa through an allosteric mechanism, whereas full-length heparin chains containing this sequence further activate the serpin to inhibit thrombin by an alternative bridging mechanism. To test whether the factor Xa specificity of allosterically activated antithrombin is encoded in the serpin reactive center loop, we mutated the factor Xa-preferred P2 Gly to the thrombin-preferred P2 Pro. Kinetic
more » ... s revealed that the mutation maximally enhanced the reactivity of antithrombin with thrombin 15-fold and decreased its reactivity toward factor Xa 2-fold when the serpin was activated by heparin pentasaccharide, thereby transforming antithrombin into an allosterically activated inhibitor of both factor Xa and thrombin. Surprisingly, the enhanced thrombin specificity of the mutant antithrombin was attenuated when a full-length bridging heparin was the activator, due both to a reduced rate of covalent reaction of the mutant serpin and thrombin and preferred reaction of the mutant serpin as a substrate. These results demonstrate that the reactive center loop sequence determines the specificity of allosterically activated antithrombin for factor Xa and that the conformational flexibility of the P2 Gly may be critical for optimal bridging of antithrombin and thrombin by physiologic heparin and for preventing antithrombin from reacting as a substrate in the bridging complex. Antithrombin is the principal serpin family protein inhibitor of coagulation proteinases in blood (1). Like other serpins, antithrombin inhibits its target proteinases by undergoing a large scale conformational change during reaction of the serpin as a regular proteinase substrate, resulting in the trapping of proteinase in a stable complex with the serpin (2). However, antithrombin differs from most other serpins in that its inhibitory activity is regulated by the polysaccharide cofactor, heparin, which acts to enhance the rate of antithrombin-proteinase reactions several thousand-fold (1, 2). Two distinct mechanisms appear to mediate the cofactor effect of heparin, the relative contribution of these mechanisms depending on the proteinase inhibited. The dominant mechanism with factor Xa as the target proteinase involves an allosteric activation of antithrombin by a sequence-specific heparin pentasaccharide (3-6). The pentasaccharide activates antithrombin by inducing conformational changes in a proteinase binding loop of the serpin known as the reactive center loop, which presumably allows the loop to optimally interact with factor Xa (7-12). With thrombin as the target proteinase, the allosteric activation mechanism appears to be a minor contributor to the heparin cofactor activity, since the pentasaccharide minimally enhances the rate of antithrombin inhibition of thrombin. Heparin chains at least 18 saccharides in length and containing the pentasaccharide are instead required to substantially accelerate thrombin inhibition by the serpin. Available evidence suggests that the longer chain heparin accelerates the inhibition reaction by an alternative bridging mechanism in which the binding of both antithrombin and thrombin to heparin promotes the interaction between serpin and proteinase in a ternary complex (6, (13) (14) (15) (16) . Based on these findings it has been implied, although without experimental proof, that the conformational changes induced in the antithrombin reactive center loop by pentasaccharide and full-length heparins allow the loop to optimally interact with factor Xa but not with thrombin because the reactive center loop sequence specifically recognizes factor Xa (2). In keeping with such a hypothesis, the P4-P1 IAGR sequence of the loop resembles the IEGR and IDGR P4 -P1 sequences in prothrombin, the natural substrate of factor Xa, whereas the loop sequence does not match well the specificity requirements of thrombin except for the P1 Arg residue (17). To test whether the IAGR reactive center loop sequence of antithrombin is responsible for the factor Xa specificity of allosterically activated antithrombin, we chose to mutate the antithrombin P2 Gly to Pro. This choice was made because (i) a P2 Pro is preferred in natural thrombin substrates (17) and (ii) replacement of a P2 Gly for Pro in synthetic tripeptide substrates produces several hundred-fold enhancements in thrombin specificity (18). While the effects of a P2 Gly 3 Pro mutation in antithrombin on thrombin inhibition were previously found to be modest, the specificity changes resulting from allosteric activation of the mutant antithrombin by heparin pentasaccharide were not examined (19, 20) . Consistent with the predictions of our hypothesis, our results show that the P2 Gly 3 Pro mutation transforms antithrombin into a serpin that can be activated by the heparin pentasaccharide to inhibit both thrombin and factor Xa with comparable enhanced rates approaching that of activated wild-type antithrombin inhibition of factor Xa. Surprisingly, the P2 Gly 3 Pro mutation converts antithrombin into a worse inhibitor of thrombin when a fulllength heparin is used to activate the serpin, due largely to the preferred reaction of the mutant antithrombin as a substrate of thrombin in the ternary bridging complex with the full-length heparin. Such results suggest that while the P2 Gly of anti-
doi:10.1074/jbc.274.40.28142 pmid:10497166 fatcat:e2h6ho5sdfc3jb75luh4azcxci