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 studies 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.