The glycosaminoglycan heparin enhances several reactions involving coagulation factor XI (FXI) including activation of FXI by factor XIIa, thrombin, and autoactivation; and inactivation of activated FXI (FXIa) by serine protease inhibitors. We examined the effect of heparin on inhibition of FXIa by the inhibitors C1-inhibitor (C1-INH) and antithrombin III (ATIII). Second order rate constants for inhibition in the absence of heparin were 1.57 ؋ 10 3 and 0.91 ؋ 10 3 M ؊1 s ؊1 for C1-INH and ATIII, respectively. Therapeutic heparin concentrations (0.1-1.0 units/ml) enhanced inhibition by ATIII 20 -55-fold compared with 0.1-7.0-fold for C1-INH. For both inhibitors, the effect of heparin over a wide range of concentrations (10 ؊1 to 10 5 units/ml) produced bellshaped curves, demonstrating that inhibition occurs by a template mechanism requiring both inhibitor and protease to bind to heparin. This implies that FXI/XIa contains structural elements that interact with heparin. Coagulation factor XI (FXI) 1 is the zymogen of a plasma serine protease that contributes to normal hemostasis by activating factor IX through limited proteolysis, in a calcium-dependent manner (1-3). The regulation of activated FXI (FXIa) appears to involve several plasma serine protease inhibitors (serpins). Initial work suggested that ␣ 1 -antitrypsin (4 -6) is the predominant plasma inhibitor of FXIa with some contribution from antithrombin III (ATIII) (5, 7, 8). Recent reports using monoclonal antibody-based enzyme-serpin complex capture techniques disagree with the earlier findings, and indicate a predominant role for C1-inhibitor (C1-INH) and a significant contribution from ␣ 2 -anti-plasmin (9). The important anticoagulant drug heparin, a highly sulfated glycosaminoglycan isolated from bovine or porcine lung and gut, enhances the inhibition of FXIa by the inhibitors ATIII (8, 10), protease nexin II (11), and C1-INH (12). Although most studies indicate that saturating concentrations of heparin enhance ATIII-mediated inhibition 20 -40-fold (8, 10, 13), a recent report indicates a considerably smaller effect and proposes that heparin's primary anticoagulant effect on FXIa is through C1-INH (12).There is evidence that FXIa inhibition by ATIII in the presence of heparin proceeds by a template mechanism in which both protease and serpin are approximated by binding to the glycosaminoglycan (13,14). This process is exemplified by heparin-enhanced inhibition of the coagulation protease thrombin by ATIII (14,15). A template mechanism has also been demonstrated for autoactivation of FXI in the presence of polyanions such as heparin and the synthetic polysaccharide dextran sulfate (16,17). The template mechanism implies that FXIa contains structural elements involved in interactions with heparin. Numerous proteins have been described that interact with heparin and other glycosaminoglycans, many of which contain clusters of basic amino acids that form positively charged binding sites for the negatively charged glycosaminoglycan (18). The nature of these...