327 Journal of Protein Chemistry, Vol. 19, No. 4, 2000 0277-8033/00/0500-0327$18.00/0 © 2000 Plenum Publishing Corporation Thrombin Inhibitor Design: X-Ray and Solution Studies Provide a Novel P1 Determinant Vicki L. Nienaber, 1,2 Paul D. Boxrud, 1,3 and Lawrence J. Berliner 1,4 Received June 6, 2000 The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was con- firmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent chan- nel beneath the S1 pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium con- centration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor. KEY WORDS: Proflavin; 6-fluorotryptamine; thrombin; X-ray crystallography; serine protease. 1. INTRODUCTION Thrombin is a trypsin-like serine protease that plays a cen- tral role in the regulation of blood coagulation. Throm- bin’s coagulant functions include cleavage of fibrinogen into insoluble fibrin in the final step of the coagulation cascade and stimulation of a number of other blood fac- tors as well as platelet aggregation. However, thrombin may also function as an anticoagulant when complexed with thrombomodulin (Fenton, 1988; Stubbs and Bode, 1993). Because of thrombin’s central role in blood coag- ulation, it has been targeted in the treatment of various he- mostatic disorders such as myocardial infarction, stroke, and pulmonary embolism (Fenton et al., 1991). As reported by Bode et al. (1989), the human α- thrombin molecule consists of two β-barrels, the catalytic triad (Ser195, His57, Asp102), and a substrate-binding groove located between the two domains. At the sub- strate-binding groove, a series of pockets defines the cleavage specificity of the protease and binds the sub- strate in an orientation ideal for enzymatic cleavage. Thrombin, a trypsin-like serine protease, prefers to cleave after positively charged lysine or arginine side chains due to a negatively charged aspartic acid located at the base of the S1 pocket. Thrombin also contains a unique apolar site at S2 that is composed partially of a nine-residue insertion loop at position 60. Berliner and Shen (1977) and Conery and Berliner (1983) described that indole analogs interact with throm- bin. These apolar ligands were found to activate thrombin amidase and esterase activity as well as fibrinogen clot- ting and under some conditions to have an inhibition phase (Berliner and Shen, 1977; Conery and Berliner, 1983). Some responses were observed to be biphasic, sug- gesting multiple binding sites which may interact cooper- atively. Because of the nature of these ligands, it has been proposed that they bind to the hydrophobic S2 pocket of thrombin (Bode et al., 1989; Malikayil et al., 1997). This 1 Department of Chemical and Physical Sciences, DuPont Merck Phar- maceutical Company, Experimental Station, Wilmington, Delaware 19880, and Departments of Chemistry and Cellular and Molecular Biochemistry, Ohio State University, Columbus, Ohio 43210. 2 Current address: Department of Structural Biology, Abbott Labo- ratories, Abbott Park, Illinois 60064. 3 Current address: Department of Pathology,Vanderbilt University School of Medicine, Nashville, Tennessee 37232. 4 To whom correspondence should be addressed; e-mail: berliner.2@osu.edu