Mechanism of Poly(acrylic acid) Acceleration of Antithrombin Inhibition of Thrombin: Implications for the Design of Novel Heparin Mimics Bernhard H. Monien, † Kai I. Cheang, ‡ and Umesh R. Desai* ,†,§ Departments of Medicinal Chemistry and Pharmacy and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23298 Received April 19, 2005 The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive ∼2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA- dependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K D of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the “bridging” mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics. Introduction The process of fibrin formation involves the sequential activation of trypsin-like serine proteinases that possess interesting similarities among them, yet simultaneously display both subtle and ingenuous differences. Fibrin formation is primarily regulated by antithrombin, a plasma glycoprotein and member of the serpin (serine proteinase inhibitor) super family of proteins. 1,2 Anti- thrombin regulates fibrin formation by primarily inhib- iting three procoagulant proteinases, factor IXa, factor Xa, and thrombin. However, antithrombin is a rather poor inhibitor of these procoagulant enzymes. Whereas most serpins inhibit their target proteinases at rates reaching the diffusion limit (10 7 -10 8 M -1 s -1 ), anti- thrombin inhibition of factor Xa and thrombin is nearly 1000-10 000-fold slower. The lethargic reaction is greatly accelerated in the presence of heparin, a natural sulfated linear poly- saccharide, which affords a surface for the rapid as- sembly of ternary antithrombin-proteinase complexes. 2-7 Under in vivo conditions, this “bridging mechanism” is responsible for most of the accelerating effect of heparin on the inhibition of all three proteinases. In another mechanism, the binding of a specific five-residue se- quence in heparin, pentasaccharide DEFGH (Figure 1), induces a conformational change in the serpin that enhances its recognition of the proteinase, resulting in an acceleration in the inhibition rate. 6,8 This mechanism is called the “conformational activation” mechanism, which causes an ∼500- and 300-fold acceleration in the inhibition of factor IXa and factor Xa, respectively. The bridging mechanism with full-length heparin polymer, in turn, induces an additional ∼600- and 170-fold * Address for correspondence: Department of Medicinal Chemistry, Virginia Commonwealth University, 410 N. 12th Street, P.O. Box 980540, Richmond, VA 23298-0540. Ph: 804-828-7328; fax: 804-827- 3664; e-mail: urdesai@vcu.edu. † Department of Medicinal Chemistry. ‡ Department of Pharmacy. § Institute for Structural Biology and Drug Discovery. Figure 1. Structures of high affinity pentasaccharide DEFGH sequence of heparin (A) and PAA (B). Sulfate and carboxylate groups in A contributing to the high affinity for antithrombin are shown in gray ovals. The table corresponding to PAAs summarizes their important properties: M R ) average mo- lecular weight; nd ) not determined; KD refers to antithrombin binding affinity at pH 7.4, I 0.05, and 25 °C; acceleration refers to the increase in antithrombin inhibition of thrombin due to PAA at pH 7.4, I 0.05, and 25 °C. Reprinted from ref 13 with permission. Copyright 2005 American Chemical Society. BATCH: jm8b07 USER: slr69 DIV: @xyv04/data1/CLS_pj/GRP_jm/JOB_i17/DIV_jm0503648 DATE: July 1, 2005 10.1021/jm0503648 CCC: $30.25 © xxxx American Chemical Society PAGE EST: 8.2 Published on Web 00/00/0000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56