Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants

Abstract

Recent development of sulfated non-saccharide glycosaminoglycan mimetics, especially sulfated pentagalloyl glucopyranoside (SPGG), as potent inhibitors of factor XIa (FXIa) (J. Med. Chem. 2013; 56:867-878 and J. Med. Chem. 2014; 57:4805-4818) has led to a strong possibility of developing a new line of factor XIa-based anticoagulants. In fact, SPGG represents the first synthetic, small molecule inhibitor that appears to bind in site remote from the active site. Considering that allosteric inhibition of FXIa is a new mechanism for developing a distinct line of anticoagulants, we have studied SPGG's interaction with FXIa with a goal of evaluating its pre-clinical relevance. Comparative inhibition studies with several glycosaminoglycans revealed the importance of SPGG's non-saccharide backbone. SPGG did not affect the activity of plasma kallikrein, activated protein C and factor XIIIa suggesting that SPGG-based anticoagulation is unlikely to affect other pathways connected with coagulation factors. SPGG's effect on APTT of citrated human plasma was also not dependent on antithrombin or heparin cofactor II. Interestingly, SPGG's anticoagulant potential was diminished by serum albumin as well as factor XI, while it could be reversed by protamine or polybrene, which implies possible avenues for developing antidote strategy. Studies with FXIa mutants indicated that SPGG engages Lys529, Arg530 and Arg532, but not Arg250, Lys252, Lys253 and Lys255. Finally, SPGG competes with unfractionated heparin, but not with polyphosphates and/or glycoprotein Ibα, for binding to FXIa. These studies enhance understanding on the first allosteric inhibitor of FXIa and highlight its value as a promising anticoagulant.

Figure 1

Chemical structures of the hydrophobic, nonsacchride, aromatic, GAG mimetics of pentagalloyl glucopyranosides (SPGGs) e.g. SPGG2 and SPGG2-OH which were used in this study.

Figure 2

Proteolytic activity of human factor XIa (FXIa), plasma kallikrein (PK), activated protein C (APC), and factor XIIIa (FXIIIa) in the presence of SPGG2 (200 μg/mL) and SPGG2-OH (1000 μg/mL) using the corresponding assays. The assays were performed using substrates appropriate for the enzymes being studied under conditions closest to the physiological condition. The ratio of the proteolytic activity of an enzyme in the presence of SPGG2 or SPGG2-OH to that in their absence was used to determine percent activity (%). See Experimental procedures for details.

Figure 3

Reversibility of SPGG2 interaction with FXIa by potential reversing agents. Shown is the in vitro restored FXIa activity (%) (inhibited by 5 μg/mL of SPGG2) in the presence of increasing concentration of polybrene (●), protamine sulfate (●), and sucrose octasulfate (○). The restored activity profiles were determined spectrophotometrically at pH 7.4 and 37 °C. Solid lines represent fits by the dose-response Eq. 2 to obtain the EC₅₀ as described in Experimental Procedures.

Figure 4

Effect of bovine serum albumin (BSA) on the inhibitory profile of SPGG2 toward FXIa. The inhibition of FXIa by SPGG2 in the presence of BSA was determined spectrophotometrically at pH 7.4 and 37 °C. Shown is a bars graph of the residual FXIa activity (%) upon inhibition by a dose of 1.5 mg/mL of SPGG2 in the presence of 0, 10, 20, and 40 mg/mL of BSA. See Experimental procedures.

Figure 5

Effect of FXI on the inhibitory profile of SPGG2 toward FXIa. Shown is the inhibition of FXIa by SPGG2 in the presence of FXI which was determined spectrophotometrically at pH 7.4 and 37 °C. Solid lines represent fits by the dose-response Eq. 1 to obtain the IC₅₀ as described in Experimental Procedures. The concentrations of FXI selected for the study were 0 (●), 113 (◇), 225 (■), and 1125 nM (△).

Figure 6

Direct inhibition of purified and recombinant FXIa (rFXIa) by SPGG2 in 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.1 %PEG8000, and 0.02% Tween80. Shown is the inhibition of purified FXIa (○), rFXIa–ABS⁻¹ (◆), rFXIa–FL (△), and rFXIa–ABS⁻² (■). Solid lines represent dose-response fits of Eq. 1 to the data to derive the IC₅₀, HS, and ΔY values. See text for details.

Figure 7

FXIa catalytic domain (PDB ID: 1ZHM) generated with PyMole showing positions of Lys and Arg residues (blue) (K529,R530, R532) involved in heparin binding and positions of Ser, His, and Asp of active site (orange). The heparin binding site in FXIa catalytic domain is the putative binding site of SPGG2.