Table 2.
Correlation between the disaccharide domain (–GlcNS3S6S-IdoA2S-) and AT-binding constant (Kd) as well as anti-FXa activity (IC50)
| Abbreviated structures | Binding affinity (Kd) to AT (nM) | Anti-FXa activity (IC50, nM) | |
|---|---|---|---|
| Fondaparinux, 5-mer | GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-OMea | 14.8 ± 1.4 nM | 12.2 nM |
| 6 – mers (hexasaccharides) | |||
| Comp 8*b | GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-pNP | 7 ± 2 nMb | 9.1 nM |
| Comp 2 | GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS3S6S-GlcA-pNP | No binding | No inhibition |
| Comp 3 | GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS3S6S-GlcA-pNP | Not measured | 11.0 nM |
| IdoA-containing 6-mer | GlcNS6S-GlcA-GlcNS3S6S-IdoA-GlcNS6S-GlcA-pNPc | 32.6 ± 5.3 nMd | 29.2 nMd |
| GlcA2S-containing 6-mer | GlcNS6S-GlcA-GlcNS3S6S-GlcA2S-GlcNS6S-GlcA-pNP4 | No bindingd | Not measured |
| 8 – mers (octasaccharides) | |||
| Comp 4 | GlcNS-GlcA-GlcNS-IdoA2S-GlcNS3S-IdoA2S-GlcNS-GlcA-pNP | No bindingc | No inhibition |
| Comp 5 | GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP-GlcNS3S6S-IdoA2S | 5.1 ± 1.4 nM | 7.7 nM |
| Comp 6 | GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-pNP | 5.6 ± 2.6 nM | 10.9 nM |
| Comp 11 * | GlcNAc6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP | 8 ± 3 nM1 | 9.4 nM |
a
Fondaparinux, an FDA-approved anticoagulant, was used as a positive control for anti-FXa activity measurement and the binding to AT. The -GlcNS3S6S-IdoA2S- disaccharide units are presented in bold face and underlined.
b
The binding constants for comp 8* and comp 11* were determined by affinity coelectrophoresis as reported in a previous publication 17.
c
Comp 4 did not bind to AT nor display anti-FXa activity, due to the fact that it does not contain 6-O-sulfo groups at the GlcNS residues at the nonreducing end 29.