Abstract 594: Characterization of Anti-thrombotic and Anti-inflammatory Properties of New Synthetic, Protamine Reversible Low Molecular Weight Heparin
Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders; however, lack of full reversibility with protamine and clearance by kidney limit treatment options. Recently we have developed a chemoenzymatic method to synthesize a new synthetic dodecasaccharide (12mer) with high anti-factor Xa (FXa) activity which was protamine reversible in vitro and in vivo.
In the current study, we further characterized the pharmacokinetic, anti-thrombotic and anti-inflammatory properties of this compound using various mouse models. First, time course (0.5, 1, 2, 4, 8 hours, n=4) and dose response (0.5, 1, 1.5, 2.0 mg/kg, n=4) studies in C57Bl/6J mice showed that FXa activity was inhibited by 12mer in vivo and the half-life of the compound injected s.c. at the dose of 1.5 mg/kg was 3 hours. 12mer was cleared from the circulation by 8 hours.
Furthermore, fibrin accumulation observed in the femoral vein 60 minutes after electrolytic injury (2.99 ± 1.16; p<0.05; normalized relative fluorescent intensity to control mouse, mean ± SD), was similarly reduced by 12mer (0.6 mg/kg, 1.63± 0.48), UFH (150 U/kg, 1.26 ± 0.35) and enoxaparin (1 mg/kg, 1.39 ± 0.85, p<0.05 for all). Importantly, protamine reversed the anti-thrombotic effect of 12mer and returned fibrin accumulation to normal levels (2.69 ± 0.87; p < 0.05 vs.12mer alone).
Finally, we investigated if 12mer can inhibit thrombotic and inflammatory effect of FXa, previously demonstrated by us in the Berkeley mouse model of sickle cell disease. Sickle mice received subcutaneous injection of saline (n=6) or 12-mer (n=6; 2.0 mg/kg) every 8 hours for 7 days. Compared to saline, 12mer reduced plasma levels of thrombin anti-thrombin complexes (8.2±1.4 vs 4.4±1.3 ng/mL; p=0.07, mean ± S.E.M), IL-6 (5.5±1.5 vs 1.1±0.4 pg/mL, p<0.05) and sVCAM1 (1.1±0.2 vs 0.6±0.07 μg/mL, p< 0.05) in sickle mice. Together, our data demonstrates that chemoenzymatic synthesis offers a promising approach toward obtaining new, protamine-reversible LMWHs with excellent anti-coagulant and anti-inflammatory properties.
Author Disclosures: K. Chandarajoti: None. E.M. Sparkenbaugh: None. N.S. Key: None. Y. Xu: None. B. Cooley: None. J. Liu: None. R. Pawlinski: None.
- © 2015 by American Heart Association, Inc.