Fondiparinux Worsens Leukocyte Endothelial Interactions Improved by Antithrombin and Increases Leukocyte Extravasation.

Background: A phase III trial of antithrombin (AT) in sepsis showed benefit only in patients who did not receive concurrent heparin. We have shown that some of the beneficial effects of AT on leukocyte-endothelial (LE) interactions in sepsis models requires the activity of heparan sulfate 3-O-sulfotransferase-1 (3-OST-1), the enzyme that is responsible for the rate-limiting step in the biosynthesis of anticoagulant heparan sulfate. Fondiparinux (FOND) is a pharmacological agent that mimics the anticoagulant heparan sulfate pentasaccharide sequence synthesized by 3-OST-1.

Objectives: We hypothesized that FOND treatment would alter LE interactions in sepsis models in mice, and AT effects in these models.

Methods: Using intravital microscopy, we evaluated the effects of proinflammatory stimuli on leukocyte rolling and firm adhesion in post-capillary venules, and extravasation, into the cremaster muscle in live C57BL/6 mice. Human AT (0.25 U/g Thrombate III), and/or heparanoid or vehicle was infused intravenously prior to proinflammatory stimuli. Venules were chosen such that in all comparisons, the average diameter (range 20 to 50 μm) and shear rate (range 200 to 800 s⁻¹) was not different between treatment groups. The number of leukocytes rolling past a defined vessel point was expressed as leukocyte rolling flux, which normalizes for leukocyte count and flow rate. The number of firmly adherent leukocytes (stationary for 30 s) was normalized for the area of vessel wall analyzed. The number of extravasated leukocytes was normalized for the area of tissue analyzed.

Results: AT treatment decreased leukocyte rolling flux (23 vs. 15%, P=0.01) induced by an injection of lipopolysaccharide (LPS at 1 μg i.p.) 2 h before observation. However, in this model, AT did not inhibit the number of firmly adherent leukocytes. When mice were pretreated with FOND (0.1 μg/g), the leukocyte rolling flux was increased compared to without heparanoid (22 vs. 25%, P<0.05) and the number of firmly adherent leukocytes was decreased (294 vs. 127 mm⁻², P<0.05), implying an inhibitory effect of FOND. However when mice were treated with AT and FOND, LE interactions were worsened: although the rolling flux decreased from 25 to 17% with AT treatment, the number of firmly adherent leukocytes increased from 127 to 305 mm⁻². Mice pretreated with danaparoid (0.025 U/g), where the pentasaccharide is rare, were not different from untreated mice, both with and without AT-pretreatment. Thus although AT improves LE interaction induced by LPS, it worsens these in the presence of FOND, as it did in 3-OST-1 deficient mice. Because we had previously shown that 3-OST-1 deficient mice had poorer survival to an LPS challenge, we wondered whether the effect of FOND was to increase leukocyte trafficking toward extravasation. Five hours after injection of IL1β (30 ng i.p.), the tissue surrounding flowing vessels in the cremaster muscle was observed for one hour for interstitial leukocytes. FOND increased the number of extravasated leukocytes in untreated mice (from 0.34 to 0.47 mm⁻², P<0.05) and IL1β-pretreated mice (from 0.32 to 0.64 mm⁻², P<0.005). The effect of AT on extravasation is currently being evaluated.

Conclusions: These studies further our knowledge of how the anticoagulant pentasaccharide sequence, mimicked by fondiparinux and lacking in 3-OST-1 deficient mice, is important in leukocyte recruitment that occurs in response to inflammation. This will allow for more rational use of mediators of this pathway such as AT in sepsis.