Investigating the susceptibility of thrombosis in sickle cell disease

Felicity Gavins, Julia Buckingham, Neil Granger

1Imperial College, London, UK, 2LSUHC, Shreveport, USA

SCD patients are more susceptible to thrombotic events and leukocytes, platelets and erythrocyte adhesion have all been implicated. This study investigated the effect of thrombus formation in the brain of mice with SCD.

As described previously (Wood, K. et al., 2004), intravital microscopy was performed in the cerebral microcirculation of anaesthetised (pentobarbitone: 50mg/kg) wild-type (WT), sickle cell transgenic (SC; βs: 25-30g) and WT/βs chimeric (used as a positive control) mice. The light/dye endothelial cell injury model (10ml/kg of 5% FITC-dextran, injected via the jugular vein) was performed in both venules and arterioles of mice. Briefly, the jugular vein (for dye administration) and trachea were canulated. A cranial window was drilled to expose the pial vessels. Photoactivation was initiated by exposing 100μm vessel length to epi-illumination with a 175-W xenon lamp (Lambda LS, Sutter) and a fluorescein filter cube (HQ-FITC, Chroma). Epi-illumination was applied continuously and the time of flow cessation was recorded (≥60s duration).

Our results demonstrated that SCD mice were more susceptible to thrombus formation in both venules (11.3 ± 1.1 min vs. 5.4 ± 0.2 min, P<0.05, n=6 mice, WT vs. SC mice respectively) and arterioles (36.3 ± 2.8 min vs. 15.39 ± 2.0 min P<0.05, n=6 mice, WT vs. SC mice respectively) vs. their WT counterparts. The positive controls, as expected, demonstrated results similar to the WT controls.

The treatment for coagulation in SCD is anti-coagulant therapy. We therefore wanted to focus on various coagulation cascades that may be involved in SCD and so we investigated both Hirudin (a specific inhibitor of thrombin whose mode of action is independent of anti-thrombin III) and anti-thrombin III. Both drugs were administered via the jugular vein, 5 min prior to onset of thrombus formation. Anti-thrombin III produced a significant increase in flow cessation time in venules only of SCD mice. However, hirudin was effective in increasing the time taken for blood flow to stop in both venules (WT+hirudin vs. SCD+hirudin: 36.4 ± 5.9 min vs. 54.7 ± 5.7. n=6 mice P<0.05) and arterioles (WT+hirudin vs. SCD+hirudin: 34.0 ± 5.4 min vs. 58.1± 6.0 n=6 mice P<0.05. Suggesting different pathways may be mediating the effects in the venules and the arterioles, such that thrombus formation is via the thrombin pathway, but it would appear that in venules, this effect is also dependent upon the anti-thrombin III pathway.

This data presented adds to the current literature suggesting a pro-inflammatory and pro-thrombogenic microcirculation is found in SCD mice. Furthermore, SCD appears to promote the formation of thrombosis. By understanding the pathways involved in SCD that contribute to the increased susceptibility of thrombosis, it may lead to therapeutic targets for this painful and life-threatening disease.

We thank the BBSRC Integrative Mammalian Biology Fund for financial support.

Wood, K. et al. Endothelial cell P-selectin mediated a proinflammatory and prothrombogenic phenotype in crerebral venules of sickle cell transgenic mice. Am J Physiol Heart Circ Physiol. 2004: H1608-1614.