Sickle Cell transgenic mice exhibit a prothrombogenic phenotype in cerebral venules

Felicity Gavins1, D. Neil Granger2, Julia Buckingham1
1Imperial College, London, United Kingdom, 2Louisiana State University Health Science Center, Shreveport, Louisiana, United States

Sickle cell disease (SCD) is chronic genetic and lifelong disease affecting the vasculature of a variety of different organs with the affected tissues assuming an inflammatory phenotype (Wood et al., 2004). Leukocyte, platelet and erythrocyte adhesion have been implicated in the occlusive events that occur with SCD. This study, investigated the effects of SCD on the time taken for thrombus formation.

Intravital microscopy was performed in the cerebral microcirculation of pentobarbitone-anaesthetised (50mg/kg) wild-type (WT), sickle cell transgenic (SC; βs: 25-30g) and WT/βs chimeric (positive control) mice (as previously described; 1). Thrombus formation was formed using light/dye (10ml/kg of 5% FITC-dextran, injected via jugular vein) endothelial cell injury model in both arterioles and venules. Briefly, the jugular vein (dye administration) and trachea were canulated. A cranial window was drilled to expose 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 time of flow cessation recorded (≥60s duration).

In contrast to WT mice, the time taken for thrombus formation was reduced by 50% in SC mice in arterioles (33.5±2.5 min vs. 16.3±3.0 min P<0.05, n=5 mice, WT vs. SC mice respectively) and venules (10.6±1.0 min vs. 5.0 ±0.3 min, P<0.05, n=5 mice, WT vs. SC mice respectively). By contrast, the average time for flow cessation to occur in WT/βs chimeric mice did not differ significantly from that in WT mice in either arterioles (45.4±3.3 min. n=4mice) and venules (11.3±0.9 min. n=4mice), suggesting the response observed in these SC mice was not due to the way in which they were generated.

We also investigated the clinical implication of anti-coagulant therapy in mice with SCD. Both Hirudin (a specific inhibitor of thrombin whose mode of action is independent of anti-thrombin III) and anti-thrombin III were administered via the jugular vein (5 min prior to onset of thrombus formation). Hirudin, produced a significant increase in the time taken for the blood flow to stop in both venules (WT+hirudin vs. SCD+hirudin: 33.5 ± 6.3 min vs. 53.5 ± 5.9. n=5 mice P<0.05) and arterioles (WT+hirudin vs. SCD+hirudin: 33.0 ± 5.4 min vs. 57.2 ± 5.8. n=5 mice P<0.05. The anti-thrombin III inhibitor was more effective in this regard in the venules of SCD mice (10.6 ± 1.0 min vs. 66.8 ± 7.6, WT Ctl vs. SCD+anti-thrombin III n=4mice. P<0.05). However, no such differences were observed in arterioles. Together these finding suggest that thrombus formation is effected via the thrombin, but is also dependent upon the anti-thrombin III pathway in venules.

This study further supports the growing evidence for a pro-inflammatory and pro-thrombogenic microcirculation in sickle cell transgenic mice, in particular the notion that SCD promotes thrombus formation. Understanding the susceptibility and pathways involved in thrombogenic situations that occur in SCD, it may lead to the identification of potential therapeutic targets in this painful and life-threatening disease.


Wood, K. et al. 2004. Am J Physiol Heart Circ Physiol. H1608-1614.


We are grateful to the BBSRC Integrative Mammalian Biology Fund for financial support.