The Protective Effect of Annexin A1 in Sickle Cell Disease

Cerebrovascular disease is a leading cause of death worldwide. It is a serious complication of sickle cell disease (SCD), an inherited autosomal recessive disorder, resulting from a single amino acid substitution in the haemoglobin β chain. Patients with SCD are more susceptible to thrombotic events, in which leukocytes, platelets and erythrocyte adhesion have all been implicated. It is known that anti-inflammatory therapies limit thrombosis and anti-thrombotic therapies reduce vascular inflammation. Therefore, we tested the therapeutic potential of the glucocorticoid inducible anti-inflammatory endogenous mediator Annexin A1 N-terminal derived peptide (AnxA1Ac2–26) in mediating thrombus formation in the brain of mice with SCD.

Intravital microscopy was performed in the cerebral microcirculation of anaesthetised (intraperitoneal (i.p.) injection of Ketamine (150mg/kg) and Xylazine (7.5 mg/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) was cannulated, and a cranial window 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 show that SC mice were more susceptible to thrombus formation in both venules (12.3 ± 3.2 min vs. 6.2 ± 0.8 min, p<0.05, n=6 mice, WT vs. SC mice respectively) and arterioles (40.4 ± 2.3 min vs. 18.21 ± 3.2 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. Treatment with AnxA1Ac2–26 (10, 100 μg/mouse. n=6 mice), administered via the jugular vein 5 min prior to onset of thrombus formation, produced a significant increase in flow cessation time in vessels of SC mice, with the most effective results being observed at the higher dose (100 μg/mouse. Venules: 10.4 ± 1.1 min and arterioles: 36.78 ± 2.7 min). We also identified a novel role for Ac2-26 in the induction of neutrophil extracellular trap (NET - decondensed chromatin decorated by granular enzymes and are released by activated neutrophils) formation in SCD.

This data 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, which can be inhibited by the administration of the anti-inflammatory endogenous mediator AnxA1Ac2–26. Further investigations are underway to determine the pathway(s) involved in the observed protective effect of Annexin A1 mimetic peptide in SCD, as this may lead to a potential therapeutic target for this painful and life-threatening disease.

We thank the NIH/NHLBI for financial support.