Anti-Thrombin III Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to the Endothelium in a Sickle Cell Mouse Model: Potential Therapeutic Application for Vaso-Occlusive Episodes

Painful vaso-occlusive episodes are a hallmark of sickle cell disease (SCD), an inherited blood disorder caused by a point mutation in the β-globin gene. Vaso-occlusive events account for the majority of SCD-related hospital admissions and contribute importantly to major organ damage and decreased survival. Therapies to prevent and treat acute vaso-occlusive episodes are limited and there is a need for new treatment options. Adhesive interactions between circulating sickle red blood cells (sRBC), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusive episodes. These cell adhesive interactions further manifest with increased local and systemic inflammation and activated coagulation evidenced in patients and mouse models of SCD.

Anti-thrombin III (ATIII) is an endogenous inhibitor of thrombin and other serine proteases in the coagulation pathway. Apart from the role in hemostasis, ATIII exerts strong anti-inflammatory properties. Plasma levels of ATIII and ATIII activity are decreased in SCD and further decline during vaso-occlusive crisis. These findings indicate that ATIII deficiency may contribute by multiple mechanisms to the pathophysiology of acute vaso-occlusion and that repletion therapy with ATIII may provide a novel, multi-targeted therapeutic approach to this difficult-to-treat complication.

In this study, we examined whether ATIII supplementation is able to reduce adhesion of sRBC and leukocytes to vascular endothelial cells in the calvarial bone marrow microvasculature in a mouse knockout-transgenic mouse model of SCD (Ryan, 1997). Homozygous SCD mice were injected intravenously with 230 U/kg ATIII (Thrombate III; Grifols, USA) or vehicle (saline). After 48 hours, the mice were subjected to 3 hours of hypoxia (8% O₂ in medical air) followed by 3 hours of reoxygenation at room air (21% O₂). Intravital microscopy analysis of the bone marrow microcirculation was performed to monitor adhesive interactions between blood cells and vascular endothelium. Leukocytes were labeled in vivo with phycoerythrin-conjugated anti-CD45 antibody which was infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery to the recipient mice. Effects of ATIII on sRBC and leukocyte adhesive interactions were compared to vehicle. Administration of ATIII to SCD mice resulted in more than 5 fold decrease in sRBC adhesion (p<0.01) compared to vehicle. Next the effects of ATIII on leukocyte flow dynamics were analyzed quantitatively. Mice pretreated with ATIII, but not those treated with vehicle, showed two-fold decrease in leukocyte rolling flux (p<0.01) and a 3-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Furthermore, ATIII improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and calculated wall shear rates. The pretreatment with ATIII was associated with decreased plasma levels of IL-1β and a trend to decreased soluble levels of vascular cell adhesion molecule 1 and thrombin-antithrombin complexes.

In conclusion, our study demonstrates significant anti-adhesive activities of ATIII, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in a mouse model of SCD. ATIII may represent a novel and effective therapeutic intervention in the treatment of acute vaso-occlusive crisis in patients with SCD.