C1-INH and atiii inhibit vaso-occlusion and vascular inflammation in sickle cell disease mice

Abstract Background. C1 esterase inhibitor (C1-INH) is a serine protease inhibitor (serpin) in plasma that regulates the complement and contact systems. C1-INH regulates the complement system, primarily by inhibiting the classical and lectin pathways. It acts by inactivating serine proteases C1r and C1s in the classical pathway and mannan-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in the lectin pathway. In addition, C1-INH inhibits activated factor XII (FXIIa), factor XI (FXIa), and kallikrein thus preventing excessive activation of coagulation via the contact pathway. Similarly, antithrombin III (ATIII), another member of the serpin family, inhibits the activity of thrombin and activated factor X (FXa) in the common pathway. ATIII inhibits thrombin-mediated activation of endothelium and the adhesion of sickle red blood cells and leukocytes to endothelium. Complement activation and thrombin have been shown in murine sickle cell disease (SCD) models to promote microvascular stasis and vascular inflammation. Because of these activities, we hypothesized that infusion of C1-INH or ATIII would prevent vaso-occlusive crises (VOC) induced by hypoxia-reoxygenation (H/R) in the Townes SCD mouse model. Methods. Townes SCD (HbSS) mice and normal HbAA control mice were implanted with dorsal skinfold chambers and challenged with H/R to induce VOC. The mice were infused with saline vehicle, purified human C1-INH or ATIII 1 h prior to H/R or 40 minutes after H/R, simulating prevention and treatment of VOC, respectively. Microvascular stasis in subcutaneous venules was measured 0.5, 1, 2, 3, and 4 h after H/R. After the final stasis measurement, a terminal blood sample was collected from the heart into 0.5 M EDTA and platelet-free plasma was isolated and snap-frozen. The lungs and kidneys were excised and placed in optimal cutting temperature medium. The lungs and kidneys were cryosectioned and examined for P-selectin and von Willebrand factor (vWF) expression on blood vessels (CD31+) in the lungs and complement activation deposits (C3 fragments and C5b9) on blood vessels in the kidneys by confocal microscopy after immunofluorescence staining.Results. Microvascular stasis was significantly decreased (P<0.0001) 1, 2, 3 and 4 h after H/R by pretreatment of HbSS mice with C1-INH or ATIII as compared to vehicle. Similarly, when C1-INH or ATIII was infused 40 minutes after H/R, stasis seen at 30 minutes post-H/R was significantly decreased (P<0.0001) at 1, 2, 3, and 4 h post-H/R as compared to vehicle. Adding enoxaparin to ATIII enhanced ATIII protection. Combining C1-INH and ATIII provided maximal benefit. These observations were mechanistically buttressed in the prevention and treatment studies by decreased expression of P-selectin (P<0.001) and vWF (P<0.005) on the pulmonary endothelium and by reduced deposits of C3 fragments (P<0.01) and C5b9 (P<0.05) in the kidneys 4 h after H/R in HbSS mice administered C1-INH or ATT as compared to vehicle. In addition, plasma IL-6 levels 4 h after H/R was decreased (P<0.001) by treatment of VOC with C1-INH or ATIII as compared to vehicle. These data suggest that inhibiting thrombo-inflammatory pathways with serpins may be a viable therapeutic strategy for VOC in SCD.