Targeted Inactivation of Murine Band 3 (AE1) Gene Produces a Hypercoagulable State Causing Widespread Thrombosis In Vivo

Abstract

Only 5% to 10% of band 3 null mice survive the neonatal period. To determine the cause of death, 3 adult and 11 newborn band 3 null mice were submitted for histopathologic examination. All but 1 pup showed evidence of thrombosis including: (1) large thrombotic lesions in the heart, which were partially organized, calcified in some fields, and endothelialized, indicating a process that developed premortem (3 of 3 adults and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver suggestive of premortem ischemic events caused by arteriolar occlusions (8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate the etiology of this hypercoagulable state, we have used the Russell’s viper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RVV clotting time of normal plasma in a dose-dependent fashion, whereas RBCs from normal mice have no effect, suggesting that the membrane of band 3 null RBCs provides a suitable surface for activation of the prothrombinase complex. Using flow cytometry, we have examined the phosphatidylserine (PS)-specific binding of fluorescein isothiocyanate (FITC)-annexin V to normal and band 3 null RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased FITC-annexin V binding was detected in band 3 null RBCs as compared with normal RBCs. Furthermore, the entire cell population of band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, suggesting that band 3 null RBCs may have increased PS exposure on the outer membrane leaflet. These findings are further supported by direct fluorescence microscopy of normal and band 3 null RBCs labeled with FITC-annexin V. Based on these observations, we postulate that the high mortality of band 3 null mice may be related to a hypercoagulable state, which appears to originate from changes in the phospholipid composition of the membrane leading to PS exposure on the outer leaflet. © 1998 by The American Society of Hematology.

Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl−/HCO3− exchanger AE1

“…Annexin binding, reflecting breakdown of PS asymmetry with exposure of PS at the cell surface, was indeed significantly larger in erythrocytes from AE1 Ϫ/Ϫ mice than in erythrocytes from AE1 ϩ/ϩ mice (Fig. 1), a finding in accordance with earlier studies (22,23). These data suggest a less stable PS asymmetry of the AE1 Ϫ/Ϫ erythrocyte cell membrane.…”

“…The enhanced PS exposure of AE1-deficient erythrocytes is reminiscent of the enhanced PS exposure of thrombocytes leading to a hypercoagulable state (22,23). PS-exposing erythrocytes may contribute to vasoocclusion by binding to receptors in the vascular wall, thereby leading to obstruction of the microcirculation (3,12).…”

Acridine orange induces translocation of phosphatidylserine to red blood cell surface

“…Because PS is well known to facilitate RBC adhesion and recognition by other cells, the AO-induced RBC intracellular interaction is not necessarily due to band-3 clustering. It is possible that band-3 clustering and PS translocation are interdependent, as previously suggested (5), but this interrelation has yet to be explored.…”

Hereditary haemolytic anaemias: unexpected sequelae of mutations in the genes for erythroid membrane skeletal proteins

“…In the alpha-spectrin-deficient mice, infarctions occur between 6 and 10 weeks of age [120]. A band 3 knock-out model [121] and the first murine model for HE [120] develop thrombi soon after birth. Amazingly, many of the mice survive these insults.…”