TARGETED INACTIVATION OF MURINE BAND-3 (AE1) GENE PRODUCES A HYPERCOAGULABLE STATE CAUSING WIDESPREAD THROMBOSIS IN-VIVO

Only 5% to 10% of band 3 null mice survive the neonatal period. To det ermine the cause of death, 3 adult and 11 newborn band 3 null mice wer e submitted for histopathologic examination. All but 1 pup showed evid ence of thrombosis including: (1) large thrombotic lesions in the hear t, which were partially organized, calcified in some fields, and endot helialized, indicating a process that developed premortem (3 of 3 adul ts and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver sugg estive of premortem ischemic events caused by arteriolar occlusions (8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate th e etiology of this hypercoagulable state, we have used the Russell's v iper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RW clotting time of normal plasma in a dose-dependent fashion, whereas RBCs from normal mice have no eff ect, suggesting that the membrane of band 3 null RBCs provides a suita ble surface for activation of the prothrombinase complex. Using flow c ytometry, we have examined the phosphatidylserine (PS)-specific bindin g of fluorescein isothiocyanate (FITC)annexin V to normal and band 3 n ull RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased F ITC-annexin V binding was detected in band 3 null RBCs as compared wit h normal RBCs. Furthermore, the entire cell population of band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, s uggesting 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 F ITC-annexin V. Based on these observations, we postulate that the high mortality of band 3 null mice may be related to a hypercoagulable sta te, which appears to originate from changes in the phospholipid compos ition of the membrane leading to PS exposure on the outer leaflet. (C) 1998 by The American Society of Hematology.