Putative Phospholipid “scramblase” of Scott Syndrome, TMEM16F/Ano6, Mediates Phosphatidylserine Exposure On Filopodia and Cell Margins of Viable Endothelial Cells.

The Scott Syndrome, a rare inherited bleeding disorder, is characterized by defective exposure of phosphatidylserine (PS) on stimulated platelets, resulting in decreased support for the prothrombinase and factor Xase complexes. Prior reports indicate delay of apoptosis-related PS exposure and decreased release of PS-rich membrane microparticles from platelets, red cells, and lymphocytes from Mrs. Scott's blood. Recent reports indicate that TMEM16F/Ano6, an 8-transmembrane domain protein homologous with Cl⁻ channels is defective in Mrs. Scott's immortalized lymphocytes and defective in another proband with Scott Syndrome. Furthermore, a gain of function mutation in this protein imparts constitutive PS exposure to immortalized lymphocytic cells. However, prior studies do not indicate the extent to which TMEM16F participates in the regulated, reversible pathway of PS exposure vs. the pre-apoptotic PS exposure or whether endothelial cells are affected. We have recently observed that TNFα-treated endothelial cells support assembly of the prothrombinase complex on filopodia and membrane margins. The prothrombinase-supporting membrane sites are characterized by PS exposure and convex curvature. We asked whether TMEM16F contributes to the PS exposure that supports prothrombinase complex assembly on filopodia and membrane margins of stimulated endothelial cells.

We utilized two polyclonal antibodies against TMEM16F peptides to localize protein expression in both resting and TNFα-treated Human Umbilical Vein Endothelial Cells (HUVECs) by confocal microscopy. TMEM16F localization was compared to PS exposure, detected by binding of fluorescein-labeled lactadherin; also prothrombinase assembly, detected by binding of fluorescein-maleimide labeled factor Va and EGRck-biotin-Alexa 647-steptavidin labeled factor Xa. TMEM16F shRNA, delivered by lentivirus vectors, was utilized to knockdown TMEM16F expression on HUVECs while control HUVECs were treated with an empty vector or with GFP-containing lentivirus vector. Endothelial cell-supported prothrombinase activity was measured over HUVECs grown in microtiter wells.

HUVECs grew to confluent monolayers on gelatin-coated cover slips. Anti-TMEM16F antibody staining of fixed, permeabilized cells showed primary localization to the nucleus and perinuclear cytoplasm. Both TMEM16F antibodies also stained small regions of the cell margins and filopodia when HUVECs were treated with TNFα, 10 ng/ml, for 24 hours. Dim TMEM16F staining co-localized with lactadherin staining, in these cells, demonstrating proximity of the protein to PS exposure. Quantitative analyses showed 57 ± 4% reduction of TEMEM16F, as judged by flow cytometry. Quantitation of TMEM16F staining of adherent cells indicated a reduction of 55 ± 5%. HUVECs bound factor Va, like lactadherin, selectively on filopodia and fibrils near the retracted edges of endothelial cells. Factor Xa co-localized the sites of factor Va, indicating formation of prothrombinese complex assembly. However, The assembly of the prothrombinase complex on TNFα-treated HUVECs treated with TMEM16F shRNA, was decreased by 60 ± 5% as judged by quantitative confocal microscopy. Prothrombinase activity was reduced by 57 ± 3% on TNFα-treated HUVECs compared to control HUVECs. PS exposure was also delayed in pre-apoptotic HUVECS treated with 10 μM A23187.

A fraction of TMEM16F is localized near, and linked to regulated PS exposure on stressed endothelial cells. Reduction of TMEM16F decreases PS exposure and focal procoagulant activity supported by these viable cells. Decreased TMEM16F also decreases the rate of pre-apoptotic PS exposure.

Shi:Brigham and Women's Hospital: Use of Lactadherin to detect phosphataidylserine, Use of Lactadherin to detect phosphataidylserine Patents & Royalties. Gilbert:Brigham and Women's Hospital: Use of Lactadherin to detect phosphataidylserine, Use of Lactadherin to detect phosphataidylserine Patents & Royalties.