Platelet Phosphatidylserine Exposure, Survival and Blood Coagulation in Mice Lacking TMEM16F

Scott Syndrome is a rare, moderately severe bleeding disorder caused by a defect in platelet, red cell, and lymphocyte phosphatidylserine exposure. The syndrome has been linked to mutations in TMEM16F, a Ca⁺⁺-activated ion channel. A moderately severe bleeding disorder in German Shephard dogs, characterized by decreased platelet phosphatidylserine exposure, has also linked to mutations in TMEM16F.

TMEM16F, is a member of a recently-identified family of calcium-activated chloride channels that are also called anoctamins. Members of the family apparently serve both as ion channels and phospholipid scrambling channels. A crystal structure of one member of the TMEM16 family shows a homo-dimeric structure of an integral membrane protein with each unit containing 10 transmembrane helices. A transmembrane hydrophilic cavity lies at the interface, and contains a cation binding site, and a slot with dimensions that could accommodate acyl chains.

Tmem16f^-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding. We have developed an independent gene-targeted (gt) Tmem16f allele generated in C57BL/6 ES cells and find results that both confirm prior reports and contrast with them.

JM8 ES cells were obtained from EUCOMM, and Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f^+/gt intercross progeny identified no surviving Tmem16f^gt/gt mice at weaning (p<0.001). However, ^+/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with ^gt/gt embryos. Blinded pathological evaluation of E17.5 ^gt/gt pups indicated reduction of ossification and angular limb changes, consistent with 2 prior reports. Thus, our results confirm previously reported bone changes, but in contrast with prior reports, indicate that Tmem16f deficiency is lethal in the C57BL/6 genetic background.

An F2 intercross of ^+/gt mice outcrossed one generation to 129x16vJ resulted in ^gt/gt mice surviving to weaning, though at only 30% of the expected Mendelian frequency. Progeny analysis points to a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3. Further efforts to localize the responsible gene(s) are underway.

Tail bleeding times for ^gt/gt were >10min, whereas littermate ^+/gt and ^+/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than ^gt/gt (p<0.05). Notably, platelets from ^+/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas ^gt/gt mice had significantly reduced PS exposure (p < 0.05). ^gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time.

Our data suggest the existence of a viability-determining genetic modifier of the TMEM16F deficiency phenotype in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover a novel regulator of hemostatic function. The observation that heterozygous deficiency leads to a PS exposure and hemostatic phenotype also suggests the possibility that heterozygous TMEM16F mutations may influence hemostasis or thrombosis in humans.