title: A TMEM16F point mutation causes an absence of canine platelet TMEM16F and ineffective activation and death-induced phospholipid scrambling.
abstract: BACKGROUND
  TMEM16F is an ion channel and calcium-dependent lipid scramblase that mediates phosphatidylserine (PS) exposure on the plasma membrane. Two disparate disease phenotypes are associated with TMEM16F loss-of-function mutations: a rare bleeding disorder (Scott syndrome) and skeletal malformations due to aberrant bone mineralization in a TMEM16F knockout mouse. We therefore undertook comparative studies of TMEM16F expression in canine Scott syndrome (CSS), an autosomal recessive platelet defect.
  
  
  OBJECTIVE
  To define anoctamin proteins and scramblase response of CSS platelets and to determine whether TMEM16F is the CSS disease gene.
  
  
  METHODS
  CSS TMEM16F cDNA and gene were sequenced and mutation detection was performed in CSS pedigrees. Platelet fractions from CSS dogs were isolated for proteomic and immunologic characterization of TMEM16F. Annexin V was used as a flow cytometric marker of induced platelet PS externalization.
  
  
  RESULTS
  A TMEM16F splice site mutation segregated with the CSS trait and TMEM16F protein was undetectable in CSS platelet membranes; however, a second anoctamin, TMEM16K, was found. Proteomic analyses revealed a network of 32 proteins that differentially cosegregated with platelet plasma membrane TMEM16F. CSS platelets had profoundly impaired scramblase response to pharmacologic and physiologic agents that increase intraplatelet calcium and conditions that induce apoptotic and necrotic cell death.
  
  
  CONCLUSIONS
  CSS platelets represent a TMEM16F-null mutant model that demonstrates a central role for TMEM16F in mediating platelet PS externalization in response to activating and death signals. Platelet TMEM16F may prove a novel drug target for modulating platelet procoagulant activity and extending platelet life span.
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
authors.forename: M B
authors.initials: MB
authors.lastname: Brooks
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Catalfamo
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
authors.forename: R
authors.initials: R
authors.lastname: MacNguyen
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
authors.forename: D
authors.initials: D
authors.lastname: Tim
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
authors.forename: S
authors.initials: S
authors.lastname: Fancher
authors.affiliation: Institute of Biotechnology, Cornell University, Ithaca, NY, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: McCardle
mesh_terms: D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001778:Blood Coagulation Disorders / Q000097:blood / Q000235:genetics / Q000473:pathology / Q000662:veterinary*; D001792:Blood Platelets / Q000378:metabolism* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000662:veterinary; D004283:Dog Diseases / Q000097:blood / Q000235:genetics* / Q000473:pathology; D004285:Dogs; D005434:Flow Cytometry / Q000662:veterinary; D020022:Genetic Predisposition to Disease; D008969:Molecular Sequence Data; D010375:Pedigree; D010641:Phenotype; D010718:Phosphatidylserines / Q000097:blood*; D049294:Phospholipid Transfer Proteins / Q000097:blood / Q000172:deficiency / Q000235:genetics*; D017354:Point Mutation; D040901:Proteomics
chemical_list: D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins
keywords: animal models; blood coagulation disorders, inherited; blood platelet disorders; phospholipid scramblase; platelet activation