title: Altered bioavailability of platelet-derived factor VIII during thrombocytosis reverses phenotypic efficacy in haemophilic mice.
abstract: Ectopic delivery of factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by concentrating the FVIIIa/FIXa enzyme-cofactor complex onto activated platelet membranes. We utilized a core rat platelet factor 4 (PF4) promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of a haemophilia A mouse (rPF4/hBDD/FVIII-/-). Platelets from rPF4/hBDD/FVIII(-/-) mice contained approximately 122 mU FVIII:C/1 x 10(9) platelets/ml with no detectable plasmatic FVIII:C, and with no effect on alpha-granule-derived platelet factor V/Va function. Paired tenase assays (+/- thrombin) confirmed that platelet (pt) FVIII (unlike platelet FV) required thrombin cleavage for complete activation. rPF4/hBDD/FVIII(-/-) mice exposed to a thrombocytotic stimulus (thrombopoietin, TPO) demonstrated a statistically-significant 66% reduction in molar ptFVIII activity with a non-significant reduction in total ptFVIII biomass. Decreased molar ptFVIII concentration correlated with loss of phenotypic correction as evaluated using a haemostatic tail-snip assay. Comparative studies using a transgenic mouse expressing human amyloid-beta-precursor protein (hAbetaPP) from the rPF4 promoter confirmed diminished hAbetaPP expression without affecting endogenous alpha-granule PF4, establishing generalizability of these observations. While Mk/platelet-released ptFVIII (unlike pFV) is proteolytically inactive, we also conclude that thrombocytotic stimuli negatively affect ptFVIII bioavailability and phenotypic efficacy, results which correlate best with molar ptFVIII concentration, and not systemically available ptFVIII.
authors.affiliation: Department of Medicine, State University of New York, Stony Brook, NY 11794-8151, USA.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Damon
authors.forename: Lesley E
authors.initials: LE
authors.lastname: Scudder
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Gnatenko
authors.forename: Varsha
authors.initials: V
authors.lastname: Sitaraman
authors.forename: Patrick
authors.initials: P
authors.lastname: Hearing
authors.forename: Jolyon
authors.initials: J
authors.lastname: Jesty
authors.forename: Wadie F
authors.initials: WF
authors.lastname: Bahou
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000235:genetics; D000818:Animals; D001777:Blood Coagulation / Q000235:genetics; D001780:Blood Coagulation Tests; D001792:Blood Platelets / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D004195:Disease Models, Animal; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D006467:Hemophilia A / Q000097:blood / Q000235:genetics / Q000628:therapy*; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009363:Neoplasm Proteins / Q000378:metabolism; D010641:Phenotype; D015539:Platelet Activation; D010976:Platelet Count; D010978:Platelet Factor 4 / Q000235:genetics; D051381:Rats; D013917:Thrombin / Q000378:metabolism; D013922:Thrombocytosis / Q000097:blood* / Q000139:chemically induced / Q000235:genetics; D013926:Thrombopoietin; D013997:Time Factors
chemical_list: D016564:Amyloid beta-Protein Precursor; D009363:Neoplasm Proteins; D010978:Platelet Factor 4; D005169:Factor VIII; D013926:Thrombopoietin; D013917:Thrombin; D015951:Factor Xa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant