title: Redistribution and hemostatic action of recombinant activated factor VII associated with platelets.
abstract: Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia.
authors.affiliation: Hemotherapy and Hemostasis Service, Hospital Clinic, Biomedical Diagnostics Center CDB, Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Lopez-Vilchez
authors.forename: Ulla
authors.initials: U
authors.lastname: Hedner
authors.forename: Carmen
authors.initials: C
authors.lastname: Altisent
authors.forename: Maribel
authors.initials: M
authors.lastname: Diaz-Ricart
authors.forename: Gines
authors.initials: G
authors.lastname: Escolar
authors.forename: Ana M
authors.initials: AM
authors.lastname: Galan
mesh_terms: D001606:Bernard-Soulier Syndrome / Q000473:pathology / Q000503:physiopathology; D001792:Blood Platelets / Q000187:drug effects* / Q000648:ultrastructure; D016022:Case-Control Studies; D002457:Cell Extracts; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D015942:Factor VIIa / Q000378:metabolism* / Q000494:pharmacology; D005434:Flow Cytometry; D006487:Hemostasis / Q000187:drug effects*; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013916:Thrombelastography; D013917:Thrombin / Q000494:pharmacology; D014019:Tissue Donors
chemical_list: D002457:Cell Extracts; D006490:Hemostatics; D011994:Recombinant Proteins; C103587:recombinant FVIIa; D015942:Factor VIIa; D013917:Thrombin