Redistribution and Hemostatic Action of Recombinant Activated Factor VII Associated with Platelets

• Published in: The American journal of pathology Vol. 178; no. 6; pp. 2938 - 2948
• Main Authors: Lopez-Vilchez, Irene, Hedner, Ulla, Altisent, Carmen, Diaz-Ricart, Maribel, Escolar, Gines, Galan, Ana M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 01.06.2011; American Society for Investigative Pathology
• Subjects: Basic Medicine; Bernard-Soulier Syndrome - pathology; Bernard-Soulier Syndrome - physiopathology; Biological and medical sciences; Blood Platelets - drug effects; Blood Platelets - ultrastructure; Case-Control Studies; Cell and Molecular Biology; Cell Extracts; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell- och molekylärbiologi; Endocytosis - drug effects; Factor VIIa - metabolism; Factor VIIa - pharmacology; Flow Cytometry; Hemostasis - drug effects; Hemostatics - pharmacology; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical and Health Sciences; Medical sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Pathology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Platelet Activation - drug effects; Protein Transport - drug effects; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Regular; Thrombelastography; Thrombin - pharmacology; Tissue Donors; Peptidases; Anatomic pathology; Platelet; Serine endopeptidases; Hemostatic; Enzyme; Hemostasis; Cause; Hydrolases; Recombinant protein; Coagulation Factor VIIa
• ISSN: 0002-9440; 1525-2191; 1525-2191
• DOI: 10.1016/j.ajpath.2011.02.026

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.