Traffic of rFVIIa through Endothelial Cells and Redistribution into Subendothelium: Implications for a Prolonged Hemostatic Effect

Original Article

Traffic of rFVIIa through Endothelial Cells and Redistribution into Subendothelium: Implications for a Prolonged Hemostatic Effect

I Lopez‐Vilchez ¹, J Tusell ², U Hedner ³, C Altisent ⁴, G Escolar 1 and AM Galan ¹ Affiliations: 1 Serv. Hemoterapia‐Hemostasia, Hospital Clinic, CDB, IDIBAPS, UB and ² Novo Nordisk Pharma SA and ³ Research and Development, Novo Nordisk A/S, Måløv, Denmark, and University of Lund and ⁴ Unitat d’Hemofilia, Hospital Universitari Vall d’Hebron


ABSTRACT

Background
Clinical evidence suggests that the hemostatic action of recombinant activated factor VII (rFVIIa) exceeds its predicted plasma life. Mechanisms involved in the long‐lasting effects of rFVIIa for prophylactic treatment of patients with hemophilia and inhibitors have not been fully elucidated.

Objectives
The traffic of rFVIIa through the endothelial cells (EC) and its redistribution into the subendothelial compartment was investigated. Its possible hemostatic action in experiments with flowing blood was also assessed.

Methods
Cultured EC and umbilical veins were exposed to rFVIIa (6 μg/mL) for up to 2 h. Immunocytochemical techniques were combined with confocal microscopy to localize rFVIIa into EC and to trace its possible redistribution into subendothelial compartments. Vessels exposed to rFVIIa were de‐endothelized and exposed to flowing blood to determine possible modifications in their hemostatic capacity.

Results
Immunocytochemical studies revealed a significantly enhanced presence of FVIIa dispersedly distributed in the cytoplasm of EC previously exposed to rFVIIa. rFVIIa relocated into nuclear and peripheral areas when prolonged incubations (24 h) were performed. Immunocytochemical studies revealed that rFVIIa localizes into the endothelium and subendothelium of incubated umbilical vessels. In perfusion studies, this rFVIIa redistributed into the subendothelium improved fibrin generation and enhanced platelet thrombus formation.

Conclusions
These results indicate that rFVIIa can be internalized and redistributed into endothelial and subendothelial compartments. This rFVIIa remains functional and promotes hemostatic activity when vessels are denuded. These findings may explain the prolonged prophylactic action of rFVIIa in some clinical conditions.

Keywords: Pharmacodynamics, endothelial cells, hemostatic action, recombinant activated factor VII traffic, prophylaxis
Correspondence: Irene López‐Vilchez, Servicio de Hemoterapia‐Hemostasia, Hospital Clinic, C/Villarroel 170, 08036 Barcelona, Spain