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 g/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 ␣ 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.

Human platelets express endothelial protein C receptor, which can be utilized to enhance localization of factor VIIa activity

“…It is therefore likely that platelet-FVIIa binding is dependent on simultaneous or coordinate interactions with multiple binding partners, which may include phospholipids, GPIba, ApoER2 or other membrane proteins. This is supported by studies showing only partial inhibition of FVIIa internalization by platelets in the presence of APC, annexin V, or antibodies to GPIba or GPVI [64]. Although the affinity of FVIIa for each of these partners may be relatively weak, simultaneous were fixed in 4% formaldehyde and spun down onto poly-L-lysine-coated glass coverslips.…”

“…A potential interaction between FVIIa and GPIba is further supported by studies showing that platelet uptake and internalization of FVIIa is decreased by~77% in platelets isolated from patients with Bernard-Soulier syndrome as compared to healthy controls. However, the control platelets showed only a 15-20% decrease in FVIIa uptake in the presence of an antibody against GPIba [64]. The potential contribution of such an interaction therefore remains unclear, particularly because thrombin, which also binds GPIba [65,66], is unable to compete with FVIIa or FVIIa-DVQ for platelet binding [17].…”

Correction of Murine Bernard–Soulier Syndrome by Lentivirus-mediated Gene Therapy

“…[7][8][9] However, there remain major concerns with recombinant factor VII treatment regarding adverse reactions, cost, and short half-life. 10 To overcome these issues, a gene therapy approach that expresses FVIIa using adeno-associated virus 8 has been tested with BSS mice and improved hemostasis was observed. 11 This strategy may also be applied to other bleeding disorders such as Glanzmann's thrombasthenia complicated by alloimmunization.…”

Alloimmunization in Congenital Deficiencies of Platelet Surface Glycoproteins: Focus on Glanzmann's Thrombasthenia and Bernard–Soulier's Syndrome

“…52,53 Enhanced thrombin generation from highconcentration rFVIIa bound to the activated platelets also improves fibrin clot structure in GT-platelet-rich plasma, 54 decreasing clot permeability and thus improving hemostasis. 55,56 rFVIIa, when added to platelet-rich plasma from normal, BSS, and FVII deficiency patients, has also been shown to be internalized to the platelet cytoplasm and redistributed to the open canalicular system and α-granules, 57 thereby improving platelet aggregation and fibrin generation in perfusion studies. Infusion of rFVIIa in hemophilia inhibitor patients has also been shown to result in a transient increase in procoagulant platelet microparticles that promote hemostasis.…”