Recombinant Activated FVII (rFVIIa) Corrects Platelet Dysfunction in Hemophilic Patients: Study on Collagen and Tissue Factor Surfaces at High Shear Rates.

Hemophilic patients suffer bleeding episodes despite having a normal bleeding time. A possible platelet dysfunction in these patients has not been deeply investigated. rFVIIa improves hemostasis of hemophilic patients, even in those who develop inhibitors. Clinical efficacy of this drug has been widely confirmed, though, its mechanism of action is not fully understood. We used the PFA-100^® with specially devised cartridges whose membrane apertures were coated with collagen alone (COL) or collagen-tissue factor (COL-TF). Blood samples from normal donors or from a group of patients with severe hemophilia A, were anticoagulated with low molecular weight heparin (LMWH). We tested the ability of rFVIIa to shorten the closure times under the previous conditions. The structure of the hemostatic plugs formed on the membrane apertures were further analyzed using light microscopy on thin cross-sections.

Closure times were statistically prolonged in blood samples from hemophilic patients tested with COL cartridges (255±22 s.vs.187±15 s in normal donors; p<0.05). Presence of TF in the apertures (COL-TF) caused a 20% shortening in closure times, both in normal donors and in hemophilic patients. Exogenous addition of 10 μg/ml rFVIIa to blood samples from hemophilic patients induced a further statistically significant reduction of closure times (p<0.05). This further reduction in closure times was not observed in blood samples drawn from normal individuals. Microscopical analysis of the plugs formed on the apertures showed that occlusive thrombi formed in the presence of TF are more compact and have higher occlusive capacity. Addition of FVIIa led to the formation of more organized platelet plugs which appeared further consolidated with fibrin strands within platelet masses.

Patients with severe hemophilia showed platelet dysfunction that could be detected with the PFA-100^® using specific cartridges. It is likely that the platelet dysfunction observed in these patients could be related to concurrent reductions in VWF that could affect platelet adhesion in these patients revealed at the very elevated shear rates used in the PFA-100^®. Under these conditions, TF deposited onto the collagen-coated apertures proved to play a significant role in the initiation of hemostasis. rFVIIa improved the recruitment of platelets on COL-TF and contributed to a partial correction of the platelet dysfunction observed in patients with hemophilia A as further confirmed by the formation of more efficient aggregates in the PFA-100. In essence, rFVIIa circumvented a pre-existent platelet adhesion defect in hemophiliac patients. The pro-hemostatic action of rFVIIa was not observed in parallel studies with blood from healthy donors, indirectly suggesting a good safety profile for this agent when hemostasis is well preserved. PFA-100 could be considered as a possible monitoring system of FVIIa when hemostasis is impaired.