División de Ciencias de la Salud Facultad de Medicina EL COMPLEJO FACTOR VIIa-FACTOR TISULAR Y SU PAPEL COMPENSATORIO EN LAS DISFUNCIONES HEMOSTÁTICAS

Background: Although the bleeding tendency in hemophilia A can be explained by a defect in coagulation, a possible platelet dysfunction has not been formally investigated. Methods: We studied collagen and TF-induced hemostasis using the blood of patients with severe hemophilia A in an in vitro set-up. Blood samples from healthy donors (n=11) or from patients (n=9) were anticoagulated with low molecular weight heparin. Perfusion studies at moderate shear rates (600s), were performed using damaged vascular segments; whereas PFA-100® with specially devised cartridges of collagen alone (COL) or collagen-tissue factor (COL-TF) were used for high shear rates (5000s ). We also tested the ability of rFVIIa to improve hemostasis. Results: Addition of rFVIIa (equivalent to 380 μg/Kg) to blood samples from healthy individuals induced an increase of fibrin formation (53.8±12.1% vs. 37.7±8.2%). In experiments performed with blood from hemophiliacs, fibrin deposition was almost absent (< 5%), but increased to 48.1±9.2% with rFVIIa. Experiments at high shear rate showed a prolonged closure times in samples from hemophilic patients using COL cartridges (255±22s vs.191±16s in healthy donors; p<0.05). Presence of TF in the apertures caused a 20% shortening in closure times in both cases. Addition of rFVIIa to hemophilic blood samples induced a further statistically significant reduction of closure times in COL-TF cartridges (p<0.05). This reduction was not observed in healthy individuals. Conclusions: Under our experimental settings, hemophilic patients showed a platelet dysfunction at high shear rate. rFVIIa, in the presence of TF, contributed to a partial correction of this effect. Studies with healthy donors, indirectly suggest no additional prothrombotic profile for rFVIIa under very high shear rate conditions. Potentially, PFA-100 could be used as a system for monitoring FVIIa in hemophilic patients. INTRODUCTION Normal hemostasis is the result of an adequate balance of platelet function interplaying with activators and inhibitors of the coagulation system. Predominance of activating factors can lead to a prothrombotic state, whereas lack or impairment of the coagulation factors can result in severe bleeding. After vessel injury, highly reactive proteins, such as collagen and Tissue Factor, are exposed to flowing blood. The TF/FVIIa complex activates the coagulation protease cascade, which leads to fibrin deposition and platelet activation though thrombin generation [1;2]. Absences of factor VIII or factor IX (hemophilia A and B respectively) are the coagulopathies that course with the most severe bleeding episodes. Interestingly, the hemorrhagic symptoms seem to be related to alteration in coagulation mechanisms since platelet functions are thought to be normal. Recombinant factor VIIa (rFVIIa) was developed for the management and prevention of bleeding episodes in patients with FVII deficiency [3], as well as in patients with hemophilia A and B who have developed inhibitors [4;5]. Additionally, in vivo and in vitro studies have evidenced that rFVIIa improves hemostasis in several coagulopathies and thrombopathies favoring the development of an hemostatic response at sites where TF is exposed [6-13]. Several studies have investigated the mechanism of action of rFVIIa in patients with hemophilia in detail [14;15]. Recent studies suggest that, in these patients, hemostatic action of rFVIIa is the result of an increase of coagulation mechanisms and a decrease of fibrinolysis due to the activation of an inhibitor of fibrinolysis such as ThrombinActivatable Fibrinolysis Inhibitor (TAFI) [16]. Previous in vitro studies from our group and others consistently found that rFVIIa can exert procoagulant effects in blood from patients with severe thrombocytopenia, major glycoprotein deficiencies [11;17;18], patients with impaired coagulation system as hemophiliacs and patients treated with oral anticoagulation [19] and cirrhotic patients [10] through an enhancement of thrombin