Studies in animal model on the thrombogenicity of a new prothrombin complex concentrate from Argentina.

Abstract

Dear Sir Prothrombin complex concentrate (PCC) is a therapeutic agent human plasma derived that contains vitamin K-dependent coagulation factors (Roberts & Eberst, 1993). These may be manufactured as three factors (II, IXandX)or four factors (II, VII, IXandX), depending on the purification procedure (Chandra & Wickerhauser, 1978; Feldman & Winkelman, 1991). Furthermore, some concentrates have also variable quantities of protein Z and physiologic coagulation inhibitor proteins C and S (Romisch et al., 1998). PCC administration has been used in the treatment of hemophilia B, severe liver failure and consumption coagulopathies as well as in the therapy for the reversal of warfarin anticoagulation in emergency settings (Lankiewicz et al., 2006). However, the therapeutic use of PCC may be accompanied by adverse events, including allergic reactions, transmission of blood-borne viruses and thrombotic episodes (Watson & Ludlam, 1997). The thrombogenic components of these concentrates have been mainly attributed to activated coagulation factors and also to the presence of coagulant-active phospholipids, zymogen overload and the lack of inhibitors antithrombin and/or protein S (Kohler, 1999). To avoid the thrombotic complications of PCC, the International Society on Thrombosis and Haemostasis (ISTH) has recommended in the past the use of heparin in the formulation of the final product (Menache, 1976). However, strong evidences support that the quality of PCC plays a crucial role in the occurrence of thromboembolic events, whichmainly dependon the purification methods used during its manufacture (Kohler, 1999). As a consequence of these observations, we developed a PCC that meets the criteria of presumed low thrombogenicity because the content of vitamin K-dependent clotting factors and inhibitors is well balanced and the thrombin and activated factors activities were negligible by in vitro assays. This study was designed to evaluate a full-dose-related response for our PCC, using the in vivo stasis model of thrombogenicity (Wessler test), adapted to rats. Different lots (n 1⁄4 3) of PCC were studied and manufactured from human plasma at the Laboratorio de Hemoderivados, Universidad Nacional de Córdoba production facility (Córdoba, Argentina). The factors II, IX andXwere isolated from cryosupernatant using ion-exchange batch chromatography, and the coagulation factors were eluted using buffer of increased ionic strength. A first step of viral inactivation was performed with solvent–detergent and then a second chromatographic step was carried out. The proteins of interest were eluted, and the protein concentration was adjusted by ultrafiltration procedure and formulated with or without heparin. Finally, the PCC was aseptically distributed in a vial and lyophilized and a second step of viral inactivation using dryheat (100 C for 30 min) was applied (Bernardi et al., 2003). PCCs were standardized according to factor IX (F IX) activity, following the European Pharmacopoeia procedures using International Standard preparations provided by the National Institute for Biological Standard and Control (Lamb et al., 1991). The method of Wessler et al. (1959) was used to test the thrombogenicity of the concentrates. Briefly, female Wistar rats (230 12 g) were anaesthetized with chloral hydrate (400 mg kg i.p.) and a 1-cm segment of the jugular vein was exposed through a midline central incision; the PCC (0 5 mL i.v. for 10 s in all the cases) or sample control was given by the cannulated femoral vein. The venous segment was tied off exactly 15 s after the beginning of infusion and the development of thrombi in the isolated segment of jugular vein was determined 10 minutes later by visual inspection using scores as reported (Wessler et al., 1959). All procedures were performed according to the guidelines established by The Animal Models Subcommittee, Scientific and Standardization Committee of the ISTH (Levi et al., 2001). PCCs (496 42 IU F IX per vial) were reconstituted according to obtain doses of 25, 50, 100 and 200 IU F IX kg in the presence or absence of heparin were used, and some vials of PCC (50 and Correspondence: Maria Eugenia Bernardi, Area de Desarrollo de Productos y Procesos, UNC-HEMODERIVADOS, Av. Valparaiso s/n, Ciudad Universitaria X5000HRA, Córdoba, Argentina. e-mail: mebernardi@hemo.unc.edu.ar