Generation and biochemical characterization of glycoPEGylated factor VIIa derivatives.
نویسندگان
چکیده
Prophylaxis with 2-4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient, recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface, TF, FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylationtrade mark technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.
منابع مشابه
Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization.
Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF,...
متن کاملPreparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates.
Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protei...
متن کاملCoagulation proteases and human cancer.
Tumours are capable of activating blood coagulation through the expression of procoagulant molecules such as tissue factor, cancer procoagulant and hepsin. Initiation of the clotting cascade results in the generation of the activated serine proteases factor VIIa, factor Xa and thrombin. These proteases act via protease-activated receptors and tissue factor to alter gene expression, thereby modu...
متن کاملInhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa.
Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace ( approximately 10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thromb...
متن کاملMechanism of factor VIIa-dependent coagulation in hemophilia blood.
The ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti-factor IX antibody-induced ("acquired") hemophilia B blood was investigated. In normal blood, both factor VIIa-tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Thrombosis and haemostasis
دوره 100 5 شماره
صفحات -
تاریخ انتشار 2008