In vitro characterization of MOD-5014, a novel long-acting carboxy-terminal peptide (CTP)-modified activated FVII.

INTRODUCTION: Recombinant FVIIa (rFVIIa) is an effective treatment for haemophilia through frequent administration. However, the short half-life of rFVIIa decreases its prophylactic ability to reduce bleeding. Carboxy-terminal peptide (CTP)-modified FVIIa (MOD-5014) is a long-acting rFVIIa developed for on-demand treatment of haemophilia using either an intravenous or subcutaneous injection with the aim of less frequent administrations, as well as for prophylactic use. AIM: The comprehensive evaluation of the activity MOD-5014 vs commercially available rhFVIIa, as well as their interaction with cofactors and inhibitors. METHODS: The in vitro characterization included clotting activity, affinity by surface plasmon resonance, cleavage of synthetic substrates, thrombin generation (TG) and rotation thromboelastometry. RESULTS: Reduced specific activity was obtained for MOD-5014 compared to rhFVIIa, while both compounds demonstrated comparable affinity to tissue factor (TF). MOD-5014 showed reduced TG when spiked at a similar concentration as rhFVIIa, suggesting that an increased concentration might be needed in a clinical setting to provide initial haemostatic effect. MOD-5014 demonstrated a slightly lower affinity for binding to activated platelets and slightly lower proteolytic activity on the platelet surface, possibly as the fusion of CTP has the potential to sterically hinder binding to both the platelet membrane and to protein substrates. Both compounds showed a similar dose-dependent stimulatory effect on clot formation, and both showed a similar deactivation pattern following incubation with TF pathway inhibitor (TFPI), antithrombin and heparin. CONCLUSION: The comparable in vitro activity of MOD-5014 and rhFVIIa paves the way for in vivo pharmacology evaluations of MOD-5014 in preparation for clinical studies.

The involvement of low-density lipoprotein in hemin transport potentiates peroxidative damage.

Hemin binds to isolated low-density lipoprotein (LDL) and thereby triggers LDL oxidation. In this study we investigated whether hemin can get together with LDL under physiological conditions. The relative affinity of three blood components to free hemin was as follows: RBCM < LDL < albumin. At physiological molar ratio of LDL/albumin all the hemin was bound to albumin. In molar excess of albumin over hemin, existing even under pathological conditions, albumin served as an efficient antioxidant for the plasma hemin-induced LDL oxidation. RBCM-embedded hemin, unlike plasma hemin, affected LDL: the mobile hemin was transferred from RBCM to LDL in the absence of albumin, whereas in the presence of albumin most of the mobile hemin finally reached the albumin but partially via LDL. Thus, a transient hemin is built up in LDL. This transient hemin triggered LDL oxidation which was not inhibited but rather promoted by albumin. The involvement of albumin in this oxidation was explained by its acting as a pump thereby increasing the transient hemin in LDL. It is suggested that increased membrane hemin level as in hemoglobinopathies and/or excess LDL in dyslipidemia provide conditions for hemin-induced LDL oxidation.

Hemoglobin induced apolipoprotein B crosslinking in low-density lipoprotein peroxidation.

Oxidative modification of human low-density lipoprotein (LDL) is thought to play a major role in the development of atherosclerosis. Free hemin, hemoglobin, myoglobin, and horseradish peroxidase (HRP) were reported in different studies as promoters of LDL lipid oxidation. Based on our previous finding that hemin induced oxidative crosslinking of the LDL protein, apolipoprotein B (apo B) (Y. I. Miller and N. Shaklai (1994) Biochem. Mol. Biol. Int. 34, 1121-1129), we compared the ability of free hemin and the above hemoproteins to induce peroxidation modification of apo B using SDS-PAGE. The levels of the final products of lipid peroxidation were determined as thiobarbituric acid-reactive substances. Hemoglobin and myoglobin were found to be as active as free hemin and all these were much more active than the classic peroxidase HRP. Moreover, the products of oxidized apo B differed: hemoglobin, myoglobin, and hemin induced mostly covalent aggregates, while HRP caused fragmentation of apo B. Hemoglobin reactivity was expressed at low H2O2 concentrations even in the absence of molecular oxygen. Desferal, along with other antioxidants, inhibited the hemoglobin-induced LDL oxidation independently of its iron-chelating property. The high peroxidative reactivity of hemoglobin is explained by its ability (unlike HRP) to transfer the oxidative equivalents from the heme active site, through the globin, to LDL. The apo B radicals thus formed are terminated, yielding intermolecular crosslinked protein. It is suggested that small amounts of the highly reactive hemoglobin in plasma, suffice to trigger LDL protein oxidation (along with its lipid oxidation), thereby inflict the atherosclerosis precondition.