Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia
Open Access
- 19 December 2016
- journal article
- Published by Wiley in Haemophilia
- Vol. 23 (2), 300-308
- https://doi.org/10.1111/hae.13110
Abstract
LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). The dissociation constant (Kd) for activated platelet binding was in the 1 μm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25–50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat/Km values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s−1/0.32 μm and 3.3 s−1/0.44 μm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103 m−1 s−1. The Kd values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kd for binding to soluble EPCR was 41 nm. Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.Keywords
Funding Information
- LFB USA
This publication has 37 references indexed in Scilit:
- N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbitsGlycobiology, 2013
- Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIaPLOS ONE, 2013
- Endothelial cell protein C receptor‐mediated redistribution and tissue‐level accumulation of factor VIIaJournal of Thrombosis and Haemostasis, 2012
- Factor VIIa binding to endothelial cell protein C receptor: Differences between mouse and human systemsThrombosis and Haemostasis, 2012
- Molecular Determinants of Phospholipid Synergy in Blood ClottingPublished by Elsevier BV ,2011
- The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated ReceptorsPublished by Elsevier BV ,2011
- Recombinant human factor VIIa (rFVIIa) cleared principally by antithrombin following intravenous administration in hemophilia patientsJournal of Thrombosis and Haemostasis, 2010
- Endothelial cell protein C receptor cellular localization and trafficking: potential functional implicationsBlood, 2009
- Prophylactic treatment of haemophilia patients with inhibitors: clinical experience with recombinant factor VIIa in European Haemophilia CentresHaemophilia, 2007
- Endothelial Cell Protein C Receptor Acts as a Cellular Receptor for Factor VIIa on EndotheliumPublished by Elsevier BV ,2007