ALBERT

Description: Background Recombinant FVIIa (NovoSeven®) is used for the treatment of spontaneous bleeds in haemophilia patients with inhibitory antibodies against FVIII or FIX. Two theories have been proposed to describe the mechanism of action of FVIIa (van't Veer C et. al., (2000) Blood 95:1330-5, Monroe DM et. al., (1998) Blood Coagulation Fibrinolysis 9(S1):S15-S20); a) competition with zymogen FVII to bind to surface exposed TF at site of injury, and b) activation of FX by FVIIa bound to phospholipids exposed on activated platelets. This study aims at generation and characterization of a novel reagent that will provide new insights into the extent of TF influence in vascular injury. Aim To engineer and characterize FVIIa variant with abrogated TF binding ability and to address influence of TF on FVIIa activity in bleeding models (in whole blood clotting). Methods Based on the x-ray crystal structure (PDB: 1DAN), the binding interface between soluble TF (sTF) and FVIIa on the FVIIa light chain was targeted for engineering a new N-glycan. FVIIa variant I69N/F71T was expressed in BHK cells. Majority of the FVIIa variant was additionally glycosylated as assessed by PNGase treatment and SDS-PAGE analysis. Low-levels of non-glycosylated FVIIa variant were removed by passing the FVIIa variant through a sTF column. The resulting FVIIa preparation was homogenous as assessed by HPLC analysis and was characterized as reported earlier (Persson E et al., (2009) FEBS J. 276:3099-109). Results We have successfully introduced a new N-glycan in FVIIa by engineering an N-glycosylation site Asn-X-Ser/Thr in the FVIIa light chain that provides steric hindrance to sTF. WT-FVIIa binds to sTF with Kd of 6 nM, I69N/F71T-FVIIa displays abrogated sTF binding ability up to 10 µM sTF. At higher concentrations, sTF may interact with FVIIa protease domain directly. Proteolytic and amidolytic activity of WT and FVIIa variant are comparable in the absence of sTF, suggesting that the additional N-glycan does not perturb the protease domain. Data from FX activation and ATIII inhibition assays show virtually no effect of sTF on the functional properties of I69N/F71T-FVIIa in presence or absence of phospholipids. Thrombelastography (TEG) assay initiated by kaolin in haemophilia like human whole blood, in the presence of a TF specific inhibitory antibody, revealed no significant differences in clotting time (R-time) and maximum thrombus generation (MTG) for WT and I69N/F71T FVIIa variants. Similar data when TEG assay was initiated by re-lipidated TF (Innovin®) showed marked differences between WT and I69N/F71T FVIIa variants suggesting loss in the TF binding ability of the FVIIa variant. Conclusions Present work demonstrates that, using exclusive mutagenesis strategy, it is possible to engineer FVIIa variant with minimal changes in the primary sequence that displays abrogated TF binding ability but intact amidolytic and proteolytic activity. Functional properties of this variant were characterized by in vitro biochemical and TEG assays. Such a variant should prove useful to delineate involvement of TF in FVIIa activity in bleeding models. Disclosures Gandhi: Novo Nordisk A/S: Employment. Grøn:Novo Nordisk A/S: Employment. Petersen:Novo Nordisk A/S: Employment. Reedtz-Runge:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Østergaard:Novo Nordisk A/S: Employment.

Description: Mammalian platelets are small, anuclear circulating cells that form tightly adherent, shear-resistant thrombi to prevent blood loss after vessel injury. Platelet thrombi that form in coronary and carotid arteries also underlie common vascular diseases such as myocardial infarction and stroke and are the target of drugs used to treat these diseases. Birds have high-pressure cardiovascular systems like mammals but generate nucleated thrombocytes rather than platelets. Here, we show that avian thrombocytes respond to many of the same activating stimuli as mammalian platelets but are unable to form shear-resistant aggregates ex vivo. Avian thrombocytes are larger than mammalian platelets, spread less efficiently on collagen, and express much lower levels of the α2bβ3 integrin required for aggregate formation, features predicted to make thrombocyte aggregates less resistant than platelets are to the high fluid shear forces of the arterial vasculature. In vivo carotid vessel injury stimulates the formation of occlusive platelet thrombi in mice but not in the size- and flow-matched carotid artery of the Australian budgerigar. These studies indicate that unique physical and molecular features of mammalian platelets enable them to form shear-resistant arterial thrombi, an essential element in the pathogenesis of human cardiovascular diseases.