ALBERT

Description: TFPI is an important inhibitor of the extrinsic coagulation pathway. It efficiently inhibits TF-FVIIa and FXa by quaternary complex formation. Plasma contains various truncated forms of TFPI which are poor inhibitors, and full length (fl)TFPI (0.3 – 0.5 nM) which is the most active TFPI in plasma. flTFPI is released from platelets upon activation, and increases flTFPI concentrations locally up to 30-fold. Most intravascular TFPI (∼80%) is associated with endothelial cells. Both endothelial forms, TFPIa and TFPIb, are similarily effective inhibitors of FX activation on the endothelial cell surface. Inhibition of TFPI in hemophilia models with blocking antibodies, aptamers or peptide inhibitors improves hemostasis and may become an option to treat hemophilia. Recently, we presented peptide inhibitors of TFPI that enhance coagulation in hemophilia models. Two optimized peptides, JBT-A7 and JBT-B5, efficiently blocked inhibitory activity of TFPI and bound to distinct binding sites. We demonstrated the crystal structure of JBT-A7, a linear TFPI inhibitory peptide composed of 20 amino acids, bound to NtermK1 (TFPI 1-83). JBT-B5, a cyclic TFPI inhibitory peptide of 23 amino acids, co-crystallized with TFPI KD1-KD2 (TFPI 22-150). Overlaying the KD1 structure in the KD1-KD2/JBT-B5 and the NTermK1/JBT-A7 complex provided atomic details for linking the two peptide entities. Binding of peptides to TFPI and TFPI fragments was studied by BioCore. The TFPI inhibitory potential of the resulting fusion peptide was tested in model systems (FXa inhibition and TF-FVIIa catalyzed FX activation) and global hemostatic assays (TF-triggered thrombin generation) using hemophilia plasma. To model situations of increased TFPI concentration, both model and plasma assays were carried out at TFPI concentrations up to 10 nM, which is 40-50-fold higher than the physiological flTFPI plasma concentration. To characterize the inhibition of platelet TFPI, we used platelets isolated from blood samples and platelet rich plasma from different donors. Binding of a biotinylated fusion peptide on living HUVE cells was assessed by fluorescence activated cell sorting (FACS) and fluorescence microscopy. Inhibition of cell surface TFPI was analyzed on cultivated HUVECs stimulated with TNFa for TF expression. We monitored FXa generation by the TFPI-dependent cell surface FX activation complex by conversion of an FXa-specific fluorogenic substrate. The overlay of the crystal structures of KD1-KD2/JBT-B5 and the NTermKD1/JBT-A7 complexes revealed non-overlapping epitopes and close proximity of the termini of both peptides. The distance could be bridged by an approximately ten amino acid linker. A fusion peptide with a 10-serine-linker was synthesized and showed highly improved dissociation in Biacore experiments and most efficiently inhibited TFPI activity in the model assays. In contrast, single peptides only partially inhibit TFPI especially at high TFPI concentrations. In thrombin generation assays using hemophilia plasma, the fusion peptide showed a substantially higher ability than the single peptides to increase the thrombin peak even at elevated TFPI. The fusion peptide efficiently inhibited TFPI released from platelets and improved thrombin generation in TFPI deficient plasma reconstituted with platelets as the only source of TFPI released upon platelet activation. The fusion peptide was also shown to bind TFPI on the surface of living HUVECs. This is consistent with its binding epitopes on KD1 and KD2 which result in inhibition of cell surface TFPI in a cell based FX activation assay. Thus, we demonstrate that a molecular fusion peptide most efficiently inhibits all physiologic forms of TFPI. X-ray structures of binary and ternary peptide TFPI complexes provided atomic details for linking two single peptides to generate a fusion peptide that most efficiently blocks TFPI in plasma, released from platelets and associated with endothelial cells. It most efficiently neutralizes TFPI even at substantially elevated concentrations occurring at sites of platelet activation. Our observations support the notion that targeting TFPI with TFPI inhibitors is a promising novel strategy to mitigate the bleeding risk in hemophilia patients. Disclosures: Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter:Baxter Innovations GmbH, Vienna, Austria: Research Funding. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Description: Abstract 4419 BAX513, a Laminaria japonica-derived fucoidan and other non-anticoagulant sulfated polysaccharides (NASPs) have been shown to improve clotting in FVIII- and FIX-deficient plasma (Liu et al. Thromb Haemost 2006; 95:68). In this study we assessed the procoagulant activities of fucoidans derived from a variety of brown sea algae species, and correlated the activity with molecular weight (MW) and degree of sulfation. Highly purified fucoidan preparations were studied in FVIII-inhibited whole blood by tissue factor-triggered thromboelastography (TEG). The procoagulant activity was characterized by calibrated automated thrombography (CAT) in FVIII- and FIX-deficient plasma and in combination with established hemophilia therapeutics. A dilute prothrombin time assay based on tissue factor pathway inhibitor supplementation (TFPI-dPT) was used to demonstrate the dose-dependent TFPI-inhibiting effect of BAX513 (EC50 = 0.18 ± 0.03 μ g/mL) in FVIII-deficient plasma. TEG in normal and FVIII-inhibited blood showed a dose-dependent procoagulant effect of most compounds where the optimal concentrations (1-100 nM) were dependent on the MW of the fucoidan. In FVIII-inhibited blood BAX513 at concentrations of ~10 nM (1.2 μ g/mL) completely normalized the TEG parameters. In contrast to sulfated fucoidans, undersulfated fucoidan hardly affected thrombin generation (TG). By CAT, the procoagulant window of NASPs in hemophilic plasma spanned more than two orders of magnitude with maximum effects being equivalent to (mU/mL) 730–940 FVIII, 32–80 FIX and 590–1230 FEIBA. NASPs combined with FVIII, FEIBA or FVIIa had an additive procoagulant effect. The optimal selection of molecular characteristics of NASPs will support the development of alternative hemophilia therapies. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Panholzer:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Description: Abstract 4420 Fucoidans are heterogeneous, polyanionic molecules with procoagulant activities in a wide concentration range. They have been described as non-anticoagulant sulfated polysaccharides (NASP) and shown to improve clotting in FVIII- and FIX-deficient plasma. In vitro characterization has suggested that fucoidans exert their procoagulant activity by inhibiting tissue factor pathway inhibitor (Liu et al. Thromb Haemost 2006; 95:68) and by accelerating thrombin-dependent FVa formation (Mutch et al. J Thromb Haemost 2007; 5 Suppl2). In our study we describe a new, previously unrecognized mechanism by which fucoidans act as procoagulant agents. The procoagulant activity of several fucoidans was characterized by calibrated automated thrombography in tissue factor (TF)-dependent experiments and by using coagulation factor-deficient plasmas. Spiking experiments with purified coagulation factors or inhibitory antibodies verified the mechanism. Stimulation of thrombin generation (TG) by fucoidans requires anionic lipid surfaces like synthetic phospholipid vesicles which contain phosphatidylserine and is TF-dependent (0-20pM). However, stimulatory activity was most pronounced in the absence of TF. Control experiments with corn trypsin inhibitor or FXII-deficient plasma excluded any involvement of the contact system. Plasmas from patients with congenital coagulation factor deficiencies were screened for TG to identify the target coagulation factor by which fucoidans exert their procoagulant activities. In the absence of TF, plasmas deficient in coagulation factors from the common pathway do not support fucoidan-mediated thrombin generation, whereas FVII-deficient plasma does. FXI was identified as the most upstream factor of the intrinsic pathway which is required for fucoidan-stimulated thrombin generation, suggesting it to be the target for the procoagulant activities of fucoidan. Spiking 30nM FXI to FXI-deficient plasma restored fucoidan-mediated TG and addition of polyclonal FXI inhibitory antibodies to normal plasma abrogated TG. Fucoidan-dependent TG did not improve when FXIa (60pM) was added to FXI-deficient plasma, suggesting activation of FXI by fucoidan. The relevance of this mechanism in hemophilia A plasma was studied by addition of low levels of FVIII (0.2-10%) resulting in a FVIII concentration-dependent increase in fucoidan-mediated TG. These results highlight the requirement of a functional intrinsic pathway for this new mechanism of fucoidans. Our findings present FXI activation at low TF concentrations as a possible mechanism for fucoidan. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Panholzer:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Description: Introduction Tissue factor pathway inhibitor (TFPI) is a three-Kunitz domain (KD1-3) protease inhibitor that downregulates the extrinsic coagulation pathway. TFPI has a double inhibitory effect; it inactivates factor Xa (FXa) by 1:1 binding via its KD2, and it prevents further FX activation by binding the tissue factor (TF) – factor VIIa (FVIIa) complex via its KD1 and the formation of a quaternary complex. Recently, we demonstrated the crystal structure of a linear TFPI inhibitory peptide composed of 20 amino acids, bound to a TFPI protein composed of N-terminus and KD1. On the other hand, a cyclic TFPI inhibitory peptide of 23 amino acids was shown to co-crystallize with TFPI KD1-KD2. Molecular fusion of the linear and cyclic peptide by an optimized linker sequence would thus target two independent epitopes and combine the antagonistic properties of the two peptides. Methods The binding properties of simultaneous interaction of the linear and cyclic peptide with TFPI were studied in Biacore experiments using immobilized human TFPI 1-160 on a CM5 chip. Measurements with the linear or cyclic peptide were done with and without prior saturation of TFPI with the linear peptide and the fusion peptide. The results were confirmed by native-PAGE analysis of peptide/KD1-KD2 mixtures, where the TFPI fragment KD1-KD2 had been incubated with either linear or cyclic peptide or both. The TFPI inhibitory effect of the linear, cyclic, and fusion peptide was assessed in several TFPI sensitive assays including inhibition of FXa, FX activation by TF/FVIIa, and thrombin generation. Calibrated automated thrombography (CAT) was performed in human hemophilia plasma triggered with low tissue factor. To model a situation of elevated plasma levels of TFPI, the assay was carried out at TFPI concentrations up to 10 nM, which is 40-fold higher than the physiological TFPI plasma concentration. Results Biacore binding studies demonstrated that binding kinetics of the cyclic peptide to TFPI 1-160 were not influenced by prior saturation of immobilized TFPI with the linear peptide and vice versa. Prior saturation of immobilized TFPI with the fusion peptide prohibited the linear and cyclic peptide from binding to TFPI, clearly demonstrating the independent binding of the two peptides to different epitopes. By native-PAGE, the linear peptide shifted the KD1-KD2 band completely, whereas the cyclic peptide shifted it only partially. In the presence of both peptides, KD1-KD2 shifted to the highest MW to charge ratio, indicating the formation of a ternary complex consisting of K1-K2, cyclic, and linear peptide. Although the linear and cyclic peptide inhibited TFPI in functional assays, fusion of the two molecular entities provided the most efficient inhibition of TFPI. This was most evident in assays involving multiple epitopes of TFPI to provide functions such as inhibition of extrinsic FX activation complex and thrombin generation, or at high TFPI concentrations. Thrombin generation assays using of 5- to 40-fold elevated TFPI showed that, separately, the two monomeric peptides are only partial inhibitors, and that a mixture of these peptides led to an improved response. However, molecular fusion of the two entities resulted in the most efficient TFPI neutralization. Thus, a synergistic effect is achieved by linking both peptides. Importantly, thrombin generation compromised by a 40-fold of normal TFPI level is normalized by fusion peptide concentrations as low as 50 nM. Summary Based on structural information, we developed a peptide inhibitor composed of two TFPI inhibitory entities. Binding studies support an independent binding mode to non-overlapping binding sites without allosteric cross-talk between binding sites. This introduces synergistic improvement of binding and functional inhibition by bivalent interaction with TFPI. This optimized fusion peptide facilitates efficient TFPI neutralization and resistance to highly increased TFPI levels. Our results further support the use of a fusion peptide in the development of subcutaneous treatment for patients with hemophilia including those with inhibitors. Disclosures Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Brandstetter:Department of Molecular Biology, University of Salzburg, Salzburg, Austria: Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Description: Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway. It inhibits FXa generation by forming a quaternary complex containing factor VIIa (FVIIa), tissue factor (TF), factor Xa (FXa), and TFPI. Two TFPI isoforms, TFPI alpha (TFPI a) and TFPI beta (TFPI b), have been identified, which differ in their C-terminal part due to alternative mRNA splicing events. TFPI a consists of three Kunitz domains (KD), while TFPI b contains two KDs and a C terminal GPI anchor linking the protein to endothelial cell surface. Deletion of the first Kunitz domain of TFPI, which is present in TFPI a and TFPI b in mice is known to be incompatible with viability due to intrauterine lethality (Huang et al., 1997). Aim: To generate transgenic humanized TFPI mice in which mouse (m)-TFPI is entirely replaced by human (hu)-TFPI, in order to facilitate analysis of specific anti hu-TFPI antagonists without interference from m-TFPI. Methods: Integration of the targeting vector, consisting of the m TFPI signal sequence, followed by the human TFPI cDNA and subsequent breeding analysis, was followed by genomic PCR. A sophisticated breeding strategy was used to entirely delete m-TFPI exon 4, which encodes KD1, in humanized transgenic mice. Expression of hu-/m-TFPI a and b mRNAs was analyzed by reverse transcription, cloning, and sequencing of the obtained DNA fragments. Protein levels of hu- and m-TFPI in plasma of transgenic and wild-type (wt) mice were analyzed using species specific ELISAs. Immunoprecipitation experiments in plasma and various mouse tissues are being performed to obtain more information on the presence and distribution of the hu-TFPI protein in transgenic mice. Results: Homozygous humanized TFPI mice were viable and exhibited no obvious abnormalities. Animals showed normal litter size with equal numbers of female and male pups. Genomic PCRs revealed proper integration of the targeting vector into the mouse chromosome and the homozygous status with the expected deletion of m-TFPI exon 4. Expression analyses of humanized TFPI mice on mRNA level demonstrated the absence of full length m-TFPI a and the presence of the humanized TFPI mRNA. Alternative spliced m-TFPI b messages lacking exons three and four were identified, likely leading to a nonfunctional protein. Full length hu-TFPI a mRNA was detected in various tissues in the humanized TFPI mice. The TFPI protein level in plasma from humanized mice was below the detection limit of the ELISA and at least ~300 fold below that for wt mice. Conclusion: Low levels of hu-TFPI may compensate the function of m-TFPI in vivo and circumvent embryonic lethality. Furthermore, we established a new mouse model which allows the regulation of physiologic and pathologic pathways to be assessed at TFPI plasma concentrations below the limit of detection. Disclosures Hoellriegl: Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

Description: Introduction Tissue factor pathway inhibitor (TFPI) is an important inhibitor of the extrinsic coagulation pathway as it inhibits factor Xa (FXa) and the tissue factor (TF) – FVIIa complex. Inhibition of TFPI with blocking antibodies, aptamers, or peptide inhibitors improves hemostasis and may become an option to treat patients with hemophilia including those with inhibitors. We developed a TFPI-antagonistic fusion peptide (FP) consisting of a linear and a cyclic peptide connected by a linker. The two peptide entities bind to different epitopes on TFPI and together synergistically inhibit TFPI. The FP was further improved by half-life extending (HL) non-covalent albumin binding. HL-FP was characterized for in vitroinhibition of TFPI, pharmacokinetics, and improvement of coagulation in animal models of hemophilia. Methods HL-FP was characterized in a set of in vitro assays for binding to and inhibition of TFPI. Interaction with immobilized TFPI was studied by BiaCore. Functional inhibition was analyzed in model assay systems such as inhibition of FXa and FX activation by TF/FVIIa and plasma assays according to the calibrated automated thrombography (CAT) protocol at low TF in hemophilia plasma. Addition of TFPI simulated conditions of potentially elevated TFPI plasma concentrations. In a single dose PK study, mice (n=6 per time point) received 400 nmol/kg of the HL-FP intravenously (i.v.) or subcutaneously (s.c.). Plasma was sampled up to 38 h after dosing and HL-FP level quantified by a compound specific LC-MS protocol. To provide an ex vivo activity measure, FVIII inhibitory antibodies were added to mouse plasma to mimic a hemophilic condition and then analyzed by calibrated automated thrombography (CAT). A 2-week repeated i.v. dose study in mice investigated TFPI accumulation due to HL-FP. HL-FP was dosed at 40, 400, and 2000 nmol/kg and mouse plasma TFPI levels determined by ELISA. The efficacy of the HL-FP was studied in a hemophilia A mouse tail cut model and in a marmoset monkey model of ex vivoimprovement of coagulation. FVIII knockout mice (n=16 per group) were dosed i.v. with 12-400 nmol/kg HL-FP in the presence of a sub-therapeutic level of recombinant FVIII (10 U/kg) and blood loss (mg) was assessed. Marmoset monkeys (N=4) received 400 nmol/kg HL-FP i.v. and plasma samples obtained 1 h after dosing were analyzed by CAT in the presence of FVIII inhibitory antibodies. Results HL-FP bound to and efficiently inhibited TFPI as demonstrated in several in vitro test systems. Binding affinity of 〈 1nM correlated well with functional inhibition of TFPI in model assays, resulting in IC50s of ~0.7nM. The HL-fusion peptide (HL-FP) efficiently inhibited plasma TFPI, which resulted in an improvement of all thrombin generation parameters in plasma of hemophilia A and B patients, with EC50s ranging from 6 to 20nM. HL-FP increased peak thrombin levels of hemophilia plasma to or slightly above a range established for individual normal plasma. Non-covalent binding to albumin substantially increased the half-life to ~4 h with ~ 50% s.c. bioavailability in mice. The ex vivo procoagulant activity determined by CAT correlated well with HL-FP plasma concentrations. In the repeated dose study, the HL-FP was well tolerated and did not accumulate TFPI, which strongly indicates that HL-FP did not interfere with TFPI clearance receptor interactions. HL-FP significantly reduced bleeding in the hemophilia mouse tail cut bleeding model at a dose as low as 40 nmol/kg. In marmoset monkeys, HL-FP efficiently improved ex vivo plasma thrombin generation, even at low peptide plasma concentrations (25- 55 nM). Summary We developed a TFPI inhibitor composed of two TFPI antagonistic peptides that completely inhibits TFPI. Introduction of an entity non-covalently bound to albumin provides intermediate half-life extension and s.c. bioavailability. This HL-FP improved coagulation and hemostasis in animal models of hemophilia and did not interfere with TFPI clearance receptor interactions. TFPI-antagonistic peptides with a prolonged half-life, resistance to elevated TFPI, and minimal interference with TFPI clearance. Our HL-FP appears to be useful in preventing bleeding in hemophilia and provides a FVIII and FIX independent approach for non-i.v. treatment. Disclosures Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Reineke:3B Pharmaceuticals GmbH, Berlin, Germany: Employment. Schiviz:Baxter Innovations GmbH, Vienna, Austria: Employment. Hoellriegl:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Description: Activated human factor IX (FIXa) exhibits very low catalytic activity towards its physiologic substrate factor X (FX), unless the cofactors, activated factor VIII (FVIIIa), procoagulant phospholipid membranes and Ca2+ are present. We were interested in the elucidation of the structural basis for the FVIIIa dependency of FIXa activity. The surface loop 99 of FIXa is important for regulation of FIXa activity. In FIXa not bound to FVIIIa, this loop is stabilized in an inactive conformation and limits access of substrate to the catalytic cleft. Aiming to improve the catalytic activity in the absence of FVIIIa, amino acid exchanges supposedly influencing the 99-loop were introduced into FIX’s catalytic domain (Hopfner KP et al. EMBO J1997;16:6626–35; Sichler K et al. JBC2003;278:4121–26). One of the 3 most promising mutants generated was FIX-Y94F/K98T (FIX-C), with 2 residues exchanged in the 99-loop. Both residues are known to restrict access of FX to the S2 and S4 substrate binding pockets. In FIX-Y94F/K98T/Y177F/I213V/E219G (FIX-L) two additional sites were targeted. By hydrogen bond formation with Asn97 and Asn100, Y177 locks the 99-loop in an inactive conformation, which is released by binding of FVIIIa to FIXa. I213 and E219 at the S1 site are conserved in FIX whereas V213 and G219 are conserved among most other trypsin-like proteases. In FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIX-M) the mutation A95aK is thought to support the interaction between loop 99 and 60, so that conformations similar to equivalent loops of FXa can be adopted. All full-length mutants and wild type FIX were expressed in HEK293 cells, purified and preactivated FIX was removed by chromatographic steps. The resulting proteins were tested for their amidolytic activity and further assessed in FX activation assays in the absence and the presence of FVIIIa. Activated partial thromboplastin time (aPTT) assays were carried out, using plasmas depleted of FVIII or FIX, and FVIII-inhibitor patient plasma. The rationally designed proteins showed not only improved catalytic properties but also some FVIIIa independent activity (table 1). In an aPTT assay, FIX-M activity was equivalent to 1.6% FVIII in FVIII-depleted plasma and to 162 mU/ml FEIBA in FVIII inhibitor patient plasma whereas the combination of mutations Y94F/K98T as exemplified in FIX-C resulted in a molecule with ∼2.5-fold higher specific activity than plasma derived FIX when assayed in FIX-depleted plasma. Characterization of rationally designed FIX proteins. Amidolytic Activity FX activation aPTT +FVIIIa −FVIIIa Ratio FIX-DP FVIII-DP FVIII-IP kcat / KM kcat / KM +FVIIIa / −FVIIIa mM−1min−1 ×10−3 mM−1min−1 mM−1min−1 ×10−4 ×103 mU/mg mU FVIII /5μg FIX mU FEIBA / 5μg FIX (Abbreviations: DP, depleted plasma; IP, inhibitor patient plasma) pdFIX 6 10 2 52 194 0 0 FIX-WT 9 9 3 37 216 1 15 FIX-C 22 12 4 29 498 15 114 FIX-L 15 32 33 10 180 12 115 FIX-M 7 9 11 8 208 16 162