ALBERT

Description: Sickle cell disease (SCD), caused by a single amino change in the β-globin gene, exhibits a complex pathophysiology. Hypoxia in the microvascular venous bed leads to inflammation of the endothelium, adhesion of neutrophils, and a decrease in neutrophil rolling and flow velocity. The cell aggregates become trapped in the vasculature through interactions with endothelial cells. The adhesive interactions of the sickle erythrocytes, leukocytes, and endothelial cells obstruct the vasculature leading to vaso-occlusion. The vaso-occlusion can result in endothelial cell dysfunction and inflammation. Soluble adhesion molecules, including soluble E-Selectin, are associated with disease severity (Kato et al, Br.J.Hematol, 2005). PF-04447943, a selective inhibitor of the cyclic guanosine monophosphate-specific phosphodiesterase-9A, is currently in clinical development for the prophylactic treatment of SCD. The Townes mouse model of SCD exhibits many of the pathological symptoms observed in patients. Previously, we demonstrated vaso-occlusion in the Townes model by intravital microscopy (IVM), and that a single dose of PF-04447943 reduced leukocyte-platelet aggregates and the adhesion of neutrophils to the endothelium. Here we examined the pharmacodynamic effects of chronic repeat dosing with PF-04447943 in male Townes sickle cell mice. Mice were dosed orally with PF-04447943 twice daily for 4 weeks in the presence and absence of hydroxyurea (HU). Treatment effects were evaluated in vivo using IVM after 4 weeks. Surgical exposition of the cremaster muscle elicits inflammation-induced vaso-occlusion in the mouse vasculature and neutrophil rolling, adhesion and neutrophil-platelet aggregates were analyzed using IVM. Administration of PF-04447943 (10 mg/kg) in combination with HU (25 mg/kg) showed a reduction in neutrophil adhesion, an increase in neutrophil rolling on the microvasculature, and a reduction in neutrophil-platelet aggregates compared to vehicle alone. A 50% reduction in neutrophil platelet aggregates was observed when 10 mg/kg of PF-04447943 was administered as a single agent (50%), but this effect was more pronounced with co-administration of PF-04447943 and HU (73%). Significant reductions (11.1%) were observed in soluble plasma E-selectin levels in mice treated with a combination of PF-04447943 and HU for 28 days compared to vehicle treated mice. Analysis of hematological parameters showed a 42% reduction in total peripheral white blood cell count in mice that received a co-administration of PF-04447943 and HU. A small but significant increase was seen in the mean corpuscular hemoglobin (MCH) as well as mean corpuscular volume (MCV). In conclusion, the phosphodiesterase-9A inhibitor PF-04447943 results in reductions in leukocyte-platelet aggregates and soluble E-selectin in a chronic treatment study in SCD mice. Long term treatment with PF-04447943 may be beneficial in improving pharmacodynamic parameters, as well as markers of inflammation that may mediate vaso-occlusion associated with SCD. All experiments were within guidelines and were reviewed and approved by Pfizer Institutional Animal Care and use Committee. Disclosures Jasuja: Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Description: BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086. AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection. METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss. RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively. CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. Disclosures Barakat: Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Description: Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA). Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Description: Introduction Deficiencies of both von Willebrand Factor (VWF) and FVIII are associated with significant bleeding phenotypes. Consequently, patients with VWD or hemophilia A commonly require replacement therapy with coagulation factor concentrates. However, as infused VWF and FVIII have relatively short plasma half-lives, patient therapy generally necessitates frequent re-dosing. Development of a long-acting rVWF therapy thus represents an important unmet clinical need. We and others have previously demonstrated that the A1A2A3 domains of VWF play a critical role in regulating macrophage-mediated clearance of VWF in vivo. Importantly, crystal structures of the A-domains have also well characterized. In this study, we sought to utilize this data to investigate the hypothesis that site-specific PEGylation within the A1A2A3 domains could be used as a novel strategy to inhibit macrophage-mediated clearance, and thereby inform development of a rVWF molecule with extended plasma half-life. Methodology Site-directed mutagenesis was used to engineer novel surface cysteine residues at selected sites within A1A2A3-VWF. Following purification and characterization, individual A1A2A3 cysteine variants were PEGylated using 40kDa PEG maleimide. Clearance of unPEGylated and PEGylated A1A2A3 variants were assessed in VWF-/- mice. VWF-macrophage interactions were quantified in vitro using differentiated THP-1 macrophages. VWF binding to LRP1 clearance receptor was assessed using both immunosorbant assays and Surface Plasmon Resonance. Results Novel single cysteine residues were introduced at stringently selected sites within A1A2A3-VWF. These sites spanned all 3 A-domains and included; S1286C, Q1353C, M1545C, L1591C, V1636C, Q1652C, V1803C and S1807C. Interestingly, the introduction of these novel cysteine residues in both the A1 and A3 domains of VWF did not alter the rate of VWF clearance compared to WT A1A2A3-VWF. Conversely however, the A2 domain was less tolerant for the insertion of cysteines, with L1591C and V1636C variants demonstrating a significantly reduced VWF plasma half-life of approx. 1.5 fold versus WT-A1A2A3 (p

Description: Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored. Thrombin generation was measured in citrated platelet poor Factor XI (FXI), Factor V (FV), FVII, von Willebrand Factor (vWF) deficient (Type 1, 2A, 2B and 3) congenital donor plasma following the in vitro addition of PF-06741086 (0, 1, 10 or 100 nM) or a human IgG1 antibody; initiated with 1 pM TF and 4 µM phospholipid. FXI, FV, and FVII donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone was also included in the analysis. In FXI deficient plasmas, a concentration-dependent increase in peak thrombin and a shortening of the lag time was observed with the addition of PF-06741086 normalizing and restoring levels to those observed in the non-hemophilic plasma. A similar response was also observed in all of the vWF deficient plasmas. In one FVII deficient plasma, an increase in peak thrombin was observed at dose of 100 nM PF-06741086, however, the lag time (20 minutes) was significantly extended, relative to healthy volunteer non-hemophilic plasma. As expected, the addition of PF-06741086 to FV deficient plasma did not increase thrombin generation at any concentration. The in vitro addition of the TFPI antibody, PF-06741086, improved thrombin generation in selected coagulation factor deficient plasmas, including vWF deficiency, to the levels observed in normal plasma. This data suggestion that the inhibition of TFPI may promote hemostasis in rare bleeding disorders such as FXI deficiency and vWF deficiencies. Disclosures Rakhe: Pfizer: Employment. Patel-Hett:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Description: Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of β-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE’s mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti–ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE–BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

Description: Hemophilia is a hereditary bleeding disorder caused by intrinsic coagulation pathway deficiencies of Factor VIII (hemophilia A) or Factor IX (hemophilia B). Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. In hemophilia patients the extrinsic pathway remains intact and thus augmentation of this pathway may circumvent the clotting deficiency in hemophilia. PF-06741086, a monoclonal antibody that binds to and neutralizes the inhibitory activity of TFPI is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. Currently, treatment of inhibitor patients is managed by bypass treatments, such as recombinant Factor VIIa (rFVIIa). The effect of PF-06741086 on thrombin generation in the presence of increasing concentrations of rFVIIa (0.0002 to 20 µg/mL) was studied in severe hemophilia A plasma. A dose-dependent increase in thrombin generation was observed over vehicle control with the addition of rFVIIa to the hemophilia plasma. Addition of a fixed concentration of PF-06741086 (16 µg/mL) in combination with rFVIIa resulted in an increase in thrombin generation including higher peak thrombin and shortening of lag time compared to rFVIIa alone. The TGA profiles with the combination of PF-06741086 and rFVIIa at 0.2, 2, and 20 µg/mL were similar suggesting a saturation of mechanism at these concentrations. The combination of PF-06741086 and rFVIIa restored thrombin generation to normal plasma levels at all rFVIIa concentrations examined. The TFPI inhibitory activity of PF-06741086 on thrombin generation in the presence and absence of rFVIIa was further studied in additional hemophilia A plasmas, including hemophilia A plasmas with inhibitors and hemophilia B plasma. All donors had less than 1% coagulation factor activity. A rFVIIa concentration of 2 µg/mL was selected because it corresponded to plasma levels that could be observed following dosing of FVIIa and because the thrombin generation response in hemophilia plasma was similar with FVIIa added to hemophilia A plasma at 0.2, 2 and 20 µg/mL. The concentration of PF-06741086 was 16 µg/mL in these studies. The effect of PF-06741086 on thrombin generation was also measured in non-hemophilic plasma which would have the full complement of coagulation factors. The addition of PF-06741086 alone or in combination with rFVIIa to hemophilia A and B plasma resulted in an increase in thrombin generation including higher peak thrombin concentration and shortening of lag time compared to addition of rFVIIa alone. In hemophilic plasma samples with inhibitors (3 - 1261 Bethesda Units), PF-06741086 alone also restored thrombin generation. A minimal additive effect in peak thrombin generation was observed with the combination of PF-06741086 (16 µg/mL) and 2 µg/mL rFVIII. The midpoint peak thrombin levels achieved with PF-06741086 alone or in combination with rFVIIa were similar to those observed in non-hemophilic plasma and did not exceed the level observed in non-hemophilic plasma dosed with PF-06741086. To summarize, use of rFVIIa in combination with PF-06741086 results in increased thrombin generation in hemophilia A, hemophilia B and inhibitor plasmas without inducing excessive coagulation. Disclosures Rakhe: Pfizer: Employment. Hett:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Description: The pathophysiology of sickle cell disease (SCD) is complex and heterogenous. Tissue hypoxia leads to endothelial activation, and formation of multicellular aggregates of red cells, leucocytes and platelets in the microvasculature. This results in a vaso-occlusion further reducing tissue perfusion. Patients with SCD experience intense pain crisis. Some SCD patients also exhibit elevated cerebral blood flow velocities in transcranial Doppler (TCD) and impaired autoregulation. Higher TCD velocities are associated with imminent risk of ischemic stroke and are an indication for transfusion for SCD patients. Similar to human SCD patients, SCD mice also exhibit an increase in cerebral perfusion (Branch et al, Blood, 2015) and irregularly dilated cerebral microvasculature compared to wild-type (WT) mice (Manci et al, Blood, 2006). Selectins play a critical role in mediating initial tethering and adhesion of hematopoietic cells with endothelial cells, and are attractive targets to treat vaso-occlusion. Rivipansel is a potent small molecule pan-selectin inhibitor with a 100 fold greater inhibitory acitivity for E-selectin over P-selectin. Previous studies have demonstrated the effect of Rivipansel (GMI-1070) in chimeric SCD mice using intravital microscopy (IVM) where bone marrow from Berkeley SCD mice was transplanted in wild type (WT) mice (Chang et al, Blood, 2010). Here, we further investigate the effect of Rivipansel on leukocyte rolling in vitro and in vivo in Townes SCD mice with corresponding changes in inflammatory markers. We also extended these studies to explore the effect of Rivipansel in a cerebral perfusion model in Townes SCD mice. The effect of Rivipansel on cell adhesion in vitro was examined using the microfluidic BioFlux shear flow system. HL-60 cells were continuously flown under physiological conditions over confluent monolayers of Chinese Hamster Ovary cells expressing E-selectin (CHO-E) or P-selectin (CHO-P) treated with Rivipansel, Immunoglobulin G (IgG) control antibody or vehicle. Rivipansel dose-dependently reduced the adhesion of HL-60 to CHO-E monolayers compared to vehicle treated cells, or CHO-P monolayers. In the Townes SCD mice, Rivipansel was evaluated using IVM of the cremaster. Surgical manipulation and the chronic inflammatory conditions in Townes mice, induce an acute vaso-occlusive condition in the post-capillary venules. Rivipansel or vehicle control were dosed at 5, 10 or 20 mgs/kg intravenously prior to surgical preparation and a second dose was administered 70 minutes later. Labeled neutrophil and platelet specific antibodies were injected to quantify neutrophil-platelet aggregates and neutrophils adhered to the vasculature. Neutrophil rolling velocity increased 2.5 fold and there was a dose dependent increase in rolling neutrophils. Rivipansel, dosed at 20 mg/kg resulted in a 69% decrease in neutrophil adhesion to the endothelium and an 85% decrease in neutrophil platelet aggregates (NPA) attached to the vasculature compared to vehicle treated mice. No effect on was observed in mice dosed a 5 mg/kg. Plasma soluble P and E-selectin levels were significantly reduced in mice dosed at 10 and 20 mg/kg compared to vehicle treated mice. Additionally, cultured primary brain endothelial cells stimulated with TNF-α demonstrated a 700 fold increase in E-selectin levels as measured by RNAseq. Based on this observation, we studied the effect of Rivipansel on cerebral perfusion studies in a TNFα induced occlusion model in SCD and WT mice where changes in cerebral microcirculation were measured in real time using laser Doppler tissue perfusion. TNFα treated SCD mice demonstrated a marked reduction in tissue perfusion in contrast to WT mice. This was rapidly reversed by intravenous injection of Rivipansel (20 mg/kg). Brains harvested from TNF treated SCD mice showed increased platelet microthrombi compared to WT mice, and treatment with Rivipansel reduced the platelet aggregates seen in the cerebral microvasculature in SCD mice. In summary, we demonstrated dose dependent decrease in leukocyte adhesion in vitro and in vivo in the Townes mice. In addition administration of Rivipansel is beneficial in improving cerebral perfusion as well improving peripheral microvascular flow in the SCD mice. All experiments were within guidelines and were reviewed and approved by Pfizer institutional animal care and use committee. Disclosures Jasuja: Pfizer: Employment. Suidan:Pfizer: Employment. Hett:Pfizer: Employment. Desai:Pfizer: Employment. Le:Pfizer: Employment. Bell:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Description: Receptor-mediated internalization appears to be the primary mean of regulating the plasma level of thrombopoietin (TPO). However, the processes that regulate Mpl internalization have not previously been described. Using the cytokine-dependent cell line BaF3, we have identified 2 distinct motifs within the cytoplasmic domain of Mpl that underlie ligand-dependent internalization. Removal of the fourth cytoplasmic tyrosine residue by deletion or truncation results in a significant decrease in maximal internalization. The remaining receptor internalization is abrogated by deletion of cytoplasmic residues 54-69, which include the core box2 region (L54L55E56I57L58) and the only dileucine motifs (L54L55 and I57L58) within the cytoplasmic domain of Mpl. Receptor internalization mediated by this latter subdomain does not require Jak2 activation. Furthermore, TPO-stimulated cellular proliferation appears to be directly correlated with receptor internalization, indicating that internalization of the TPO/Mpl complex may be essential for normal signal transduction. Finally, we have demonstrated that upon removal of TPO from the supernatant, Mpl promptly reappears on the cell surface, suggesting that a pool of intracellular Mpl can be rapidly recycled to the cell surface. These data help identify the receptor motifs involved in TPO-induced internalization of Mpl and suggest that Mpl translocation may be necessary for normal cellular proliferation. (Blood. 2003;102:102-108)

Description: Hemophilia A and B are X-linked genetic disorders resulting from functional deficiencies of the intrinsic coagulation plasma proteins Factor VIII (FVIII) or Factor IX (FIX), respectively. An approach to achieving hemostatic pharmacology in hemophilia is to augment the extrinsic cascade is to neutralize Tissue Factor Pathway Inhibitor (TFPI). TFPI is a multi-Kunitz (K) domain inhibitor which binds to and inhibits Factor Xa via the K2 domain and Factor VIIa/Tissue Factor activity (K1 domain). Marstacimab is a fully human monoclonal antibody that binds to and neutralizes TPFI activity and is under development for treatment of hemophilia. To support lead identification during late discovery, five anti-tissue factor pathway inhibitor (TFPI) antibodies (EC50