An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Inflammasome Activation Triggers Blood Clotting and Host Death through Pyroptosis.

Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy.

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.

Mouse venous thrombosis upon silencing of anticoagulants depends on tissue factor and platelets, not FXII or neutrophils.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes.

BACKGROUND: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. METHODS: APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. RESULTS: APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. CONCLUSIONS: Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.

Regions of conformational flexibility in the proprotein convertase PCSK9 and design of antagonists for LDL cholesterol lowering.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol levels by binding to the liver LDL receptor (LDLR) and promoting its degradation. Therefore, PCSK9 has become a compelling new therapeutic target for lipid lowering and the prevention of cardiovascular disease. PCSK9 contains two regions of conformational flexibility, the N-terminal regions of the prodomain and of the catalytic domain. The recognition that the latter region, the so-called P' helix, is able to transition from an α-helical to a disordered state gave rise to new strategies to develop small molecule inhibitors of PCSK9 for lipid lowering. In the ordered state the P' helix is buried in a groove of the PCSK9 catalytic domain located next to the main LDLR binding site. The transition to a disordered state leaves the groove site vacated and accessible for compounds to antagonize LDLR binding. By use of a groove-directed phage display strategy we were able to identify several groove-binding peptides. Based on structural information of PCSK9-peptide complexes, a minimized groove-binding peptide was generated and utilized as an anchor to extend towards the adjacent main LDLR binding site, either by use of a phage-displayed peptide extension library, or by appending organic moieties to yield organo-peptides. Both strategies led to antagonists with pharmacologic activities in cell-based assays. The intricate bipartite mechanism of the potent organo-peptide inhibitors was revealed by structural studies, showing that the core peptide occupies the N-terminal groove, while the organic moiety interacts with the LDLR binding site to create antagonism. These findings validate the PCSK9 groove as an attractive target site and should inspire the development of a new class of small molecule antagonists of PCSK9.

Heterodimers of the transcriptional factors NFATc3 and FosB mediate tissue factor expression for 15(S)-hydroxyeicosatetraenoic acid-induced monocyte trafficking.

Tissue factor (TF) is expressed in vascular and nonvascular tissues and functions in several pathways, including embryonic development, inflammation, and cell migration. Many risk factors for atherosclerosis, including hypertension, diabetes, obesity, and smoking, increase TF expression. To better understand the TF-related mechanisms in atherosclerosis, here we investigated the role of 12/15-lipoxygenase (12/15-LOX) in TF expression. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the major product of human 15-LOXs 1 and 2, induced TF expression and activity in a time-dependent manner in the human monocytic cell line THP1. Moreover, TF suppression with neutralizing antibodies blocked 15(S)-HETE-induced monocyte migration. We also found that NADPH- and xanthine oxidase-dependent reactive oxygen species (ROS) production, calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation, and interactions between nuclear factor of activated T cells 3 (NFATc3) and FosB proto-oncogene, AP-1 transcription factor subunit (FosB) are involved in 15(S)-HETE-induced TF expression. Interestingly, NFATc3 first induced the expression of its interaction partner FosB before forming the heterodimeric NFATc3-FosB transcription factor complex, which bound the proximal AP-1 site in the TF gene promoter and activated TF expression. We also observed that macrophages from 12/15-LOX-/- mice exhibit diminished migratory response to monocyte chemotactic protein 1 (MCP-1) and lipopolysaccharide compared with WT mouse macrophages. Similarly, compared with WT macrophages, monocytes from 12/15-LOX-/- mice displayed diminished trafficking, which was rescued by prior treatment with 12(S)-HETE, in a peritonitis model. These observations indicate that 15(S)-HETE-induced monocyte/macrophage migration and trafficking require ROS-mediated CaMKIV activation leading to formation of NFATc3 and FosB heterodimer, which binds and activates the TF promoter.

Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody.

High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.

Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor.

PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 µm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 µm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the ß-strand and a discontinuous short α-helix, and it engaged in the same ß-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.

Excess of heme induces tissue factor-dependent activation of coagulation in mice.

An excess of free heme is present in the blood during many types of hemolytic anemia. This has been linked to organ damage caused by heme-mediated oxidative stress and vascular inflammation. We investigated the mechanism of heme-induced coagulation activation in vivo. Heme caused coagulation activation in wild-type mice that was attenuated by an anti-tissue factor antibody and in mice expressing low levels of tissue factor. In contrast, neither factor XI deletion nor inhibition of factor XIIa-mediated factor XI activation reduced heme-induced coagulation activation, suggesting that the intrinsic coagulation pathway is not involved. We investigated the source of tissue factor in heme-induced coagulation activation. Heme increased the procoagulant activity of mouse macrophages and human PBMCs. Tissue factor-positive staining was observed on leukocytes isolated from the blood of heme-treated mice but not on endothelial cells in the lungs. Furthermore, heme increased vascular permeability in the mouse lungs, kidney and heart. Deletion of tissue factor from either myeloid cells, hematopoietic or endothelial cells, or inhibition of tissue factor expressed by non-hematopoietic cells did not reduce heme-induced coagulation activation. However, heme-induced activation of coagulation was abolished when both non-hematopoietic and hematopoietic cell tissue factor was inhibited. Finally, we demonstrated that coagulation activation was partially attenuated in sickle cell mice treated with recombinant hemopexin to neutralize free heme. Our results indicate that heme promotes tissue factor-dependent coagulation activation and induces tissue factor expression on leukocytes in vivo. We also demonstrated that free heme may contribute to thrombin generation in a mouse model of sickle cell disease.

Mutational tail loss is an evolutionary mechanism for liberating marapsins and other type I serine proteases from transmembrane anchors.

Human and mouse marapsins (Prss27) are serine proteases preferentially expressed by stratified squamous epithelia. However, mouse marapsin contains a transmembrane anchor absent from the human enzyme. To gain insights into physical forms, activities, inhibition, and roles in epithelial differentiation, we traced tail loss in human marapsin to a nonsense mutation in an ancestral ape, compared substrate preferences of mouse and human marapsins with those of the epithelial peptidase prostasin, designed a selective substrate and inhibitor, and generated Prss27-null mice. Phylogenetic analysis predicts that most marapsins are transmembrane proteins. However, nonsense mutations caused membrane anchor loss in three clades: human/bonobo/chimpanzee, guinea pig/degu/tuco-tuco/mole rat, and cattle/yak. Most marapsin-related proteases, including prostasins, are type I transmembrane proteins, but the closest relatives (prosemins) are not. Soluble mouse and human marapsins are tryptic with subsite preferences distinct from those of prostasin, lack general proteinase activity, and unlike prostasins resist antiproteases, including leupeptin, aprotinin, serpins, and α2-macroglobulin, suggesting the presence of non-canonical active sites. Prss27-null mice develop normally in barrier conditions and are fertile without overt epithelial defects, indicating that marapsin does not play critical, non-redundant roles in development, reproduction, or epithelial differentiation. In conclusion, marapsins are conserved, inhibitor-resistant, tryptic peptidases. Although marapsins are type I transmembrane proteins in their typical form, they mutated independently into anchorless forms in several mammalian clades, including one involving humans. Similar pathways appear to have been traversed by prosemins and tryptases, suggesting that mutational tail loss is an important means of evolving new functions of tryptic serine proteases from transmembrane ancestors.

Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma.

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Interferon-gamma-mediated tissue factor expression contributes to T-cell-mediated hepatitis through induction of hypercoagulation in mice.

UNLABELLED: Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF. CONCLUSIONS: Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.

Tissue factor promotes activation of coagulation and inflammation in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer.

Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.

Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.