Daniel Kirchhofer

Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH2) was determined to be 266 ± 8, 320 ± 22, and 467 ± 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379–389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivoclearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25–43% of the albumin half-life in these animals. These half-lives exceed those of a Fab′2 and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.

Crystal structure of the HGF β-chain in complex with the sema domain of the met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF‐dependent signaling requires proteolysis from an inactive single‐chain precursor into an active α/β‐heterodimer. We show that the serine protease‐like HGF β‐chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven‐bladed β‐propeller, where the bottom face of blades 2 and 3 binds to the HGF β‐chain ‘active site region’. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF β binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF β‐chains brings two HGF β:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.

Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2

Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer and promotes tumor progression and metastasis. We generated a soluble form of hepsin comprising the entire extracellular domain to show that it efficiently converts single‐chain hepatocyte growth factor (pro‐HGF) into biologically active two‐chain HGF. Hepsin activity was potently inhibited by soluble forms of the bi‐Kunitz domain inhibitors HAI‐1B (IC50 21.1 ± 2.7 nM) and HAI‐2 (IC50 1.3 ± 0.3 nM). Enzymatic assays with HAI‐1B Kunitz domain mutants (R260A and K401A) further demonstrated that inhibition was due to Kunitz domain‐1. The results suggest a functional link between hepsin and the HGF/Met pathway, which may contribute to tumor progression.

Local Protease Signaling Contributes to Neural Tube Closure in the Mouse Embryo

We report an unexpected role for protease signaling in neural tube closure and the formation of the central nervous system. Mouse embryos lacking protease-activated receptors 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, and Par2 was expressed selectively along the line of closure. Ablation of G(i/z) and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often coexpressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

Monocyte tissue factor-dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin.

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

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.

Inhibition of tissue factor limits the growth of venous thrombus in the rabbit.

Summary.  Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF‐free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF‐bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen‐coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP‐1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I‐fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti‐TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen‐coated coverslips demonstrated the presence of TF‐bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot‐bound TF and that blocking the TF activity can inhibit thrombus propagation.

Levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice

Summary.  Background: Tissue factor (TF) is present in blood in various forms, including small membrane vesicles called microparticles (MPs). Elevated levels of these MPs appear to play a role in the pathogenesis of thrombosis in a variety of diseases, including sepsis. Objective: Measure levels of MP TF activity and activation of coagulation in control and endotoxemic mice. Materials and methods: MPs were prepared from plasma by centrifugation. The procoagulant activity (PCA) of MPs was measured using a two‐stage chromogenic assay. We also measured levels of thrombin‐antithrombin and the number of MPs. Results: Lipopolysaccharide (LPS) increased MP PCA in wild‐type mice; this PCA was significantly reduced by an anti‐mouse TF antibody (1H1) but not with an anti‐human TF antibody (HTF‐1). Conversely, in mice expressing only human TF, MP PCA was inhibited by HTF‐1 but not 1H1. MPs from wild‐type mice had 6‐fold higher levels of PCA using mouse factor (F)VIIa compared with human FVIIa, which is consistent with reported species‐specific differences in FVIIa. Mice expressing low levels of human TF had significantly lower levels of MP TF activity and TAT than mice expressing high levels of human TF; however, there were similar levels of phosphatidylserine (PS)‐positive MPs. Importantly, levels of MP TF activity in wild‐type mice correlated with levels of TAT but not with PS‐positive MPs in endotoxemic mice. Conclusion: These results suggest that the levels of TF‐positive MPs can be used as a biomarker for evaluating the risk of disseminated intravascular coagulation in endotoxemia.

Pro-urokinase-type plasminogen activator is a substrate for hepsin.

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1′) peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 × 105 m–1 s–1) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ± 1.9 nm uPA h–1, which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.

Laminin-332 Is a Substrate for Hepsin, a Protease Associated with Prostate Cancer Progression

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the β3 chain of Ln-332. N-terminal sequencing identified the cleavage site at β3 Arg245, in a sequence context (SQLR245↓LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.