Phage display is widely used for the selection of target-specific peptide sequences. Presentation... more Phage display is widely used for the selection of target-specific peptide sequences. Presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. Here, we present a detailed analysis of the effects of valency, linker choice, and receptor density on binding affinity of a multivalent architecture, using streptavidin (SA) as model multivalent receptor. For surfaces with low receptor densities, the SA binding affinity of multivalent dendritic phage peptide constructs increases over 2 orders of magnitude over the monovalent species (e.g., K(d,mono) = 120 μM vs K(d,tetra) = 1 μM), consistent with previous work. However, the affinity of the SA-binding phage presenting the exact same peptides was 16 pM when dense receptor surfaces used for initial phage display were used in assays. The phage affinity for SA-coated surfaces weakens severely toward the nanomolar regime when surface density of SA is decreased. A similarly strong dependence in this respect was observed for dendritic phage analogues. When presented with a dense SA-coated surface, dendrimer display affords up to a 10(4)-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The perspective offered here suggests that in vivo targeting schemes might best be served to conduct ligand selection under physiologically relevant receptor density surfaces, either by controlling the receptor density placed at the selection surface or by using more biologically relevant intact cells and tissues.
matrices, read depth and variant frequencies. We applied the heterogeneity estimation procedure t... more matrices, read depth and variant frequencies. We applied the heterogeneity estimation procedure to the genomes of pathogenic and opportunistic pathogenic bacteria newly sequenced by us, including 6 genomes of Klebsiella pneumoniae, 6 genomes of Acinetobacter baumannii, 4 genomes of Mycoplasma hominis, 4 genomes of M. tuberculosis and 3 genomes of Staphylococcus aureus. As a result, we found that the genomes of M. hominis are extremely heterogeneous (more than 4 times comparing to K. pneumoniae), while M. tuberculosis and S. aureus are slightly less heterogeneous, and A. baumannii and K. pneumoniae have substantially homogenous genomes. We plan to extensively study the available pathogenic genomes to re-assess their sequencing quality, reconstruct minor clonal variants based on NGS data, and get insights regarding the origins of their variability.
Rationale: The leukocyte response in acute inflammation is characterized by an initial recruitmen... more Rationale: The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. Objective: To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. Methods and Results: Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of nonclassical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium, where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates formyl-peptide receptor 2 on classical monocytes, resulting in monocytic β 1 - and β 2...
Multiple, simultaneous interactions are often used in biology to enhance the affinity and specifi... more Multiple, simultaneous interactions are often used in biology to enhance the affinity and specificity of binding, an effect that is known as multivalency.1 The principle of multivalency has been recognized as an important strategy for the development of (semi)synthetic ligands with high affinity ...
The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenes... more The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links α2-AP to fibrin to protect it from early lysis. This was exploited to develop an α2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using 111In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and 111In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl3) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with 111In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16...
The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) w... more The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1µM EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94Å (9.4nm) to 85Å above the phospholipid surface (assuming κ2 = 2/3). An identical increase in energy transfer between the APC active si...
Arteriosclerosis, thrombosis, and vascular biology, Jan 19, 2017
The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagula... more The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleati...
C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway ... more C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vic...
Phage display is widely used for the selection of target-specific peptide sequences. Presentation... more Phage display is widely used for the selection of target-specific peptide sequences. Presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. Here, we present a detailed analysis of the effects of valency, linker choice, and receptor density on binding affinity of a multivalent architecture, using streptavidin (SA) as model multivalent receptor. For surfaces with low receptor densities, the SA binding affinity of multivalent dendritic phage peptide constructs increases over 2 orders of magnitude over the monovalent species (e.g., K(d,mono) = 120 μM vs K(d,tetra) = 1 μM), consistent with previous work. However, the affinity of the SA-binding phage presenting the exact same peptides was 16 pM when dense receptor surfaces used for initial phage display were used in assays. The phage affinity for SA-coated surfaces weakens severely toward the nanomolar regime when surface density of SA is decreased. A similarly strong dependence in this respect was observed for dendritic phage analogues. When presented with a dense SA-coated surface, dendrimer display affords up to a 10(4)-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The perspective offered here suggests that in vivo targeting schemes might best be served to conduct ligand selection under physiologically relevant receptor density surfaces, either by controlling the receptor density placed at the selection surface or by using more biologically relevant intact cells and tissues.
matrices, read depth and variant frequencies. We applied the heterogeneity estimation procedure t... more matrices, read depth and variant frequencies. We applied the heterogeneity estimation procedure to the genomes of pathogenic and opportunistic pathogenic bacteria newly sequenced by us, including 6 genomes of Klebsiella pneumoniae, 6 genomes of Acinetobacter baumannii, 4 genomes of Mycoplasma hominis, 4 genomes of M. tuberculosis and 3 genomes of Staphylococcus aureus. As a result, we found that the genomes of M. hominis are extremely heterogeneous (more than 4 times comparing to K. pneumoniae), while M. tuberculosis and S. aureus are slightly less heterogeneous, and A. baumannii and K. pneumoniae have substantially homogenous genomes. We plan to extensively study the available pathogenic genomes to re-assess their sequencing quality, reconstruct minor clonal variants based on NGS data, and get insights regarding the origins of their variability.
Rationale: The leukocyte response in acute inflammation is characterized by an initial recruitmen... more Rationale: The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. Objective: To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. Methods and Results: Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of nonclassical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium, where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates formyl-peptide receptor 2 on classical monocytes, resulting in monocytic β 1 - and β 2...
Multiple, simultaneous interactions are often used in biology to enhance the affinity and specifi... more Multiple, simultaneous interactions are often used in biology to enhance the affinity and specificity of binding, an effect that is known as multivalency.1 The principle of multivalency has been recognized as an important strategy for the development of (semi)synthetic ligands with high affinity ...
The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenes... more The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links α2-AP to fibrin to protect it from early lysis. This was exploited to develop an α2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using 111In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and 111In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl3) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with 111In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16...
The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) w... more The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1µM EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94Å (9.4nm) to 85Å above the phospholipid surface (assuming κ2 = 2/3). An identical increase in energy transfer between the APC active si...
Arteriosclerosis, thrombosis, and vascular biology, Jan 19, 2017
The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagula... more The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleati...
C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway ... more C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vic...
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Papers by T. Hackeng