- Email: [email protected]
Complement receptor C3aR modulates platelet function and thrombosis
1160
Abstracts / Immunobiology 217 (2012) 1129–1222
86
88
Complement receptor C3aR modulates platelet function and thrombosis
Compstatin induces allosteric changes in C3 and C3b and changes their ligand binding pattern
Reinhard J. Sauter 1 , Manuela Fahrleitner 1 , Rebecca Schleicher 1 , Bjoern Kraemer 1 , Meinrad Gawaz 1 , John D. Lambris 2 , Harald F. Langer 1
Hui Chen, Daniel Ricklin, John D. Lambris
1 Department of Cardiovascular Medicine, Section Cardioimmunology, Eberhard Karls-Universität Tübingen, Germany 2 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Beyond its role in innate immunity, complement mediates a wide range of functions in tissue remodeling. Although activation of platelets is crucial during tissue injury and remodeling in multiple diseases, the exact mechanisms how platelet functions are modulated by complement components remain elusive. We found that platelets express the complement receptor C3aR. Stimulation of isolated platelets with C3a peptide enhanced ADP induced aggregation of platelets using aggregometry and resulted in increased adhesion of platelets to fibrinogen under arterial flow conditions. Moreover, platelets isolated from C3aR−/− mice revealed reduced spreading on fibrinogen. In vivo using C3a receptor knockout mice and C5a receptor knockout mice we could demonstrate that specifically lack of the C3a receptor resulted in reduced thrombus formation after vascular injury in an intravital microscopy setting. Accordingly, C3aR-deficient mice displayed increased bleeding time in tail bleeding time experiments, which could not be explained by a difference in peripheral platelet count, and after reperfusion of C3aR+/+ platelets into C3aR−/− mice prolonged bleeding time associated with C3aR deficiency was reversed. In an experimental stroke model, C3aR−/− mice displayed reduced infarct volume, a difference that was abolished in the absence of platelets. In conclusion, these data indicate a new and surprising function of the anaphylatoxin receptor C3aR on platelets for platelet function and thrombosis.
Compstatin is a cyclic 13-residue peptide that potently prevents activation of the human complement system by inhibiting the conversion of C3 to C3b. Structural and biophysical studies previously located the binding site of this inhibitor at the interface of macroglobulin domains 4 and 5 (MG4/5) of the beta chain of C3/C3b. However, the exact molecular mechanism of inhibition has not yet been fully resolved. In the study presented here, we utilized a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and orthogonal techniques to resolve this important aspect. By incubating C3 and C3b with either active compstatin or an inactive analog, we could confirm that the inhibitor occupies the same binding site on both proteins in solution. More importantly, though, HDX-MS analysis also revealed that incubation with active compstatin induced structural changes in C3 and C3b that are located distant from the compstatin binding site, thereby indicating the possibility of allosteric effects. Indeed, a functional implication of such global conformational changes was supported by surface plasmon resonance (SPR)-based binding studies, which revealed significant changes in the binding pattern of C3b towards key ligands such as Factor H or the bacterial inhibitor SCIN in the presence of compstatin. On the other hand, computational studies and SPR analyses also indicated that steric hindrance of the interaction between the C3 substrate and the alternative pathway convertase contribute to the inhibitory effect of compstatin. Our study therefore not only sheds light into the mechanism of action of a clinically relevant complement inhibitor, but also reveals surprising insight into the conformational flexibility of C3/C3b and the possibility of allosteric inhibition as a therapeutic concept.
http://dx.doi.org/10.1016/j.imbio.2012.08.087
http://dx.doi.org/10.1016/j.imbio.2012.08.089
87
89
Complement- and platelet-mediated shuttling of blood-borne bacteria to CD8A+ dendritic cells
Control of arthritis by local synthesis of recombinant antibody neutralizing C5
Steven Broadley, Patricia Graef, Ann Plaumann, Amelie Seidlmeier, Andreas Warnisch, Dirk H. Busch, Admar Verschoor
Paolo Macor 1 , Paolo Durigutto 1 , Federica Ziller 1 , Luca De Maso 1 , Fabio Fischetti 1 , Roberto Marzari 1 , Daniele Sblattero 2 , Francesco Tedesco 1
Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany
1
Department of Life Sciences, University of Trieste, Trieste, Italy Department of Medical Sciences and IRCAD, University of Eastern Piedmont, Novara, Italy 2
The acquisition of pathogen-derived antigen by dendritic cells (DCs) is a key event in the generation of cytotoxic CD8+ T cell responses. In mice, the intracellular bacterium Listeria monocytogenes is directed from the blood to splenic CD8a+ DCs. L. monocytogenes rapidly associates with platelets in the bloodstream in a manner dependent on GPIb and complement C3. Platelet association targets a small but significant portion of L. monocytogenes to splenic CD8a+ DCs, diverting bacteria from swift clearance by other, less immunogenic phagocytes. Thus, an effective balance is established between maintaining sterility of the circulation and induction of antibacterial immunity by DCs. Our focus is on the mechanisms involved in the association between L. monocytogenes and platelets, and its influence on the ensuing immune response and bacterial clearance. http://dx.doi.org/10.1016/j.imbio.2012.08.088
Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have recently addressed this issue by developing a recombinant molecule containing a neutralizing antibody to C5 and a homing peptide for inflamed synovium. This recombinant molecule was effective in controlling animal models of arthritis. We now propose an alternative approach consisting in the intraarticular injection of a DNA vector encoding for the recombinant minibody MB12/22 neutralizing C5 to allow local production of the antibody in sufficient amount to be effective in preventing joint inflammation in rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong
Abstracts / Immunobiology 217 (2012) 1129–1222
86
88
Complement receptor C3aR modulates platelet function and thrombosis
Compstatin induces allosteric changes in C3 and C3b and changes their ligand binding pattern
Reinhard J. Sauter 1 , Manuela Fahrleitner 1 , Rebecca Schleicher 1 , Bjoern Kraemer 1 , Meinrad Gawaz 1 , John D. Lambris 2 , Harald F. Langer 1
Hui Chen, Daniel Ricklin, John D. Lambris
1 Department of Cardiovascular Medicine, Section Cardioimmunology, Eberhard Karls-Universität Tübingen, Germany 2 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Beyond its role in innate immunity, complement mediates a wide range of functions in tissue remodeling. Although activation of platelets is crucial during tissue injury and remodeling in multiple diseases, the exact mechanisms how platelet functions are modulated by complement components remain elusive. We found that platelets express the complement receptor C3aR. Stimulation of isolated platelets with C3a peptide enhanced ADP induced aggregation of platelets using aggregometry and resulted in increased adhesion of platelets to fibrinogen under arterial flow conditions. Moreover, platelets isolated from C3aR−/− mice revealed reduced spreading on fibrinogen. In vivo using C3a receptor knockout mice and C5a receptor knockout mice we could demonstrate that specifically lack of the C3a receptor resulted in reduced thrombus formation after vascular injury in an intravital microscopy setting. Accordingly, C3aR-deficient mice displayed increased bleeding time in tail bleeding time experiments, which could not be explained by a difference in peripheral platelet count, and after reperfusion of C3aR+/+ platelets into C3aR−/− mice prolonged bleeding time associated with C3aR deficiency was reversed. In an experimental stroke model, C3aR−/− mice displayed reduced infarct volume, a difference that was abolished in the absence of platelets. In conclusion, these data indicate a new and surprising function of the anaphylatoxin receptor C3aR on platelets for platelet function and thrombosis.
Compstatin is a cyclic 13-residue peptide that potently prevents activation of the human complement system by inhibiting the conversion of C3 to C3b. Structural and biophysical studies previously located the binding site of this inhibitor at the interface of macroglobulin domains 4 and 5 (MG4/5) of the beta chain of C3/C3b. However, the exact molecular mechanism of inhibition has not yet been fully resolved. In the study presented here, we utilized a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and orthogonal techniques to resolve this important aspect. By incubating C3 and C3b with either active compstatin or an inactive analog, we could confirm that the inhibitor occupies the same binding site on both proteins in solution. More importantly, though, HDX-MS analysis also revealed that incubation with active compstatin induced structural changes in C3 and C3b that are located distant from the compstatin binding site, thereby indicating the possibility of allosteric effects. Indeed, a functional implication of such global conformational changes was supported by surface plasmon resonance (SPR)-based binding studies, which revealed significant changes in the binding pattern of C3b towards key ligands such as Factor H or the bacterial inhibitor SCIN in the presence of compstatin. On the other hand, computational studies and SPR analyses also indicated that steric hindrance of the interaction between the C3 substrate and the alternative pathway convertase contribute to the inhibitory effect of compstatin. Our study therefore not only sheds light into the mechanism of action of a clinically relevant complement inhibitor, but also reveals surprising insight into the conformational flexibility of C3/C3b and the possibility of allosteric inhibition as a therapeutic concept.
http://dx.doi.org/10.1016/j.imbio.2012.08.087
http://dx.doi.org/10.1016/j.imbio.2012.08.089
87
89
Complement- and platelet-mediated shuttling of blood-borne bacteria to CD8A+ dendritic cells
Control of arthritis by local synthesis of recombinant antibody neutralizing C5
Steven Broadley, Patricia Graef, Ann Plaumann, Amelie Seidlmeier, Andreas Warnisch, Dirk H. Busch, Admar Verschoor
Paolo Macor 1 , Paolo Durigutto 1 , Federica Ziller 1 , Luca De Maso 1 , Fabio Fischetti 1 , Roberto Marzari 1 , Daniele Sblattero 2 , Francesco Tedesco 1
Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany
1
Department of Life Sciences, University of Trieste, Trieste, Italy Department of Medical Sciences and IRCAD, University of Eastern Piedmont, Novara, Italy 2
The acquisition of pathogen-derived antigen by dendritic cells (DCs) is a key event in the generation of cytotoxic CD8+ T cell responses. In mice, the intracellular bacterium Listeria monocytogenes is directed from the blood to splenic CD8a+ DCs. L. monocytogenes rapidly associates with platelets in the bloodstream in a manner dependent on GPIb and complement C3. Platelet association targets a small but significant portion of L. monocytogenes to splenic CD8a+ DCs, diverting bacteria from swift clearance by other, less immunogenic phagocytes. Thus, an effective balance is established between maintaining sterility of the circulation and induction of antibacterial immunity by DCs. Our focus is on the mechanisms involved in the association between L. monocytogenes and platelets, and its influence on the ensuing immune response and bacterial clearance. http://dx.doi.org/10.1016/j.imbio.2012.08.088
Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have recently addressed this issue by developing a recombinant molecule containing a neutralizing antibody to C5 and a homing peptide for inflamed synovium. This recombinant molecule was effective in controlling animal models of arthritis. We now propose an alternative approach consisting in the intraarticular injection of a DNA vector encoding for the recombinant minibody MB12/22 neutralizing C5 to allow local production of the antibody in sufficient amount to be effective in preventing joint inflammation in rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong