Mechanism and therapy of hereditary angioedema typ III and role of the contact system in inflammatory diseases

Combinations of proinflammatory and procoagulant reactions are associated with a variety of disorders affecting the cardiovascular system. Vascular leakage contributes to the pathology of conditions such as, sepsis, allergy and anaphylactic reactions. Edema formation is the result of extravasated proteins and fluid and the peptide hormone bradykinin is considered to be one of the key mediators in the regulation of vascular leakage. Bradykinin is produced by the kallikrein-kinin system, which consists of factor XII, plasma prekallikrein, high molecular weight kininogen and C1 esterase inhibitor. Activated factor XII generates active prekallikrein (kallikrein), which cleaves kininogen, leading to the liberation of bradykinin. Activation of mast cells during allergic reactions mediates inflammatory responses, which cause increased vascular permeability. We report a new mechanism by which mast cell-released heparin increases vascular leakage. Upon allergen challenge mast cells release the negatively charged polysaccharide heparin that efficiently activates factor XII and initiates the kallikrein-kinin system. Heparin-driven kallikrein-kinin system activation culminates in bradykinin formation causing excessive vascular leakage in mice that are deficient in C1 esterase inhibitor, the major endogenous inhibitor of factor XII and kallikrein. These findings also have implications in anaphylactic and allergic diseases and we show that the factor XII– driven kallikrein-kinin system critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell mediated diseases. Hereditary angioedema (HAE) is a rare inherited disease that is characterized by acute swelling that involves the skin, extremities and mucosa. HAE types I and II are caused by deficiency in or dysfunctional C1 esterase inhibitor. In contrast, a third HAE variant exists in patients that have normal C1 esterase inhibitor (HAE III). HAE III is associated with a single point mutation at residue Thr309 in factor XII. However, the mechanism of HAE III was unknown. This study characterizes the mechanism and therapy of HAE III. HAE III patientplasma and recombinant Thr309 mutated factor XII result in a double band or in a band with a lower molecular weight than wild-type factor XII in Western blotting. This is the consequence of a loss of glycosylation. The mutation in factor XII causes excessive activation of the kallikrein-kinin system resulting in enhanced production of bradykinin. Addition of C1 esterase inhibitor dose-dependently blocked bradykinin production in HAE types I and II, but not in HAE III. We generated a fully humanized antibody (3F7) that specifically interferes with activated factor XII proteolytic activity. 3F7 inhibits activated factor XII-driven cleavage of high molecular weight kininogen in a dose dependent manner and interferes with aberrant kallikrein-kinin system-triggered bradykinin formation in HAE III plasma. We reconstituted factor XII deficient mice with recombinant human mutated factor XII and established an HAE III transgenic mouse that expresses human Thr309-mutated factor XII in the liver using Tet-off transgenic technology. Intravital confocal scanning microscopy and tracer extravasation-based methods show excessive bradykinin-mediated vascular leakage in both F12 mice reconstituted with mutated factor XII and in HAE III transgenic mice when challenged with factor XII-contact activator. Both a kallikrein inhibitor and 3F7 reduce edema in HAE III associated leakage in mice. This study characterizes the mechanism of HAE III and establishes factor XII inhibition as a novel therapeutic strategy to interfere with excessive vascular leakage in HAE III and potentially, other causes of edema. LIST OF SCIENTIFIC PAPERS The thesis is based on the following publications and manuscript: I. Björkqvist, J., Lecher, B. , Maas, C. , and Renné, T. Zinc-dependent contact system activation induces vascular leakage and hypotension in rodents. Biological Chemistry, 2013, 394, 1195-204. II. Oschatz, C. , Maas, C. , Lecher, B. , Jansen, T. , Björkqvist, J., Tradler, T. , Sedlmeier, R. , Burfeind, P., Cichon, S. , Hammerschmidt, S. , Müller-Esterl, W. , Wuillemin, WA. , Nilsson, G. , and Renné, T. Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo. Immunity, 2011, 34, 258-68. III. Sala-Cunill, A. , Björkqvist, J., Senter, R. , Guilarte, M. , Cardona, V. , Labrador, M. , Nickel, KF. , Butler, L. , Luengo, O. , Kumar, P., Labberton, L. , Long, A. , Di Gennaro, A. , Kenne, E. , Jämsä, A. , Krieger, T. , Schlüter, H. , Fuchs, T. , Flohr, S. , Hassiepen, U. , Cumin, F. , McCrea, K. , Maas, C. , Stavrou, E. , and Renné, T. Plasma contact system activation drives anaphylaxis in severe mast cell–mediated allergic reactions. Journal of Allergy and Clinical Immunology, 2014, doi: 10.1016/j.jaci. 2014.07. 057 IV. Björkqvist, J., Oschatz, C. , Lewandrowski, U. , Schönig, K. , Noethen, M. , Sickmann, A. , Panousis, C. , Maas, C. , and Renné, T. Defective glycosylation of coagulation factor XII causes hereditary angioedema type III. Submitted Manuscript. V. Larsson, M. , Rayzman, V. , Nolte, MW. , Nickel, KF. , Björkqvist, J., Jämsä, A. , Hardy, MP. , Fries, M. , Schmidbauer, S. , Hedenqvist, P., Broomé, M. , Pragst, I. , Dickneite, G. , Wilson, MJ. , Nash, AD. , Panousis, C. , and Renné, T. A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk. Science Translational Medicine, 2014, 6, 222ra17 Publications by the author, which are not included in the thesis Björkqvist, J., Jämsä, A. , and Renné, T. Plasma kallikrein: the bradykininproducing enzyme. Thrombosis and Haemostasis, 2013, 110, 399-407 Björkqvist, J., Sala-Cunill, A. , and Renné, T. Hereditary angioedema: a bradykinin-mediated swelling disorder. Thrombosis and Haemostasis, 2013, 109, 368-74 Björkqvist, J., Nickel, KF. , Stavrou, E. , and Renné, T. In vivo activation and functions of the protease factor XII. Thrombosis and Haemostasis, 2014, doi: 10.1160/TH14-04-0311. Schürmann, D. , Herzog, E. , Raquet, E. , Nolte, MW. , May, F. , Müller-Cohrs, J., Björkqvist, J., Dickneite, G. , and Pragst, I. C1-esterase inhibitor treatment: preclinical safety aspects on the potential prothrombotic risk. Thrombosis and Haemostasis, 2014, doi: 10.1160/TH13-06-0469. Hansson, KM. , Björkqvist, J., Deinum, J. Addition of prothrombin to plasma can result in a paradoxical increase in activated partial thromboplastin time. Blood Coagulation and Fibrinolysis, 2014, doi: 10.1097/MBC.0000000000000161. Hansson, KM. , Björkqvist, J., Deinum, J. The Effect of Recombinant and Plasma Derived Prothrombin on Prothrombin Time (PT) in Human Plasma. International Journal of Laboratory Hematology, 2014, doi: 10.1111/ijlh.12293. CONTENTS