CERIC Center of Excellence for Research on Inflammation and Cardiovascular disease

Thomas Renné | Featured Publications

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection

Linda Labberton, Ellinor Kenne, Andy T. Long, Katrin F. Nickel, Antonio Di Gennaro, Rachel A. Rigg, James S. Hernandez, Lynn Butler, Coen Maas, Evi X. Stavrou & Thomas Renné. Nature Communications 2016 6;7:12616

Background


Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII.

Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III

Jenny Björkqvist, Steven de Maat, Urs Lewandrowski, Antonio Di Gennaro, Chris Oschatz, Kai Schönig, Markus M. Nöthen, Christian Drouet, Hal Braley, Marc W. Nolte, Albert Sickmann, Con Panousis, Coen Maas, and Thomas Renné. Journal of Clinical Investigation 2015 Jul 20. pii: 77139. doi: 10.1172/JCI77139

Background


Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.

Plasma contact system activation drives anaphylaxis in severe mast cell-mediated allergic reactions

Anna Sala-Cunill, Jenny Björkqvist, Riccardo Senter, Mar Guilarte, Victoria Cardona, Moises Labrador, Katrin F. Nickel, Lynn Butler, Olga Luengo, Parvin Kumar, Linda Labberton, Andy Long, Antonio Di Gennaro, Ellinor Kenne, Anne Jämsä, Thorsten Krieger, Hartmut Schlüter, Tobias Fuchs, Stefanie Flohr, Ulrich Hassiepen, Frederic Cumin, Keith McCrae, Coen Maas, Evi Stavrou, and Thomas Renné. Journal of Allergy and Clinical Immunology pii: S0091-6749(14)01111-7. doi: 10.1016/j.jaci.2014.07.057

Background


Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mast cell–derived mediators into the circulation.

Objectives and Methods


We report here that a plasma protease cascade, the factor XII–driven contact system, critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects.

Results


Deficiency in or pharmacologic inhibition of factor XII, plasma kallikrein, high-molecular-weight kininogen, or the bradykinin B2 receptor, but not the B1 receptor, largely attenuated allergen/IgE-mediated mast cell hyperresponsiveness in mice. Reconstitutions of factor XII null mice with human factor XII restored susceptibility for allergen/IgE-mediated hypotension. Activated mast cells systemically released heparin, which provided a negatively charged surface for factor XII autoactivation. Activated factor XII generates plasma kallikrein, which proteolyzes kininogen, leading to the liberation of bradykinin. We evaluated the contact system in patients with anaphylaxis. In all 10 plasma samples immunoblotting revealed activation of factor XII, plasma kallikrein, and kininogen during the acute phase of anaphylaxis but not at basal conditions or in healthy control subjects. The severity of anaphylaxis was associated with mast cell degranulation, increased plasma heparin levels, the intensity of contact system activation, and bradykinin formation.

Conclusions


In summary, the data collectively show a role of the contact system in patients with anaphylaxis and support the hypothesis that targeting bradykinin generation and signaling provides a novel and alternative treatment strategy for anaphylactic attacks.

A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk

Magnus Larsson, Veronika Rayzman, Marc W. Nolte, Katrin F. Nickel, Jenny Björkqvist, Anne Jämsä, Matthew P. Hardy, Marion Fries, Stefan Schmidbauer, Patricia Hedenqvist, Michael Broomé, Ingo Pragst, Gerhard Dickneite, Michael J. Wilson, Andrew D. Nash, Con Panousis, and Thomas Renné. Science Translational Medicine 6(222):222ra17 (2014).
Currently used anticoagulants prevent thrombosis but increase bleeding. We show an anticoagulation therapy without bleeding risk based on a plasma protease factor XII function-neutralizing antibody. We screened for antibodies against activated factor XII (FXIIa) using phage display and demonstrated that recombinant fully human antibody 3F7 binds into the FXIIa enzymatic pocket. 3F7 interfered with FXIIa-mediated coagulation, abolished thrombus formation under flow, and blocked experimental thrombosis in mice and rabbits. We adapted an extracorporeal membrane oxygenation (ECMO) cardiopulmonary bypass system used for infant therapy to analyze clinical applicability of 3F7 in rabbits. 3F7 provided thromboprotection as efficiently as heparin, and both drugs prevented fibrin deposition and thrombosis within the extracorporeal circuit. Unlike heparin, 3F7 treatment did not impair the hemostatic capacity and did not increase bleeding from wounds. These data establish that targeting of FXIIa is a safe mode of thromboprotection in bypass systems, and provide a clinically relevant anticoagulation strategy that is not complicated by excess bleeding.

Time-dependent degradation and tissue factor addition mask the ability of platelet polyphosphates in activating factor XII–mediated coagulation

Katrin Faye Nickel, Henri M. Spronk, Nicola J. Mutch, and Thomas Renné. Blood 122(23):3847-9 (2013).
Platelets are known to promote coagulation in a factor XII (FXII)-dependent manner. Consistent with earlier studies that identified synthetic platelet-size polyphosphates (polyP) as FXII activators in vitro, we have shown that platelet polyP of 60 to 100 phosphate subunit lengths activate FXII in vivo, thus contributing to thrombus propagation. Independent laboratories have confirmed these findings and have shown that the ability of polyP to generate FXIIa in vitro increases with the chain length of the polymer. Inhibition of polyP interferes with thrombin receptor agonist peptide (TRAP)-activated platelet driven-coagulation.