Mechanisms and regulation of the polyphosphate/factor XII-driven contact system in thrombosis and hemostasis (2016)

<p>Blood coagulation leading to fibrin formation is essential to prevent loss of blood (hemostasis), but can also contribute to occlusion of vessels (thrombosis). Thrombosis causes pulmonary embolism, myocardial infarction and stroke, which are together the most common cause of death in the developed world. Current anticoagulation therapy for prevention or treatment of thromboembolic events is sufficient, however results in an increase in potentially life threatening bleedings.</p><p>Two distinct pathways initiate fibrin formation: one pathway is triggered by tissue factor exposed on damaged vessel walls (extrinsic pathway) and the other by blood-borne factors (intrinsic pathway). The intrinsic pathway starts by activation of blood coagulation factor XII. The findings that factor XII appears to be involved in pathologic thrombus formation, and that factor XII deficiency is not associated with abnormal bleeding has led to a significant interest for factor XII in the scientific community. Inhibition of factor XII seemed to offer a selective and safe strategy for preventing thrombotic diseases.</p><p>Coagulation factor XII becomes activated by contact with negatively charged surfaces. In previous studies it is shown that platelets initiated factor XII-driven coagulation in vivo and this activation is driven by polyphosphate. Polyphosphate is an inorganic polymer that has been identified as a potent procoagulant and proinflammatory mediator in vitro and in vivo. Polyphosphate initiated fibrin formation by the factor XII-driven intrinsic pathway of coagulation. An infusion of polyphosphate induced lethal pulmonary emboli in mice, while a deficiency in factor XII or pharmacological inhibition of factor XII activity protected animals from this pathological clot formation. These data showed that polyphosphate driven factor XII activation is a necessary mechanism in platelet-driven thrombosis in vivo.</p><p>In this thesis we localize polyphosphate on activated platelets and establish and characterize recombinant tools to neutralize procoagulant polyphosphate activities based on a bacterial exopolyphosphatase. Exopolyphosphatase is an enzyme, which specifically breaks down polyphosphate. Using mutagenesis we identify a specific polyphosphate-binding mutant based on the binding domain of the exopolyphosphatase, resulting in a dual strategy of binding and degrading polyphosphate to interfere with procoagulant polyphosphate activities. Both strategies block the ability of polyphosphate to activate factor XII in different in vitro assays. Furthermore pretreatment of mice with exopolyphosphatase or the specific polyphosphate-binding mutant interferes with arterial thrombosis and protects from lethal pulmonary thromboembolism induced by platelet activity. Moreover the bleeding time of these mice are completely normal, showing that targeting of polyphosphate does not interfere with hemostasis. Finally, we use the specific polyphosphate-binding mutant as a probe to analyze polyphosphate on cells and in human samples. We present the first assay to analyze polyphosphate in human platelet-rich plasma offering the opportunity of analyzing a possible thrombotic biomarker in future clinical trials.</p><h3>List of scientific papers</h3><p>I. Polyphosphate nanoparticles on the platelet surface trigger contact system activation. Johan J.F. Verhoef, Arjan D. Barendrecht, Katrin F. Nickel, Kim Dijkxhoorn, Steven de Maat, Ellinor Kenne, Linda Labberton, Owen McCarty, Raymond Schiffelers, Antoni P. Hendrickx, Huub Schellekens, Marcel H. Fens, Thomas Renné, Coen Maas. [Submitted]</p><p>II. 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 M. Butler, Coen Maas, Evi X. Stavrou, Thomas Renné. Nature Communications 2016;7:12616 <br><a href="https://doi.org/10.1038/ncomms12616">https://doi.org/10.1038/ncomms12616</a><br><br> </p><p>III. A flow cytometry-based assay for procoagulant platelet polyphosphate Linda Labberton, Andy T. Long, Sandra J. Gendler, Christine Snozek, Evi X. Stavrou, Katrin F. Nickel, Coen Maas, Stefan Blankenberg, James S. Hernandez, Thomas Renné. [Submitted]</p><p>IV. The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis. Katrin F. Nickel, Göran Ronquist, Florian Langer, Linda Labberton, Tobias A. Fuchs, Carsten Bokemeyer, Guido Sauter, Markus Graefen, Nigel Mackman, Evi X. Stavrou, Gunnar Ronquist, Thomas Renné. Blood 2015;126(11):1379-1389 <br><a href="https://doi.org/10.1182/blood-2015-01-622811">https://doi.org/10.1182/blood-2015-01-622811</a><br><br> </p>