Enhancing thrombin-mediated activation of factor XI: a novel therapeutic approach to control bleeding? (LSBR 1702)
Project leader: Prof. Dr. Joost C.M. Meijers (Plasma Proteins, Sanquin Research)
PhD student: Ms. Awital Bar Barroeta, (April 2018 – April 2022)
Technician: J. Arnoud Marquart (May 2018 – May 2020)
Haemostasis is a tight balance between coagulation and bleeding. Disruption of this balance will either lead to over-coagulation (thrombosis) or under-coagulation (bleeding). The coagulation cascade is a protein network central to haemostasis that is involved in maintaining this tight balance. Factor XI (FXI) is one of the proteins in this network. When it is activated by thrombin it plays a supporting role in haemostasis, preventing the degradation of a formed clot. However, FXI has also been shown to play a role in thrombosis, mainly related to activation by factor XIIa (FXIIa). FXI functions by activating factor IX (FIX). In our research we looked at the correlation between the structure of FXI and its function. The structure of inactive FXI is known, and it looks like a cup on a saucer. We have found that when FXI is activated a binding space for factor IX becomes more accessible. This corresponds with the protein model we made of activated in which the “cup” moves 180 degrees with respect to the saucer revealing the FIX binding site. We have also set up a protein assay in which we can monitor the binding between FXI and thrombin. In this assay both proteins are labelled with fluorescent tags that will only emit light at a desired wavelength if the tags are close to one another, i.e. upon protein binding. We have shown that the assay can quantify the interaction between FXI and thrombin and is sensitive enough to monitor any changes in the system induced by control molecule. Moreover, the assay is sensitive and robust enough to be optimised for use on a high-throughput scale. Lastly, we have developed nanobodies, which are the isolated heavy chain domains of a camelid antibody, that target FXI. We have characterised six nanobodies that can effectively inhibit its function. Five of these nanobodies prevent FIX from binding to FXI by binding to the same binding site on FXI, rendering it impossible to perform its function in the coagulation cascade. The sixth nanobody, competes with the interaction between FXI and high-molecular weight kininogen, which controls the FXI localisation needed for activation, and can thereby prevent FXI activation. Moreover, the binding site of this nanobody sheds more light on the binding conformation of HK on FXI. These nanobodies could be used as lead compounds in drug development.