Final Activity Report Summary - BI-FXIII-B (Stabilization of blood coagulation factor XIIIA in blood plasma: identification of the binding domain(s) involved in the binding with FXIII-A on FXIII-B) Normal haemostasis is a delicate balance between the natural procoagulant and anticoagulant system. The former system is responsible for the generation of a plug if the integrity of the vessel wall is disrupted. It is characterised as a cascade in which a series of enzyme reactions lead to the generation of the final effector enzyme, thrombin. Thrombin cleaves an abundant plasma protein, fibrinogen to form fibrin, which will polymerize and form the haemostatic plug. Thrombin activates factor XIII to FXIIIa, which stabilises fibrin with covalent crosslinking of its chains. Circulating FXIII consists of two potentially active A subunits and two protective B subunits (A2B2). During activation, the B subunits dissociate and the A subunits, from which thrombin cleaves off a 37 amino acid activation peptide, will achieve active conformation. Without the protective effect of FXIII-B, FXIII-A is unstable and is slowly eliminated from the circulation. This phenomenon can be of a great importance as it is a potential drug target. A novel type of anticoagulant might be useful for fine-tuning of FXIII-A levels. The main scientific goal of this project was to identify the previously unknown binding sites on FXIII-B, which participate in the binding of FXIII-B to FXIII-A. A recombinant approach was used. From a liver cDNA library, a fragment containing the intronless coding region of FXIII-B was cloned using overlapping Polymerase chain reaction (PCR) methodology. In this method, the full-length cDNA of FXIII-B was divided into three fragments, which were amplified individually using oligonucleotide primers and the resulted PCR products were re-amplified using the outer primers only. Final result of this second round was a PCR product containing the fragments together. In order to avoid the introduction of errors during the PCR, a high-fidelity Pfu DNA polymerase was used instead of Taq throughout the whole amplification process. Using different primer sets the full-length cDNA of FXIII-B could be generated in pure and highly concentrated form, i.e. ready to be cloned. This fragment was cloned then into a plasmid vector using two different restriction enzymes. The plasmid was a mammalian expression vector, which made the protein production in mammalian cell line possible. The vector contained two short peptide tags, a V5 and a His-tag in-frame; therefore the expressed proteins were in fusion with these tags in their C-terminal. The former made the detection and the latter the purification of the recombinant proteins possible. The integrity and error-free nature of the cloned cDNA was verified using sequencing. No mutation was found in the full-length FXIII-B clone and the assay proved that the cloning process was successful. Using this clone as a template, a series of new PCR assays were set up. In these methods, using different primer pairs, deletion mutants were generated. FXIII-B consists of 10 so-called Sushi (CCP, complement control protein or SCR, short consensus repeat) domains. Using the same PCR approach, 2, 4, 6 and 8 of these domains were deleted of the FXIII-B cDNA clone and inserted into the same expression vector. Sequence of the inserts was verified using DNA sequence analysis. After the cloning the transient expression studies were optimised. In the optimised transfection assay, we used COS-7 cell line and DEAE-dextran transfection method. 72 hours after the transfection, the recombinant medium was collected and the rFXIII-B mutants were partially purified and analysed using immunoblotting. All clones could be expressed. These proteins were most likely to be folded correctly as judged by reduced and non-reduced SDS-PAGE. In conclusion, in addition to the full-length FXIII-B, 4 recombinant mutants were constructed, expressed and partially purified using mammalian transient system. After scaling up, these mutants will be used in purified form in the binding studies with FXIII-A.
