A preliminary study to find novel interaction partners for the coagulation factor XIII (FXIII) B subunit free form
S. Singh1, J. Dodt2, A. Reuter2, V. Ivaskevicius1, J. Oldenburg1, A. Biswas1 (1Bonn, Germany, 2Langen, Germany)
Bleeding disorders, coagulation and fibrinolytic factors
Date: 17.02.2017,
Time: 17:15 - 18:15
Objective: To improvise upon an immuno-affinity based purification strategy in order to pull down and detect putative novel interaction partners of the coagulation FXIII subunit B in its free form.
Methods: Full length FXIIIB subunit was derived from two sources: A) Commercial recombinant FXIIIB (Zedira, Darmstadt, Germany) B) Expressed, isolated and purified from HEK293T mammalian cell lines using monoclonal antibodies(Mab) specific for the B subunit. FXIII B from both sources were immobilized on α-FXIIIB monoclonal antibodies (raised in mice) using Pierce co-immunoprecipitation kit (Pierce biotechnology, Thermoscientific, Rockford, USA). Three set of experiments were designed: 1) α-FXIIIB bound rFXIIIB exposed to FXIII deficient plasma (Affinity biological) 2) only α-FXIIIB exposed to FXIII deficient plasma 3) only the immobilization membrane (without α-FXIIIB or rFXIIIB). Eluates from these three set up were run on an SDS PAGE and the resulting band were analyzed by peptide mass fingerprinting
Results: Amongst the bands that were attributed exclusively to the free B subunit two major types of proteins and their individual subunits/chains could be identified: 1) All three fibrinogen chains, i.e. α, β and γ chains were found to co-precipitate with the free form of the B subunit 2) The Complement protein C1qA and C1qb were also found to be co-precipitated with free B.
Conclusion: Recent reports have shown that the fibrinogen γ chain might be interacting with the free B subunit further confirming our findings. The α and β chains might have just co-precipitated alongwith the γ chain or might in fact also interact with the B subunit. The C1q complex might be interacting with the B subunit as well and this might represent one of the pleiotropic roles of the B subunit in the complement system. However both observations require further studies.