Summary
Antiserum from rabbits immunised with pure human fibrinogen was affinity purified
on immobilised fibrin fragment E (FFE). This FFE antibody (Ab) induced significant
growth inhibition of a human cancer xenograft in mice and suppression of tumour angiogenesis,
leaving no formed vessels and only CD31-staining endothelial fragments in place. Tubule
formation of HUVEC on MatrigelTM was also significantly inhibited by FFE Ab. Since
MatrigelTM is fibrin-free, this effect implicated a different FFE Ab binding site
than FFE. Flow cytometry of HUVEC showed that FFE Ab bound to HUVEC, but with a broad
range of 55–98 %. Immunofluorescent staining of HUVEC explained this range, since
FFE Ab was seen not to bind to human umbilical vein endothelial cells (HUVEC) directly
but instead to a matrix protein variably adherent to HUVEC. This protein was identified
as fibronectin (FN) by appearance, staining with FN Ab, and by a FN knockdown study.
Neither HUVEC nor matrix reacted with fibrin D-dimer (DD) Ab. Immunofluorescent stains
of HUVEC matrix with FFE and FN Ab’s showed that these Ab’s bound to the same epitopes
on FN, as also seen on Western blots of purified FN. These findings indicate the presence
of an antigenic determinant in fibrinogen/FFE that is homologous with an epitope(s)
in FN recognised by FFE Ab, and critical for angiogenesis in this xenograft. The FN
epitope(s) remains to be identified, but the present findings can be used for the
selection of the appropriate clones from mice immunised with fibrinogen which can
facilitate this identification, and which may also be of clinical use.
Keywords
Fibrin fragment E (FFE) - FFE antibody - fibronectin - angiogenesis
