Ca concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.

Bonazza, Klaus; Rottensteiner, Hanspeter; Schrenk, Gerald; Fiedler, Christian; Scheiflinger, Friedrich; Allmaier, Günter; Turecek, Peter; Friedbacher, Gernot

FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca. Recombinant full-length FVIII, the purified heavy chain, light chain and B-domain as well as B-domain-deleted rFVIII were analysed in buffers of different Ca concentrations by atomic force microscopy. In the absence of Ca, FVIII appeared as a globular molecule, whereas at high amounts of Ca up to 50-nm long tail structures emerged. These tails could be identified as unravelled B-domains, as images of isolated B-domains showed the same morphology and heavy chains which include the B-domain were also rich of tails, whereas the isolated light chains and B-domain-deleted FVIII lacked any deviation from a globular shape. The images further suggested that the B-domain interacts with the light chain particularly at low Ca concentrations. Our results show a Ca-regulated conformational change of the B-domain in the context of full-length rFVIII. As the B-domain tightly associated with the core of the FVIII molecule under low Ca-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.

BLOOD coagulation factor VIII; CONFORMATIONAL analysis; ATOMIC force microscopy; CALCIUM; STRUCTURE-activity relationships; THROMBOSIS

Analytical & Bioanalytical Chemistry, 2015, Vol 407, Issue 20, p6051

1618-2642

Academic Journal

10.1007/s00216-015-8778-z