Inactivation of FVIII by Plasmin: Molecular Conformational Changes

Reduced SDS PAGE failed to detect proteolysis of the 200,000 subunit when purified FVIII was slowly inactivated by plasmin until less than 1% of the VIIIc was left. Graphs of VIIIc loss show highly repeatable discontinuities (8 expts.), suggesting a conformational change, as do transitorily increased then partially decreased VIII R:AG levels. The mobility of FVIII as judged by crossed-immunoelectrophoresis remained unchanged, but the area under the ‘arcs’ increased dramatically. Crossed-immunoelectrophoresis plates were washed, the ‘arcs’ cut out, melted and subjected to reduced SDS PAGE. Only the 200,000, IgG ‘H’ and ‘L’ bands were seen; the latter increased markedly in contrast to the 200,000 band. Also SDS denatured digestion aliquots were treated with a trace of mercaptoethanol and applied to running SDS PA gels. A rise, then fall in the 200,000 monomer:dimer ratio showed that during inactivation FVIII is more thoroughly ‘disorganised’ by SDS. 2.75 % PAGE (1) or SDS PAGE in similar gels (2) show that a linear relationship obtains between the log of the FVIII oligomer no. and their relative mobilities up to oligomers ‘8’ and ‘6’ respectively; above this new linear relationships obtain. When FVIII is briefly inactivated by a higher concentration of plasmin the ‘inflection point’ in (1) is lost and later reappears at ‘5’; in (2) it shifts to oligomer no. ‘3’ long after inactivation. These results show that during inactivation by plasmin FVIII undergoes a molecular ‘opening-out’ to reveal more antigenic sites before detectable breakdown of the 200,000 subunits and that changes occur in the native oligomeric series.