Summary
Highly purified factor VIII was incubated for up to 24 hours in the presence of plasmin,
and the biological activities and peptide structure of the digestion products determined
at intervals. Procoagulant activity (VIIIC) was rapidly lost, but 17-32% of the initial
ristocetin cofactor (VIIIR: WF) activity remained after 24 hours. Immunoelectrophoresis
showed a progressive increase in rocket height and a concomitant increase in electrophoretic
mobility of the factor-VIII-related antigen (VIIIR: AG). Crossed immunoelectrophoresis
of the 24- hour digest showed three distinct precipitin arcs, of which the major one,
with intermediate anodal mobility, gave reactions of non-identity with the other two.
On sepharose gel chromatography the 24-hour digest gave three peaks: peak II contained
about 80% of the residual VIIIR: WF and resolved on SDS-polyacrylamide gels into a
series of peptides with apparent molecular weights between 125,000 and 185,000; these
were reduced by mercaptoethanol to fragments of 15,000-80,000 daltons, a 65,000 dalton
fragment being particularly strongly PAS positive. We conclude that large molecular
size is not a prerequisite for VIIIR :WF activity, and that the presence of factor-VIII
breakdown products may be a cause of misleading results in the determination of VIIIR:AG
by immunoelectrophoresis.
