The activity of factor VIIIa was enhanced and stabilized by treatment of factor VIII with the crosslinker disuccinimidyl suberate. The activity was > 200-fold higher compared with that of native factor VIIIa and was stable for at least 15 days at 4 degrees C and pH 7.2. The crosslinked factor VIIIa was purified by immunoaffinity chromatography and gel filtration. Electrophoretic analysis revealed high-molecular-mass (approximately 150 kDa) molecules as well as the three bands characteristic of native factor VIIIa. Thus crosslinking appeared to yield molecules stabilized by intra- and/or inter-subunit crosslinks. The material was further fractionated using immobilized von Willebrand factor and the factor VIIIa activity could be ascribed to trimers containing only intra-subunit crosslinks. Moreover, reduction of crosslinked factor VIIIa produced using dithiobis(succinimidylpropionate) suggested that the molecules containing intersubunit crosslinks had not been cleaved by thrombin at arginine 372.