Studies on rabbit factor VIII

Abstract

It was reported that there were two forms of factor VIII procoagulant activity in rabbit plasma. In this work we studied the properties of these two forms, high molecular weight factor VIII (HMW FVIII) and low molecular weight factor VIII (LMW FVIII). The results were as follows.1) Rabbit plasma was chromatographed on 6% agarose at physiologic ionic strength. In agreement with previous reports, the factor VIII activity eluted in the void volume and in early fractions of the third protein peak.2) At -5°C, ice-cold 53.3% ethanol was added to rabbit plasma to a final concentration of 3%. The resulting cryoethanol precipitate was dissolved in barbital-saline buffer, then chromatographed on 6% agarose. The factor VIII activity was detected only in the void volume, suggesting that rabbit HMW FVIII was concentrated in the cryoethanol precipitate but LMWF VIII was not.3) Chromatography of rabbit plasma adsorbed with Al(OH)3 showed no activity of LMV FVIII, indicating that LMW FVIII was removed from plasma by Al(OH)3.4) Chromatography of rabbit HMW FVIII in the presence of 0.25M Ca2+ demonstrated that factor VIII activity was separated from the void volume and eluted later. From the elution position the molecular weight of Ca2+-dissociated LMW FVIII subcomponent seemed to be somewhat higher than that of native LMW FVIII in rabbit plasma. After the removal of Ca2+, the late-eluting fractions having factor VIII activity were mixed with void volume fractions and the mixture was rechromatographed at physiologic ionic strength. The FVIII activity was found in the void volume, which indicated that LMW FVIII subcomponent was recombined with void volume protein. These results were similar to those reported for human factor VIII.5) When rabbit serum was chromatographed on 6% agarose, the factor VIII activity was not found in the void volume but found in the same elution position in which rabbit plasma LMW FVIII was detected by chromatography using the same column. This indicated following three possibilities:a) During blood coagulation HIM FVIII was consumed but LMW FVIII wag not.b) LMW FVIII was produced from HMW FVIII and remained in serum.c) Procoagulant activity of later fractions was due to procoagulant active substance, which might be factor Xa or thrombin, other than LMW FVIII.