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Previous studies demonstrated that plasma from dogs homozygous for von Willebrand’s disease (VWD) without detectable factor VUI-related antigen (VIII:RAg) contained moderate levels of a stable factor VIH-coagulant (VIII:C), which had lower apparent molecular weight and combined with the VIII:RAg of canine hemophilic plasma. We have now compared this VWD-VIII:C to the CaCl2-separated form of VIII:C prepared from normal canine factor VIII. The VWD plasma was fractionated at room temperature by 6% agarose gel filtration in 0.15M NaCl and 0.24M CaCl2 buffers, pH 7.25; the VIII:C eluted in each buffer with a relative elution volume (Ve/Vo) of 1.4. Isolated lower molecular weight VIII:C stabilized with albumin (5 mg/ml) and dialysed free of Ca++ was prepared from normal purified canine factor VIII by elution in 0.24M CaCl2 buffer. This CaCl2~separated VIII:C was rechromatographed in both buffers as above and had a Ve/Vo of 1.9. When the VWD and CaCl2 forms of VIII:C were each combined with canine hemophilic plasma and gel filtered in 0.15M NaCl buffer, the VIII:C’s now appeared at the void volume. These mixtures with 1/10 volume 2.4M CaCl2 added before or after combination were also analyzed in 0.24M CaCl2 buffer. The CaCl2-VIII:C eluted with a Ve/Vo of 1.9 regardless of the order of 2.4M CaCl2 addition, but the VWD-VIII:C eluted with Ve/Vo’s of 1.4 when CaCl2 was added before mixing and 1.9 when added afterwards.  Thus, when mixed with hemophilic plasmas both the CaCl2 and VWD forms of VIII:C exhibited similar combination and separation characteristics. However, the finding that the CaCl2-VIII:C is of apparent lower molecular weight than the VWD form suggests that the CaCl2-separated VIII:C is different from the naturally occurring form of VWD (VIII: RAg-free) VIII:C.

Comparison Of Naturally Occurring And CaCl2-Separated Canine Factor VIII-Coagulants Free Of FACTOR VIII-Related Antigen