Stabilization Of FVIII: C By Calcium

Abstract

The well-known two phase decay curve of Factor VIII which has been attributed to enzyme degradation is not altered by the addition of specific protease inhibitors. However, complete stability of Factor VIII, for at least 24 hours, is accomplished when physiological levels of calcium are maintained. Addition of calcium plus heparin to whole blood at 0, 2, and even 4 hours, effectively restores Factor VIII activity to 0 time values and maintains this activity throughout the next 24 hours. The restoration of activity noted after 2 and 4 hours.of incubation is very rapid with the Factor VIII activity increasing within 15 minutes of addition of calcium. Data obtained from column chromatography indicates that the molecular distribution of Factor VIII between high and low molecular weight forms is affected both by time and by the level of calcium present in blood. When blood is collected either into heparin or a mixture of CPD plus heparin plus calcium chloride, the elution profile indicates that Factor VIII activity is equally distributed between high and low molecular weight forms, both at 0 and 24 hours with the total activity remaining unchanged during this time interval. However, when Factor VIII activity at 0 time is in the high molecular weight form with less than 10% in the low molecular weight form. By 24 hours there is virtually no low molecular weight activity remaining in this citrated plasma. The data demonstrate an absolute requirement for calcium in the maintenance of Factor VIII: C activity and suggests that the relative lability of Factor VIII during the first phase of the decay curve is due to the loss of VIII: C activity when calcium is not present at physiological concentrations to stabilize the molecule.