Preparation of Modified Bovine Factor VIII with Enhanced Biological Activity Using Insoluble-trypsin Columns

Abstract

SummaryCrude bovine Factor VIII preparations have been modified by passage over a column of trypsin-agarose. Factor VIII prepared without an early adsorption step utilizing either barium sulfate or aluminum hydroxide gel generated significant Factor Xa activity by passage over such a column and was unsuitable for further characterization. Factor VIII prepared using adsorption with barium sulfate contained no Factor Xa activity after passage over trypsin-agarose. This trypsin-modified material showed a decreased molecular size as judged by behavior on gel filtration and an increased rate of reaction with bovine Factor IXa.

Effects of thrombin treatment of preparations of factor VIII and the Ca2+-dissociated small active fragment.

“…A decrease in the apparent molecular size of factor VIII has recently been shown to accompany activation with trypsin (30). The enzymatic activation of factor VIII is probably complex and numerous speculations are possible.…”

The Role of the Liver in Biosynthesis of the Non-Vitamin K-Dependent Clotting Factors

“…Further complicating the issue is the fact that certain of these factors can undergo changes which may or may not affect biologic activity but greatly affect its half-life. For example, Factor VIII, which has been modified by insoluble trypsin such that no change in one-stage activity occurs [107] has a drastically reduced in vivo half-life [61]. In this regard it is interesting to note that the procoagulant fragment of calcium-dissociated Factor VIII is rapidly cleared [3].…”