APC Cleavage of Factor VIIIa in the Presence of Mutant Factor IXa

by: Steve Sayles

In this experiment a single-stage clotting assay was employed to monitor the interaction between factor VIIIa and factor IXa during APC (activated protein C) cleavage. Factor IXa can provide factor VIIIa protection from APC cleavage. IXa mutants with mutations in their EGF1 and 330 domains were reacted with factor VIIIa. These two domains of factor IX have been shown to play a key role in the formation of the factor Xase complex. The FVIIEGF1 had its EGF1 domain replaced by the EGF1 domain of factor VII. In the PCEGF1 mutant the EGF1 domain was replaced by protein C. The third factor IX form contained a mutation in which arginine was inserted to the 330 helix present in factor IX. Analysis of the reactions containing these three mutations revealed that the FVIIEGF1 mutant provided protection to factor VIII that was analogous to that offered by wild type factor IX. The PCEGF1 and R333Q mutant forms of factor IX did not appear to offer protection to factor VIII from APC cleavage suggesting this region of factor IX may be important in the interaction between factor VIIIa and factor IXa.

Research Advisor: Lynn M. O'Brien, Ph.D., Nazareth College