Activated
protein C (APC) binds to its substrates
activated factor V (FVa) and
activated factor VIII (FVIIIa) with a basic exosite that consists of loops 37, 60, 70 and the
autolysis loop. These loops have a high density of basic residues, resulting in a positive charge on the surface of APC. Many of these residues are important in the interaction of APC with FVa and FVIIIa. The current study focused on the function of the
autolysis loop in the interaction with FVIIIa. This loop was previously shown to interact with FVa, and it inhibits APC inactivation by plasma
serpins. Charged residues of the
autolysis loop were individually mutated to
alanine and the activity of these mutants was assessed in functional FVIIIa inactivation assays. The
autolysis loop was functionally important for FVIIIa inactivation. Mutation of R306, K311 and R314 each resulted in significantly reduced FVIIIa inactivation. The inactivating cleavages of FVIIIa at R336 and R562 were affected equally by the mutations.
Protein S and FV stimulated cleavage at R562 more than cleavage at R336, independent of mutations in the
autolysis loop. Together, these results confirmed that the
autolysis loop plays a significant role as part of the basic exosite on APC in the interaction with FVIIIa.