Effects of Single Point Mutations within Proposed Copper Binding Sites on Specific Activity and Inter-Chain Affinity of Factor VIII.

Copper ions appear important for the structural integrity and the function of factor VIII. Reconstitution studies have shown that Cu²⁺ increases specific activity of factor VIII as well as increases affinity between heavy chain (HC) and light chain (LC). Based on the ceruloplasmin homology, the existence of three Cu²⁺ binding sites has been proposed. These include a type 1 site in HC (A1 domain) coordinated by C310, H315, H267, and M320, a type 1 site in LC (A3 domain) coordinated by C2000, H1954, H2005, and M2010, and type 2 site spanning A1 and A3 domains coordinated by H99 and H1957 with possible other additional residues. We rationalized that point mutations within a putative Cu²⁺ coordination sites would diminish Cu²⁺ binding at that site, as detected by changes in factor VIII specific activity and/or inter-subunit affinity. We produced several mutant factor VIII proteins bearing point mutation of C310S, H315A, C2000S, H1954A, H99A, or H1957A using a B-domainless human factor VIII vector. Each mutant was stably expressed and purified by SP-sepharose, which bound factor VIII primarily though LC. Western blotting indicated a reduction in the relative amount of HC in C310S and H99A factor VIII forms, suggesting that inter-chain affinity was reduced by these mutations. EDTA-treated factor VIII forms in the presence of Ca²⁺ were titrated with Cu²⁺ and activity was monitored using a factor Xa generation assay. The concentration of free Cu²⁺ was controlled by the presence of EDTA and Ca²⁺ based on the known values for Cu²⁺-EDTA and Ca²⁺-EDTA affinities. The activity regain observed for wild type factor VIII following titration with Cu²⁺ yielded a Kd = 11.5 ± 2.2 fM. All of the mutants tested retained a high affinity response to Cu²⁺, suggesting that single point mutations were not sufficient to eliminate Cu²⁺ binding. However, while Kd values for H1957A (10.3 ± 2.2 fM), H99A (2.1 ± 1.9 fM), H315A (6.7 ± 3.1 fM), and C310S (12.7 ± 3.4 fM) retained similar or slightly reduced affinity for Cu²⁺, the Kd values for C2000S (304 ± 105 fM) and H1954A (365 ± 105 fM) showed a significant reduction in Cu²⁺ affinity. When factor VIIIa subunits were titrated with Cu²⁺ and monitored by intrinsic fluorescence, only purified A1 subunit showed a saturable effect of the signal (Kd = 11.9 ± 4.9 fM), indicating that this subunit contained a high affinity Cu²⁺ binding site. Taken together, these results suggest that occupancy of the type 1 Cu²⁺ binding site in A3 contributes to the Cu²⁺-dependent increase in specific activity of factor VIII and that the type 1 site in A1 and/or the type 2 site contributes to HC-LC association.