pH-dependent association of factor VIII chains: Enhancement of affinity at physiological pH by Cu2+

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5–6.75 (Kd ∼4 nM), whereas a Kd ∼20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 μM total Cu2+) on maximal activity regenerated was negligible at pH 6.25–8.0. However, this level of Cu2+ increased the inter-chain affinity by ∼5-fold at pH 7.25. This effect resulted from an ∼1.5-fold increased association rate constant (kon) and an ∼3-fold reduced dissociation rate constant (koff). High affinity (Kd = 5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.