Effects of Copper on the Structure and Function of Factor VIII Subunits:  Evidence for an Auxiliary Role for Copper Ions in Cofactor Activity

Abstract

Factor VIII, a divalent metal ion-dependent heterodimer, contains a single copper atom, but the role of this metal in the structure and function of the cofactor is unclear. Earlier results showed that the dissociated heavy and light chains of factor VIII could be recombined in the presence of Ca(II) or Mn(II) but not Cu(II) to yield functional protein [Fay, P. J. (1988) Arch. Biochem. Biophys. 262, 525-531]. Inclusion of Cu(I) or Cu(II) inhibited the Mn(II)- or Ca(II)-dependent reconstitution of factor VIII with an IC50 approximately 10 micro M. The heavy chain was the susceptible subunit with inhibition by copper ion resulting from its reduced affinity for light chain. On the other hand, Mn(II)-dependent factor VIII reconstitutions performed with Cu(II) light chain and native heavy chain occurred at an accelerated rate (approximately 10-fold) and yielded an enhanced activity ( approximately 50%), likely reflecting an increased specific activity of the heterodimer. Cu ions enhanced the activity of EDTA-treated factor VIII in the presence of Ca(II) but not in its absence, suggesting that EDTA-treated factor VIII is not equivalent to separated subunits and that copper ions are auxiliary to ions that mediate reconstitution. Conformational analyses showed that the ellipticities and extrinsic fluorescence of both subunits were differentially affected by Cu(II) and Mn(II). These structural effects were fully reversed by EDTA. The metal ions had little if any effect on the conformation of intact factor VIII or the A1/A3-C1-C2 dimer. Mn(II) and Cu(II) stabilized the factor VIII light chain, and the latter stabilized the A1 subunit derived from the heavy chain, yielding similar thermal denaturation profiles that were distinct from that observed for the Ca(II)-stabilized subunits. Thus both subunits of factor VIII bind copper ions, and the effects of this binding differ from the interactions observed with Ca(II) or Mn(II). These data support a model where copper in factor VIII likely functions to increase specific activity of the heterodimer rather than directly mediating the intersubunit interaction.

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

“…This contention was further supported by the similarity in activity values obtained in the absence and presence of Cu 2+ . These results are in conflict with past observations suggesting that Cu 2+ made modest contributions to the specific activity of factor VIII [13,16]. We suggest these earlier observations in fact resulted from Cu 2+ -dependent enhancement of the inter-chain affinity to yield a greater fraction of reconstituted factor VIII heterodimers rather than a direct effect of Cu 2+ on specific activity.…”

“…Factor VIII activity is regenerated by combining the isolated subunits in the presence of Ca 2+ or Mn 2+ [9][10][11]. In addition, low levels of Cu + or Cu 2+ enhance this effect [12,13]. Thus, it was thought that the linkage of HC and LC by a metal ion (Ca 2+ , Mn 2+ , or Cu 2+ ) formed an active heterodimer.…”

The A1 and A2 Subunits of Factor VIIIa Synergistically Stimulate Factor IXa Catalytic Activity

“…5, authentic factor VIIIa-like activity (Ͼ100 nM factor Xa/min/nM factor IX) required reassociation of the three individual subunits. Although Ca(II) alone was sufficient to promote formation of significant factor VIIIa activity, the presence of Cu(II) during the association of A1 and A3-C1-C2 subunits enhanced the specific activity of the cofactor by ϳ2-fold, consistent with our earlier findings (23). The factor VIIIa reconstituted under these conditions showed a similar k cat (ϳ160 nM factor Xa/min/nM factor IXa) as observed earlier for native factor VIIIa (ϳ200 nM factor Xa/min/nM factor IXa, Ref.…”

A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes

“…EDTA is also known to mediate the dissociation of FVIII subunits (19). The inhibitory effect of EDTA was, however, not due to EDTA-induced FVIII dissociation, because dissociation of FVIII subunits requires higher EDTA concentrations and longer incubation periods than that used in our assays (19).…”