Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and Inter-Subunit Affinity

Abstract

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.

Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa

“…Two-stage Chromogenic Factor Xa Generation Assay-The rate of conversion of factor X to factor Xa was monitored in a purified system (19) according to methods previously described (20,21). Briefly, factor VIII (1 nM) in buffer containing 20 mM HEPES, pH 7.2, 0.1 M NaCl, 0.01% Tween 20, 0.01% bovine serum albumin, 5 mM CaCl 2 , and 10 M PS/PC/PE vesicles (Buffer A) was activated with 20 nM ␣-thrombin for 1 min.…”

Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa

“…The rate of conversion of FX to FXa was monitored in a purified system (25) according to methods previously described (26,27). FVIII (1 nM) in 20 mM HEPES, 0.1 M NaCl, 5 mM CaCl 2 , 0.01% Tween 20, 0.01% BSA, pH 7.2 (HEPES buffer), containing 20 μM PSPCPE vesicles (PS:PC:PE = 3:2:5) was activated with 30 nM α-thrombin for 1 min.…”

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

“…Experimental evidence has demonstrated that both factor V and VIII bind a single copper atom (31,32). A functional role for copper in factor V or Va has not yet been ascertained, but, in factor VIII, a type II copper leads to Ϸ100-fold affinity between the factor VIII subunits (33). In our structure, ligands to the Cu 2ϩ include His-1802, His-1804 (both predicted), and Asp-1844 in a trigonal planar coordination geometry.…”