Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Factor VIIIa is essential for blood coagulation and is reconstituted from subunits.
• Previous work showed a truncated Factor A1 fragment (A1(37-336)) has reduced affinity for Factor A2 but retains some activity.
• Understanding subunit interactions is key to Factor VIIIa function.

Purpose of the Study:

• To identify specific regions of Factor A1 that interact with Factor A2.
• To characterize the pH-dependence of these interactions.
• To elucidate the structural basis of Factor VIIIa reconstitution.

Main Methods:

• Limited tryptic digestion of Factor A1 to generate fragments.
• Assays to measure inhibition of Factor VIIIa reconstitution.
• Fluorescence energy transfer to study A1-A2 interactions.
• Covalent cross-linking to confirm direct A1-A2 binding.

Main Results:

• Two A1 fragments, A1(37-121) and A1(221-336), significantly inhibited Factor VIIIa reconstitution at pH 6.0.
• These inhibitory effects were pH-dependent, with reduced inhibition at pH 7.2.
• Direct binding and cross-linking were observed between A2 and A1(37-121), but not A1(221-336).

Conclusions:

• Factor A1 possesses an extended, pH-dependent A2-interactive surface involving regions 37-121 and 221-336.
• The interaction surface is conformationally labile and stabilized upon binding to the A3-C1-C2 dimer.
• These findings provide insights into the mechanism of Factor VIIIa assembly and function.