THE ROLE OF CLEAVAGE OF THE LIGHT-CHAIN AT POSITIONS ARG(1689) OR ARG(1721) IN SUBUNIT INTERACTION AND ACTIVATION OF HUMAN BLOOD-COAGULATION FACTOR-VIII

The role of Factor VIII Light chain cleavage in Factor VIII activation and subunit interaction was investigated. Purified Factor VIII was di ssociated into its separate subunits, and the isolated light chain was cleaved by thrombin at position Arg(1689) or by Factor Xa at position Arg(1721). These Factor VIII light chain derivatives then were used f or reconstitution with purified Factor VIII heavy chain to obtain hete rodimers that were exclusively cleaved within the Light chain, Intact and cleaved light chain could effectively be reassociated with heavy c hain, with concomitant regain of Factor VIII cofactor function. The as sociation rate constant of Factor Xa-cleaved light chain was found to be 3-fold lower than that of thrombin-cleaved or intact light chain, s uggesting a role of the region Ser(1690)-Arg(1721) in subunit assembly . Dissociation rate constants, however, were independent of Factor VII I light chain cleavage. Low ionic strength was observed to promote ass ociation but to destabilize the Factor VIII heterodimer. At high ionic strength, Factor VIII dissociation was extremely slow (k(off) approxi mate to 10(-5) s(-1)) for all Factor VIII light chain derivatives, ind icating that Factor VIII light chain cleavage is not related to Factor VIII dissociation. Furthermore, Factor VIII light chain cleavage does not affect enzyme-cofactor assembly, since the various light chain de rivatives proved equally efficient in binding to Factor IXa (K-d appro ximate to 15 nM). Studies in a purified Factor X-activating system dem onstrated that thrombin and Factor Xa activate Factor VIII to the same extent. However, Factor Xa differed from thrombin in that it cleaved at Arg(1721) rather than at Arg(1689). Reassociated heterodimers of Fa ctor VIII heavy chain and intact light chain did not promote Factor X activation. In contrast, heterodimers that contained cleaved light cha in exhibited substantial Factor VIIIa activity. These data demonstrate that a single cleavage at either Arg(1689) or Arg(1721) converts the inactive Factor VIII heterodimer into an active cofactor of Factor IXa .