The role of Factor VIII light chain cleavage in Factor VIII activation and subunit interaction was investigated. Purified Factor VIII was dissociated into its separate subunits, and the isolated light chain was cleaved by thrombin at position Arg or by Factor Xa at position Arg. These Factor VIII light chain derivatives then were used for reconstitution with purified Factor VIII heavy chain to obtain heterodimers that were exclusively cleaved within the light chain. Intact and cleaved light chain could effectively be reassociated with heavy chain, with concomitant regain of Factor VIII cofactor function. The association rate constant of Factor Xa-cleaved light chain was found to be 3-fold lower than that of thrombin-cleaved or intact light chain, suggesting a role of the region Ser-Arg in subunit assembly. Dissociation rate constants, however, were independent of Factor VIII light chain cleavage. Low ionic strength was observed to promote association but to destabilize the Factor VIII heterodimer. At high ionic strength, Factor VIII dissociation was extremely slow (k 10 s) for all Factor VIII light chain derivatives, indicating 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 derivatives proved equally efficient in binding to Factor IXa (K 15 nM). Studies in a purified Factor X-activating system demonstrated that thrombin and Factor Xa activate Factor VIII to the same extent. However, Factor Xa differed from thrombin in that it cleaved at Arg rather than at Arg. Reassociated heterodimers of Factor VIII heavy chain and intact light chain did not promote Factor X activation. In contrast, heterodimers that contained cleaved light chain exhibited substantial Factor VIIIa activity. These data demonstrate that a single cleavage at either Arg or Arg converts the inactive Factor VIII heterodimer into an active cofactor of Factor IXa.

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