Intracellular accumulation of factor VIII induced by missense mutations Arg593 -> Cys and Asn(618)-> Ser explains cross-reacting material-reduced haemophilia A

Patients with cross-reacting material (CRM)-reduced haemophilia A exhibit r educed levels of factor VIII antigen. In this study, we determined the mole cular basis of the genetic defect in the factor VIII gene induced by either the Arg(593)-->Cys or the Asn(618)-->Ser missense mutation, identified in two CRM-reduced haemophilia A patients. We introduced either the Arg(593)-- >Cys or the Asn(618)-->Ser mutation into a B-domain-deleted factor VIII cDN A and expressed the modified cDNAs in C127 cells. Reduced levels of factor VIII activity and factor VIII antigen in conditioned medium of transfected cells indicated that the secretion of both factor Vm variants was impaired. The ratio of factor VIII antigen present in cell extract to that in condit ioned medium was 1.9 and 2.4 times higher for rFVIII-R593C and rFVIII-N618S , respectively, than for rFVIII. Metabolic labelling and immunoprecipitatio n of transfected cells revealed that rFVIII-R593C and rFVIII-N618S persiste d somewhat longer inside the cell than factor rFVIII. Intracellular accumul ation and subsequent degradation of factor VIII-R593C and factor VIII-N618S may explain the reduced levels of both factor VIII activity and antigen in plasma of mild haemophilia A patients with corresponding genetic defects.