Summary. Patients with cross-reacting material (CRM)-reduced haemophilia A exhibit reduced levels of factor VIII antigen. In this study, we determined the molecular basis of the genetic defect in the factor VIII gene induced by either the Arg 593 !Cys or the Asn 618 !Ser missense mutation, identi®ed 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 cDNA and expressed the modi®ed 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 VIII variants was impaired. The ratio of factor VIII antigen present in cell extract to that in conditioned medium was 1´9 and 2´4 times higher for rFVIII-R593C and rFVIII-N618S, respectively, than for rFVIII. Metabolic labelling and immunoprecipitation of transfected cells revealed that rFVIII-R593C and rFVIII-N618S persisted somewhat longer inside the cell than factor rFVIII. Intracellular accumulation 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.

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A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Substitution of Arg2307 by Gln in factor VIII has been found to be associated with mild to moderate haemophilia A [Gitschier, Wood, Shuman and Lawn (1986) Science 232, 1415-1416]. We have introduced this particular point mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNA in C127 cells. Cells expressing the resulting protein, termed des-(868-1562)-factor VIII-R2307Q, were compared with those expressing the previously characterized des-(868-1562)-factor VIII. No immunoreactive material could be detected in the conditioned medium of cells transfected with des-(868-1562)-factor VIII-R2307Q cDNA using assays specific for the factor VIII light chain and the factor VIII heavy chain. Analysis of metabolically labelled cells transfected with des-(868-1562)-factor VIII-R2307Q cDNA revealed that this mutant protein is synthesized at a level similar to des-(868-1562)-factor VIII. In contrast to des-(868-1562)-factor VIII, metabolically labelled des-(868-1562)-factor VIII-R2307Q was not encountered in the conditioned medium of the transfected cells, indicating that the mutant protein is not secreted from the cell. Inspection of the intracellular localization of the two proteins in the cell employing morphological analysis, endoglycosidase H and experiments with inhibitors of glucosidases I and II was consistent with localization of des-(868-1562)-factor VIII and des-(868-1562)-factor VIII-R2307Q in the endoplasmic reticulum. Taken together, our data indicate that the Arg2307-->Gln mutation results in aberrant intracellular trafficking of factor VIII, which may explain the low levels of factor VIII antigen in the plasma of haemophilia A patients that carry this particular point mutation.