CHARACTERIZATION OF DES-(741-1668)-FACTOR-VIII, A SINGLE-CHAIN FACTOR-VIII VARIANT WITH A FUSION SITE SUSCEPTIBLE TO PROTEOLYSIS BY THROMBIN AND FACTOR XA

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 Arg(1648), as Arg(740) of the heavy chain is f used to Ser(1669) 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 thrombi n and factor Xa, and the same result was obtained for heavy chain clea vage. In contrast, des-(741-1668)factor VIII cleavage by thrombin at p osition Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg(1721). The rate of cleavage at position Arg1689 by thrombin was also lower than t hat at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were simi lar to the rates obtained when plasma-derived factor VIII was activate d by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-f actor VIII proceeded at a higher rate by factor Xa than by thrombin. T hese results indicate that factor VIII activation is strongly associat ed with cleavage at position Arg(1689) or Arg(1721). For the interacti on between des-(741-1668)-factor VIII and von Willebrand factor, a K-d value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar t o that of heterodimeric factor VIII. The affinity of single-chain des( 741-1668)-factor VIII for factor Ma was found to be 27 +/- 6 nM. The i n vivo recovery and half-life of des-(741-1668)-factor VIII were asses sed in guinea pigs. Upon infusion of des-(741-1668)factor VIII at a do sage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in fact or VIII activity was obtained. The same recovery was determined for wi ld-type factor VIII. The half-life of des-(741-1668)-factor VIII was f ound to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recomb inant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its f usion site, binds with high affinity to von Willebrand factor and fact or IXa, and behaves like heterodimeric recombinant factor WI in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may p rove useful for the treatment of bleeding episodes in patients with ha emophilia A.