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

Abstract

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.

Kinetics of Factor VIII Light‐Chain Cleavage by Thrombin and Factor Xa

“…Secondly, a factor VIII variant lacking the sequence 771-1666 activated at a normal rate compared with plasma factor VIII [36]. Thirdly, we have previously described that activation of the variant des-(741-1668)-factor VIII lacking the entire B-domain is similar to that of plasma factor VIII [9]. Thus, it is very unlikely that B-domain sequences contribute to activation of factor VIII.…”

Intracellular accumulation of factor VIII induced by missense mutations Arg593→Cys and Asn618→Ser explains cross‐reacting material‐reduced haemophilia A

“…The presence of the point mutations in the constructed plasmids was veri®ed by sequence analysis using an Applied Biosystems 373a automated DNA sequencer. Mouse ®broblasts (C127 cells) were stably transfected with either the plasmids pCLBdB695-R593C or pCLB-dB695-N618S as described previously (Donath et al, 1995). Stably transfected C127 cells expressing the various factor VIII variants were cultured in Iscove's medium supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and 100 mg/ml hygromycin.…”

Enhanced Thrombin Sensitivity of a Factor VIII-Heparin Cofactor II Hybrid

“…The specific activity of the purified factor VIII ranged from 2000 to 4500 units/mg which is similar to that of factor VIII preparations derived from plasma. Activation of des-(868 -1562)-factor VIII-HCII and des-(868 -1562)-factor VIII by thrombin was monitored as described previously (24,27). To prevent feedback activation of factor VIII by activated factor X, acetylated factor X was used in our studies (29).…”

“…Tissue Culture and Transfection-Mouse fibroblasts were transfected with pCLB-dB695-HCII and pCLB-dB695 as described previously (27). Production of factor VIII was monitored by measuring both factor VIII activity and factor VIII antigen.…”