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Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

ABSTRACT: The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

SUBMITTER: Newell JL

PROVIDER: S-EPMC2670113 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

Newell Jennifer L JL Fay Philip J PJ

The Journal of biological chemistry 20090224 17

The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor ...[more]

PMID: 19240027

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Project description: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.

Dataset Information

Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

Publications

Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

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