Submitted October 18, 2004
Accepted February 1, 2005
Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site
Keiji Nogami, Qian Zhou, Hironao Wakabayashi, and Philip J Fay*
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
* Corresponding author; email: Philip_Fay{at}urmc.rochester.edu.
Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) which possessed ~1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an ~80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed cleavage occurred at His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate as compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Km (apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested a ~70-fold reduced Vmax for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P1Arg, and this property likely reflects the severity of the hemophilia A phenotype.
