Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site
Year: 2005
Type: article
Abstract: 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) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Michaelis-Menten constant (K(m), apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (V(max)) 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 P(1)Arg, and this property likely reflects the severity of the hemophilia A phenotype.
Source: Blood
Institutions: University of Rochester Medicine, University of Rochester
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2021770322