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Blood, 1 June 2005, Vol. 105, No. 11, pp. 4362-4368. Prepublished online as a Blood First Edition Paper on February 10, 2005; DOI 10.1182/blood-2004-10-3939. This Article Full Text Full Text (PDF) All Versions of this Article: 2004-10-3939v1 105/11/4362    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Nogami, K. Articles by Fay, P. J. Search for Related Content PubMed PubMed Citation Articles by Nogami, K. Articles by Fay, P. J. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY 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 From the Departments of Biochemistry and Biophysics and Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY. 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 (Km, apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (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. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J. L. Newell and P. J. Fay Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., August 31, 2007; 282(35): 25367 - 25375. [Abstract] [Full Text] [PDF] F. Varfaj, H. Wakabayashi, and P. J. Fay Residues Surrounding Arg336 and Arg562 Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C J. Biol. Chem., July 13, 2007; 282(28): 20264 - 20272. [Abstract] [Full Text] [PDF]

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