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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-03-0843. This Article Full Text (PDF) All Versions of this Article: 2002-03-0843v1 100/8/2820    most recent Alert me when this article is cited Alert me if a correction is posted 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 Myles, T. Articles by Leung, L. L Search for Related Content PubMed PubMed Citation Articles by Myles, T. Articles by Leung, L. L Social Bookmarking                   What's this? Submitted March 19, 2002 Accepted May 2, 2002 The structural requirements for the activation of human factor VIII by thrombin Timothy Myles*, Thomas H Yun, and Lawrence L Leung Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA * Corresponding author; email: tmyles{at}stanford.edu. The coagulation factors V (FV) and VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Natural variants of these factors frequently lead to severe bleeding disorders. To understand the mechanisms of activation of FVIII by thrombin, we used a bank of mutant thrombins to define residues important for its activation. From the initial screening of 53 mutant thrombins for the activation of human recombinant FVIII, we mapped thrombin mutants with 50% or less activity to anion binding exosite-I (K21A, H66A, K65A, R68A, R70A and Y71A) and anion binding exosite-II (R98A), the Na+ binding site (E229A, R233A, D234A and D193A/K196A) and the 50-insertion loop (W50A), which were similar to our results for the activation of FV. The role of these residues for cleavage at Arg372 and Arg1689 was investigated using plasma FVIII. Anion binding exosite-I appears to be important for cleavage at both sites while the anion binding exosite-II residue R98A is important for cleavage at Arg372 alone. The E229A mutant, which contributes to the Na+ binding site, and the 50-insertion loop mutant W50A have severely impaired cleavage at both Arg372 and Arg1689. This suggests that the integrity of the active site and the Na+ bound form of thrombin are both important for its procoagulant activity against FVIII. 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