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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-03-0843.
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Blood, 15 October 2002, Vol. 100, No. 8, pp. 2820-2826
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Structural requirements for the activation of human factor VIII
by thrombin
Timothy Myles,
Thomas H. Yun, and
Lawrence L. K. Leung
From the Division of Hematology, Stanford University
School of Medicine, CA.
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
(Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and
anion-binding exosite-II (Arg98Ala), the Na+-binding site
(Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the
50-insertion loop (Trp50Ala), 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, whereas the
anion-binding exosite-II residue Arg98Ala is important for cleavage at
Arg372 alone. The Glu229Ala mutant, which contributes to the
Na+-binding site, and the 50-insertion loop mutant W50A
have severely impaired cleavage at Arg372 and Arg1689. This suggests
that the integrity of the active site and the Na+-bound
form of thrombin are important for its procoagulant activity against
FVIII. Detailed mutagenic analysis of thrombin can assist in
understanding the pathogenesis of bleeding disorders and may lead to
the rational design of selective thrombin inhibitors.
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