Structural requirements for the activation of human factor VIII by thrombin

Abstract

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. (Blood. 2002;100:2820-2826)

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

“…These results indicate that although alternate P1 residues exist in the R1689Q mutant, the P1 residue at 1689 does not impair thrombin binding. This result supports the contention that exosite binding is a significant contributor to thrombin binding to factor VIII (12,28).…”

“…This latter observation was based upon results showing that mutations at Arg-740, impairing this cleavage, significantly reduced cleavage rates at the two other P1 sites. Thrombin-catalyzed activation of factor VIII is dependent upon interactions involving the anion binding exosites of the proteinase (11,12). Exosite binding is believed to determine substrate affinity, whereas subsequent active site docking primarily affects V max (13).…”

Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII

“…The apparent K i value for R740Q factor VIII was 9.6 Ϯ 0.2 nM, which is similar to the K m (apparent) of wild-type factor VIII. These results suggest the R740Q mutation does not appreciably impair binding of thrombin to substrate factor VIII and are consistent with the activation of procofactor being largely driven by exosite interactions (28,29).…”

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…The concentration of active thrombin molecules was determined by titration with D-phenylalanylprolylarginyl chloromethyl ketone (PPACK; Calbiochem) using the chromogenic substrate H-D-Val-Leu-Arg-p-nitroanilide (S-2266, Chromogenix). A comparison of the catalytic activities of the purified WT and mutant thrombins toward the chromogenic substrate H-D-Phe-PipArg-p-nitroanilide (S-2388, Chromogenix) has been described in detail (36,38,39). Dextran sulfate (average M r 500,000) and cyanogen bromide-activated Sepharose 4B-agarose were purchased from Sigma.…”

“…Utilizing a collection of thrombin mutants generated by alanine scanning site-directed mutagenesis, we previously mapped the interactions of thrombin with some of its key substrates, including protein C, thrombin-activable fibrinolysis inhibitor, and thrombomodulin (36), fibrinogen (37), FV (38), and FVIII (39). In this study, we used this collection of thrombin mutants to identify the key residues on thrombin required for FXI activation.…”