Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Keiji Nogami,Lawrence L.K Leung,Timothy Myles,Philip J Fay,Qian Zhou,Hironao Wakabayashi

doi:10.1074/jbc.m412778200

Keiji Nogami, Lawrence L.K Leung + Show 4 more

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https://doi.org/10.1074/jbc.m412778200

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Abstract

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.

Highlights

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor
The results from the two binding assays are mutually supportive. These results suggest that thrombin binding to the A1 subunit is dependent upon reaction conditions, with the fast form binding this subunit with high affinity, whereas thrombin binding the A2 subunit occurs at a moderate affinity site independent of occupancy of the Naϩ site in thrombin
Effects of A2 N- and C-terminal, Acidic-rich Peptides on Factor VIII Activation and Cleavage by Thrombin—Based upon the observations that the affinity of thrombin for A2 subunit appears independent upon Naϩ occupancy of the enzyme, likely reflects binding to a single site, and relies heavily on the anion-binding exosite II for this interaction, we focused a series of studies on the interaction of thrombin with this subunit

Summary

Introduction

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain Peptides encompassing these clustered acidic regions, residues 373–395 and 719 –740, blocked thrombin cleavage of the isolated heavy chain at Arg372 and Arg740 and inhibited A2 binding to thrombin Ser205 Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. This mutant was resistant to cleavage at Arg740, whereas cleavage at Arg372 was not affected These data suggest the acidic region comprising residues 389 –394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg740 during procofactor activation. The serine proteinases, thrombin and factor Xa, function as a positive feedback amplifier of the coagulation cascade through the specific cleavage of factor VIII (5) Both enzymes convert this procofactor to the active cofactor, factor VIIIa, following limited proteolysis. Whereas interaction with exosite I appears essential for factor VIII activation through cleavages of Arg372 and Arg1689, exosite II seems to have a more limited role in facilitating cleavage at Arg372 (17)

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