title: The connecting segment between both epidermal growth factor-like domains in blood coagulation factor IX contributes to stimulation by factor VIIIa and its isolated A2 domain.
abstract: The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu(84)-Thr(87) and the factor IX-specific loop Asn(89)-Lys(91) were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn(89)-Lys(91) resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu(84)-Thr(87) between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa.
authors.affiliation: Department of Plasma Proteins, Sanquin Research at CLB, 1066 CX Amsterdam, The Netherlands.
authors.forename: Patrick H N
authors.initials: PH
authors.lastname: Celie
authors.forename: Gunny
authors.initials: G
authors.lastname: Van Stempvoort
authors.forename: Caroline
authors.initials: C
authors.lastname: Fribourg
authors.forename: Leon J
authors.initials: LJ
authors.lastname: Schurgers
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Lenting
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
mesh_terms: D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D005164:Factor IX / Q000737:chemistry / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000577:Amides; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D015944:Factor VIIIa; D005164:Factor IX