title: Residues Phe342-Asn346 of activated coagulation factor IX contribute to the interaction with low density lipoprotein receptor-related protein.
abstract: When blood coagulation factor IX is converted to activated factor IX (factor IXa), it develops enzymatic activity and exposes the binding sites for both activated factor VIII and the endocytic receptor low density lipoprotein receptor-related protein (LRP). In the present study we investigated the interaction between factor IXa and LRP in more detail, using an affinity-purified soluble form of LRP (sLRP). Purified sLRP and full-length LRP displayed similar binding to factor IXa. An anti-factor IX monoclonal antibody CLB-FIX 13 inhibited factor IXa.sLRP complex formation. Both the antibody and a soluble recombinant fragment of LRP (i.e. cluster IV) interfered with factor IXa amidolytic activity, suggesting that the antibody and LRP share similar binding regions near the active site of factor IXa. Next, a panel of recombinant factor IXa variants with amino acid replacements in the surface loops bordering the active site was tested for binding to antibody CLB-FIX 13 and sLRP in a solid phase binding assay. Factor IXa variants with mutations in the region Phe(342)-Asn(346), located between the active site of factor IXa and factor VIII binding helix, showed reduced binding to both antibody CLB-FIX 13 and sLRP. Surface plasmon resonance analysis revealed that the variant with Asn(346) replaced by Asp displayed slower association to sLRP, whereas the variant with residues Phe(342)-Tyr(345) replaced by the corresponding residues of thrombin showed faster dissociation. Recombinant soluble LRP fragment cluster IV inhibited factor IXa-mediated activation of factor X with IC(50) values of 5 and 40 nm in the presence and absence of factor VIII, respectively. This inhibition thus seems to occur via two mechanisms: by interference with factor IXa.factor VIIIa complex assembly and by direct inhibition of factor IXa enzymatic activity. Accordingly, we propose that LRP may function as a regulator of blood coagulation.
authors.affiliation: Department of Plasma Proteins, Sanquin Research at CLB, 1066 CX Amsterdam, The Netherlands.
authors.forename: Jakub
authors.initials: J
authors.lastname: Rohlena
authors.forename: Joost A
authors.initials: JA
authors.lastname: Kolkman
authors.forename: Ria C
authors.initials: RC
authors.lastname: Boertjes
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Lenting
mesh_terms: D000991:Antithrombins / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005164:Factor IX / Q000737:chemistry* / Q000378:metabolism; D005170:Factor X / Q000737:chemistry / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D000991:Antithrombins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D001216:Asparagine; D005164:Factor IX; D005170:Factor X; D005982:Glutathione Transferase; D015951:Factor Xa