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Thromb Haemost 2007; 98(06): 1170-1181
DOI: 10.1160/TH07-05-0353
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII

Andrey G. Sarafanov
1   Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA;
,
Evgeny M. Makogonenko
1   Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA;
,
Olav M. Andersen
2   Max-Delbrueck Center for Molecular Medicine, Berlin, Germany;
,
Irina A. Mikhailenko
3   Department of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA
,
Natalya M. Ananyeva
1   Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA;
,
Alexey V. Khrenov
1   Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA;
,
Midori Shima
4   Department of Pediatrics, Nara Medical University, Nara, Japan
,
Dudley K. Strickland
3   Department of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA
,
Evgueni L. Saenko
1   Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA;
› Author Affiliations Financial support: This study was supported by NIH grants HL 66101 and HL 72929, and Judith Graham Postdoctoral Research Fellowship grant from National Hemophilia Foundation.
Further Information

Publication History

Received 15 May 2007

Accepted after resubmission 22 September 2007

Publication Date:
30 November 2017 (online)

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Summary

Catabolism of coagulation factorVIII (FVIII) is mediated by lowdensity lipoprotein receptor-related protein (LRP). The ligandbinding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains. This study was aimed to identify specific complementtype repeats of LRP involved in interaction with the A2 site and to probe their functional importance in A2 catabolism. We generated individual LRP clusters II, III and IV, along with nine overlapping CR triplets encompassing clusters II and IV in a baculovirus expression system and studied their interaction with isolated A2. In surface plasmon resonance (SPR) assay, A2 bound to clusters II and IV with KDs 22 and 39 nM, respectively, and to the majority of CR triplets with affinities in the range of KDs 25–90 nM. Similar affinities were determined for A2 interaction with a panel of CR doublets overlapping cluster II (CR 3–4, 4–5, 5–6 6–7 and 7–8). These LRP fragments inhibited the binding of 125I-A2 to LRP in solid-phase assay,LRP-mediated internalization of 125I-A2 in cell culture and 125I-A2 clearance from the mouse circulation. Point mutations of critical A2 residues of the LRPbinding site resulted in differential reduction or abolishment of its binding to LRP fragments. We conclude that A2 interacts with LRP via multiple binding sites spanning CR 3–8 in cluster II and CR 23–29 in cluster IV, and the minimal A2-binding unit of LRP is formed by two adjacent CR.

Keywords

Coagulation factor VIII - low-density lipoprotein receptor-related protein
 

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