Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation

Sara Calzavarini; Bruno O. Villoutreix; Barbara Lunghi; Ruzica Livaja; Francesco Bernardi; Björn Dahlbäck

doi:10.1160/TH12-10-0780

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2013; 110(01): 31-38
DOI: 10.1160/TH12-10-0780

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation

Sara Calzavarini^§

¹Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy

²Inserm UMR-S 973, University Paris Diderot, Paris, France

³Department of Laboratory Medicine, Clinical Chemistry, University of Lund, University Hospital, Malmö, Sweden

,

Bruno O. Villoutreix

²Inserm UMR-S 973, University Paris Diderot, Paris, France

,

Barbara Lunghi

¹Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy

,

Ruzica Livaja

³Department of Laboratory Medicine, Clinical Chemistry, University of Lund, University Hospital, Malmö, Sweden

,

Francesco Bernardi

¹Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy

,

Björn Dahlbäck

³Department of Laboratory Medicine, Clinical Chemistry, University of Lund, University Hospital, Malmö, Sweden

› Author Affiliations

Abstract

Summary

Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients.

Keywords

Factor V deficiency - factor V structure - A domains - genotype-phenotype relationship

Full Text

References

References
1 Duckers C, Simioni P, Rosing J. et al. Advances in understanding the bleeding diathesis in FV deficiency. Br J Haematol 2009; 146: 17-26.
2 Kane WH, Davie EW. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 1988; 71: 539-555.
3 Zeibdawi AR, Grundy JE, Lasia B. et al. Coagulation factor Va Glu-96-Asp-111: a chelator-sensitive site involved in function and subunit association. Biochem J 2004; 377: 141-148.
4 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine Factor V and Factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-10962.
5 Suzuki K, Dahlback B, Stenflo J. Thrombin-catalyse d activation of human coagulation factor V. J Biol Chem 1982; 257: 6556-6564.
6 Shen L, Dahlbäck B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 1994; 269: 18735-18738.
7 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-1008.
8 Lunghi B, Scanavini D, Ulazzi L. et al. Expression and characterization of a new factor V mutation (R1698W) found in combination with FV Leiden -Reports from the 18th International Congress on Thrombosis. Pathophysiol Haemost Thromb 2003; 33: 48.
9 Delev D, Pavlova A, Heinz S. et al. Factor 5 mutation profile in German patients with homozygous and heterozygous factor V deficiency. Haemophilia 2009; 15: 1143-1153.
10 Villoutreix BO, Sperandio O. In silico studies of blood coagulation proteins: from mosaic proteases to nonenzymatic cofactor inhibitors. Curr Opin Struct Biol 2010; 20: 168-179.
11 Villoutreix BO, Dahlback B. Structural investigation of the A domains of human blood coagulation factor V by molecular modeling. Protein Sci 1998; 07: 1317-1325.
12 Pellequer JL, Gale AJ, Getzoff ED. et al. Three-dimensional model of coagulation factor Va bound to activated protein C. Thromb Haemostasis 2000; 84: 849-857.
13 Sorensen K W, Nicolaes GA, Villoutreix BO. et al. Functional properties of recombinant factor V mutated in a potential calcium-binding site. Biochemistry 2004; 43: 5803-5810.
14 Song J, Talbot K, Hewitt J. et al. Differential contributions of Glu96, Asp102 and Asp111 to coagulation factor V/Va metal ion binding and subunit stability. Biochem J 2009; 422: 257-264.
15 Steen M, Miteva M, Villoutreix BO. et al. Factor V New Brunswick: Ala221Val associated with FV deficiency reproduced in vitro and functionally characterized. Blood 2003; 102: 1316-1322.
16 Vincent LM, Tran S, Tran-Fadulu V. et al. Dysregulation of alternative splicing of coagulation factor V results in bleeding disorder, east Texas type. Blood. 2006 108. 57A-58A (Abstract 180).
17 Lunghi B, Iacoviello L, Gemmati D. et al. Detection of new polymorphic markers in the factor V gene: Association with factor V levels in plasma. Thromb Haemost 1996; 75: 45-48.
18 Kaufman RJ. Vectors used for expression in mammalian cells. Methods Enzymol 1990; 185: 487-511.
19 Norstrøm E, Thorelli E, Dahlbäck B. Functional characterization of recombinant FV Hong Kong and FV Cambridge. Blood 2002; 100: 524-530.
20 Steen M, Tran S, Autin L. et al. Mapping of the Factor Xa Binding Site on Factor Va by Site-directed Mutagenesis. J Biol Chem 2008; 283: 20805-20812.
21 Sun YH, Tran S, Norstrøm EA. et al. Enhanced rate of cleavage at Arg-306 and Arg-506 in coagulation factor Va by Gla domain-mutated human-activated protein C. J Biol Chem 2004; 279: 47528-47535.
22 Autin L, Steen M, Dahlbäck B. et al. Proposed structural models of the prothrombinase (FXa-FVa) complex. Proteins 2006; 63: 440-450.
23 Yin S, Ding F, Dokholyan NV. Eris: an automated estimator of protein stability. Nature Methods 2007; 04: 466-467.
24 Nicolaes GA, Villoutreix BO, Dahlbäck B. Partial glycosylation of Asn2181 in human factor V as a cause of molecular and functional heterogeneity. Biochemisty 1999; 38: 13584-13591.
25 Pemberton S, Lindley P, Zaitsev V. et al. A Molecular Model for the triplicated A Domains of Human Factor VIII Based on the Crystal Structure of Human Ceruloplasmin. Blood 1997; 97: 2413-2421.
26 Chi Ki, Ngo J, Huang M, Roth DA. et al. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure 2008; 16: 597-606.
27 Wakabayashi H, Varfaj F, Deangelis J. et al. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface. Blood 2008; 112: 2761-2769.

Supplementary Material

Online Supplementary Material (PDF)