Modelling and expression studies of two novel mutations causing factor V deficiency

Daniel Delev; Anna Pavlova; Stefan Heinz; Mathias Costa Blaise; Tamir Chandra; Bernd Poetsch; Erhard Seifried; Johannes Oldenburg

doi:10.1160/TH08-02-0106

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 100(05): 766-772
DOI: 10.1160/TH08-02-0106

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Modelling and expression studies of two novel mutations causing factor V deficiency

Daniel Delev

¹Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

,

Anna Pavlova

¹Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

,

Stefan Heinz

²Institute for Transfusion Medicine and Immunohaematology, Red Cross Blood Donor Service, Baden-Wuerttemberg-Hessen, University Clinic Frankfurt, Germany

,

Mathias Costa Blaise

³Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, USA

,

Tamir Chandra

⁴Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK

,

Bernd Poetsch

¹Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

,

Erhard Seifried

²Institute for Transfusion Medicine and Immunohaematology, Red Cross Blood Donor Service, Baden-Wuerttemberg-Hessen, University Clinic Frankfurt, Germany

,

Johannes Oldenburg

¹Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

› Author Affiliations

Abstract

Summary

Human coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations,Tyr91Asn and Asp2098Tyr,found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation.Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu²⁺ binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.

Keywords

Factor V deficiency - novel mutations - MD simulation - transient expression studies

Full Text

References

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