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Functional characterization of Factor V-Ile359Thr, a novel mutation associated with thrombosis.

Steen, Mårten LU ; Norström, Eva LU ; Tholander, Ann-Louise LU ; Bolton-Maggs, Paula H B; Mumford, Andrew; McVey, John H; Tuddenham, Edward G D and Dahlbäck, Björn LU (2004) In Blood 103(9). p.3381-3387
Abstract
A missense mutation, FV-IIe359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-IIe359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306GIn/IIe359Thr/Arg679GIn, FV-IIe359Thr/Arg506GIn/Arg679GIn, and FV-Asn357GIn/IIe359Thr). The FV-IIe359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-IIe359Thr migrated more slowly, while the... (More)
A missense mutation, FV-IIe359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-IIe359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306GIn/IIe359Thr/Arg679GIn, FV-IIe359Thr/Arg506GIn/Arg679GIn, and FV-Asn357GIn/IIe359Thr). The FV-IIe359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-IIe359Thr migrated more slowly, while the FV-Asn357GIn/IIe359Thr was indistinguishable from FV-wild type (FV-WT), indicating that FV-IIe359Thr was expressed with an additional carbohydrate chain. Activated protein C (APC)-mediated inactivation in an FVa degradation assay showed that the IIe359Thr mutation significantly reduced the cleavage at Arg306 both in the presence and absence of protein S, whereas the cleavage at Arg506 was unaffected. When tested in an FVIIIa degradation assay, the FV-IIe359Thr variant exhibited equally low APC cofactor activity as FV Leiden (FVArg506GIn). In conclusion, the IIe359Thr mutation appears to affect anticoagulation by 2 mechanisms, impeding the APCmediated down-regulation of the FVa molecule and additionally being a poor APC cofactor for the down-regulation of FVIIIa. These findings explain the association of the FV-IIe359Thr mutation with thrombosis. (C) 2004 by The American Society of Hematology. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Blood
volume
103
issue
9
pages
3381 - 3387
publisher
American Society of Hematology
external identifiers
  • pmid:14695241
  • wos:000221565600033
  • scopus:1942425200
ISSN
1528-0020
DOI
10.1182/blood-2003-06-2092
language
English
LU publication?
yes
id
a698cce2-5ba3-4f25-8daf-6e93bf12468a (old id 119363)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14695241&dopt=Abstract
date added to LUP
2007-07-27 09:23:11
date last changed
2017-07-30 03:44:16
@article{a698cce2-5ba3-4f25-8daf-6e93bf12468a,
  abstract     = {A missense mutation, FV-IIe359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-IIe359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306GIn/IIe359Thr/Arg679GIn, FV-IIe359Thr/Arg506GIn/Arg679GIn, and FV-Asn357GIn/IIe359Thr). The FV-IIe359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-IIe359Thr migrated more slowly, while the FV-Asn357GIn/IIe359Thr was indistinguishable from FV-wild type (FV-WT), indicating that FV-IIe359Thr was expressed with an additional carbohydrate chain. Activated protein C (APC)-mediated inactivation in an FVa degradation assay showed that the IIe359Thr mutation significantly reduced the cleavage at Arg306 both in the presence and absence of protein S, whereas the cleavage at Arg506 was unaffected. When tested in an FVIIIa degradation assay, the FV-IIe359Thr variant exhibited equally low APC cofactor activity as FV Leiden (FVArg506GIn). In conclusion, the IIe359Thr mutation appears to affect anticoagulation by 2 mechanisms, impeding the APCmediated down-regulation of the FVa molecule and additionally being a poor APC cofactor for the down-regulation of FVIIIa. These findings explain the association of the FV-IIe359Thr mutation with thrombosis. (C) 2004 by The American Society of Hematology.},
  author       = {Steen, Mårten and Norström, Eva and Tholander, Ann-Louise and Bolton-Maggs, Paula H B and Mumford, Andrew and McVey, John H and Tuddenham, Edward G D and Dahlbäck, Björn},
  issn         = {1528-0020},
  language     = {eng},
  number       = {9},
  pages        = {3381--3387},
  publisher    = {American Society of Hematology},
  series       = {Blood},
  title        = {Functional characterization of Factor V-Ile359Thr, a novel mutation associated with thrombosis.},
  url          = {http://dx.doi.org/10.1182/blood-2003-06-2092},
  volume       = {103},
  year         = {2004},
}