Advanced

Novel APC-cleavage sites in FVa providing insights into mechanisms of action of APC and its cofactor protein S.

Tran, Sinh LU and Dahlbäck, Björn LU (2010) In Journal of Thrombosis and Haemostasis 8(1). p.129-136
Abstract

BACKGROUND: Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined.

OBJECTIVE: To identify and characterize the additional APC-cleavage sites in FVa.

METHODS: The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV... (More)

BACKGROUND: Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined.

OBJECTIVE: To identify and characterize the additional APC-cleavage sites in FVa.

METHODS: The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV variants were expressed and their sensitivity for APC characterized functionally and with Western blotting.

RESULTS: The additional APC-cleavage sites were located at Lys309, Arg313, Arg316, Arg317 and Arg505. FVa-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q (denoted 8M-FVa) was APC resistant. To investigate individual sites, they were mutated back using 8M-FV as a template. The kinetics of APC-degradation of these variants demonstrated that protein S was equally efficient in enhancing the APC effect for all the novel sites.

CONCLUSIONS: Multiple APC-cleavage sites close to Arg306 and a single site close to Arg506 were identified. Protein S was equally efficient as APC cofactor for all novel sites. The stimulation by protein S of the Arg505 cleavage argues against a specific protein S-mediated stimulation of cleavage at Arg306 due to relocation of the APC active site closer to the membrane.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Amino Acid Sequence, Arginine, Factor Va, Humans, Kinetics, Lysine, Mutagenesis, Site-Directed, Mutation, Phospholipids, Protein C, Protein Processing, Post-Translational, Protein S, Structure-Activity Relationship, Substrate Specificity, Journal Article, Research Support, Non-U.S. Gov't
in
Journal of Thrombosis and Haemostasis
volume
8
issue
1
pages
8 pages
publisher
Federation of European Neuroscience Societies and Blackwell Publishing Ltd
external identifiers
  • wos:000272864200022
  • pmid:19874463
  • scopus:73049099489
ISSN
1538-7933
DOI
10.1111/j.1538-7836.2009.03657.x
language
English
LU publication?
yes
id
8c79869d-fc2e-4d64-bdff-aa7e87406975 (old id 1499874)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/19874463?dopt=Abstract
date added to LUP
2009-11-03 09:32:32
date last changed
2018-05-29 11:25:41
@article{8c79869d-fc2e-4d64-bdff-aa7e87406975,
  abstract     = {<p>BACKGROUND: Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined.</p><p>OBJECTIVE: To identify and characterize the additional APC-cleavage sites in FVa.</p><p>METHODS: The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV variants were expressed and their sensitivity for APC characterized functionally and with Western blotting.</p><p>RESULTS: The additional APC-cleavage sites were located at Lys309, Arg313, Arg316, Arg317 and Arg505. FVa-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q (denoted 8M-FVa) was APC resistant. To investigate individual sites, they were mutated back using 8M-FV as a template. The kinetics of APC-degradation of these variants demonstrated that protein S was equally efficient in enhancing the APC effect for all the novel sites.</p><p>CONCLUSIONS: Multiple APC-cleavage sites close to Arg306 and a single site close to Arg506 were identified. Protein S was equally efficient as APC cofactor for all novel sites. The stimulation by protein S of the Arg505 cleavage argues against a specific protein S-mediated stimulation of cleavage at Arg306 due to relocation of the APC active site closer to the membrane.</p>},
  author       = {Tran, Sinh and Dahlbäck, Björn},
  issn         = {1538-7933},
  keyword      = {Amino Acid Sequence,Arginine,Factor Va,Humans,Kinetics,Lysine,Mutagenesis, Site-Directed,Mutation,Phospholipids,Protein C,Protein Processing, Post-Translational,Protein S,Structure-Activity Relationship,Substrate Specificity,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {1},
  pages        = {129--136},
  publisher    = {Federation of European Neuroscience Societies and Blackwell Publishing Ltd},
  series       = {Journal of Thrombosis and Haemostasis},
  title        = {Novel APC-cleavage sites in FVa providing insights into mechanisms of action of APC and its cofactor protein S.},
  url          = {http://dx.doi.org/10.1111/j.1538-7836.2009.03657.x},
  volume       = {8},
  year         = {2010},
}