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Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma.

Krisinger, Michael LU ; Guo, Li Jun LU ; Salvagno, Gian Luca LU ; Guidi, Gian Cesare; Lippi, Giuseppe and Dahlbäck, Björn LU (2009) In The FEBS Journal 276. p.6586-6602
Abstract
Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and... (More)
Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
The FEBS Journal
volume
276
pages
6586 - 6602
publisher
Wiley-Blackwell
external identifiers
  • WOS:000271057200017
  • PMID:19817854
  • Scopus:70350435294
ISSN
1742-464X
DOI
10.1111/j.1742-4658.2009.07371.x
language
English
LU publication?
yes
id
93f7ebc7-fdb2-4f56-808a-a7d129cd6258 (old id 1500452)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/19817854?dopt=Abstract
date added to LUP
2009-11-03 14:37:51
date last changed
2017-01-01 07:43:08
@article{93f7ebc7-fdb2-4f56-808a-a7d129cd6258,
  abstract     = {Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.},
  author       = {Krisinger, Michael and Guo, Li Jun and Salvagno, Gian Luca and Guidi, Gian Cesare and Lippi, Giuseppe and Dahlbäck, Björn},
  issn         = {1742-464X},
  language     = {eng},
  pages        = {6586--6602},
  publisher    = {Wiley-Blackwell},
  series       = {The FEBS Journal},
  title        = {Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma.},
  url          = {http://dx.doi.org/10.1111/j.1742-4658.2009.07371.x},
  volume       = {276},
  year         = {2009},
}