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Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Nicolaes, Gerry A. F.; Bock, Paul E.; Segers, Kenneth; Wildhagen, Karin C. A. A.; Dahlbäck, Björn LU and Rosing, Jan (2010) In Journal of Biological Chemistry 285(30). p.22888-22898
Abstract
Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K-D of 0.11 +/- 0.05 nM and competed with active site-labeled Oregon Green activated coagulation factor X... (More)
Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K-D of 0.11 +/- 0.05 nM and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was > 100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
285
issue
30
pages
22888 - 22898
publisher
ASBMB
external identifiers
  • wos:000279999900022
  • scopus:77954943563
ISSN
1083-351X
DOI
10.1074/jbc.M110.131029
language
English
LU publication?
yes
id
4bc5a985-e656-47f5-bc9d-f98531a1993e (old id 1657441)
date added to LUP
2010-08-31 10:11:34
date last changed
2018-05-29 12:16:09
@article{4bc5a985-e656-47f5-bc9d-f98531a1993e,
  abstract     = {Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K-D of 0.11 +/- 0.05 nM and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was > 100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.},
  author       = {Nicolaes, Gerry A. F. and Bock, Paul E. and Segers, Kenneth and Wildhagen, Karin C. A. A. and Dahlbäck, Björn and Rosing, Jan},
  issn         = {1083-351X},
  language     = {eng},
  number       = {30},
  pages        = {22888--22898},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C},
  url          = {http://dx.doi.org/10.1074/jbc.M110.131029},
  volume       = {285},
  year         = {2010},
}