The regulation of factor Va activity by activated protein C -Importance of the individual activated protein C cleavage sites in factor Va
Author
Summary, in English
The subject of this thesis is the activated protein C (APC)-mediated inactivation of factor Va. The activated form of factor V (FVa) is a procoagulant cofactor for Factor Xa (FXa) in the conversion of prothrombin to thrombin. The procoagulant function of FVa is down regulated by APC through proteolytic cleavage at three sites, Arg306, Arg506 and Arg679. The cleavage at Arg506 is kinetically favored, while the cleavage at Arg306 is needed for full loss of procoagulant activity. The cleavage at Arg679 is believed to play a minor role for the loss of FVa activity. Numerous plasma components have been reported to influence the inactivation of FVa. Factor V variants were constructed recombinantly to evaluate the importance of each of the three APC cleavage sites in the FVa inactivation and how they are influenced by numerous plasma components.
The stimulation of FVa inactivation by protein S, originally believed to only involve the Arg306 cleavage, was in this thesis shown to affect all three cleavage sites. The inhibition by FXa was confirmed to selectively involve the Arg506 cleavage and protein S was shown to counteract the inhibition, due to competitive binding of protein S and FXa to FVa. The prothrombin inhibition was shown to involve all three cleavage sites.
Three natural occurring mutations in the FV gene were expressed and characterized. In FV Hong Kong and FV Cambridge the amino acid at the 306 site is substituted. Unlike the more common mutation that affects the Arg506 site (FV Leiden), these mutations were shown not to cause a serious APC-resistance phenotype and the FVa inactivation was not seriously impaired. In addition to its procoagulant function, intact FV has an anticoagulant function, as it works as a cofactor for APC in the degradation of Factor VIIIa. The APC cofactor activity is severely impaired in FV Leiden, but our studies show that in FV Hong Kong and FV Cambridge this activity is only partly hampered. FV Liverpool is a mutation found in two brothers with thrombotic tendency. The mutation causes the position of an additional carbohydrate in FV. We showed that the FVa inactivation and FV cofactor function were impaired due to impaired binding of APC and protein S to FVa.
The stimulation of FVa inactivation by protein S, originally believed to only involve the Arg306 cleavage, was in this thesis shown to affect all three cleavage sites. The inhibition by FXa was confirmed to selectively involve the Arg506 cleavage and protein S was shown to counteract the inhibition, due to competitive binding of protein S and FXa to FVa. The prothrombin inhibition was shown to involve all three cleavage sites.
Three natural occurring mutations in the FV gene were expressed and characterized. In FV Hong Kong and FV Cambridge the amino acid at the 306 site is substituted. Unlike the more common mutation that affects the Arg506 site (FV Leiden), these mutations were shown not to cause a serious APC-resistance phenotype and the FVa inactivation was not seriously impaired. In addition to its procoagulant function, intact FV has an anticoagulant function, as it works as a cofactor for APC in the degradation of Factor VIIIa. The APC cofactor activity is severely impaired in FV Leiden, but our studies show that in FV Hong Kong and FV Cambridge this activity is only partly hampered. FV Liverpool is a mutation found in two brothers with thrombotic tendency. The mutation causes the position of an additional carbohydrate in FV. We showed that the FVa inactivation and FV cofactor function were impaired due to impaired binding of APC and protein S to FVa.
Department/s
Publishing year
2003
Language
English
Document type
Dissertation
Publisher
Björn Dahlbäck, The Wallenberg Laboratory, Division of Clinical Chemistry, University Hospital, Malmö, Lund University, S-20502 Malmö, Sweden,
Topic
- Medicinal Chemistry
Keywords
- extracellulära vätskor
- Hematologi
- extracellular fluids
- Haematology
- activated protein C resistance
- activated protein C
- Blood coagulation
- Factor V
Status
Published
Research group
- Clinical Chemistry, Malmö
Supervisor
- [unknown] [unknown]
ISBN/ISSN/Other
- ISBN: 91-628-5637-5
Defence date
8 May 2003
Defence time
10:15
Defence place
Aulan vid medicinkliniken, MAS, ingång 35, plan 1
Opponent
- Guido Tans (Professor)