| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Protein Science, Vol 6, Issue 9 2016-2027, Copyright © 1997 by Cold Spring Harbor Laboratory Press
ARTICLE |
J. O. EGAN, M. KALAFATIS and K. G. MANN
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont 05405
Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg(306), Arg(506), and Arg(679). Site-directed mutagenesis of human factor V cDNA was used to substitute Arg(306) -> Ala (rfVa(306A)) and Arg(506) -> Gln (rfVa(506Q)). Both the single and double mutants (rfVa(306A/506Q)) were constructed. The activation of these procofactors by {alpha}-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVa(PLASMA)) in HBS Ca(2+) +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVa(WT)) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVa(PLASMA). The rfVa(306A) and rfVa(506Q) mutants were each inactivated at rates slower than rfVa(WT) and fVa(PLASMA). Following a 90-min incubation with APC, rfVa(306A) and rfVa(506Q) retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa(306A/506Q), was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVa(WT), rfVa(306A), rfVa(506Q), and rfVa(306A/506Q) were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVa(WT) and rfVa(306A) inactivation in the presence of protein S were unchanged, indicating cleavage at Arg(506) is not affected by protein S. The initial rate of rfVa(506Q) inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg(306). Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg(306) and Arg(506) are required for efficient factor Va inactivation.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Yegneswaran, Y. Kojima, P. M. Nguyen, A. J. Gale, M. J. Heeb, and J. H. Griffin Factor Va Residues 311 325 Represent an Activated Protein C Binding Region J. Biol. Chem., September 28, 2007; 282(39): 28353 - 28361. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Toso and R. M. Camire Role of Hirudin-like Factor Va Heavy Chain Sequences in Prothrombinase Function J. Biol. Chem., March 31, 2006; 281(13): 8773 - 8779. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. van der Neut Kolfschoten, R. J. Dirven, H. L. Vos, G. Tans, J. Rosing, and R. M. Bertina Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679 J. Biol. Chem., February 20, 2004; 279(8): 6567 - 6575. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Rezende, R. E. Simmonds, and D. A. Lane Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex Blood, February 15, 2004; 103(4): 1192 - 1201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. G. Mann and M. Kalafatis Factor V: a combination of Dr Jekyll and Mr Hyde Blood, January 1, 2003; 101(1): 20 - 30. [Full Text] [PDF]
|
|
|
|
|
|
E. Norstrom, E. Thorelli, and B. Dahlback Functional characterization of recombinant FV Hong Kong and FV Cambridge Blood, June 28, 2002; 100(2): 524 - 530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yegneswaran, M. D. Smirnov, O. Safa, N. L. Esmon, C. T. Esmon, and A. E. Johnson Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor. A FLUORESCENCE RESONANCE ENERGY TRANSFER STUDY J. Biol. Chem., February 26, 1999; 274(9): 5462 - 5468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Yang, J. Cui, A. Rehumtulla, A. Yang, M. Moussalli, R. J. Kaufman, and D. Ginsburg The Structure and Function of Murine Factor V and Its Inactivation by Protein C Blood, June 15, 1998; 91(12): 4593 - 4599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Long, D. Lu, R.-L. Xie, and M. Kalafatis Human Protein S Cleavage and Inactivation by Coagulation Factor Xa J. Biol. Chem., May 8, 1998; 273(19): 11521 - 11526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Smirnov, O. Safa, L. Regan, T. Mather, D. J. Stearns-Kurosawa, S. Kurosawa, A. R. Rezaie, N. L. Esmon, and C. T. Esmon A Chimeric Protein C Containing the Prothrombin Gla Domain Exhibits Increased Anticoagulant Activity and Altered Phospholipid Specificity J. Biol. Chem., April 10, 1998; 273(15): 9031 - 9040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Williamson, K. Brown, R. Luddington, C. Baglin, and T. Baglin Factor V Cambridge: A New Mutation (Arg306right-arrowThr) Associated With Resistance to Activated Protein C Blood, February 15, 1998; 91(4): 1140 - 1144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Undas, E. B. Williams, S. Butenas, T. Orfeo, and K. G. Mann Homocysteine Inhibits Inactivation of Factor Va by Activated Protein C J. Biol. Chem., February 2, 2001; 276(6): 4389 - 4397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Friedrich, G. A.F. Nicolaes, B. O. Villoutreix, and B. Dahlback Secondary Substrate-binding Exosite in the Serine Protease Domain of Activated Protein C Important for Cleavage at Arg-506 but Not at Arg-306 in Factor Va J. Biol. Chem., June 15, 2001; 276(25): 23105 - 23108. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
