|
 Inactivation of Factor Va by Activated Protein C
(APC) Proteolytic inactivation of factor Va by APC is
represented, where: PS=protein S, PCPS=phospholipid vesicles (or cellular surface) and Ca++=calcium
ions. The inactivation of factor Va to form factor Vai results from proteolysis of the
factor Va heavy chain (94K) at three specific sites by APC (solid arrows) (1,2). The
location of these cleavage sites in the factor Va heavy chain are as follows: (human:
R306, R506, & R679) and (bovine: R306, R505, & R662). Complete inactivation of the
cofactor molecule requires cleavage at the Arginine-306 position. Cleavage at Arginine-306
by activated protein C occurs only in the presence of membrane, and requires prior
cleavage of the heavy chain at Arginine-505. Proteolysis of the factor Va light chain by
APC occurs only in the bovine molecule and is not required for inactivation (dashed
arrow).
Activated protein C (APC) is an anticoagulant serine protease derived from the two chain, vitamin K-dependent zymogen,
protein C (3-7). A complex between
alpha-thrombin and thrombomodulin
catalyzes a single cleavage at Arg-12 (Arg-14 in bovine) in the heavy chain of protein C, to generate
activated Protein C. Several non-physiologically relevant proteases such as
RVV-X activator, trypsin, and PROTAC are also capable of activating protein C.
APC functions as an anticoagulant which catalyzes the proteolytic inactivation of the cofactors,
factors Va and VIIIa, leading to inhibition of the prothrombinase and factor Xase complexes. The inactivation of factors Va and VIIIa is both Ca2+ and phospholipid dependent. The vitamin K dependent cofactor,
protein S, moderately increases this rate of inactivation by forming a 1:1 complex with APC (Kd=6x10-9M) (8).
Several factors attenuate the anticoagulant activity of APC. Factor Xa protects factor Va from proteolysis by APC by competing for a similar binding site on factor Va. Thrombin has also been proposed as a regulator of APC by proteolytic inactivation of protein S. In addition, APC is regulated by a circulating heparin-dependent protein C inhibitor (PAI-3), a circulating heparin-independent protein C inhibitor, a platelet-derived protein C inhibitor, and
PAI-1. The complexes formed between APC and both types of PAI have been reported to account for increased fibrinolysis observed upon infusion of APC or the generation of APC in vivo.
In addition to our standard APC preparation, an active site-blocked form containing Dansyl-EGR-chloromethlyketone is also available.
Activated protein C is prepared from purified protein C by activation with thrombin followed by ion exchange chromatography (4). APC is supplied in 50% (vol/vol) glycerol/H2O and should be stored at
-20oC. Purity is determined by SDS-PAGE analysis and activity is measured using a chromogenic substrate assay. All production lots of APC are also tested for their ability to prolong the aPTT of normal human plasma, as required for the APC resistance assay (10,11). The results of this test are provided for each lot, as an aPTT (+/- APC) ratio (10nM APC).
Properties of
Activated Protein C
| Localization: |
Plasma |
| Mode of action: |
Anticoagulant, inactivates factors Va and VIIIa |
| Molecular weight: |
56,200 (human) (5)
52,650 (bovine) (5) |
| Extinction coefficient: |
| E |
|
= 14.5 (human) (9) |
|
|
= 13.7 (bovine) (9) |
|
| Isoelectric point: |
4.4-4.8 (human) (9)
4.2-4.5 (bovine) (9) |
| Structure: |
two chains, Mr=35,000 and 21,000, disulfide linked, NH2-terminal
gla domain two EGF domains |
| Percent carbohydrate: |
23 % (human) (5)
14 % (bovine) (5) |
| Post-translational modifications: |
eleven gla residues (bovine), nine gla residues (human), one
β-hydroxyaspartate |
|
Catalog
Number |
Description |
|
HCAPC-0080 |
Human Activated Protein C (Compliment
fluorogenic substrate(s): HTI Catalog # SN-54
and SN-59) |
|
HCAPC-DEGR |
Human Activated Protein C
- DEGR |
|
BCAPC-1080 |
Bovine Activated Protein C |
|
BCAPC-DEGR |
Bovine Activated Protein C
- DEGR |
|
MCAPC-5080 |
Mouse Activated Protein C (indefinitely
backordered) |
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References
1. Kalafatis, M., and Mann, K.G., J. Biol. Chem., 268, 27246 (1993).
2. Kalafatis, M., et al., J. Biol. Chem., 269, 31869 (1994).
3. Esmon, C.T., Progress in Thromb. and Hemostas., 10, 25 (1984).
4. Esmon, C.T., J. Biol. Chem., 264, 4743 (1989).
5. Kisiel, W., et al., Methods Enzymol., 80, 320 (1981).
6. Stenflo, J., Semin. in Thromb. and Hemostas., 10, 109 (1984).
7. Marlar, R.A., Semin. in Thromb. and Hemostas., 11, 387 (1985).
8. Walker, F.J., et al., J. Biol. Chem., 256, 11128 (1981).
9. Discipio, R.G., et al., Biochemistry, 18, 899 (1979).
10. Dahlback, B., and Hildebrand, B., Proc. Natl. Acad. Sci. USA, 91, 1396 (1994).
11.Svensson, P.J., and Dahlback, B., New Engl. J. Med., 330, 517 (1994).
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