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Activated Protein C

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).

PURCHASING AND PRODUCT INFORMATION

 

Catalog Number

HCAPC-0080

HCAPC-DEGR

 

BCAPC-1080

BCAPC-DEGR

 

Description

Human Activated Protein C 

Human Activated Protein C - DEGR (active site blocked)

Bovine Activated Protein C

Bovine Activated Protein C - DEGR (active site blocked)

Size

50 µg

50 µg

 

50 µg

50 µg

 

Formulation

50% (vol/vol) glycerol/H2O

20 mM Tris, 150 mM NaCl, pH 7.4

 

50% (vol/vol) glycerol/H2O

20 mM Hepes, 150 mM NaCl, pH 7.4

 

Storage

-20oC (DEGR versions -80oC)

Purity

>95% by SDS-PAGE

NOT tissue/cell culture grade

Activity Determination

Chromogenic assay

Shelf Life (properly stored)

12 months

Compliment fluorogenic substrate(s): HTI Catalog # SN-54 and SN-59

Sample Gel Information:

Gel: Novex 4-12% Bis-Tris

Load: Human aPC, 1 µg per lane

Buffer: MOPS

Standard: SeeBluePlus 2; Myosin (191 kDa), Phosphorylase B (97 kDa), BSA (64 kDa), Glutamic Dehydrogenase (51 kDa), Alcohol Dehydrogenase (39 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (19 kDa), Lysozyme (14 kDa)

 

Sample publications referencing our Activated Protein C:

  1. Hwang, K., Arthritis Rheum. 2003 June ; 48(6): 1622每1630. (used as capture in ELISA)

  2. Raife, T., et al., J Clin Invest. 1994 April; 93(4): 1846每1851. (used as standard in aPC generation)

  3. Schuepbach, R., et al., Blood. 2008 March 1; 111(5): 2667每2673. (PAR1 cleavage)

  4. Adams, T., Hockin, M., Mann, K., Everse, S., Proc Natl Acad Sci U S A. 2004 June 15; 101(24): 8918每8923. (bovine aPC used to inactivate bovine Va)

  5. Gale, A. et al., J. Biol. Chem. (2008) VOL. 283, NO. 24, pp. 16355每16362. (Inactivation of VIIIa)

This publication list is not all encompassing, and is only meant to provide limited examples of how Haematologic Technologies' products are used.  We encourage you to search the literature for other examples pertinent to your experimentation, and to contact us with any technical questions.

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SAMPLE DATA SHEET

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Overview of Activated Protein C

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 (by hylton). 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
1 %
1 c m, 280 nm
= 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

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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