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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 MCAPC-5080*	Description Human Activated Protein C  Human Activated Protein C - DEGR Bovine Activated Protein C Bovine Activated Protein C - DEGR Mouse Activated Protein C  (Mouse aPC is indefinitely backordered)	Size 50 µg 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 50% (vol/vol) glycerol/H2O
Storage -20oC (DEGR versions -80oC)	Purity >95% by SDS-PAGE	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: Hwang, K., Arthritis Rheum. 2003 June ; 48(6): 1622–1630. (used as capture in ELISA) Raife, T., et al., J Clin Invest. 1994 April; 93(4): 1846–1851. (used as standard in aPC generation) Schuepbach, R., et al., Blood. 2008 March 1; 111(5): 2667–2673. (PAR1 cleavage) Adams, T., Hockin, M., Mann, K., Everse, S., Proc Natl Acad Sci U S A. 2004 June 15; 101(24): 8918–8923. (bovine aPC used to inactivate bovine Va) 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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*Due to mouse plasma quality issues our mouse proteins are not being sold until further notice.  At the present time we cannot give a completion date.  Thank you.

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 -20^oC. 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

PURCHASING AND PRODUCT INFORMATION

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). 

 Please inquire about products not listed.  We can also custom purify proteins from other species.