Protein C, Activated (APC) Bovine

Inactivation of Factor Va by Activated Protein C (APC): 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 (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.\n\nActivated protein C (APC) is an anticoagulant serine protease derived from the two chain, vitamin K-dependent zymogen, protein C (3-7). A complex between a-thrombin and thrombomodulin catalyzes a single cleavage at Arg-12 (Arg-14 in bovine) in the heavy chain of protein C, to generate APC. Several non-physiologically relevant proteases such as RVV-X activator, trypsin, and PROTAC are also capable of activating protein C. \n\nAPC 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=6x10e-9M) (8).\n\nSeveral 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. \n\nIn addition to our standard APC preparation, an active site-blocked form containing Dansyl-EGR-chloromethlyketone is also available.\n\nStorage and Stability: May be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.\n\nAdditional Specifications:\nActivity: 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).\n \nLocalization: Plasma \n\nMode of Action: Anticoagulant, inactivates factors Va and VIIIa\n\nExtinction Coefficient: E1%1cm, 280nm=13.7 (9)\n\nIsoelectric Point: 4.2-4.5 (9)\n\nStructure: Two chains, Mr=35,000 and 21,000, disulfide linked, NH2-terminal gla domain two EGF domains\n\nPercent Carbohydrate: 14% (5)\n\nPost-translational Modifications: Eleven gla residues, one b-hydroxyaspartate

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SPECIFICATIONS

Catalog Number

P9081

Size

50ug

Form

Supplied as a liquid in 50% glycerol/H2O.

Purity

(same/more than)95%. Activated protein C is prepared from purified protein C by activation with thrombin followed by ion exchange chromatography (4). Purity is determined by SDS-PAGE analysis. \n

References

1. Kalafatis, M., and Mann, K.G., J. Biol. Chem., 268, 27246 (1993).\n2. Kalafatis, M., et al., J. Biol. Chem., 269, 31869 (1994).\n3. Esmon, C.T., Progress in Thromb. and Hemostas., 10, 25 (1984).\n4. Esmon, C.T., J. Biol. Chem., 264, 4743 (1989).\n5. Kisiel, W., et al., Methods Enzymol., 80, 320 (1981).\n6. Stenflo, J., Semin. in Thromb. and Hemostas., 10, 109 (1984).\n7. Marlar, R.A., Semin. in Thromb. and Hemostas., 11, 387 (1985).\n8. Walker, F.J., et al., J. Biol. Chem., 256, 11128 (1981).\n9. Discipio, R.G., et al., Biochemistry, 18, 899 (1979).\n10. Dahlback, B., and Hildebrand, B., Proc. Natl. Acad. Sci. USA, 91, 1396 (1994).\n11. Svensson, P.J., and Dahlback, B., New Engl. J. Med., 330, 517 (1994).