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Thromb Haemost 1993; 69(05): 441-447
DOI: 10.1055/s-0038-1651630
Original Article
Coagulation
Schattauer GmbH Stuttgart

A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo[*]

Carolyn L Orthner
The Plasma Derivatives Laboratory, American Red Cross, Biomedical Research and Development Division, Rockville, MD, USA
,
Billy Kolen
The Plasma Derivatives Laboratory, American Red Cross, Biomedical Research and Development Division, Rockville, MD, USA
,
William N Drohan
The Plasma Derivatives Laboratory, American Red Cross, Biomedical Research and Development Division, Rockville, MD, USA
› Author Affiliations
Further Information

Publication History

Received 16 September 1992

Accepted after revision 22 January 1993

Publication Date:
25 July 2018 (online)

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Summary

Activated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.

The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

*A preliminary report of this work was presented at the 33rd Annual Meeting of the American Society of Hematology, Denver, CO; Blood 1991; 78: Suppl. 1, P 219a (Abstr 865).


 

  • References

  • 1 Clouse LH, Comp PC. The regulation of hemostasis: the protein C system. N Engl J Med 1986; 314: 1298-1304
    CrossrefPubMedGoogle Scholar
  • 2 Esmon CT. The role of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-4746
    PubMedGoogle Scholar
  • 3 Kisiel W. Human plasma protein C. Isolation, characterization, and mechanism of activation by α-thrombin. J Clin Invest 1979; 64: 761-769
    CrossrefPubMedGoogle Scholar
  • 4 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochem Biophys Acta 1979; 571: 333-342
    CrossrefPubMedGoogle Scholar
  • 5 Suzuki J, Stenflo J, Dahlback B, Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
    PubMedGoogle Scholar
  • 6 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-489
    PubMedGoogle Scholar
  • 7 Eaton DL, Rodriguez HR, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25: 505-512
    CrossrefPubMedGoogle Scholar
  • 8 Suzuki K, Nishioka J, Kusumoto H, Hashimoto S. Mechanism of inhibition of activated protein C by protein C inhibitor. J Biochem 1984; 95: 187-195
    CrossrefPubMedGoogle Scholar
  • 9 Heeb MJ, Griffin JH. Physiologic inhibition of human activated protein C by α1-antitrypsin. J Biol Chem 1988; 263: 11613-11616
    PubMedGoogle Scholar
  • 10 Berger Jr H, Kirstein CG, Orthner CL. Pharmacokinetics of activated protein C in guinea pigs. Blood 1991; 77: 2174-2184
    PubMedGoogle Scholar
  • 11 Heeb MJ, Gruber A, Griffin JH. Identification of divalent metal iondependent inhibition of activated protein C by α2-macroglobulin and α2-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin. J Biol Chem 1991; 266: 17606-17612
    PubMedGoogle Scholar
  • 12 Hoogendoorn H, Toh CH, Nesheim ME, Giles AR. α2-macroglobulin binds and inhibits activated protein C. Blood 1991; 78: 2283-2290
    PubMedGoogle Scholar
  • 13 Emerick SC, Murayama H, Yan SB, Long GL, Harms C, Marks CA, Mattler LE, Hsu CA, Comp PC, Esmon NL, Esmon CT, Bang NU. Preclinical pharmacology of activated protein C. In: The Pharmacology and Toxicology of Proteins. Holeenberg JS, Winkelhake JL. (eds) New York: Alan R Liss, Inc,; 1987: 351-367
    Google Scholar
  • 14 Orthner CL, Ralston AH, McGriff JD, Gee DM, Drohan WN. Large scale purification and preclinical studies of activated protein C (APC) as an antithrombotic agent. Blood 1990; 76 (Suppl. 01) 517a
    PubMedGoogle Scholar
  • 15 Okajima K, Koga S, Kaji M, Inoue M, Nakagaki T, Funatsu A, Okabe H, Takatsuki K, Aoki N. Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects. Thromb Haemostas 1990; 63: 48-53
    PubMedGoogle Scholar
  • 16 Jackson CV, Frank J, Crowe G, Craft T, Sundboom J, Grinnell B, Yan B, Smith G. Activated recombinant, human protein C (ra-PC) is an effective adjunct to coronary artery thrombolysis in the anesthetized dog. J Mol Cell Cardiol 1989; 21 (Suppl. 02) 116
    PubMedGoogle Scholar
  • 17 Jackson CV, Frank J, Crowe G, Sundboom J, Grinnell B, Yan B, Smith G. Anticoagulant and antithrombotic efficacy of activated, recombinant, human protein C (ra-PC) determined in a canine model of coronary artery thrombosis. J Mol Cell Cardiol 1989; 21 (Suppl. 02) 117
    PubMedGoogle Scholar
  • 18 Griffin JH, Hanson SR, Gruber A, Kelly AB, Harker LA. Antithrombotic effect of combining activated protein C with urokinase in a primate model of arterial thrombosis. Thromb Haemostas 1989; 62: 1512a
    PubMedGoogle Scholar
  • 19 Gruber A, Griffin JH, Harker LA, Hanson SR. Inhibition of plateletdependent thrombus formation by human activated protein C in a primate model. Blood 1989; 73: 639-642
    PubMedGoogle Scholar
  • 20 Orthner CL, Drohan WN, Oliver W, Berger Jr H. Prevention of arterial reocclusion in a canine model of thrombolysis by human activated protein C. In preparation.
    PubMedGoogle Scholar
  • 21 Orthner CL, Madurawe RD, Velander WH, Drohan WN, Battey FD, Strickland DK. Conformational changes in an epitope localized to the NH2-terminal region of Protein C. J Biol Chem 1989; 264: 18781-18788
    PubMedGoogle Scholar
  • 22 Orthner CL, Ralston AH, McGriff JD, Gee DM, Drohan WN. The large-scale production and properties of an immunoaffinity-purified human activated protein C concentrate from human plasma. In preparation.
    PubMedGoogle Scholar
  • 23 Wickerhauser M, Williams C. Isolation of antithrombin III without interfering with ethanol fractionation system. Thromb Haemostas 1979; 42: 168a
    PubMedGoogle Scholar
  • 24 Goding JW. Monoclonal Antibodies. Principles and Practice. New York: Academic Press; 1983: 188
    Google Scholar
  • 25 Kisiel W, Hanahan DJ. Purification and characterization of human factor II. Biochim Biophys Acta 1973; 304: 103-113
    CrossrefPubMedGoogle Scholar
  • 26 Nordenman B, Nystrom C, Bjork I. Eur J Biochem. 1977 78. 195-203
    PubMedGoogle Scholar
  • 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1979; 227: 680-685
    PubMedGoogle Scholar
  • 28 Orthner CL, Highsmith FA, Tharakan J, Madurawe RD, Morcol T, Velander WH. Comparison of the performance of immunosorbents prepared by site-directed or random coupling of monoclonal antibodies. J Chromatogr 1991; 558: 55-70
    CrossrefPubMedGoogle Scholar
  • 29 Banner DW, Hadváry P. Crystallographic analysis at 3.0 – A resolution of the binding to human thrombin of four active site-directed inhibitors. J Biol Chem 1991; 266: 20085-20093
    PubMedGoogle Scholar
  • 30 Ranby M, Sundell IB, Nilsson TK. Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal tPA activity. Thromb Haemostas 1989; 62: 917-922
    PubMedGoogle Scholar
  • 31 Griffin JH, Mosher DF, Zimmerman TS, Kleiss AJ. Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood 1989; 60: 261-264
    PubMedGoogle Scholar
  • 32 Mannucci PM, Vigano S. Deficiencies of protein C, an inhibitor of blood coagulation. Lancet 1982; 463-467
    PubMedGoogle Scholar
  • 33 Teitel JM, Bauer KA, Lau HK, Rosenberg RD. Studies of the prothrombin activation pathway utilizing radioimmunoassays for the F2/F1+2 fragment and thrombin-antithrombin complex. Blood 1982; 59: 1086-1097
    PubMedGoogle Scholar
  • 34 Nossel HL, Yudelman I, Canfield RE, Butler Jr VP, Spanondis K, Wilner GD, Qureshi GD. Measurement of fibrinopeptide A in human blood. J Clin Invest 1974; 54: 43-53
    CrossrefPubMedGoogle Scholar
  • 35 Lollar P, Owen WG. Clearance of thrombin from circulation in rabbits by high-affinity binding sites on endothelium. J Clin Invest 1980; 66: 1222-1230
    CrossrefPubMedGoogle Scholar
  • 36 Bauer KA, Kass BL, Beeler DL, Rosenberg RD. Detection of protein C activation in humans. J Clin Invest 1984; 74: 2033-2041
    CrossrefPubMedGoogle Scholar
  • 37 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340-2348
    PubMedGoogle Scholar
  • 38 Laurell M, Carlson TH, Stenflo J. Monoclonal antibodies against the heparin-dependent protein C inhibitor suitable for inhibitor purification and assay of inhibitor complexes. Thromb Haemostas 1988; 60: 334-339
    PubMedGoogle Scholar
  • 39 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of Protein C. J Biol Chem 1982; 257: 859-864
    PubMedGoogle Scholar
  • 40 Doyle MF, Mann KG. Multiple active forms of thrombin. IV. Relative activities of meizothrombins. J Biol Chem 1990; 265: 10693-10701
    PubMedGoogle Scholar
  • 41 Mannucci PM, Boyer C, Tripodi A, Vigano-D’Angélo S, Wolf M, Valsecchi C, D’Angelo A, Meyer D, Larrieu MJ. Multicenter comparison of five functional and two immunological assays for protein C. Thromb Haemostas 1987; 57: 44-48
    PubMedGoogle Scholar