Evaluation of Coagulometric Assays in the Assessment of Protein C Anticoagulant Activity; Variable Sensitivity of Commercial APTT Reagents to the Cofactor Effect of Protein S

Summary

In vivo expression of protein C activity is dependent on the availability of the activated protein C (APC) cofactor protein S. In the clinical laboratory, measurement of protein C anticoagulant activity is mostly performed in modified APTT assays. We have evaluated 13 commercial APTT reagents for their sensitivity to the cofactor effect of protein S by comparing APC-dependent clotting time prolongations in normal plasma and in protein S depleted plasma. In normal plasma, the sensitivities of the APTT reagents to the anticoagulant effect of APC were markedly different and correlated with the sensitivity of reagents to factor V and VIII. Reagents containing soy phosphatides appeared more sensitive than reagents containing phospholipid of animal origin. Analysis of dose-response curves obtained in normal plasma distinguished one group of reagents showing clotting time prolongations linearly related to the APC concentrations, a second group showing a log-linear relationship and a third group showing a log-log relationship. In protein S depleted plasma, sensitivity of APTT reagents to APC was in general proportional to that observed in normal plasma. However, for some reagents dose-response curves were qualitatively different in normal and in protein S depleted plasma. With all the APTT reagents, APC-dependent clotting time prolongations corresponding to 30-80% of APC anticoagulant activity observed in normal plasma, were observed in protein S depleted plasma. At variance, in a modified Xa one-stage clotting assay, negligible clotting time prolongations were observed in protein S depleted plasma, indicating that over 90% of the APC anticoagulant activity was protein S dependent in this assay system. Dilution of a relative insensitive APTT reagent effectively increased its sensitivity to the cofactor effect of protein S, suggesting that different phospholipid content and/or composition might be responsible for the different sensitivity of APTT reagents to protein S.

These results question the validity of APTT based assays for the identification of qualitative protein C abnormalities with defective interaction with protein S.

Keywords

Protein C - Protein S - Protein C coagulometric assays

• References

• 1 Clouse LH, Comp PC. The regulation of hemostasis: the protein C system. N Engl J Med 1986; 314: 1298-1304
• 2 Esmon CT. The regulation of natural anticoagulant pathway. Science 1987; 225: 1348-1352
• 3 Bertina RM, Broekmans AW, Van Es-Krommerhoek C, Van Wijngaarden A. The use of a functional and immunological assay for plasma protein C in the study of the heterogeneity of protein C deficiency. Thromb Haemostas 1984; 51: 01-05
• 4 Comp PC, Nixon RR, Esmon CT. Determination of functional levels of protein C, an antithrombotic protein, using thrombin-thrombomodulin complex. Blood 1984; 63: 15-21
• 5 Sala N, Owen WG, Collen D. Functional assay of protein C in human plasma. Blood 1984; 63: 671-675
• 6 Francis RB, Seyfert U. Rapid amidolytic assay of protein C in whole plasma using an activator from the venom of Agkistrodon Contortrix. Am J Clin Pathol 1987; 87: 619-625
• 7 McCall F, Conkie JA, Walker ID, Davidson JF. Measurement of protein C in plasma-a fully automated assay. Thromb Res 1987; 45: 681-685
• 8 Odegaard OR, Try K, Andersson TR. Protein C: an automated activity assay. Haemostasis 1987; 17: 109-113
• 9 Francis RB, Patch MJ. A functional assay for human protein C in plasma. Thromb Res 1984; 36: 481-483
• 10 Francis RB. A simplified PTT based Protein C activity assay using the thrombin-thrombomodulin complex. Thromb Res 1986; 37: 337-344
• 11 Hickton CM, Felding P, Ikeda K, Martisson G, Nilsson IM. A functional assay of protein C in human plasma. Thromb Res 1986; 41: 501-508
• 12 Martinoli JL, Stocker K. Fast functional protein C assay using PROTAC, a novel protein C activator. Thromb Res 1986; 43: 253-264
• 13 Rapaport SI, Zivelin A, Donnelly K. A plasma protein C activity assay suitable for a clinical laboratory. Its use to measure activity in hereditary and acquired deficiency states. Am J Clin Pathol 1987; 87: 491-497
• 14 Vukovich TC, Knobl PN, Bauer K, Tragi KH, Deutsch E. Quantitative determination of human protein C. Evaluation of a “fast” functional assay in comparison to a “traditional” functional and an immunological assay. Thromb Res 1988; 49: 169-179
• 15 Lobermann H, Kolde HJ, Deubel R, Peter R, Tourte E, Becker U. Determination of protein C in plasma. Behring Inst Mitt 1986; 79: 112-120
• 16 Vigano'-D'Angelo S, Comp PC, Esmon CT, D'Angelo A. Relationship between protein C antigen and anticoagulant activity during oral anticoagulation and in selected disease states. J Clin Invest 1986; 77: 416-425
• 17 Vigano'-D'Angelo S, Esmon CT, Comp PC, D'Angelo A. Measurement of protein C and protein S in plasma samples. In: Biotechnology in Clinical Medicine. Albertini A, Lenfant C, Paoletti R. (eds). Raven Press Ltd; New York: 1987: 163-173
• 18 Espana F, Estelles A, Aznar J, Gilabert J. Assay of protein C in human plasma: comparison of amidolytic, coagulation and immunological assays. Thromb Res 1986; 44: 771-782
• 19 Vinazzer H, Pangraz U. Protein C: comparison of different assays in normal and abnormal plasma samples. Thromb Res 1987; 46: 01-08
• 20 Mannucci PM, Boyer C, Tripodi A, Vigano'-D'Angelo S, Wolf M, Valsecchi C, D'Angelo A, Meyer D, Larrieu M. Multicenter comparison of five functional and immunological assays for protein C. Thromb Haemostas 1987; 57: 44-48
• 21 Walker F. Regulation of activated protein C by protein S. J Biol Chem 1981; 256: 11128-11131
• 22 Francis RB, Patch MJ. A comparative study of commercial PTT-reagents in a PTT-based functional protein C assay. Thromb Res 1984; 36: 481-483
• 23 Dahlbäck B. Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-846
• 24 Steam DJ, Kurosawa S, Sims PJ, Esmon LN, Esmon CT. The interaction of a calcium dependent monoclonal antibody with protein C activation peptide region. Evidence for obligatory calcium binding to both antigen and antibody. J Biol Chem 1988; 263 (02) 826-832
• 25 D'Angelo A, Vigano'-D'Angelo S, Esmon CT, Comp PC. Acquired deficiencies of protein S, Protein S activity during oral anticoagulation in liver disease and in disseminated intravascular coagulation. J Clin Invest 1988; 81: 1445-1454
• 26 Ingram GI C, Brozovic CM, Slater NP. Bleeding disorders 1982; Chapter 10: 251
• 27 Walker FJ. Regulation of activated protein C by protein S. A possible function for bovine protein S. J Biol Chem 1980; 255: 5521-5524
• 28 Mannucci PM, Tripodi A. Laboratory screening of inherited thrombotic syndromes. Thromb Haemostas 1987; 57: 247-251
• 29 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-342
• 30 Fulchner CA, Gardiner JE, Griffin JH, Zimmermann TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its homology with factor V. Blood 1984; 63r: 486-489