Subscribe to RSS
Download PDF
DOI: 10.1055/s-0038-1647916
Specificity of Antisera to Human Fibrinopeptide A Used in Clinical Fibrinopeptide A Assays[*]
Publication History
Received:
06 August 1975
accepted:
06 August 1975
Publication Date:
02 July 2018 (online)
Summary
Distinction between fibrinopeptide A (FPA) and larger polypeptides containing the FPA sequence is critical for the interpretation of clinical results with FPA immunoassay methods. Therefore, the immunochemical reactivity of 14 rabbit anti-FPA sera with six different FPA containing antigens was studied in detail. Antigens tested included: fibrinogen; fragment E of fibrinogen; the amino-terminal disulfide knot of fibrinogen; Aα 1 (Ala)-51 (Met); Aα l(Ala)-23(Arg); and, FPA. Synthetic partial sequences of FPA were also tested. The 14 FPA-specific antisera were divided into 3 distinct categories with: I, FPA immuno-reactivity of larger polypeptides containing FPA approximately 1/100 of FPA on a molar basis; II, FPA immunoreactivity of the larger polypeptides intermediate between I and III; and III, FPA immunoreactivity of the larger polypeptides approximately equal to that of FPA on a molar basis. The antigenic determinants of a category I antiserum (R 2) are included in Aα 7(Asp)-16(Arg) with Asp(7), Phe(8) and Arg(16) being essential. When attached to FPA, the sequence Gly(17)-Arg(23) decreases the immunoreactivity of FPA with category I antisera 100-fold.
The practical consequence of these findings is that, when category III antisera are employed, both FPA and larger FPA-containing polypeptides are equally immunoreactive. Since thrombin treatment of the larger polypeptides does not alter their immunoreactivity, category III antisera cannot discriminate between FPA and the larger polypeptides. On the other hand, with category I antisera, although the immunoreactivity of FPA itself is unaltered by thrombin treatment, larger polypeptides [e.g., Aα l(Ala)-23(Arg)] show a 100-fold increase in immunoreactivity following thrombin treatment and thus can readily be identified and separately quantitated. It is concluded that antisera with the specificity of category I are essential for the specific and accurate measurement of FPA, and for its distinction from larger FPA-containing polypeptides, in clinical plasma samples.
* Presented at the Vth Congress of the International Society on Thrombosis and Haemostasis, Paris, July, 1975.
-
Reference
- 1 Bilezikian S. B, Nossel H. L, Butler Jr. V. P, Canfield R. E. 1975; Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes. Journal of Clinical Investigation 56: 438.
- 2 Blombäck B, Blombäck M, Edman P, Hessel B. 1966; Human hbrinopeptides. Isolation, characterization and structure. Biochimica Biophysica Acta 115: 371.
- 3 Blombäck B, Blombäck M, Henschen A, Hessel B, Iwanaga S, Woods K. R. 1968; N-terminal disulfide knot of human fibrinogen. Nature 218: 130.
- 4 Blombäck B, Hessel B, Iwanaga S, Reuterby J, Blombäck M. 1972; Primary structure of human fibrinogen and fibrin. I. Cleavage of fibrinogen with cyanogen bromide. Isolation and characterization of NH2-terminal fragments of the a (“A”)-chain. Journal of Biological Chemistry 247: 1496.
- 5 Butler Jr. V. P, Nossel H. L, Wilner G. D, Canfield R. E, Blombäck B, Hessel B, Blombäck M, Kockum C, Gerrits G. B. J, Budzynski A. Z, Marder V. J, Cronlund M. 1976 Immunochemical studies of human fibrinopeptide A. III. Characterization of various antisera and antigens. Submitted for publication.
- 6 Canfield R. E, Dean J, Nossel H. L, Butler Jr. V. P, Wilner G. D. 1976 Reactivity of fibrinogen and fibrinopeptide A containing fibrinogen fragments with antisera to fibrinopeptide A. Biochemistry. In press.
- 7 Gerrits W. B. J, Flier O. Th. N, Van Der Meer J. 1974; Fibrinopeptide A immunoreactivity in human plasma. Thrombosis Research 5: 197.
- 8 Iwanaga S, Wallen P, Gröndahl N. J, Henschen A, Blombäck B. 1969; On the primary structure of human fibrinogen. Isolation and characterization of N-terminal fragments from plasmic digests. European Journal of Biochemistry 8: 189.
- 9 Kowalska-Loth B, Gärlund B, Egberg N, Blombäck B. 1973; Plasmic degradation products of human fibrinogen II. Chemical and immunological relation between fragment E and N-DSK. Thrombosis Research 2: 423.
- 10 Merrifield R. B. 1969; Solid-phase peptide synthesis. Advances in Enzymology and Related Areas of Molecular Biology 32: 221.
- 11 Nossel H. L, Younger L. R, Wilner G. D, Procupez T, Canfield R. E, Butler Jr. V. P. 1971; Radioimmunoassay of human fibrinopeptide A. Proceedings of the National Academy of Sciences (U.S.A.) 68: 2350.
- 12 Nossel H. L, Yudelman I, Canfield R. E, Butler Jr. V. P, Spanondis K, Wilner G. D, Qureshi G. D. 1974; Measurement of fibrinopeptide A in human blood. Journal of Clinical Investigation 54: 43.
- 13 Nossel H. L, Ti M, Soland T, Spanondis K, Canfield R. E, Butler Jr. V. P. 1975 Specific patterns of fibrinogen proteolysis in clinical syndromes revealed by fibrinopeptide assays (Abstract). Proceedings of the Vth Congress of the International Society on Thrombosis and Haemostasis, 434.
- 14 Schick A. F, Singer S. J. 1961; On the formation of covalent linkages between two protein molecules. Journal of Biological Chemistry 236: 2477.
- 15 Wallen P. 1971; Plasmic degradation of fibrinogen. Scandinavian Journal of Haematology, Suppl 13: 3.
- 16 Wilner G. D, Nossel H. L, Canfield R. E, Butler Jr. V. P. 1976 Immunochemical studies of human fibrinopeptide A. Using synthetic peptide homologues. Biochemistry. In press.