A study of the carboxy-terminal A-alpha chain polypeptides of human fibrinogen

Conkie, James Antony (1979) A study of the carboxy-terminal A-alpha chain polypeptides of human fibrinogen. PhD thesis, University of Glasgow.

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Abstract

1. Human fibrin(ogen), the clottable protein of plasma, is the final substrate in both the coagulation and the fibrinolytic enzyme systems. The subunit structure of this protein consists of three pairs of polypeptide chains, Aalpha, Bbeta and gamma chains, which are joined together by disulphide bonds. Using antisera to the individual polypeptide chains of fibrinogen, immunological investigation should provide more information about the degradation products of fibrinogen than if anti-fibrinogen antiserum alone was used. 2. The immunological behaviour of fibrinogen and its derivatives has been investigated largely by means of an antiserum to one of the individual polypeptide chains of fibrinogen - the Aalpha chain. Using the relatively simple and qualitatively informative, analytical techniques of immunoprecipitation in agarose gel and gel electrophoresis, significant observations have been made on the immunological reactions of anti-Aalpha antiserum. 3. For the preparation of immunogen, fibrinogen was precipitated from human plasma and purified to fraction 1-4. Fibrinogen was reduced by dithiothreitol and alkylated with iodoacetic acid. Separation of the individual carboxymethylated Aalpha, Bbeta and gamma polypeptide chains was achieved by ion-exchange chromatography. Studies on the solubility properties of the carboxymethylated Aalpha polypeptide chains of fibrinogen in various buffers suggested alkaline barbitone (pH 8.6) as the buffer of choice for immunodiffusion experiments, 4. After further purification of the Aalpha chain by polyacrylamide gel electrophoresis, antiserum to the Aalpha chain was raised in rabbits. Anti-Aalpha. antisera, was heat-decompleraented and absorbed with Bbeta polypeptide chains. On immunodiffusion, absorbed anti-Aalpha. antiserum reacted with Aalpha, but not with Bbeta or gamma chains. In addition, the antiserum reacted with purified fibrinogen and plasma fibrinogen, showing a reaction of identity with the Aalpha chain. 5. The progress of plasmin degradation of fibrinogen was examined by immunoelectrophoresis. When anti-Aalpha antiserum was used, the appearance of two intersecting precipitin arcs confirmed that early cleavage of the Aalpha chain of fibrinogen had occurred. These two precipitin arcs persisted throughout the digestion, A 26,000 molecular weight polypeptide, isolated by gel filtration from a late digest, corresponded in electro- phoretic mobility to the more cathodal arc of the late digest of fibrinogen. This polypeptide was named Aalpha-related antigen (molecular weight 26,000), Aalpha-RA(26,000). 6. Examination of the various stages of plasmin degradation of fibrinogen by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed the progressive appearance of six low molecular weight polypeptides as the digestion proceeded. One of these, Aalpha RA(26,000), was isolated and its immunological behaviour confirmed its Aalpha chain origin. Two other polypeptides of slightly higher molecular weight were isolated in semi-purified form; it appeared likely that they also had an Aalpha chain origin and were precursors of Aalpha RA(26,000). None of these polypeptides reacted with commercial anti-fibrinogen antiserum, and reacted only weakly with unabsorbed antifibrinogen antiserum, Anti-Aalpha antiserum did not react with purified fibrinogen fragments Y, B or E, but a faint reaction was obtained with fragment X. 7. Urolcinase activation of the plasma fibrinolytic system in vitro resulted in a limited degree of fibrinogenolysis. The extent of such plasma fibirinogen degradrastion may not be readily detect, by assays employing anti-fibrinogen anti-serum, but qualitative examination of the serum from such motivated plasma revealed an increase in alpha-related antigen(s). The serum Aalpha related antigen(s) were not immunologically identical to Aalpha-RA(26,000). 8. Activation of the plasma fibrinolytic system in vivo by venous occlusion of the arm also resulted in a limited degree of fibrinogenolysis. In the limited number of cases studied, an increase in the level of Aalpha-related antigen(s) was demonstrated in the post-occlusion serum samples by the techniques of single radial immunodiffusion and Laurell electrophoresis. Further refinement of these techniques may constitute the basis for an assay of in vivo fibrinogenolysis. 9. The site of origin of the fragment Aalpha -HA(26,000) was located in a central region of the Aalpha polypeptide chain of fibrinogen; the antigenic determinant sites of the fragment were contained in the Aalpha, remnant of the high solubility fibrinogen, fraction I-9 but not in the COOH-terminal polypeptides released from the Aalpha chain of fibrinogen by ancrod cleavage. 10. From the results of immunodiffusion with anti-Bbeta and anti-gamma antisera, some cross-contamination of the Bbeta and gamma chain preparations was likely. Neither antisera reacted with the Aalpha chains, and only anti-3beta anti-serum reacted weakly with whole fibrinogen. A method for the immunological examination of the Aalpha, Bbeta and gamma chains of plasma fibrinogen, using small volumes of plasma, has been suggested.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J F Davidson
Keywords: Biochemistry
Date of Award: 1979
Depositing User: Enlighten Team
Unique ID: glathesis:1979-72405
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12
URI: http://theses.gla.ac.uk/id/eprint/72405

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