Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg→His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

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FIBRINOGEN BIRMINGHAM; A NEW CONGENITAL HETERODIMERIC DYSFIBRINOGENEMIA WITH DEFECTIVE FIBRINOPEPTIDE A RELEASE (Aα 16 Arg→His)

10.1055/s-0038-1643338 ◽

1987 ◽

Author(s):

K R Siebenlist ◽

J T Prchal ◽

M W Masesson

Keyword(s):

Amino Acid ◽

Amino Acid Analysis ◽

Hplc Analysis ◽

Biochemical Characterization ◽

Clinical Manifestations ◽

Severe Bleeding ◽

Fibrinopeptide A ◽

History Of ◽

Bleeding Episodes

Aα 16 Arg→His substitutions are common forms of congenital dysfibrinogenemias. Clinical manifestations range from asymptomatic to moderate hemorrhagic tendencies. Biochemical characterization of one such heterozygotic individual (Fibrinogen Louisville, Galanakis, etal. Ann NY Acad Sci 408:644,1983) indicated that only homodimeric fibrinogen molecules (i.e., containing either normal or abnormal Aα chains) were present. We isolated fibrinogen from the plasma of a 23 year old patient with a history of easy bruising and several recent moderate to severe bleeding episodes. Coagulability with reptilase was 677 (65-70%; n=5) whereas with thrombin (Ha) it approached 100%, depending directly upon the time of incubation with enzyme. HPLC analysis of Ila-induced fibrinopeptide release demonstrated the presence of an abnormal A-peptide (A*), amounting to 50% of the total, which was released more slowly than the normal A-peptide (A). Amino acid analysis of A* demonstrated the absence of Arg and the presence of His. Carboxypeptidase digestion confirmed the structure of A* as Aα 16 Arg-→ His. The clot and the soluble clot liquor resulting from reptilase treatment were separated and each was then further treated with Ilato release A*. HPLC analysis indicated that 31% of the total A* present in the sample was associated with the reptilase clot and 697 remained in the clot liquor. This distribution of A* suggests that Fibrinogen Birmingham, unlike Fibrinogen Louisville, contains heterodimeric molecules that are incorporated into the reptilase clottable fraction. This finding is consistent with a process of random hepatic assembly of dimeric fibrinogen molecules in a heterozygotic individual.

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FIBRINOGEN LONG BEACH

10.1055/s-0038-1643337 ◽

1987 ◽

Author(s):

H Pirkle ◽

I Theodor ◽

P Vukasin ◽

B Nandi ◽

D Miyada ◽

...

Keyword(s):

Amino Acid ◽

Gel Electrophoresis ◽

Amino Acid Analysis ◽

Cleavage Site ◽

Fibrinopeptide A ◽

Long Beach ◽

Thrombin Cleavage ◽

Complete Failure

We find that, in addition to a normal fibrinopeptide A (FPA), fibrinogen Long Beach releases an abnormal FPA whose HPLC behavior suggests and whose amino acid analysis confirms the substitution of His for Arg at position 16, the thrombin cleavage site. However, this dysfibrinogen displays properties strikingly different from those previously reported to be associated with Aa 16 Arg → His substitutions. These properties include a limited release of normal FPA (18-20%) by both thrombin and batroxobin, an essentially full release of abnormal FPA (40-50%) by thrombin (1.5 NIH u/ml) in contrast to a 7-8% release of abnormal FPA by batroxobin, and a complete failure of highly purified batroxobin to clot the dysfibrinogen while generating a fragment which migrates between the B8 and y chains on SDS gel electrophoresis. The basis for these differing properties is not yet clear.

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Chemotaxonomic studies on davaineid tapeworms, based on amino acid analysis

Journal of Helminthology ◽

10.1017/s0022149x00025207 ◽

1984 ◽

Vol 58 (4) ◽

pp. 325-326 ◽

Cited By ~ 2

Author(s):

Anjali Bhalya ◽

Amita Seth ◽

V. N. Capoor ◽

Sandeep K. Malhotra

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Analysis ◽

Gallus Gallus ◽

Gallus Domesticus ◽

Gallus Gallus Domesticus ◽

The Family

ABSTRACTTwelve amino acids were detected in six davaincid ecstodes, Davainea hewetensis Dhawan & Capoor (1972) collected from one Gallus gallus domesticus L. at Allahabad, India. Chemotaxonomic studies within the family Davaineidac have shown the presence of cystine and hydroxyproline only in D. hewetensis, although phenylalanine and proline occur in all species examined previously.

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Bovine Platelet Proteins II. Purification of Platelet Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653556 ◽

1969 ◽

Vol 21 (03) ◽

pp. 419-427 ◽

Cited By ~ 5

Author(s):

N. O Solum ◽

S Łopaciuk

Keyword(s):

Analytical Ultracentrifugation ◽

Insoluble Fraction ◽

Disc Electrophoresis ◽

Plasma Fibrinogen ◽

Starch Gel Electrophoresis ◽

Freezing And Thawing ◽

Insoluble Material ◽

High Resolution Power ◽

Starch Gel ◽

Platelet Proteins

Summary1. Platelet fibrinogen has been purified from washed bovine platelets. The procedure was based on the methods for purification of plasma fibrinogen by fractionated precipitations and extractions with ethanol and glycine below 0°, and precipitation of proteins by dimethylformamide at 0°.2. The platelet extract obtained by freezing and thawing of the cells, freed from insoluble material by centrifugation at 23,000 x g for 30 min, contained 0.22 ±0.003mg fibrinogen per 109 platelets. Total protein of this fraction was 0.77 ±0.08 mg per 109 platelets whereas that of the insoluble fraction was 0.79 ±0.09 mg per 109 platelets.3. The most purified platelet fibrinogen fraction contained 91-98% of the protein in a thrombin-clottable state. The yield was approx. 20%. It showed homogeneity in analytical ultracentrifugation, in immunoelectrophoresis using an antiserum produced by immunization of rabbits against platelet extract, and in starch gel electrophoresis using a discontinuous system of Tris HCl and borate buffers offering a high resolution power towards the platelet proteins. Polyacrylamide disc electrophoresis revealed two to three faint lines behind the main fibrinogen line. At least one such line was also observed with purified plasma fibrinogen.

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An ESR study of the peroxidase reaction catalyzed by human methaemoglobin and methaemoglobin-haptoglobin complex

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19910923 ◽

1991 ◽

Vol 56 (4) ◽

pp. 923-932

Author(s):

Jana Stejskalová ◽

Pavel Stopka ◽

Zdeněk Pavlíček

Keyword(s):

Hydrogen Peroxide ◽

Ascorbic Acid ◽

Amino Acid ◽

Peroxidase Activity ◽

Amino Acid Analysis ◽

Superoxide Radical ◽

Esr Spectra ◽

The Other ◽

Delocalized Electron

The ESR spectra of peroxidase systems of methaemoglobin-ascorbic acid-hydrogen peroxide and methaemoglobin-haptoglobin complex-ascorbic acid-hydrogen peroxide have been measured in the acetate buffer of pH 4.5. For the system with methaemoglobin an asymmetrical signal with g ~ 2 has been observed which is interpreted as the perpendicular region of anisotropic spectrum of superoxide radical. On the other hand, for the system with methaemoglobin-haptoglobin complex the observed signal with g ~ 2 is symmetrical and is interpreted as a signal of delocalized electron. After realization of three repeatedly induced peroxidase processes the ESR signal of the perpendicular part of anisotropic spectrum of superoxide radical is distinctly diminished, whereas the signal of delocalized electron remains practically unchanged. An amino acid analysis of methaemoglobin along with results of the ESR measurements make it possible to derive a hypothesis about the role of haptoglobin in increasing of the peroxidase activity of methaemoglobin.

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