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Thromb Haemost 1988; 60(02): 153-159
DOI: 10.1055/s-0038-1647021
Original Article
Schattauer GmbH Stuttgart

Early Alpha Chain Crosslinking in Human Fibrin Preparations

Joan H Sobel
The Department of Medicine, College of Physicians & Surgeons of Columbia University, New York NY, USA
,
Christiane A Thibodeau
The Department of Medicine, College of Physicians & Surgeons of Columbia University, New York NY, USA
,
Robert E Canfield
The Department of Medicine, College of Physicians & Surgeons of Columbia University, New York NY, USA
› Author Affiliations
Further Information

Publication History

Received 30 November 1987

Accepted after revision 09 May 1988

Publication Date:
28 June 2018 (online)

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Summary

Purified fibrinogen preparations were clotted under crosslinking conditions and the evolution of α polymers was examined by Western blotting (SDS-PAGE). Monoclonal antibodies that bind to two localized regions within the COOH-terminal portion of the (A)α chains of fibrinogen (F-103, Aα #259-276; F-102, Aα #540-554) were used for immunodetection. Three crosslinked components (100K, 168K, 210K) that each exhibited coincident F-102 and F-103 immunoreactivities were evident as early as γ dimer formation under the in vitro conditions employed. Initial events in the a chain crosslinking process appeared to include interactions between relatively intact chains and a variety of degraded ones that shared the structure Aα #1-276, but differed in the extent to which regions between residues — #276 and — #539 were preserved. Degraded fibrinogen molecules whose Aα chains all terminated before Aα #540-554 were isolated from purified fibrinogen preparations by immunoaffinity chromatography on F-102 Sepharose. Immunoblotting data obtained for the crosslinking capacity of these degraded molecules indicated that early crosslinked components (95 K, 205 K) could form even in the absence of intact a chain partners. This crosslinking, moreover, could progress to the polymer stage. These findings demonstrate that some early crosslinking activity is localized exclusively within regions NH2-terminal to Aα — #540-554 and suggest that fibrinogen molecules with partially degraded Aα chains, which are likely to be circulating under many pathophysiologic conditions, can undergo fibrin stabilization through crosslinking despite a loss of at least 70 COOH-terminal Aα chain residues.

Keywords

Alpha chain crosslinking - Crosslinked fibrin - Crosslinking sites - Monoclonal antibodies
 

  • References

  • 1 Matacic SS, Loewy AG. The identification of isopeptide crosslinks in insoluble fibrin. Biochem Biophys Res Commun 1968; 30: 356-362
    CrossrefPubMedSearch in Google Scholar
  • 2 Mc Kee PA, Mattock P, Hill RL. Subunit structure of human fibrinogen, soluble fibrin, and crosslinked insoluble fibrin. Proc Natl Acad Sci 1970; 66: 738-744
    CrossrefPubMedSearch in Google Scholar
  • 3 Schwartz ML, Pizzo SV, Hill RL, Mc Kee PA. The effect of fibrin stabilizing factor on the subunit structure of human fibrin. J Clin Invest 1971; 50: 1506-1513
    CrossrefPubMedSearch in Google Scholar
  • 4 Gaffney PJ, Whitaker AN. Fibrin crosslinks and lysis rates. Thromb Res 1979; 14: 85-94
    CrossrefPubMedSearch in Google Scholar
  • 5 McDonagh Jr RP, Mc Donagh J, Duckert F. The influence of fibrin crosslinking on the kinetics of urokinase-induced clot lysis. Br J Haematol 1971; 21: 323-332
    CrossrefPubMedSearch in Google Scholar
  • 6 Pisano JJ, Bronzert TJ, Peyton MP, Finlayson JS. ε-(γ-glutamyl)lysine crosslinks: Determination in fibrin from normal and Factor XHI-deficient individuals. Ann NY Acad Sci 1972; 202: 98-113
    CrossrefPubMedSearch in Google Scholar
  • 7 Takagi T, Doolittle RF. Amino acid sequence studies of the a chain of fibrinogen. Location of four-plasmin attack points and a covalent crosslinking site. Biochemistry 1975; 14: 5149-5156
    CrossrefPubMedSearch in Google Scholar
  • 8 Fretto LJ, Ferguson EW, Steinman HM, Mc Kee PA. Localization of the α-chain crosslink acceptor sites of human fibrin. J Biol Chem 1978; 253: 2184-2195
    PubMedSearch in Google Scholar
  • 9 Doolittle RF, Cassman KG, Cottrell BA, Friezner SJ. Amino acid sequence studies on the a chain of human fibrinogen. Isolation and characterization of two linked α-chain cyanogen bromide fragments from fully crosslinked fibrin. Biochemistry 1977; 16: 1715-1719
    CrossrefPubMedSearch in Google Scholar
  • 10 Fretto LJ, Mc Kee PA. Structure of a polymer from in vitro and in vivo highly crosslinked human fibrin. J Biol Chem 1978; 253: 6614-6622
    PubMedSearch in Google Scholar
  • 11 Corcoran DH, Ferguson EW, Fretto LJ, Mc Kee PA. Localization of a crosslink donor site in the a chain of human fibrin. Thromb Res 1980; 19: 883-888
    CrossrefPubMedSearch in Google Scholar
  • 12 Sobel JH, Ehrlich PH, Birken SB, Saffran AJ, Canfield RE. Monoclonal antibody to the region of fibronectin involved in crosslinking to human fibrin. Biochemistry 1983; 22: 4175-4183
    CrossrefPubMedSearch in Google Scholar
  • 13 Mosesson MW, Sherry S. The preparation and properties of human fibrinogen of relatively high solubility. Biochemistry 1966; 5: 2829-2835
    CrossrefPubMedSearch in Google Scholar
  • 14 Mosesson M, Galanakis D, Finlayson JS. Comparison of human plasma fibrinogen subfractions and early plasmic fibrinogen derivatives. J Biol Chem 1974; 249: 4656-4664
    PubMedSearch in Google Scholar
  • 15 Mills D, Karpatkin S. The non-plasmin, proteolytic origin of human fibrinogen heterogeneity. Biochim Biophys Acta 1971; 251: 121-125
    CrossrefPubMedSearch in Google Scholar
  • 16 Mosesson MW, Finlayson JS, Umfleet RA, Galanakis D. Human fibrinogen heterogeneities. I. Structural and related studies of plasma fibrinogens which are high solubility catabolic intermediates. J Biol Chem 1972; 247: 5210-5219
    PubMedSearch in Google Scholar
  • 17 Galanakis DK, Mosesson MW, Stathakis NE. Human fibrinogen heterogeneities: Distribution and charge characteristics of chains of Aα origin. J Lab Clin Med 1978; 92: 376-386
    PubMedSearch in Google Scholar
  • 18 Ferguson EW. High-resolution two-dimensional electrophoretic analysis of fibrinogen digestion by plasmin. J Lab Clin Med 1980; 96: 710-721
    PubMedSearch in Google Scholar
  • 19 Liu CY, Sobel JH, Weitz JI, Kaplan KL, Nossel HL. Immunologic identification of the cleavage products from the Aα and Bβ chains in the early stages of plasmin digestion of fibrinogen. Thromb Haemostas 1986; 56: 100-106
    PubMedSearch in Google Scholar
  • 20 Finlayson JS, Mosesson MW, Bronzert TJ, Pisano JJ. Human fibrinogen heterogeneities II. Crosslinking capacity of high solubility catabolic intermediates. J Biol Chem 1972; 247: 5220-5222
    PubMedSearch in Google Scholar
  • 21 Finlayson JS, Mosesson MW. Crosslinking of a chain remnants in human fibrin. Thromb Res 1973; 2: 467-478
    CrossrefPubMedSearch in Google Scholar
  • 22 Pizzo SV, Schwartz ML, Mattock P, Hill RL, Mc Kee PA. Correlation of clottability of purified human fibrinogen with its digestion by purified human plasmin. J Lab Clin Med 1970; 76: 892-893
    PubMedSearch in Google Scholar
  • 23 Ehrlich PH, Sobel JH, Moustafa ZA, Canfield RE. Monoclonal antibodies to a chain regions of human fibrinogen that participate in polymer formation. Biochemistry 1983; 22: 4184-4192
    CrossrefPubMedSearch in Google Scholar
  • 24 Sobel JH, Thibodeau CA, Gawinowicz Kolks MA, Canfield RE. Immunochemical characterization of crosslinked derivatives isolated from alpha chain oligomers formed during early stages of fibrin crosslinking. Thromb Heamostas 1988; 60: 160-169
    PubMedSearch in Google Scholar
  • 25 Lipinska I, Lipinski B, Gurewich V, Hoffman KD. Fibrinogen heterogeneity in cancer, in occlusive vascular disease, and after surgical procedures. Am J Clin Pathol 1976; 65: 958-966
    PubMedSearch in Google Scholar
  • 26 Koehn JA, Canfield RE. Purification of human fibrinopeptides by high performance liquid chromatography. Anal Biochem 1981; 116: 349-356
    CrossrefPubMedSearch in Google Scholar
  • 27 Lorand L, Gotoh T. Fibrinoligase: The fibrin stabilizing factor system. Methods Enzymol 1970; 19: 770-782
    PubMedSearch in Google Scholar
  • 28 Bruck C, Portetelle D, Glineur C, Bollen A. One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-Gel Blue chromatography. J Immunol Methods 1982; 53: 313-319
    CrossrefPubMedSearch in Google Scholar
  • 29 Laemmli UK, Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 1973; 80: 575-599
    CrossrefPubMedSearch in Google Scholar
  • 30 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedSearch in Google Scholar
  • 31 Francis CW, Marder VJ, Martin SE. Plasmic degradation of crosslinked fibrin I. Structural analysis of the particulate clot and identification of new macromolecular soluble complexes. Blood 1980; 56: 456-464
    PubMedSearch in Google Scholar
  • 32 Francis CW, Markham Jr RE, Marder VJ. Demonstration of in situ fibrin degradation in pathologic thrombi. Blood 1984; 63: 1216-1224
    PubMedSearch in Google Scholar
  • 33 Watt KW K, Cottrell BA, Strong DD, Doolittle RF. Amino acid studies on the a chain of human fibrinogen. Overlapping sequences providing the complete sequence. Biochemistry 1979; 24: 5410-5416
    PubMedSearch in Google Scholar
  • 34 Lottspeich F, Henschen A. Amino acid sequence of human fibrin. Preliminary note on the completion of the C-terminal part of the a chain sequence. Hoppe-Seyler’s Z Physiol Chem 1978; 359: 1451-1455
    PubMedSearch in Google Scholar
  • 35 Kant J, Lord ST, Crabtree GR. Partial mRNA sequences of human Aα Bβ and γ fibrinogen chains: Evolutionary and functional implications. Proc Natl Acad Sci USA 1983; 80: 3953-3957
    CrossrefPubMedSearch in Google Scholar
  • 36 Rixon MW, Chan W-Y, Davie EW, Chung DW. Characterization of a complementary deoxyribonucleic acid coding for the α chain of human fibrinogen. Biochemistry 1983; 22: 3237-3244
    CrossrefPubMedSearch in Google Scholar
  • 37 Cottrell BA, Doolittle RF. The amino acid sequence of a 27-residue peptide released from the α chain carboxy-terminus during the plasmic degradation of human fibrinogen. Biochem Biophys Res Commun 1976; 71: 754-761
    CrossrefPubMedSearch in Google Scholar
  • 38 Holm B, Nilsen DW T, Kierulf P, Godal HC. Purification and characterization of 3 fibrinogens of different molecular weight obtained from normal human plasma. Thromb Res 1985; 37: 165-176
    CrossrefPubMedSearch in Google Scholar
  • 39 Lipinska I, Lipinski B, Gurewich V. Fibrinogen heterogeneity in human plasma. Electrophoretic demonstration and characterization of two major fibrinogen components. J Lab Clin Med 1974; 84: 509-515
    PubMedSearch in Google Scholar
  • 40 Galanakis DK, Mosesson MW. Human fibrinogen heterogeneities: Determination of the major Aα chain derivatives in blood. Thromb Res 1983; 31: 403-413
    CrossrefPubMedSearch in Google Scholar
  • 41 Weinstein MJ, Deykin D. Quantitative abnormality of an Aα chain molecular weight form in the fibrinogen of cirrhotic patients. Br J Haematol 1978; 40: 617-630
    CrossrefPubMedSearch in Google Scholar
  • 42 Holm B, Godal HC. Quantitation of the three normally occurring plasma fibrinogens in health and during so called “acute phase” by SDS electrophoresis of fibrin obtained from EDTA-plasma. Thromb Res 1974; 35: 279-290
    PubMedSearch in Google Scholar
  • 43 Weisel JW, Stauffacher C, Bullitt E, Cohen C. A model for fibrinogen: Domains and sequence. Science 1985; 230: 1388-1391
    CrossrefPubMedSearch in Google Scholar
  • 44 Medved LV, Gorkun OV, Privalov PL. Structural organization of C-terminal parts of fibrinogen Aα-chains. FEBS 1983; 160: 291-295
    CrossrefPubMedSearch in Google Scholar
  • 45 Holm B, Brosstad K, Kierulf P, Godal HC. Polymerization properties of two normally circulating fibrinogens, HMW and LMW. Evidence that the COOH-terminal end of the a chain is of importance for fibrin polymerization. Thromb Res 1985; 39: 595-606
    CrossrefPubMedSearch in Google Scholar
  • 46 Weisel JW, Papsun D. Involvement of the COOH-portion of the COOH-terminal portion of the alpha chain of fibrin in the branching of fibers to form a clot. Thromb Res 1987; 47: 155-163
    CrossrefPubMedSearch in Google Scholar
  • 47 Holm B, Nilsen DW T, Godal HC. Half life and incorporation into venous thrombi of the normally occuring plasma fibrinogen fractions HMW and LMW. Thromb Res 1986; 41: 57-66
    PubMedSearch in Google Scholar