Fibrin Detected in Plasma of Patients with Disseminated intravascuiar Coagulation by Fibrin-specific Antibodies Consists Primarily of High Molecular Weight Factor XIIIa-crosslinked and Plasmin-modified Complexes Partially Containing Fibrinopeptide A

Summary

Pooled plasma from 40 patients with severe disseminated intravascuiar coagulation (DIC) secondary to septic conditions was subjected to gel permeation chromatography on Sephacryl S-500 HR after sample pretreatment with KSCN for dissociation of non-covalent fibrin complexes. Fibrin antigen in eluates was detected by an array of ELISA tests, using two monoclonal antibodies against fibrin degradation product D-dimer, a monoclonal antibody against an epitope generated by plasmin cleavage of the D-domain, and an antibody against the neo-N- terminus of the α-chain of fibrin exposed by cleavage of fibrinopeptide A. Tag antibodies were a polyclonal antibody against the fibrinogen/ fibrin D-domain, a POD-conjugated version of the monoclonal antibody against fibrin α-chain neo-N-terminus, and a polyclonal antibody against fibrinopeptide A. Most fibrin-related material present in the pooled DIC plasma was of higher molecular mass than fibrinogen. Fibrin polymers were reactive with antibodies against D-dimer, plasmin cleaved D-domain, and fibrin α-chain neo-N-terminus. Part of the polymers reacted with antibodies against fibrinopeptide A, indicating presence of fibrinogen or desA-fibrin monomer within the covalently linked complex. In conclusion, the primary analytes detected by monoclonal antibodies for D-dimer, plasmin-specific epitopes of fibrin degradation products, as well as sites exposed by fibrinopeptide cleavage in plasma from patients with disseminated intravascuiar coagulation are high molecular weight factor XIIIa-crosslinked fibrin complexes, containing plasmin-cleaved D-domains, intact fibrin monomer units, and fibrinogen or desA-fibrin monomer.

• References

• 1 Elms MJ, Bunce IH, Bundesen PG, Rylatt DB, Webber AJ, Masci PP, Whitaker AN. Measurement of crosslinked fibrin degradation products – an immunoassay using monoclonal antibodies. Thromb Haemost 1983; 50: 591-594
• 2 Whitaker AN, Elms MJ, Masci PP, Bundesen PG, Rylatt DB, Webber AJ, Bunce IH. Measurement of cross linked fibrin derivatives in plasma: an immunoassay using monoclonal antibodies. J Clin Pathol 1984; 37: 882-887
• 3 Carr MEJ. Disseminated intravascular coagulation: pathogenesis, diagnosis, and therapy. J Emerg Med 1987; 05: 311-322
• 4 Whitaker AN, Rowe EA, Masci PP, Gaffney PJ. Identification of D-dimerE complex in disseminated intravascular coagulation. Thromb Res 1980; 18: 453-459
• 5 Carr JM, McKinney M, McDonagh J. Diagnosis of disseminated intravascular coagulation. Role of D-dimer. Am J Clin Pathol 1989; 91: 280-287
• 6 Bredbacka S, Blombäck M, Wiman B, Pelzer H. Laboratory methods for detecting disseminated intravascular coagulation (DIC): new aspects. Acta Anaesthesiol Scand 1993; 37: 125-130
• 7 Ott P, Astrup L, Jensen RH, Nyeland B, Pedersen B. Assessment of Ddimer in plasma: diagnostic value in suspected deep venous thrombosis of the leg. Acta Med Scand 1988; 224: 263-267
• 8 Mirshahi M, Soria C, Soria J, Faivre R, Kieffer Y, Bassand JP, Toulemonde F, Caen J. Changes in plasma fibrin degradation products as a marker of thrombus evolution in patients with deep vein thrombosis. Thromb Res 1988; 51: 295-302
• 9 Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Stevens P, Brill Edwards P, Panju A, Patel A. Evaluation of a soluble fibrin assay in patients with suspected deep vein thrombosis. Thromb Haemost 1995; 74: 833-836
• 10 Speiser W, Mallek R, Koppensteiner R, Stumpflen A, Kapiotis S, Minar E, Ehringer H, Lechner K. D-dimer and TAT measurement in patients with deep venous thrombosis: utility in diagnosis and judgement of anticoagulant treatment effectiveness. Thromb Haemost 1990; 64: 196-201
• 11 Boneu B, Bes G, Pelzer H, Sié P, Boccalon H. D-Dimers, thrombin antithrombin III complexes and prothrombin fragments 1+2: diagnostic value in clinically suspected deep vein thrombosis. Thromb Haemost 1991; 65: 28-31
• 12 Bounameaux H, de MoerlooseP, Perrier A, Reber G. Plasma measurement of D-dimer as diagnostic aid in suspected venous thromboembolism: an overview [see comments]. Thromb Haemost 1994; 71: 01-06
• 13 Mao SJ, Rechtin AE, Krstenansky JL, Jackson RL. Characterization of a monoclonal antibody specific to the amino terminus of the alpha-chain of human fibrin. Thromb Haemost 1990; 63: 445-448
• 14 Scheefers Borchel U, Müller Berghaus G, Fuhge P, Eberle R, Heimburger N. Discrimination between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide. Proc Natl Acad Sci USA 1985; 82: 7091-7095
• 15 Lill H, Spannagl M, Trauner A, Schramm W, Schuller D, Ofenloch Haehnle B, Draeger B, Naser W, Dessauer A. A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo. Blood Coagul Fibrinolysis 1993; 04: 097-102
• 16 Dempfle CE, Dollman M, Lill H, Puzzovio D, Dessauer A, Heene DL. Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site ‘A’: effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN. Blood Coagul Fibrinolysis 1993; 04: 79-86
• 17 Kudryk B, Rohoza A, Ahadi M, Chin J, Wiebe ME. A monoclonal antibody with ability to distinguish between NH2-terminal fragments derived from fibrinogen and fibrin. Mol Immunol 1983; 20: 1191-1200
• 18 Matsuda M, Terukina S, Yamazumi K, Maekawa H, Soe G. A monoclonal antibody that recognizes the NH2-terminal conformation of fragment D. Excerpta medica 1990; 892: 43-48
• 19 Kario K, Matsuo T, Kabayashi H, Matsuo M, Yamamoto K, Sakurai G, Baba M. Rapid quantitative evaluation of plasma D-dimer levels in thrombotic states using an automated latex photometric immunoassay. Thromb Res 1992; 66: 179-189
• 20 Kraus M, Aillaud E. Nephelometric determination of plasma D-dimer. Ann Hematol 1994; 68 Suppl 02 A110
• 21 Gogstad GO, Dale S, Brosstad F, Brandsnes O, Holtlund J, Mork E, Gartner E, Borch SM. Assay of D-dimer based on immunofiltration and staining with gold colloids. Clin Chem 1993; 39: 2070-2076
• 22 Gron B, Bennick A, Filion Myklebust C, Matsueda GR, Nieuwenhuizen W, Brosstad F. Soluble, cross-linked fibrin(ogen) hybrid oligomers do not stimulate t-PA conversion of plasminogen. Thromb Res 1992; 66: 231-238
• 23 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 24 Shainoff JR, Dardik BN. Role of fibrinogen in fibrin transport: chromatographic studies. Thromb Res 1980; 17: 491-500
• 25 Dardik BN, Shainoff JR. Kinetic characterization of a saturable pathway for rapid clearance of circulating fibrin monomer. Blood 1985; 65: 680-688
• 26 Rylatt DB, Blake AS, Cottis LE, Massingham DA, Fletcher WA, Masci PP, Whitaker AN, Elms M, Bunce I, Webber AJ. et al. An immunoassay for human D dimer using monoclonal antibodies. Thromb Res 1983; 31: 767-778
• 27 Dempfle CE, Hafner G, Lestin HG, Tӧpfer G, Adema E, Hubbuch A. Multizentrische Evaluierung von Tina-quant a D-Dimer. J Lab Med 1996; 20: 31-37
• 28 Zamarron C, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator. J Biol Chem 1984; 259: 2080-2083
• 29 Lijnen HR, Van HoefB, de CockF, Collen D. Effect of fibrin-like stimulators on the activation of plasminogen by tissue-type plasminogen activator (t-PA)-studies with active site mutagenized plasminogen and plasmin resistant t-PA. Thromb Haemost 1990; 64: 61-68
• 30 Wiman B, Ranby M. Determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay. Thromb Haemost 1986; 55: 189-193
• 31 Dempfle CE, Pfitzner SA, Dollman M, Huck K, Stehle G, Heene DL. Comparison of immunological and functional assays for measurement of soluble fibrin. Thromb Haemost 1995; 74: 673-679
• 32 Graeff H, Hafter R, Bachmann L. Subunit and macromolecular structure of circulating fibrin from obstetrics patients with intravascuiar coagulation. Thromb Res 1979; 16: 313-328
• 33 Francis CW, Marder BJ, Martin SE. Plasmic degradation of crosslinked fibrin, I. Structural analysis of the particulate clot and indentification of new macromolecular soluble complexes. Blood 1980; 56: 456-464
• 34 Adema E, Gebert U. Pooled patient samples as reference material for D-dimer. Thromb Res 1995; 80: 85-88
• 35 Brenner B, Francis CW, Marder VJ. The role of soluble cross-linked fibrin in D dimer immunoreactivity of plasmic digests. J Lab Clin Med 1989; 113: 682-688
• 36 Gron B, Bennick A, Filion MyklebustC, Brosstad F. Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer. Blood Coagul Fibrinolysis 1993; 04: 447-454
• 37 Gron B, Filion MyklebustC, Bennick A, Nieuwenhuizen W, Matsueda GR, Brosstad F. Early cross-linked fibrin in human plasma contains alphapolymers with intact fibrinopeptide A. Blood Coagul Fibrinolysis 1992; 03: 731-736