A kringle-specific monoclonal antibody.
Kringle domains are found in several plasma proteins of blood coagulation and fibrinolysis. A murine monoclonal antibody, designated alpha HII-5, was produced against a synthetic peptide representing residues 216-231 of human prothrombin kringle 2. The sequence of the hexadecapeptide (Glu-Asn-Phe-Cys-Arg-Asn-Pro-Asp-Gly-Asp-Glu-Glu-Gly-Val-Gly-Cys) is conserved in several kringle-containing proteins, represents a predicted region of high local hydrophilicity in prothrombin kringle 2, and contains the anionic (Asp-223 and Asp-225) residues that contribute to lysine binding by plasminogen kringle 4. In a solution-phase immunoassay, antibody alpha HII-5 bound prothrombin and the kringle 5 light chain fragment of plasminogen (miniplasminogen), but not plasminogen or plasmin. In contrast, using a solid-phase assay with antigen immobilized onto a surface (polystyrene microtiter plates, glass, or nitrocellulose) antibody alpha HII-5 specifically bound prothrombin, plasminogen, recombinant tissue plasminogen activator (tPA), and the apo(a) subunit of lipoprotein(a). By immunoblotting analysis antibody alpha HII-5 bound determinants on prothrombin fragment 2 and plasminogen kringle 5. These observations suggest that a subset of kringle domains on plasma proteins, including prothrombin kringle 2 and plasminogen kringle 5, contains a homologous antigenic determinant in the region of the kringle lysine-binding site. In contrast to prothrombin kringle 2, the homologous peptide site on plasminogen is not available for antibody binding except when plasminogen is adsorbed to a nonphysiological surface, or when kringles 1-4 are removed.
Church
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Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405.
Messier
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Ouellette
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Potts
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D007118:Immunoassay / Q000379:methods; D015151:Immunoblotting; D018082:Kringles / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000276:immunology; D011516:Prothrombin / Q000276:immunology*; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D011516:Prothrombin; D010958:Plasminogen