A Kringle-Specific Monoclonal Antibody

Abstract

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.

Immunologic and hematologic properties of antibodies to prothrombin and plasminogen in a mouse model

“…1,16 There are also other reports supporting the concept of cross-reactive antikringle antibodies. Church and his group 18 immunised mice with a synthetic peptide from prothrombin kringle 2 containing a highly conserved region of the kringle proteins, and showed that a monoclonal antibody to this peptide recognised plasminogen kringle 5 in solid phase, not in liquid phase. The same monoclonal antibody bound other kringle proteins t-PA, apo(a), prothrombin and intact plasminogen in the solid phase.…”

Antibodies to prothrombin crossreact with plasminogen in patients developing myocardial infarction

“…Church et al (1994 ) have shown that monoclonal antibodies raised against the kringle 2 of prothrombin also recognize the kringle 5 of plasminogen, but not the other four kringle units. In our preliminary experiments we found that antiprothrombin antibodies were not inhibited by preparations of plasminogen containing either kringles 1–3 or 4, but did react with the whole molecule (data not shown).…”