Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases.
A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.
Mitkevich
O V
OV
Institute of Experimental Cardiology, Russian Cardiology Research Center, Moscow, Russia.
Sobel
J H
JH
Shainoff
J R
JR
Vlasik
T N
TN
Kalantarov
G F
GF
Trakht
I N
IN
Streltsova
Z A
ZA
Samokhin
G P
GP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D018604:Epitope Mapping; D005340:Fibrinogen / Q000276:immunology*; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology*; D010449:Peptide Mapping / Q000379:methods; D013927:Thrombosis / Q000097:blood; D011503:Transglutaminases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010446:Peptide Fragments; C101356:fibrinogen Aalpha (529-39); D005340:Fibrinogen; D011503:Transglutaminases; D003488:Cyanogen Bromide