Development of a radioimmunoassay for the fibrinogen-derived peptide B beta 1-42

The peptide B beta 1-42 is the initial cleavage product of plasmin-mediated proteolysis of the NH2-terminal region of fibrinogen or fibrin I, while beta 15-42 is the major fragment released by plasmin degradation of fibrin II. Numerous studies have described the measurement of plasma B beta 1-42 levels as an index of plasmin activity. Previous assays were indirect and included quantitation of thrombin-increasable fibrinopeptide B immunoreactivity (TIFPB) or measurement of beta 15-42 with an antiserum (132) which cross-reacted with B beta 1-42. We report on a new antiserum (R142) directed against B beta 1-42 which does not cross-react with beta 15-42 or fibrinopeptide B. Employing this antiserum, a specific assay for B beta 1-42 was developed. This assay was used to measure plasmin-mediated B beta 1-42 release from fibrinogen and its subsequent proteolysis by thrombin. The selectivity constant (Kcat/Km) for thrombin cleavage of the B beta 14-15 bond of B beta 1-42 was 10(5)-fold less than that for proteolysis of the same bond in the intact fibrinogen molecule, thus explaining the stability of this peptide in the presence of thrombin activity in the blood. Similarly, the selectivity constant for plasmin cleavage of the B beta 21-22 bond of B beta 1-42 was 140-fold less than that for proteolysis of the B beta 42-43 bond of fibrinogen, indicating that secondary plasmin attack of the B beta 1-42 molecule is not physiologically important. The specific B beta 1-42 assay provided excellent recovery of peptide added to blood or plasma. Comparison of B beta 1-42 levels with TIFPB values in 37 patient samples yielded good correlation over a wide range of levels (r2 = 0.91). The median plasma B beta 1-42 level in 15 normal individuals was 1.2 pmol/mL. This is similar to the previously reported normal range for TIFPB (1 to 4 pmol/mL) but is higher than the normal level of 0.4 pmol/mL reported with the assay employing antiserum 132. This discrepancy reflects rapid removal of Arg (B beta 42) by plasma carboxypeptidase activity resulting in 50% loss of B beta 1-42 immunoreactivity with antiserum 132 but no loss with R142. To circumvent this problem, we have developed a beta 15-42 antiserum (R154) which, like antiserum 132, cross-reacts with B beta 1-42. However, B beta 1-42/beta 15-42 immunoreactivity with R154 is stable in the presence of carboxypeptidase activity.