Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes.

Abstract

A B S T R A C T Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A(a) and B (P) chains. A radioimmunoassay capable of measuring the A peptide in human blood as an index of thrombin action in vivo has been described previously. This paper describes the development of a radioimmunoassay for FPB and the use of both assays in the demonstration of distinctive patterns of cleavage of the amino terminal ends of the A(a) and B(P) chains of fibrinogen by various enzymes.Antisera were raised in rabbits to a synthetic analogue of FPB coupled to bovine serum albumin. FPB analogue was coupled to desaminotyrosine and radiolabeled with 'I by the chloramine-T technique. The radiolabeled peptide was bound by the antiserum, and binding was inhibited by synthetic or native FPB. Unbound tracer was separated from bound tracer by charcoal adsorption. Plasmin cleaved a segment(s) of the B (f) chain which included FPB but cleaved no detectable FPA-containing material for the first 2 h of incubation. In the case of plasmin-treated fibrinogen, the dialysates had been further treated with thrombin before being assayed for FPA and FPB. Trypsin rapidly cleaved both peptides, the B before the A. Reptilase cleaved only FPA in 24 h. ACC cleaved FPB at a rapid rate, with a slower cleavage of FPA. The distinctive cleavage patterns produced by the serine proteases may be useful in interpreting the levels of FPA and FPB measured in human blood and in studying the generation of FPA and FPB in clinical blood samples.

The generation of fibrinopeptide A in clinical blood samples: evidence for thrombin activity.

“…Preparation of the antisera to the fibrinopeptides has been previously described (3,4,10 The FPA radioimmunoassay was performed as previously described (4 (18 ml), the needle was withdrawn from the vein and removed from the syringe. The blood in the syringe was mixed by inversion and 4.5 ml blood was added at 1-min intervals to each of four tubes containing 0.5 ml of 0.15 M NaCl solution with 500 U of heparin and 500 U of Trasylol.…”

“…lb and 2, and Table IV). To test the postulate that in vitro FPA release resulted from thrombin action, FPA and FPB cleavage (10). In 17 patients the generation of FPA was significantly greater than that of FPB (Table V).…”

“…The development ofa radioimmunoassay (3) has permitted measurement of FPA levels in clinical blood samples as an index ofthrombin action in vivo (4)(5)(6)(7)(8)(9). A radioimmunoassay for FPB has also been developed and together with the FPA assay, used to measure the relative cleavage rates of small fibrinopeptide containing fractions from fibrinogen by different enzymes in buffer solutions (10).…”

Measurement of desarginine fibrinopeptide B in human blood.

“…The exact way in which this balance controls the biochemical structure of fibrin in thrombin is not known. With isolated reagents thrombin cleaves fibrinogen in two stages (1)(2)(3). In the first stage fibrinopeptide A is released and fibrin I formed.…”

“…First fibrinopeptide A and fibrin I are formed and then fibrinopeptide B (B,3 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] and fibrin II. Since it is postulated that fibrin II is important in the genesis of thrombosis, it is of interest to measure fibrinopeptide B in peripheral blood samples.…”