Fibrinopeptide B, human (FPB,human)

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

Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A (alpha) and B (beta) 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 (alha) and B (beta) 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 couple to desaminotyrosine and radiolabeled with 125I 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. The senistivity of the assay was such that 50% inhibition of binding of the tracer was caused by 1.25 ng of the native FPB. Fibrinogen was treated with thrombin, plasmin, trypsin, Reptilase, and an extract of the venom from Ancistrodon contortrix contortrix (ACC). After ethanol precipitation and centrifugation, dialysates of enzymatically altered fibrinogen were assayed for FPA and FPB. The action of thrombin on fibrinogen resulted in a rapid release of FPA and a slower release of FPB. Plasmin cleaved a segment(s) of the B (beta) 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 slowere 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.

Immunochemical studies of antisera to human fibrinopeptide-B

The immunochemical specificity of rabbit antisera to human fibrinopeptide-B (FPB) has been studied by comparing the relative abilities of FPB and of various proteins and peptides containing the NH2-terminal segment of the B beta-chain of human fibrinogen to inhibit the binding of a radioiodinated FPB derivative by each of seven anti-FPB sera. Anti-FBP sera varied in the extent to which they cross-reacted with fibrinogen, the NH2-terminal disulfide knot of fibrinogen (N-DSK), B beta 1(Pyr)-118(Met), B beta 1(Pyr)-42(Arg), and desarginyl-FPB. Anti-FPB sera have been identified that discriminate effectively between FPB and larger FBP-containing peptides; such antisera can be used to measure FPB in the absence of the larger peptides or to demonstrate the presence of larger peptides such as B beta 1(Pyr)-42(Arg) in extracts of clinical plasma samples by means of an increase in FPB immunoreactivity following thrombin treatment. One anti-FPB serum has been identified that is capable of detecting desarginyl-FPB, and this antiserum has been used in the development of a radioimmunoassay for desarginyl-FPB. Thus, by precisely defining the specificity of anti-FPB sera, it has been possible to identify antisera that are useful, not only in the measurement of FPB, but also in the detection of other important related molecules, such as B beta 1(Pyr)-42(Arg) and desarginyl-FPB. The immunochemical detection of these FPB-related peptides should provide useful information concerning the action of proteolytic enzymes, such as plasmin on the NH2-terminal segment of the B beta-chain of fibrinogen, and of carboxypeptidase-B on free FPB, in human plasma.

[Fibrinopeptide A (FPA), fibrinopeptide B (FPB) and fibrinopeptide Bbeta(15-42) (FPBbeta15-42)]

[Fibrinopeptide A (FPA) and fibrinopeptide B beta 15-42 (FPB beta 15-42)]

Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions

Fibrin polymerisation is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant γD364H -fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to γD364H -fibrinogen, we examined two variant fibrinogens with substitutions altering knob 'A', Aα17A- and Aα17C-fibrinogen. We examined thrombin- or batroxobin-catalysed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for γ D364H-fibrinogen. Thrombin-catalysed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than for γD364H -fibrinogen. No variants polymerised with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerisation was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes 'b'. SEM showed that the variant clots from Aα17A- and γD364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas Aα17C -fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions.