Fibrinopeptide B

Using Table 5.6 (see p. 156), propose a sequence for glu-fibrinopeptide B based on the product-ion MS-MS spectrum shown in Figure 4.20. 

Figure 51-4. Diagrammatic representation (not to scaie) of fibrinogen showing pairs of Aa, B 3, and y chains iinked by disuifide bonds. (FPA, fibrinopeptide A FPB, fibrinopeptide B.)...

Fig. 3. Fibrinogen molecule with its two A a, two B 3, and two -y chains. Thrombin (Ha) acts on the A a chain to generate fibrinopeptide A (FPA) and the a-chain. It also cleaves the B 3 chain to generate fibrinopeptide B (FPB) and the p-chain. S-S represents disulfide bonds. Altogether, 29 disulfide bonds hold together the six polypeptide chains that make up the fibrinogen molecule.

After the early discovery of a tyrosine 0-sulfate residue in bovine fibrinopeptide B, 15 this posttranslational modification which occurs ubiquitously in proteins was also detected in a series of biologically active peptides such as the neurohormones of the gastrin/cholecysto-kinin (CCK) family of peptides, phyllokinin, Leu-enkephalin, and the thrombin inhibitor hirudin listed in Table 1. 

Substitution of particular amino acids in the fibrinopeptides and at thrombin s cleavage sites in recombinant fibrinogen has been used to understand thrombin specificity, the relative rates of fibrinopeptide cleavage, and effects on polymerization. If fibrinopeptide B is replaced with a fibrinopeptide-A-like peptide, fibrinopeptide release is similar to release from the Aa chain (Mullin et al., 2000). Thus, the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. 

There is evidence for the involvement of the N-terminal B/3 chain in fibrin polymerization (Pandya et at, 1985 Siebenlist et al., 1990). A recombinant fibrinogen with His substituted for Arg at the B/3 thrombin-cleavage site led to a 300-fold decrease in the rate of fibrinopeptide B release, whereas the rate of fibrinopeptide A release was normal (Moen et al., 2003). As a consequence, thrombin- or batroxobin-catalyzed or desA monomer polymerization was impaired, due to the histidine substitution itself. Thus, it appears that the N-terminus of the B/3 chain is involved in the lateral aggregation of normal desA protofibrils. 

Mutations of the Bj3 chain are less common. Substitution of Cys for Gly at amino acid 15 results in prolonged polymerization from delayed release of fibrinopeptide B (Yoshida et al., 1991). Mutation of Ala68 to Thr results in defective binding of thrombin to fibrin and consequent thrombosis (Koopman et al., 1992). Deletion of residues 9-72, which includes a cysteine that is normally part of a disulfide bond, results in delayed release of both fibrinopeptides A and B (Liu et al., 1985). 

Dyr, J. E., Blomback, B., Hessel, B., and Komalik, F. (1989). Conversion of fibrinogen to fibrin induced by preferential release of fibrinopeptide B. Biochim Biophys. Acta. 990, 18-24. 

Mihalyi, E. (1988a). Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B. Biochem. 27, 967-976. 

Appropriate standard peptides for calibration with monoisotopic molecular masses covering, for example, the mass range 500-3000 Da (27). Glu-fibrinopeptide-B is also used as external calibrant for MS" experiments (28). 

Top) peptide ladder sequencing principle. Phenyl isothiocyanate (PITC) produces phenylthiohydantoin (PTH) of the terminal amino acid and a new peptide with one less amino acid. Phenyl isocyanate (PIC), in low quantity, produces N-terminal phenylcarba-mate (PC) from a small fraction of each peptide. (Bottom) example of sequencing of [Glu1]fibrinopeptide B. Reproduced (modified) from Chait B.T., Wang R., Beavis R.C. and Kent S.B.H., Science, 262, 89, 1993, with permission. 

Figure 3 A ladder sequence through 6 cycles of [Glul] fibrinopeptide B carried out on a total of 17.5 picomoles of starting peptide.

Figure 5 Quantitative derivatisation of 50 fmol [Glul] fibrinopeptide B with a C-5 quaternary linked tag. The derivatisation was performed on the peptide sample already spotted onto the target slide.

---