Modified form of the fibrinogen B[beta] chain (des-Gln B[beta]), a potential long-lived marker of pancreatitis.
On activation by thrombin, cleavage of the A and B peptides from the respective N termini of the A[alpha] and B[beta] chains initiates the polymerization process. The newly exposed Gly-Pro-Arg and Gly-His-Arg sequences dock with preformed binding sites located in homologous regions of the D domains of neighboring molecules. This binding leads to the formation of a half-staggered bimolecular array of fibrin molecules, and these protofibrils subsequently condense both longitudinally and laterally to form the clot matrix (3).
The circulating fibrinogen molecule displays a vast amount of genetic and acquired variation, and these pre- and posttranslational modifications have important effects on function (4, 5). There are alternative transcripts for both the A[alpha] and [gamma] chains, with the A[alpha] being phosphorylated nonstoichiometrically (1) and both the B[beta] and [gamma] chains containing biantennary oligosaccharide side chains that terminate either with 2 sialic acids or with 1 sialic acid and 1 galactose residue (5). The existence of common genetic polymorphisms for the A[alpha] (Thr/Ala312) and B[beta] chains (Arg/Lys448 and Pro/Leu235) further confound comparisons of mass measurements, particularly when comparing B[beta]-chain masses between individuals (6). This inherent B[beta]-chain variation is further complicated by variable phosphorylation of the proline residue at position 31 (1).
Notwithstanding these limitations, the mean mass of the major B[beta]-chain component reported for 6 individuals with typical coagulation profiles was 54 200 Da, with an additional peak at +291 Da corresponding to the disialylated isomer (5). During our investigations of the molecular basis of dysfibrinogenemia and hypofibrinogenemia, we noted an additional B[beta] peak at -130 Da in a control sample from a patient with pancreatitis. We describe the structure of this isoform and our measurements of its proportion in relation to the activity of pancreatic amylase, a marker of pancreatic disease.
Materials and Methods
MEASUREMENT OF AMYLASE ACTIVITY
We measured the activity of pancreatic amylase in the plasma with an Architect c8000 chemistry analyzer (Abbott Laboratories) with Abbott reagents and with protocols that use an antibody inhibitor of salivary amylase in a colorimetric assay with a protected p-nitrophenyl-maltoheptaoside substrate.
FIBRINOGEN ISOLATION AND CHAIN SEPARATION
Fibrinogen was isolated from heparin-anticoagulated plasma by precipitation with 23% saturated ammonium sulfate and washing 3 times with 25% saturated ammonium sulfate (7). The starting plasma solution and the washing solutions all contained 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L [epsilon]-aminocaproic acid, 10 mmol/L EDTA, and 5 mmol/L cysteamine hydrochloride.
Purified fibrinogen (or fibrin) was dissolved to a concentration of 5-10 g/L in 8 mol/L urea containing 100 mmol/L Tris-HCl, pH 8.0, and 15 mmol/L dithiothreitol. The solution was incubated at 37[degrees]C for 3 h, and then either 2 [micro]L (analytical) or 50 [micro]L (preparative) was loaded onto a Jupiter C4 column (250 mm x 4.6 mm; Phenomenex), and the protein solution was fractionated with a linear gradient of 61%-81% solvent B over 20 min (5). Solvent B was 600 mL/L acetonitrile and 0.5 mL/L trifluoroacetic acid in water; solvent A was 0.5 mL/L trifluoroacetic acid. The column was monitored at 215 run; peak crests and the bulk of the material were collected separately.
ELECTROSPRAY IONIZATION MASS SPECTROMETRY
Twenty microliters of the fibrin or fibrinogen chain peak crests were injected into the ion source of a VG Platform II quadrupole instrument (Micromass) at a flow rate of 5 [micro]L/min (5). The system was operated in positive-ion mode, the probe was charged at +3000 V, and the source temperature was maintained at 60[degrees]C. The m/z range of 850-1600 was scanned for 2.5 s with an interscan time of 100 ms and a cone voltage ramp of 40-65 V. At least 80 scans were collected and averaged for each run. The raw uncalibrated data were processed with MassLynx Mass Spectrometry software (Waters Corporation) and transformed onto a true molecular-mass scale with the maximum entropy algorithm.
PREPARATION OF FIBRIN 0 CHAINS AND FIBRINOPEPTIDES
We redissolved the purified fibrinogen (0.7 mg) in 300 [micro]L of 25 mmol/L Tris-HCl and 25 mmol/L NaCl, pH 7.4, and added 3 U bovine thrombin. After 25 min, the resulting clot was recovered by winding around a glass rod; the supernatant was retained for fibrinopeptide isolation. The clot was solubilized in 8 mol/L urea, and the individual chains were separated as described above for fibrinogen. The fibrinopeptide-containing supernatant was heated to 96[degrees]C for 5 min to inactivate thrombin and to precipitate any residual soluble fibrin. After centrifugation, the fibrinopeptides were chromatographed on a Nova-Pak [C.sub.18] column (150 mm x 3.9 mm; Waters Corporation). A linear gradient of 33%-56% solvent B was applied over 12 min. Solvent B was 500 mL/L acetonitrile in 49 mmol/L K[H.sub.2]P[O.sub.4], pH 2.9; solvent A was 49 mmol/L K[H.sub.2]P[O.sub.4], pH 2.9 (7). The A and B peptides were dried at 55[degrees]C under nitrogen. The peptides were redissolved in water, and the phosphate salts were removed by extracting the peptides onto [C.sub.18] resin (8). After elution with 600 mL/L acetonitrile, we analyzed the peptides by electrospray ionization (ESI)  mass spectrometry (MS).
Isolated B[beta] chains (approximately 200 [micro]g) were dried under nitrogen and redissolved in 50 [micro]L 50 mmol/L N[H.sub.4]HC[O.sub.3] containing 10 [micro]g trypsin. After overnight incubation at 37[degrees]C, the digest was dried, redissolved in 500 mL/L acetonitrile and 20 mL/L formic acid in water, and analyzed by ESI MS (6).
Isolated B[beta] chains (approximately 200 [micro]g) were redissolved in a 25-[micro]L volume of 700 mL/L formic acid that contained 200 [micro]g CNBr. After overnight incubation at room temperature, the mixture was dried under reduced pressure over NaCH. We added 25 [micro]L water, vortex-mixed the suspension, transferred the soluble peptides to a new tube, and added 25 [micro]L acetonitrile and 1 [micro]L formic acid. We injected 20 [micro]L of this solution directly into the mass spectrometer.
CARBOXYPEPTIDASE A TREATMENT
We redissolved fibrinogen to 5 g/L in 50 mmol/L N[H.sub.4]HC[O.sub.3] containing 0.18 g/L bovine carboxypeptidase A (CpA) and incubated the fibrinogen solution overnight at room temperature (9). We then reprecipitated the fibrinogen with 25% saturated ammonium sulfate, separated the chains by reversed-phase HPLC, and measured their masses by ESI MS.
Purified fibrinogen (500 [micro]L of an approximately 2.0 g/L solution) was dialyzed over 16 h against 5 250-mL changes of buffer (20 mmol/L HEPES and 150 mmol/L NaCl, pH 7.4). After dialysis, we measured the fibrinogen concentration from the difference in absorbance at 280 nm and 320 nm and with an absorptivity of 15.1; we then adjusted the concentration to 0.444 g/L with dialysis buffer. We added 180 [micro]L of the fibrinogen solution (containing 80 [micro]g fibrinogen) in triplicate to wells of a black isoplate and then added 20 [micro]L 20 U/L human [alpha]-thrombin. The absorbance at 350 nm was monitored at 12-s intervals during a 1-h period.
Fibrinogen was purified from the plasma of 9 patients with indications of pancreatitis (pancreatic amylase activities from 114-1826 U/L) and from 6 individuals with typical amylase activities (<56 U/L). We detected no differences between the 2 groups in gel patterns after polyacrylamide gel electrophoresis with SDS (data not shown). On nonreducing gels, both groups showed the expected 340- and 305-kDa bands associated with fully intact [(A[alpha], B[beta], [gamma]).sub.2] molecules and molecules with 1 cleaved A[alpha] chain, respectively; on reducing gels, both groups showed the same pattern of A[alpha] (64 kDa), B[beta] (52 kDa), and [gamma] chains (48 kDa).
When the individual fibrinogen chains were separated by reversed-phase HPLC, the 2 groups again showed similar elution profiles; a typical pattern is shown in Fig. 1. When we analyzed the individual peak crests directly by ESI MS, however, the B[beta]-chain peak showed marked alterations in the proportions of the different isoforms (Fig. 2). The major signal at 54 194 (13) Da [mean (SD), n = 15] represents the previously characterized monosialylated form of the glycoprotein chain, and the species at 54 487 (13) Da is its disialylated derivative (5). The masses of both of these peaks were quantitatively decreased by 129 Da in samples with high amylase activities, and paired typical and altered (-129 Da) signals were clearly visible in plasma samples with intermediate amylase activities. The presence of the same modification in the 2 B[beta] chains with different biantennary oligosaccharide termini suggested an alteration in the polypeptide itself rather than in the carbohydrate structure.
To help locate the modification site, we incubated purified fibrinogen with thrombin and separated the soluble fibrinopeptides from the resulting fibrin polymer. Our reversed-phase HPLC analysis of the fibrinopeptides derived from the N termini showed a typical pattern of A and B peptides, and mass analysis confirmed the predicted peptide masses and sequences (data not shown).
We redissolved the fibrin clot in 8 mol/L urea containing 15 mmol/L dithiothreitol, separated the individual fibrin chains by reversed-phase HPLC, and analyzed the [beta] peak by MS (Fig. 3). As expected, both sets of [beta] chains showed the characteristic decrease of 1535 Da corresponding to the loss of fibrinopeptide B; however, the fact that the [beta] chain from a high-amylase individual had a mass 130 Da lower than its typical counterpart suggested a protein modification of the C terminus rather than the N terminus.
[FIGURE 1 OMITTED]
We used tryptic peptide mapping to confirm this supposition and to obtain a more accurate measurement of the decrease in mass. Maps for chains with high and low degrees of modification were very similar, but those for an individual with high amylase activity lacked the peak at 1032 m/z that was seen in the control individual (Fig. 4). This signal at 1032 m/z represents the M+1H ion of T-53 (IRPFFPQQ), the C-terminal peptide of the B[beta] chain, and loss of its C-terminal glutamine (128 Da) would cause the peptide to appear at 904 m/z. The presence of some 904 m/z ions in the control reflects partial modification of the B[beta] chains, because previous measurements of the intact chains in this control had shown them to be 30% modified.
[FIGURE 2 OMITTED]
Because the B[beta] chain has a methionine residue conveniently located 9 residues in from the C terminus, we used CNBr digestion to confirm the putative pruning of the terminal glutamine residue. The predicted C-terminal fragment (KIRPFFPQQ) would have an M+2H ion at 581.2 m/z, and its truncated counterpart would have a corresponding ion at 517.1 m/z. A direct analysis of the water-soluble CNBr peptides showed that although the control had a dominant signal for the full-length peptide at 581 m/z, the predominant form in the digest from the high-amylase individual was the truncated form (KIRPFFPQ) at 517 m/z (Fig. 4). The presence of both peaks in each spectrum was because the fibrinogen sample from the control individual was 9% modified, whereas the patient's fibrinogen was 77% modified.
The data to this point provide compelling evidence that the degree of C-terminal modification of the fibrinogen B[beta] chain was loosely related to pancreatic disease, and because such disease is associated with increased plasma activities of amylase, lipase, and various proteases, CpA in particular, we investigated the effect of this exoprotease on fibrinogen. After incubating native fibrinogen with CpA, we isolated the B[beta] chains and analyzed them by ESI MS. CpA incubation produced a pattern identical to that identified in the patients with acute pancreatitis: there was complete conversion of both the mono- and disialylated isoforms to their des-Gln forms (Fig. 2).
We then examined the kinetics of thrombin-catalyzed fibrin polymerization to determine whether the modification affected fibrinogen function. In triplicate experiments, purified fibrinogen with high (85%) and low (5%) degrees of modification showed similar lag times, maximum velocity values, and final turbidities (data not shown), and these results were well within the range of nonpathologic variation (10).
[FIGURE 3 OMITTED]
An approximate molecular-mass decrease of 130 Da was observed for both major isoforms of the fibrinogen B[beta] chain derived from individuals with high pancreatic amylase activities. Tryptic and CNBr peptide mapping located the modification site to the C terminus and yielded a more precise value of 128 Da for the actual decrease in mass. This decrease can be entirely accounted for by the loss of the C-terminal glutamine residue. Incubating native fibrinogen with CpA reproduced the in vivo findings exactly, with only the terminal glutamine residue in the KIRPFFPQQ sequence being cleaved. A similar proteolytic modification has been reported for serum albumin in association with pancreatic pseudocysts (11). The principal modified form, des-Leu albumin, lacked the C-terminal leucine residue, and incubation with CpA reproduced the cleavage that had occurred in vivo (9).
Although typical baseline CpA activities are very low (12), increased activity has been demonstrated in plasma samples from patients with pancreatitis (13-16). CpA, the major carboxypeptidase produced by the pancreas, preferentially cleaves uncharged C-terminal residues (17); however, a number of CpAs with slightly different specificities have recently been identified in several mammalian species (18). Whereas CpA 1 prefers branched aliphatic amino acids at the C terminus, CpA 2 cleaves bulky aromatic residues (19, 20). The bovine CpA we used has a combined specificity for both aliphatic and aromatic amino acids (17), but we presume that CpA 1 is the modifying enzyme in humans because of the more aliphatic character of the glutamine residue. Cleavage of the penultimate glutamine is probably prevented by the neighboring proline.
Previous searches for novel biomarkers of pancreatic inflammation have focused on the enzymes released from necrotic cells (e.g., amylase, lipase, and CpA), and little attention has been paid to the products generated by these enzymes. The results of the present study and the previous study on albumin (11) provide valuable information on modifications that affect 2 of the major plasma proteins during pancreatitis; however, further research on the kinetics of the process and on the correlation between the proportion of the variant polypeptide and the course of the disease is necessary to evaluate the diagnostic benefit of this type of marker. This consideration is particularly important because of the differences in the half-lives of the causative enzymes and their circulating protein substrates. Fig. 5 shows a plot of the percentage of des-Gln B[beta] as a function of pancreatic amylase activity. Of note is that one of the controls with 30% modification had a typical activity of 25 U/L. Given that the half-life of CpA is approximately 2.5 h (21) and that of fibrinogen is approximately 4 days, this result may indicate an individual recovering from acute pancreatic injury, because the control samples were all from individuals for whom analyses of pancreatic amylases had been requested. Removing this outlier suggests a typical mean percentage of approximately 5%, a proportion consistent with previous (unpublished data) observations made during an investigation of functionally abnormal fibrinogens associated with clotting abnormalities.
Quantification of des-Gln B[beta] by fibrinogen purification, HPLC, and protein ESI MS may seem impracticable in a routine diagnostic setting, but such an approach may be useful in a research context. The development of a monoclonal antibody capable of recognizing the truncated molecule should be possible, however, and such an antibody could easily be incorporated into a routine assay. In addition, with the increasing availability of MS technology in clinical laboratories, a "bottom up" approach that uses tryptic or CNBr digests with signature-ion quantification by direct-injection MS could be feasible. Either fibrinogen pelleted from an ammonium sulfate solution or a washed fibrin clot could be a convenient fibrin(ogen) source.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
To assess the possible consequences of the truncation, we examined fibrin polymerization, because the functionally important D domain contains C termini of both the B[beta] and [gamma] chains. A comparison of fibrin polymerization kinetics revealed no significant differences, however. Given that the very end of the B[beta] chain has no defined function and that the important C terminus of the [gamma] chain seems to be intact in patient-derived fibrinogen (data not shown), this result is not surprising.
Grant/funding support: None declared.
Financial disclosures: None declared.
Received September 12, 2007; accepted September 13, 2007. Previously published online at DOI: 10.1373/clinchem.2007.093179
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 Nonstandard abbreviations: ESI, electrospray ionization; MS, mass spectrometry; CpA, carboxypeptidase A.
DAVID SCHMIDT and STEPHEN O. BRENNAN *
Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand.
* Address correspondence to this author at: Molecular Pathology Laboratory, Canterbury Health Laboratories, PO Box 151, Christchurch 8140, New Zealand. Fax 64-3-3640545; e-mail firstname.lastname@example.org.
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|Title Annotation:||Proteomics and Protein Markers|
|Author:||Schmidt, David; Brennan, Stephen O.|
|Date:||Dec 1, 2007|
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