Clinical Chemistry 53: 2105-2111, 2007.
First published October 5, 2007; 10.1373/clinchem.2007.093179
(Clinical Chemistry. 2007;53:2105-2111.)
© 2007 American Association for Clinical Chemistry, Inc.
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Proteomics and Protein Markers |
Modified Form of the Fibrinogen Bβ Chain (des-Gln Bβ), a Potential Long-Lived Marker of Pancreatitis
David Schmidt and
Stephen O. Brennana
1 Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch, New Zealand.
aAddress correspondence to this author at: Molecular Pathology Laboratory, Canterbury Health Laboratories, PO Box 151, Christchurch 8140, New Zealand. Fax 64-3-3640545; e-mail steve.brennan{at}chmeds.ac.nz.
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Abstract
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Background: During an investigation of genetic variants of fibrinogen, we observed a novel form of the Bβ chain, with a mass decrease of approximately 128 Da, in one of the controls. The plasma sample originated from an individual who had experienced acute pancreatitis a week earlier but whose serum amylase activity had returned to normal. We investigated the structure of the modified fibrinogen and explored its relationship to pancreatic disease.
Method: Fibrinogen was isolated from the plasma of 9 individuals with increased pancreatic amylase activity (114–1826 U/L) and presumed pancreatitis and from 6 control individuals with amylase activities <56 U/L. Fibrinogen (or fibrin) Bβ chains were isolated by reversed-phase HPLC and analyzed directly by electrospray ionization mass spectrometry. Tryptic and CNBr peptide mapping and thrombin treatment pinpointed the location of the 128-Da loss in mass.
Results: The acquired fibrinogen Bβ chain modification was attributable to the loss of its C-terminal glutamine residue. Incubating purified fibrinogen with pancreatic carboxypeptidase A (CpA) produced an identical modification. The des-Gln Bβ fibrinogen accounted for >80% of the Bβ chains in 3 of the individuals with increased amylase but only approximately 5% of the Bβ chains in control samples.
Conclusion: Pancreatic CpA activity is used as an index of acute pancreatic disease, but given that the circulatory half-lives of fibrinogen and CpA are approximately 4 days and only 2.5 h, respectively, measuring des-Gln Bβ fibrinogen, the in vivo product of CpA activity, could provide clinicians with retrospective evidence of disease.
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Introduction
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Fibrin is the primary protein of blood clots, and its correct deposition is essential for preserving the integrity of the hemovascular system. Its precursor, fibrinogen, is a 340-kDa glycoprotein composed of 6 polypeptide chains [i.e., (A
, Bβ, 
)2] linked by 29 disulfide bonds. The molecule has a symmetrical trinodal structure with a central E domain linked to 2 peripheral D domains in a linear D-E-D configuration. The E domain contains the N termini of all 6 chains, and the outer D domains are formed from the C-terminal regions of the Bβ and chains(1)(2).
On activation by thrombin, cleavage of the A and B peptides from the respective N termini of the A and Bβ 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 and chains, with the A being phosphorylated nonstoichiometrically(1) and both the Bβ and 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 (Thr/Ala312) and Bβ chains (Arg/Lys448 and Pro/Leu235) further confound comparisons of mass measurements, particularly when comparing Bβ-chain masses between individuals(6). This inherent Bβ-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 Bβ-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 Bβ 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.
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Materials and Methods
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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 
-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 °C for 3 h, and then either 2 µL (analytical) or 50 µL (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 nm; 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 µL/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 °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 β chains and fibrinopeptides
We redissolved the purified fibrinogen (0.7 mg) in 300 µL 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 °C for 5 min to inactivate thrombin and to precipitate any residual soluble fibrin. After centrifugation, the fibrinopeptides were chromatographed on a Nova-Pak C18 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 KH2PO4, pH 2.9; solvent A was 49 mmol/L KH2PO4, pH 2.9(7). The A and B peptides were dried at 55 °C under nitrogen. The peptides were redissolved in water, and the phosphate salts were removed by extracting the peptides onto C18 resin(8). After elution with 600 mL/L acetonitrile, we analyzed the peptides by electrospray ionization (ESI)1
mass spectrometry (MS).
trypsin digestion
Isolated Bβ chains (approximately 200 µg) were dried under nitrogen and redissolved in 50 µL 50 mmol/L NH4HCO3 containing 10 µg trypsin. After overnight incubation at 37 °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).
CNBR digestion
Isolated Bβ chains (approximately 200 µg) were redissolved in a 25-µL volume of 700 mL/L formic acid that contained 200 µg CNBr. After overnight incubation at room temperature, the mixture was dried under reduced pressure over NaOH. We added 25 µL water, vortex-mixed the suspension, transferred the soluble peptides to a new tube, and added 25 µL acetonitrile and 1 µL formic acid. We injected 20 µL of this solution directly into the mass spectrometer.
carboxypeptidase a treatment
We redissolved fibrinogen to 5 g/L in 50 mmol/L NH4HCO3 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.
fibrin polymerization
Purified fibrinogen (500 µL 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 µL of the fibrinogen solution (containing 80 µg fibrinogen) in triplicate to wells of a black isoplate and then added 20 µL 20 U/L human -thrombin. The absorbance at 350 nm was monitored at 12-s intervals during a 1-h period.
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Results
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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, Bβ, )2 molecules and molecules with 1 cleaved A chain, respectively; on reducing gels, both groups showed the same pattern of A (64 kDa), Bβ (52 kDa), and 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 Bβ-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 Bβ chains with different biantennary oligosaccharide termini suggested an alteration in the polypeptide itself rather than in the carbohydrate structure.
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Figure 1. Reversed-phase HPLC profile of reduced fibrinogen chains showing separation of A, Bβ, and chains.
Bβ peak crests were collected for direct mass analysis, and the remainder of the crest and bulk material was dried for mass mapping.
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Figure 2. Transformed ESI MS profiles of isolated fibrinogen Bβ chains.
(A), typical profile of a sample from a control individual with an amylase activity of 23 U/L; the paired peaks represent the mono- and disialylated isoforms of the glycoprotein chain. (B), sample from an individual with an amylase activity of 114 U/L. (C), sample from an individual with an amylase activity of 1671 U/L. (D), isolated Bβ chains from fibrinogen treated with CpA. The ordinate shows the response relative to the most intense peak in the spectrum. The amylase activity and the percentage of modified Bβ chains are indicated in each panel. ND, amylase activity not determined.
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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 β peak by MS (Fig. 3
). As expected, both sets of β chains showed the characteristic decrease of 1535 Da corresponding to the loss of fibrinopeptide B; however, the fact that the β 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.
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Figure 3. Transformed mass spectrum of fibrin β chains isolated from thrombin-treated fibrinogen.
(A), sample from a control individual with 3% of β chains modified. (B), sample from an individual with >90% of β chains modified. After removal of the N-terminal fibrinopeptide (1553 Da), the mass decrease of approximately 131 Da is still evident in the C-terminal portion of the protein. The amylase activity and the percentage of modified β chains are indicated in each panel. ND, amylase activity not determined.
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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 Bβ 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 Bβ chains, because previous measurements of the intact chains in this control had shown them to be 30% modified.
Because the Bβ 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 Bβ 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 Bβ 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).
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Discussion
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An approximate molecular-mass decrease of 130 Da was observed for both major isoforms of the fibrinogen Bβ 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)(14)(15)(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 Bβ 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.
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Figure 5. Graph showing variation in the percentage of des-Gln Bβ chains as a function of pancreatic amylase activity.
The regression line illustrates the correlation between the established marker and the novel marker.
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Quantification of des-Gln Bβ 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.
To assess the possible consequences of the truncation, we examined fibrin polymerization, because the functionally important D domain contains C termini of both the Bβ and chains. A comparison of fibrin polymerization kinetics revealed no significant differences, however. Given that the very end of the Bβ chain has no defined function and that the important C terminus of the chain seems to be intact in patient-derived fibrinogen (data not shown), this result is not surprising.
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Acknowledgments
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Grant/funding support: None declared.
Financial disclosures: None declared.
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Footnotes
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1 Nonstandard abbreviations: ESI, electrospray ionization; MS, mass spectrometry; CpA, carboxypeptidase A. 
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Abstract
Introduction
) arising from the C-terminal peptide is missing and has been replaced by the ion at 904 m/z (
). CNBr digests of Bβ chains from individuals with low (C) and high (D) levels of modification. The M+2H ion of the C-terminal peptide (KIRPFFPQQ) is largely missing at 581 m/z (