Identification of á´ -amino acid-containing peptides in human serum.

Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen ß-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome.

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.

Oncostatin M receptor ß and cysteine/histidine-rich 1 are biomarkers of the response to erythropoietin in hemodialysis patients.

Biomarkers that evaluate the response to erythropoietic-stimulating agents largely measure inflammation and iron availability. While these are important factors in modifying an individual's response to these agents, they do not address all aspects of a poor response. To clarify this, we isolated peptides in the serum of good and poor responders to erythropoietin in order to identify biomarkers of stimulating agent response. Ninety-one candidate biomarker targets were identified and characterized using mass spectrometry, of which tandem mass spectroscopy provided partial amino-acid sequence information of 17 different peptides for 16 peptide masses whose abundance significantly differed between poor and good responders. The analysis concluded that three peptides associated with a poor response were derived from oncostatin M receptor ß (OSMRß). The 13 serum peptides associated with a good response were derived from fibrinogen α and ß, coagulation factor XIII, complement C3, and cysteine/histidine rich 1(CYHR1). Poor response was most strongly associated with the OSMRß fragment with the largest molecular weight, while a good response was most strongly associated with CYHR1. Immunoblots found the abundance of intact OSMRß and CYHR1 significantly differed between good and poor responders. Thus, two measurable biomarkers of the response to erythropoietic-stimulating agents are present in the serum of treated patients.

Nanostructured diamond-like carbon on digital versatile disc as a matrix-free target for laser desorption/ionization mass spectrometry.

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.

Use of a double resonance electron capture dissociation experiment to probe fragment intermediate lifetimes.

The relative abundances of fragment ions in electron capture dissociation (ECD) are often greatly affected by the secondary and tertiary structures of the precursor ion, and have been used to derive the gas-phase conformations of the protein ions. In this study, it is found that resonance ejection of the charge reduced molecular ion during ECD resulted in significant changes in many fragment ion populations. The ratio of the ion peak intensities in the double resonance (DR)-ECD to that in the normal ECD experiment can be used to calculate the lifetime of the radical intermediates from which these fragments are derived. These lifetimes are often in the ms range, a time sufficiently long to facilitate multiple free radical rearrangements. These ratios correlate with the intramolecular noncovalent interactions in the precursor ion, and can be used to deduce information about the gas-phase conformation of peptide ions. DR-ECD experiments can also provide valuable information on ECD mechanisms, such as the importance of secondary electron capture and the origin of c./z ions.

Increased fibrinolytic activity and body cavity coagula.

When a large volume of coagulum remains in the body cavity after trauma or surgery, secondary fibrinolysis occurs, which disturbs the hemostatic balance and results in rebleeding. To better understand this condition, we conducted a clinical study on patients with and without coagula and an experimental study on fibrinolytic activity in a rat model. The results of the clinical study showed that when coagula existed in the body cavity, the blood levels of the fibrin degradation products D-dimer and fibrinopeptide Bbeta15-42 remained high compared with when subjects were under similar stress but without the presence of coagula. In the experimental studies, fibrinolytic activity of the omentum, measured by the fibrin plate method, was higher in rats with hemoperitoneum. This suggests that increased fibrinolytic activity may lead to rebleeding from the area of transient hemostasis when coagulum is present in the body cavity.

Electrospray ionisation mass spectrometry facilitates detection of fibrinogen (Bbeta 14 Arg --> Cys) mutation in a family with thrombosis.

We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.

Pretreatment fibrinogen levels are associated with response to chemotherapy in patients with small cell carcinoma of the lung: Department of Veterans Affairs Cooperative Study 188.

Small cell carcinoma of the lung (SCCL) responds commonly to combination chemotherapy but resistance to therapy follows. Prior reports have suggested that a relationship may exist between plasma fibrinogen levels and response to therapy in SCCL. This study was designed to determine the possible predictive value of the fibrinogen level for tumor response (chemoresistance) in SCCL. Pretreatment fibrinogen levels were correlated with outcome and response to therapy in a cohort of 119 previously untreated patients with SCCL who were admitted to VA Cooperative Study 188. Higher pretreatment fibrinogen levels at diagnosis correlated significantly with more advanced stage of disease at entry (P < 0.001) and with reduced overall survival (P = 0.030). In addition, higher pretreatment fibrinogen levels were correlated significantly with a reduced likelihood of achieving subsequent disease regression with combination chemotherapy (P = 0.005). Because several clinical trials have shown that anticoagulant therapy improves tumor response rates and survival of SCCL, we postulate that tumor cell thrombin generation not only promotes SCCL growth but may also be primarily responsible for both increased fibrinogen levels and for resistance to chemotherapy. These findings provide incentive for studies of thrombin effects on the development of multidrug resistance, and for new clinical trials of more potent and specific inhibitors of thrombin that may further improve tumor response and survival in SCCL.

Biocompatibility of a heparin-bonded membrane oxygenator (Carmeda MAXIMA) during the first 90 minutes of cardiopulmonary bypass: clinical comparison with the conventional system.

We clinically compared a heparin-bonded Carmeda MAXIMA membrane oxygenator to a nonheparin-bonded MAXIMA in 20 patients undergoing coronary artery bypass grafting or valve replacement. Reductions of fibrinogen, factor XII, and high molecular weight kininogen were greater in the MAXIMA group. Serum C3a and free hemoglobin were lower in the Carmeda group. The level of C4a, though remarkably lower than that of C3a, was higher in the Carmeda group then in the MAXIMA group. Both oxygenators performed well in terms of blood gas exchange. We conclude that the heparin-bonded Carmeda oxygenator offers superior biocompatibility during cardiopulmonary bypass.

Application of a monoclonal antibody to estimate rabbit fibrinopeptide A released by habutobin.

We reported previously that habutobin, one of the type A thrombin-like enzymes, releases fibrinopeptide A alone from rabbit fibrinogen. To evaluate the effective action of habutobin in experiments using rabbit for the treatment of thrombosis, we attempted to develop an immunological method for measuring the fibrinopeptide A level in the circulating blood of rabbit. The purified rabbit fibrinopeptide A was coupled to keyhole limpet hemocyanin and BALB/c mice were immunized with the resultant fibrinopeptide A-hemocyanin conjugate. The spleen cells of an immunized mouse were fused with myeloma cells (P3-X63-Ag8-U1). As a result, one hybridoma (a-F-7) was selected, which secreted an antibody against rabbit fibrinopeptide A. Using this monoclonal antibody, we developed a competitive enzyme-linked immunoassay for estimating rabbit fibrinopeptide A. It was able to measure rabbit fibrinopeptide A contained in bentonite defibrinated plasma. This competitive enzyme-linked immunoassay should be useful for determining the fibrinopeptide A level in the circulating blood of rabbits, using plasma defibrinated by bentonite.

Immunoelectrophoretic and immunohistochemical characterizations of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudo-intimas.

Cadaveric aortic intimas with uncomplicated atherosclerosis were examined to determine the distribution and polypeptide chain composition of fibrinogen-related protein. Immunohistochemical staining showed deposits rich in fibrinopeptides A and B. The deposits were usually disseminated throughout intimas of moderate thickness < 0.7 mm, but were distributed focally in elongate patches bounded both lumenally and medially by deposit-free tissue in thick atheromas. Saline extracts generally showed undegraded monomers and dimers by electrophoresis. The residual protein contained A alpha and gamma-chains that were cross-linked predominantly (>80%) into unresolved high M(r) (>200 kd) derivatives, whereas B beta-chains were left non-cross-linked, as occurs in late stages of cross-linking by transglutaminases. The resolved components had electrophoretic mobilities corresponding to characteristic products of both factor XIIIa and tissue-transglutaminase. A greater incorporation of alpha- rather than gamma-chains into cross-linked products implicated tissue-transglutaminase as contributing heavily. By contrast, vascular graft pseudo-intimas and a cadaveric clot were rich in degraded fibrin devoid of fibrinopeptide A, and cross-linked in patterns typical of XIIIa with gamma 2 dimers constituting the principal product. The findings indicate that the fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue-transglutaminase without being converted to fibrin.

Activation of coagulation and fibrinolysis during surgery, analyzed by molecular markers.

Activation of hemostasis during surgery was investigated in 30 elective cases, who underwent either gastric (group G) or hepatic (group H) resection by a serial determination of various molecular markers such as fibrinopeptide A (FPA), fibrinopeptide B beta 15-42 (B beta 15-42) D-dimer, thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 plasmin inhibitor complex (PIC). In both groups, the values of FPA and TAT were significantly elevated intraoperatively, indicating an occurrence of hypercoagulable state. The degree of the elevation was more marked in group H, probably due to greater tissue damage during hepatic resection. Also in both groups, the values B beta 15-42 and PIC were significantly increased during surgery, while the amount of D-dimer was within normal range in most cases, indicating the occurrence of the primary fibrinolysis. These findings are compatible with our previous observations on the postoperative changes in hemostasis. There were statistically significant but variable correlations between the values of fibrinopeptides and the enzyme-inhibitor complexes. The absolute values of the molecular markers of fibrinolysis were always higher than those of coagulation, suggesting that a considerable amount of plasmin, rather than thrombin, is released by surgical tissue damages.

Fibrinogen Geneva, a new case of A alpha 16 Arg----Cys dysfibrinogenaemia.

A new congenital variant of fibrinogen, from which only half the normal amount of fibrinopeptide A can be released by thrombin, was found in three members of a family having no major bleeding or thrombotic tendency. Following carboxamidomethylation of the reduced fibrinogen chains, an abnormal peptide was cleaved by thrombin from the amino terminus of the A alpha-chain (A* alpha 1-19) and isolated by reversed phase high-performance liquid chromatography. Amino acid analysis indicated the presence of carboxymethyl cysteine in this A alpha-chain fragment which in normal fibrinogen is devoid of cysteine. We conclude that fibrinogen Geneva is another fibrinogen variant with the substitution A alpha 16 Arg----Cys.

Induction of marked thrombin activity by pharmacologic concentrations of plasminogen activators in nonanticoagulated whole blood.

Thrombin activity reflected by increased plasma concentrations in vivo of fibrinopeptide A (FPA) increases when streptokinase (SK) is administered to patients with acute myocardial infarction. Although procoagulant effects have been found in vitro, the use of anticoagulated plasma limits the extent to which the phenomena observed can be viewed to implicate procoagulant effects in vivo. Accordingly, we characterized the procoagulant effects of SK and tissue plasminogen activator (t-PA) in nonanticoagulated whole blood. Blood was collected from normal volunteers by venipuncture (No. 19 gauge steel needle) directly into polypropylene tubes containing either t-PA, SK, SK and heparin, t-PA and heparin, or saline. The concentration of FPA after 10 min of incubation with saline was 150 +/- 46 nM (n = 14)(SE). In contrast, in blood incubated with SK FPA was consistently and markedly increased after 10 min: 2318 +/- 416 nM (100 IU/ml SK) and 10,889 +/- 1156 nM (1000 IU/ml SK) (p less than 0.001 compared with control). Less marked elevations of FPA occurred after 10 min in blood incubated with t-PA (3171 +/- 604 nM with 2500 ng/ml t-PA, p less than 0.01 compared with 1000 IU/ml SK). Increases in FPA were less than 100 nM in blood incubated with activators and heparin. The extent to which plasminogen was activated, as measured by the release of the B beta 1-42 fibrinopeptide, was related to the magnitude of elevation of FPA. Procoagulant activity induced by extensive plasminogen activation may contribute to undesirable effects in vivo, such as a propensity to recurrent thrombosis or delayed fibrinolysis.

In vivo measurements of fibrin formation and fibrinolysis in operable breast cancer.

Fibrin formation and fibrinolysis were estimated in 89 breast cancer patients by measurement in plasma of Fibrin Fragment B beta 15-42 and Fibrinopeptide A (FPA), serum Fibrin(ogen) Degradation Products (FDPs) and plasminogen activator by Fibrin Plate Lysis Assay. Results were compared with (a) 26 patients with benign breast diseases; and (b) 45 healthy factory workers. FPA, FDP and B beta 15-42 levels were elevated in both breast cancer patients and benign disease patients, but there were no significant differences between these two groups. Cancer stage, patient age and smoking habits did not affect these results, but Oestrogen Receptor (ER) positive patients had higher B beta 15-42 values than ER negative patients (p = 0.017). These results show that fibrin formation is enhanced preoperatively in patients with either benign or malignant breast disease. The fibrinolytic response to activated coagulation may be relatively deficient in breast cancer. The roles of malignancy, stress and other factors in the causation of these abnormalities require further assessment.

Alterations in coagulation and fibrinolysis associated with cardiopulmonary bypass during open heart surgery.

The effect of heparin as an anticoagulant was examined and the extent of fibrinolytic activity during cardiopulmonary bypass (CPB) was measured. Twenty patients undergoing valve replacement or aortocoronary bypass surgery were studied. Fibrinopeptide A (FPA) levels gradually became elevated as CPB proceeded, and antithrombin III (AT III) decreased during CPB. This indicates that despite the use of heparin, the coagulation system was activated, leading to fibrin formation in the microcirculation. On the other hand, fibrinopeptide B (FPB beta 15-42) also increased to four times the preoperative value at two hours on CPB. Intrinsic fibrinolytic activity, as determined by the activity of kaolin-activated euglobulin, was transiently increased only at the beginning of CPB. The C1 inactivator-resistant fibrinolytic activity and tissue plasminogen activator antigen (t-PA;Ag) increased sharply during CPB and reached maximum levels one hour after the start of CPB, indicating that enhanced fibrinolytic activity during CPB is predominantly of extrinsic origin as the result of t-PA release from the vascular walls. It is concluded from the above findings that thrombin activity continues during CPB. Enhanced fibrinolytic activity during CPB appears to be important because t-PA activates plasminogen predominantly where fibrin is formed, leading to dissolution of the microthrombi formed during CPB.

Effects of intermittent pneumatic leg compression for prevention of postoperative deep venous thrombosis with special reference to fibrinolytic activity.

The mechanism of intermittent pneumatic leg compression for prevention of postoperative deep venous thrombosis was investigated. The incidence of postoperative deep venous thrombosis was studied using iodine-125 fibrinogen in 64 patients with malignant disease who had intermittent pneumatic leg compression for 48 hours postoperatively. Changes in euglobulin lysis time and B beta 15-42 peptide were investigated before and after operation in 16 patients with benign disease, in 27 patients with malignant disease who did not have postoperative intermittent pneumatic leg compression, and in another 29 patients with malignant disease who had postoperative intermittent pneumatic leg compression. The overall incidence of deep venous thrombosis was 6.25 percent. A prolongation of euglobulin lysis time was found postoperatively in all three groups, which was significant in malignant disease groups, although less significant in the group with intermittent pneumatic leg compression when compared with the benign disease group. Preoperatively, a significant increase in B beta 15-42 peptide was found in patients with malignant disease when compared with patients with benign disease. Postoperatively, the B beta 15-42 level increased in the same pattern in all groups and no significant differences in the levels were found among them. A significant shortening of euglobulin lysis time by intermittent pneumatic leg compression, in addition to its hemodynamic effects, is considered an important factor in the prevention of postoperative deep venous thrombosis.