OBJECTIVETo establish a high-performance capillary electrophoresis (HPCE)-based method for detection of trace amount of urinary fibrinopeptide A and B (FPA and FPB, respectively) as the specific molecular markers of thrombus formation in vivo.

METHODSThe HPCE system consisted of a 25 cm x 50 microm (inner diameter) coated capillary column, 0.1 mol/L phosphoric acid buffer (pH 2.5) and a UV-detector (wavelength at 190 nm). To improve the sensitivity and reproducibility, solid-phase extraction of FPA and FPB in the urine was performed using a Sep-pak C18 column, with a synthetical fibrinopeptide B-Tyr (FPB-Tyr) as the internal standard.

RESULTSWith this HPCE method, optimal separations of FPA, FPB and FPB-Tyr was achieved within 16 min, with the migration time of 7.28 min, 14.31 min and 15.22 min, respectively. The adjusted peak area ratios of FPA or FPB and the internal standard showed good linearity with the corresponding concentrations of FPA or FPB spiked in the urine(R>0.99). Under the above chromatography conditions, the minimum detection concentration of FPA and FPB in untreated urine was 30 microg/L and 40 microg/L, respectively, and the assay precision and recovery of FPA and FPB were acceptable.

CONCLUSIONThe method we established is reliable and specific for separation and identification of fibrinopeptides and other bioactive peptides.

Electrophoresis, Capillary ; methods ; Fibrinopeptide A ; urine ; Fibrinopeptide B ; urine ; Humans ; Reproducibility of Results

BACKGROUND AND PURPOSE: Arginine esterase(Ancrod), a thrombin-like enzyme, purified from the venoms of Agkistrodon halys, has known to cleave fibrinopeptide A from the fibrinogen and lead to circulation of soluble noncross-linked "ancrodfibrin', which stimulates endogenous T-PA release.Many authors have suggested clinical applicability of this enzyme,but clinical studies on its fibrinolytic action has been insufficient.Thus we studied the influence of this enzyme on fibrinolytic activity in cerebral infarction. METHOD: We observed the change of euglobulin fibrinolytic activity, t-PA antigen, t-PA activity, fibrinopeptide A, fibrinogen, FDP and D-dimer, during 12 hours after a bolus intravenous administration of 0.25 unit of the arginine esterase to the 9 patients with cerebral infarction. RESULT:There was no change of the euglobulin fibrinolytic activity, fibrinopeptide A and t-PA Ag but there was significant increase in both t-PA activity and FDP, D-dimer and significant decrease in fibrinogen. CONCLUSION: Our result suggest that arginine esterase converts fibrinogen to a fibrin polymer which has a increased susceptibility to lysis by plasmirl This enzyme seems to amplify T-PA activity through the consequent increase in FT)P, because there is no increase in the euglobulin fibrinolytic activity, fibr'mopeptide A and t-PA Ag suggesting direct T-PA release. Arginine esterase, having action of effective defibrinogenation and safe fibrinolysis,could be used for the thrombus related disease.
Administration, Intravenous ; Agkistrodon ; Arginine ; Cerebral Infarction ; Fibrin ; Fibrinogen ; Fibrinopeptide A ; Humans ; Polymers ; Snake Venoms* ; Snakes* ; Thrombosis ; Venoms

BACKGROUND: Ethanol gelation test (EGT) is one of the paracoagulation test used to detect the activation of coagulation and formation of fibrin monomer complexes in the fibrinolytic process. Many patients with infectious diseases such as dengue haemorrhagic fever can develop disseminated intravacular coagulation (DIC), which should be diagnosed properly as soon as possible for the management of the patients. To diagnose the coagulation activation and DIC usually the laboratory has to perform the coagulation test, including fibrinopeptide A and D-dimer test. Many laboratories in rural areas in Indonesia do not have the facilities to do such test, and the cost will not be affordable by most of the patients. The aim for the study is to evaluate the EGT as a screening test to detect coagulation activation and DIC, the correlation of D-dimer and EGT. Method: Sixty citrated plasma were obtained from patients in Clinical Pathology Laboratory Cipto Mangunkusumo Hospital for D-dimer test. D-dimer were performed using Nycard Kit with cut off point of 300 ng/dl. The EGT were performed using the method described by Breen. Positive test could be observed by the clot formation. RESULTS: The result of the within-run test for normal and abnormal plasma for EGT showed good results. The plasma was stalell until day 22. The EGT was positive for all the plasma with D-dimer >700 ng/ml. The sensitivity for EGT was 81.6%, specificity 81.8%, positive predictive value 95.2% and negative predictive value 50%. Conclusion: EGT could be used as a screening test for thrombin activity in coagulation activation in rural laboratories with minimal facilities.
Communicable Diseases ; Dacarbazine ; Dengue ; Disseminated Intravascular Coagulation* ; Ethanol* ; Fever ; Fibrin ; Fibrinopeptide A ; Humans ; Indonesia ; Mass Screening ; Pathology, Clinical ; Plasma ; Sensitivity and Specificity ; Thrombin

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections. Qingfei oral liquid (QFOL), a traditional Chinese medicine, is widely used in clinical treatment for RSV-induced pneumonia. The present study was designed to reveal the potential targets and mechanism of action for QFOL by exploring its influence on the host cellular network following RSV infection. We investigated the serum proteomic changes and potential biomarkers in an RSV-infected mouse pneumonia model treated with QFOL. Eighteen BALB/c mice were randomly divided into three groups: RSV pneumonia model group (M), QFOL-treated group (Q) and the control group (C). Serum proteomes were analyzed and compared using a label-free quantitative LC-MS/MS approach. A total of 172 protein groups, 1009 proteins, and 1073 unique peptides were successfully identified. 51 differentially expressed proteins (DEPs) were identified (15 DEPs when M/C and 43 DEPs when Q/M; 7 DEPs in common). Classification and interaction network showed that these proteins participated in various biological processes including immune response, blood coagulation, complement activation, and so forth. Particularly, fibrinopeptide B (FpB) and heparin cofactor II (HCII) were evaluated as important nodes in the interaction network, which was closely involved in coagulation and inflammation. Further, the FpB level was increased in Group M but decreased in Group Q, while the HCII level exhibited the opposite trend. These findings not only indicated FpB and HCII as potential biomarkers and targets of QFOL in the treatment of RSV pneumonia, but also suggested a regulatory role of QFOL in the RSV-induced disturbance of coagulation and inflammation-coagulation interactions.
Animals ; Biomarkers ; blood ; Chromatography, Liquid ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fibrinopeptide B ; analysis ; genetics ; Gene Expression Regulation ; drug effects ; Heparin Cofactor II ; analysis ; genetics ; Lung ; pathology ; Mice, Inbred BALB C ; Proteome ; drug effects ; Proteomics ; Respiratory Syncytial Virus Infections ; blood ; drug therapy ; Respiratory Syncytial Viruses ; drug effects ; Tandem Mass Spectrometry