Determination of the trace levels of urinary fibrinopeptides by high-performance capillary electrophoresis / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>With 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.</p><p><b>CONCLUSION</b>The method we established is reliable and specific for separation and identification of fibrinopeptides and other bioactive peptides.</p>

Hemostatic markers of coagulopathy in acute promyelocytic leukemia

Hemostatic disorders are leading causes of death in patients with acute myeloid leukemia [AML] and particularly those with acute prom yelocytic leukemia [APL]. A contribution of fibrinolytic mechanisms has been claimed in the patho genesis of APL coagulopathy but investigations of the fibrinolytic activity of prom yelocytes have yielded conflicting results, sometimes based on reports of scattered [single] cases. The aim of this work is to study the changes of the different markers of thrombin generation and fibrinolysis in patients with APL and those with other AML subtypes [non APL-A ML], and to clarify the patho genesis of coagulopathy in patients with APL compard with those with non APL -A ML. The study included blood samples of 15 patients with APL and 25 patients with non APL-A ML, as well as 20 apparently healthy children with matched age and sex as a control group. Cases and controls were all subjected to the following investigations: pro thrombin concentration [PC], activated partial thromboplastin time [APTT], thrombin-anti thrombin complex [TAT], prothrombin fragment 1+2 [PF1+2], fibrinopeptide A [FPA], D-dimer, fibrinogen level, plasminogen activator inhibitor [PAI] and alpha 2-antiplasmin [alpha 2-AP]. As regards the markers of thrombosis PC was significantly lower in APL and AML in comparison to controls and in the same time it was significantly lower in APL in comparison to AML. PT, APTT, TAT, PF1+2, FPA and D-dimer levels in plasma of both APL and AML were significantly higher than controls and also it was found that these markers were significantly higher in APL than AML. About the fibrinolytic markers, fibrinogen was significantly lower in the cases of APL and AML than controls and it was found to be significantly lower in APL than AML. PAI and alpha 2-AP were significantly lower in APL and AML than controls but there was no significant difference between APL and AML. In the APL group a positive correlation was found between bone marrow promyelocyte% and D-dimer [r= 0.718, P< 0.003**] and between TAT and prepheral absolute promyelocyte [x10/L] [r=0.677, P< 0.006**]. In conclusion, acute myeloid leukemia in children, either APL or non-APL causes some changes in hemostatic mechanisms leading to acute DIC which was proved by the following tests: * Procoagulant activation which is proved by prolongation of APTT, PT, increased TAT, prothrombin fragment 1+2, fibrinopeptide A plasma levels as well as decreased prothrombin concentration and fibrinogen levels. * Fibrinolytic activation proved by decreased plasminogen activator inhibitor, alpha 2-antiplasmin plasma levels as well as increased plasma level of D-dimer which is a marker of plasmin activation. * Inhibitor consumption, proved by the study showed that the aforementioned laboratorial abnormalities were aggravated among cases with APL than those with non-APL-AML So DIC can be detected before bleeding

The Use of Ethanol Gelation Test to Screen the Activation of Coagulation and Disseminated Intravascular Coagulation / 임상검사와정도관리

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.

Prognostic significance of hypercoagulability in acute Ischemic stroke

Considerable evidences suggest an important role for hypercoagulability as a contributor to stroke outcome. The levels of many hemostatic markers change at the onset of stroke, howevere, it is still unsetteled whether those abnormalities pre-existant O. secondary to the acute event. This prospective study was undertaken in order to evaluate the prognostic significance of the hemostatic markers; beta-thromboglobulin [beta TG], fibrinopeptide-A [FPA] and protein-C [PC] activity.40 consecutive ischemic stroke patients were included during the first 48 hours of the onset. Stroke clinical examination scale [SCES] and CT brain scan were assessed. In order to assay the hemostatic markers, blood samples were drawn within 48 hours, after one week and after six weeks of the stroke onset. Plasma were prepared and tested for platelet activation, signed by beta TG and coagulation activation signed by FPA and PC activity. For comparison, 20 patients with nonvascular diseases were tested for these markers. Within 48 hours of the stroke onset significant elevation of betaTG and FPA associated with significant decrease of PC activity was observed in stroke patients compared to the controls. Observable reduction of betaTG and FP A with gradual elevation of PC activity occurred after one week. Further reduction of betaTG and FPA, and increase of PC activity occurred after six weeks of the stroke onset. Moreover, marked activation of the platelet and coagulation system was observed among patients who had severe stroke, extensive infarction as well as those who died during the study in comparison to patients with less severe stroke, small sized infarction and the survivors respectively. In conclusion, marked activation of platelet and coagulation occurs in the acute phase of ischemic stroke with subsequent decreament. The initial changes are more obvious in the patients with extensive infarction and poor prognosis. This may rise the importance of the hemostatic markers; betaTG, FPA and PC activity as prognostic factors rather than factors contributing only, to the thrombotic event

Effects of antithrombin III and antivenom on procoagulant activity of Russell's viper venom in a whole blood model.

The procoagulant activities of Russell's viper venom were assessed in an in vitro whole blood model. Sequential samplings showed that the generation of fibrinopeptide A (FPA), a marker of thrombin activity, and platelet factor 4 (PF4), a marker of platelet activity, exhibited bi-phasic kinetics with an initial slow phase followed by a rapid phase of secretion. In the presence of Russell's viper venom, the generation of both FPA and PF4 was accelerated with the bi-phasic kinetics of PF4 being maintained while that of FPA completely disappeared. Administration of either antivenom (1,600 ng) or 10 IU antithrombin III (AT-III) had no antagonistic effect against the venom but combination of both resulted in a significant prolongation of both FPA and PF4 release (p < 0.05). High dose AT-III (20 IU) resulted in normalization of both FPA and PF4 kinetics and serial levels of both parameters were lower than those treated with the combined regimen, although these were not statistically significant. Unlike the untreated venom activated whole blood, there was no clot formation following treatment with either the combined regimen or high dose AT-III. The results of this study suggested that the effect of Russell's viper venom on the clotting cascade is more potent and direct than that on platelet activity. There were complementary effects between antivenom and AT-III is controlling of both FPA and PF4 release induced by the venom. Furthermore, in this in vitro experiment, AT-III alone when administered in a sufficient dose, abolished the procoagulant effects of Russell's viper venom.

Effect of Snake Venom: Arginine-esterase on the Fibrinolytic Activity

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.

Fibrinopéptido A y cardiopatía isquémica aguda: valor diagnóstico y pronóstico: comunicación preliminar / Fibrinepectide A and acute ischaemic heart: diagnostic and prognostic value: preliminary communication

Debido a que la cardiopatía isquémica continúa siendo una de las primeras causas de mortalidad en nuestro país y a nivel mundial, es lógico entender el auge que han tenido los trabajos que involucren marcadores paraclínicos del fenómeno de la coagulación, debido al papel fundamental de ésta, en la fisopatología de dicha enfermedad, de allí nuestro interés en la investigación del rol que desempeña el fibrinopéptido A en el diagnóstico y pronóstico de la cardiopatía isquémica aguda. Por lo cual se realizó un estudio prospectivo, en donde se estudiaron 17 pacientes con cardiopatía isquémica aguda (13 angor y 4 IM), que ingresaron a la UCC del Hospital "Domingo Luciani", durante el lapso agosto-septiembre del año 94, a todos ellos se les tomó muestra al ingreso, a las 6 y a las 12 horas de su llegada al hospital, para derteminar los niveles plasmáticos de Fibrinopéptido A (marcador de la actividad de la trombina), así como también estudios paraclínicos convencionales (ECG, Enzimas Cardíacas, Rx de Tórax, ematología completa, Urea Creatinina, PTT, PT, Plaquetas). Analizando las relaciones existentes entre las diferentes variables mediante la técnica de Anova y T. Test; se evidencia que existe diferencia estadísticamente significativa entre el grupo control y los pacientes con cardiopatía isquémica aguda (IM y Angor) en los niveles de fibrinopéptido A, dando valores de P=0.036 demostrando de esta manera, la utilidad de este marcador en la cardiopatía isquémica aguda

Levels of plasma fibrinopeptide-A during surgery and the severity of the operation.

There is evidence to suggest that the rise of fibrinopeptide A (FPA) during surgery is influenced by tissue thromboplastin released during tissue damage. To investigate whether FPA correlates with the severity of the damage of the operation, 39 patients were recruited and venous blood samples were taken pre-operatively, during skin incision, during bowel manipulation and post-operatively for the assay of FPA. The operations are grouped as minor (A), moderate (B), major (C) and very major (D). The peak FPA levels occurred during bowel manipulation in every degree of operations, and ranged between 6.0 to 22.2 pmol/ml. There was a tendency that peak FPA values rose according to the severity of the surgery, however only very major operations (D) are significantly higher when compared with minor operations (A) (p < 0.01). There was no good correlation between peak FPA levels and length of skin incision (p = 0.83, r = 0.04) as well as peak FPA levels and duration of operation (p = 0.90, r = 0.03). Significantly higher levels of FPA in very major operation (D) was due to more excessive tissue damage during surgery, while due to relatively minimal tissue injury, size of skin incision correlated poorly with FPA.

Fibrinopeptide A: an indicator of increased thrombin activity in diabetes mellitus

To determine the role of Fibrinopeptide [FPA] in vascular complication in diabetics, it is estimated by a sensitive RIA technique in7 control subjects and 28 diabetics classified according to their line of treatment into IDD and NIDD who were further subdivided into those with vascular complications and those without vascular complications. Results showed that diabetics as a whole had a significantly higher mean values of both fasting and postprandial FPA compared to controls. A significant difference for fasting FPA was noticed between non complicated IDD and NIDD [P < .05], between complicated IDD and non complicated NIDD [P < 0.01] and between non complicated NIDD and complicated NIDD [P < 0.01]. Postprandial FPA was significantly higher in non complicated IDD, non complicated NIDD and complicated NIDD compared to controls [P > 0.05, P > 0.01 and P < 0.05 respectively]. Uncomplicated diabetics had a significantly higher mean fasting FPA level [P < 0.05] compared to complicated but not regarding to postprandial FPA. Finally a significant positive correlation was observed between fasting FPA versus fasting blood glucose in non complicated NIDD [r = 0.85, P < 0.05] and complicated NIDD [r = 0.86, P < 0.05], also between postprandial FPA and both fasting FPA [r = 0.94, P < 0.01] and fasting blood glucose [r = 0.91, P < 0.01] in complicated NIDD. It may be concluded that elevated FPA level in diabetes is considered as a sensitive specific parameter of in vivo thrombin activation in diabetes [Hypercoagulable state]. This elevation is associated with diabetes more than being associated with vascular complication

Study of fibrinopeptide A, fibrin monomer and fibrin degradation products in non-insulin dependent diabetes mellitus

The study was carried out on 20 non-insulin dependent diabetics and 10 healthy adult control subjects. The work aimed at estimation of fibrinopeptide A [FPA], fibrin monomer [FM] and fibrin degradation products [FDP] in non-insulin dependent diabetic patients with and without vascular complications, in addition to euglobulin clot lysis time [EGCLT], blood sugar, serum triglycerides, serum cholesterol, HDL-cholesterol and LDL-cholesterol. Serum fibrinopeptide-A [FPA] level was elevated in diabetics with vascular complications, reflecting augmented thrombin activity. EGCLT showed significant prolongation in diabetics with vascular complications. Pathological levels of FDP were detected in three diabetic patients [30%] with vascular complications. The significance of these findings were then analyzed and discussed