Fibrinopeptide A Concentrations Research Articles

Urinary fibrinopeptide-A as a predictive biomarker of exacerbation in asthma

BackgroundAsthma is a prevalent and potentially life-threatening disease associated with exacerbation and costly hospital admissions. The coagulation cascade is up-regulated in severe asthma and increased fibrinogenesis in the airway may precede exacerbation in moderate asthma. ObjectiveA longitudinal prospective study to test the hypothesis that levels of urinary fibrinopeptide A (FP-A), a marker of coagulation, increase prior to an exacerbation of asthma. Methods24 non-smoking participants with moderate to severe asthma were recruited and followed to exacerbation and to recovery for up to 8 weeks afterwards. Baseline measurements included spirometry, full blood count, atopic status and plasma markers of coagulation. Participants provided daily Peak Expiratory Flow (PEF) readings and three urine samples per week for analysis of FP-A, a specific marker of activation of coagulation. A novel method to concentrate urinary FP-A for immunodetection and quantification was developed. Participants were followed up until exacerbation, when baseline measurements were repeated, and monthly thereafter for 2 months or to recovery. Measurements and main findings17 participants exacerbated during the study. Significantly increased concentrations of plasma D-dimer (0.25(0.2–0.42) vs 0.21(0.12–0.29) μg FEU/ml, p = 0.02) were found at exacerbation. A peak in urinary FP-A concentration was detected on average 4.2 ± 2 days prior to exacerbation and was significantly (p < 0.05) higher than at exacerbation or 7 days later. Urinary FP-A concentrations correlated positively with time to recovery and negatively (p < 0.01) with IgE concentration. ConclusionFP-A is detectable in urine several days before the onset of an asthma exacerbation indicating disordered coagulation preceding asthma exacerbations.

Assessment of some plasma fibrinolytic proteins in sickle cell anemia patients in steady state and in vaso-occlusive crises

Background: The clinical manifestations of vaso-occlusion result from a dynamic combinations of abnormalities in hemoglobin (Hb) structure and functions, red blood cell membrane integrity, erythrocyte density, endothelial activation, microvascular tone, inflammatory mediators and coagulation factors. Objective: The objective of the following study is to determine the changes in plasma concentration of fibrinolytic proteins among sickle cell anemia (SCA) patients in two clinical states; steady state and vaso-occlusive crises (VOC) and compare with HbAA controls and also to determine if any, the clinical relevance of these proteins in evaluating these patients in the different clinical state. Materials and Methods: A total of 25 (14M: 11F) HbSS subjects in VOC, 24 (13M: 11F) HbSS subjects in a steady state and 30 (17M: 13F) healthy HbAA volunteers matched for age and sex with the subjects were recruited for the study. Hematological parameters, i.e., full blood count, fibrinolytic proteins concentration including plasma concentration of D-dimer, plasminogen, fibrinogen (FBG), tissue plasminogen activator (tPA) and fibrinopetide-A were determined. Result: Plasma fibrinopeptide A (FPA) concentration of 680.99 ΁ 411.37 ng/ml for SCA subjects in VOC and 449.67 ΁ 310.01 ng/ml in steady state subjects were significantly increased (P Conclusion: The significant increase in FPA concentration observed among subjects compared to controls may have been confounded by hyperleucocytosis especially polymorphonuclear leucocytes commonly associated with SCA. Thus, the clinical relevance of these plasma proteins in evaluating these patients in either of the clinical states of this disorder is doubtful.

Effects of local heparin administration on coronary thrombin activity during percutaneous transluminal coronary angioplasty.

Simultaneously obtained blood samples from the coronary sinus and systemic arterial circulation were analyzed for antithrombin III (ATIII) activity and fibrinopeptide A (FpA) concentration in nine patients undergoing elective PTCA in order to determine the effects of locally delivered heparin. Samples were obtained at the following designated times: prior to the administration of systemic heparin (period I); 5 min following a loading dose of systemic heparin (period II); 5 min following the final balloon inflation but prior to local delivery (period III); and 5 min following the administration of 4,000 units of unfractionated heparin using a local delivery catheter system (period IV). We found consistent increases in both systemic arterial (P = 0.006) and coronary sinus (P = 0.0002) ATIII activity with systemic heparinization designed to prolong the activated clotting time to 300 sec. However, local delivery of heparin further increased coronary sinus ATIII activity (P = 0.003, period III vs. period IV). FpA concentration decreased in both systemic arterial (P < 0.0001) and coronary sinus (P < 0. 0001) samples following systemic heparinization. Moreover, local delivery of heparin further decreased coronary sinus FpA concentration (P = 0.04). Thus, on a background of intense anticoagulation during PTCA, the local delivery of 4,000 units of unfractionated heparin confers incremental antithrombotic activity. Cathet. Cardiovasc. Intervent. 48:84-88, 1999.

Determinants of rebound thrombin activity after cessation of heparin in patients undergoing coronary interventions.

This study was designed to characterize hemostatic activation (using fibrinopeptide A (FPA), a marker of thrombin activity, and beta-thromboglobulin (BTG), a marker of platelet activation) sequentially in the coronary and peripheral circulation in patients during percutaneous coronary intervention (PCI) and several hours after PCI and discontinuation of heparin therapy. Heparin administered during PCI is known to nonuniformly suppress thrombin activity in the coronary. Persistent elevations of FPA in coronary sinus (CS) blood during PCI have been associated with subsequent ischemic events. As a related consideration, rebound thrombin activity has been demonstrated in peripheral blood samples several hours after cessation of heparin therapy in patients with acute coronary syndromes. Accordingly, we hypothesized that increased thrombin activity occurs in the coronary circulation after PCI and is induced by cessation of intravenous heparin to facilitate vascular sheath removal. Such a rebound prothrombotic effect, may contribute to suboptimal outcomes after PCI. In 21 patients undergoing PCI, heparin-bonded catheters were employed to obtain sequential CS and femoral vein (FV) blood samples for measurement in the coronary and peripheral circulation of plasma FPA, a marker of thrombin activity in vivo, and BTG released by platelets during degranulation. Following heparin administration samples were obtained immediately prior to (base) and during (start and end) PCI. Late samples were obtained several hours after PCI (284 +/- 46 min, mean +/- SD) following the cessation of heparin and prior to planned vascular sheath removal. Mean FPA concentration in CS blood was low at baseline (3.82 +/- 2.09 ng/ml) and did not increase during PCI. Mean FPA concentration in CS blood increased significantly several hours after cessation of heparin (3.42 +/- 2.36 vs. 7.82 +/- 9.98, end vs. late, P < 0.001). In contrast, mean FPA concentration in FV blood was highest at baseline following vascular sheath insertion, decreased during PCI (69%, P < 0.05, base vs. end), and trended upward after PCI and cessation of heparin. Mean FPA values were higher at all times in FV compared with CS blood samples and were not concordant after PCI. Elevation of coronary circulation FPA after PCI was maximal in patients with myocardial infarction within 7 days (13.7 +/- 12.4 vs. 5.6 +/- 7.9 ng/ml, P = 0.08), but was not influenced by heparin treatment prior to PCI, a history of unstable angina, or coronary stent placement during PCI (9 of 21 patients). BTG values showed less variation than did FPA values, and cessation of heparin after PCI was not associated with an increase in BTG in CS or FV blood samples. An increase in thrombin activity occurs in the coronary circulation after PCI following discontinuation of heparin. The extent of increased thrombin activity was greatest in patients with recent myocardial infarction and was not exacerbated by coronary stent placement during PCI. This phenomenon may contribute to the important minority of ischemic complications early after PCI.

Association of heparin-resistant thrombin activity with acute ischemic complications of coronary interventions.

Acute thrombosis is thought to contribute to abrupt coronary occlusion during percutaneous coronary revascularization despite the administration of heparin and aspirin. This study was designed to detect the presence of heparin-resistant thrombin activity and to define its relationship to the acute ischemic complications of coronary interventions. Plasma levels of fibrinopeptide A (FPA) and prothrombin fragment 1.2 (F1.2), markers of thrombin and factor Xa activity, respectively, were measured in the coronary sinus with heparin-bonded catheters in 58 patients undergoing coronary interventions. Activated coagulation times were maintained > 300 seconds by the Hemochron method. Mean FPA levels decreased significantly, from 7.0 +/- 0.9 nmol/L before the procedure to 5.2 +/- 0.5 nmol/L after the heparin bolus and to 2.9 +/- 0.2 nmol/L after the procedure (P = .0001). In 26 patients (45%), FPA levels remained above the threshold for suppression angioplasty of thrombin activity determined during angiography in 7 patients without coronary artery disease (> 3.0 nmol/L). FPA concentrations after coronary interventions were increased in patients with intracoronary thrombus (P = .01), abrupt coronary occlusion (P = .06), postprocedural non-Q-wave myocardial infarction (P = .04), and clinically unsuccessful procedures (P = .04). F1.2 levels were relatively low before the procedures and did not change significantly. Heparin administration suppresses thrombin activity in most but not all patients undergoing coronary interventions. Heparin-resistant thrombin activity is associated with angiographic evidence of intracoronary thrombus and ischemic complications of coronary interventions.

The role of the protein C-thrombomodulin system and fibrinolysis during cardiovascular surgery: Influence of acute preoperative plasmapheresis

Objectives: To assess the benefits of withdrawn autologous plasma, the objective of this study was to investigate whether withdrawal of acutely performed platelet-rich or platelet-poor plasmapheresis allays changes in the protein C-thrombomodulin and fibrinolytic systems after retransfusion secondary to cardiopulmonary bypass (CPB). In addition, the study attempted to determine the influence of acute plasmapheresis (APP) on the protein C-thrombomodulin and fibrinolytic systems as well as on homologous blood consumption and perioperative blood loss in elective aortocoronary bypass patients. Design: The investigation was scheduled as a prospective, randomized, unblinded study. Setting: This single investigation was conducted in the Department of Anaesthesiology and Intensive Care Medicine at a university in Germany. The study protocol was approved by the Ethics Committee of the hospital, and informed consent was obtained. Participants: Sixty male patients scheduled for elective coronary artery bypass grafting with extracorporeal circulation were included in the study. Interventions: APP was performed between induction of anesthesia and incision, collecting either 10 mL/kg of autologous platelet-poor plasma (PPP patients, group 1; n = 20) or the same amount of platelet-rich plasma (PRP patients, group 2; n = 20). Patients of group 3 ( n = 20) had no APP (control group). All patients were maintained on their usual regimen of cardiac drugs until the morning of surgery. To preserve hemodynamic stability and restore the intravascular oncotic pressure, the separated plasma was replaced by infusion of an equal amount of hydroxyethyl starch solution (HES) (6% HES, molecular weight 2 × 10 5, substitution rate 0.5%). In all operations, the same surgical procedure was chosen. For all patients, induction and maintenance of anesthesia were similar, consisting of weight-related doses of fentanyl (35 μg/kg), midazolam (0.65 mg/kg), and pancuronium bromide (0.15 mg/kg). The lungs of all patients were mechanically ventilated during the first 5 hours after the end of the operation. Measurements and Main Results: All patients had serial coagulation studies including antithrombin (AT) III-activity, prekallikrein, factor XII, and immunologic tests such as thrombin-antithrombin III (TAT), fibrinopeptide A (FPA), protein C and S (PC and PS), thrombomodulin (TM), tissue-plasminogen-activator (t-PA), plasminogen-activator-inhibitor (PAI 1), fibrinopeptide Bβ 15–42 (FPBβ 15–42), D-dimers, and hemoglobin and platelet counts determined intraoperatively and postoperatively. Chest tube drainage and transfusion requirements were recorded. APP had no negative effects on the quality of PPP and PRP plasma. The platelet count of the withdrawn plasma was 28 ± 12 × 10 9/L (PPP group) and 245 ± 36 × 10 9/L (PRP group). At the end of the operation (after retransfusion of autologous plasma) and on the morning of the first postoperative day, platelet counts were significantly higher ( p > 0.05) in the PRP than in the PPP and control groups. Plasma concentrations of TAT and FPA increased (ranging from +185% to +340% from baseline values) and AT III-activity, PC, PS, and TM antigen decreased (ranging from −8% to −55% from baseline values) to a different extent for all three groups throughout CPB. t-PA-activity increased with a maximum at the end of CPB (PPP group, 6.9 ± 1.5 IU/mL; PRP group, 3.8 ± 0.8 IU/mL; control group, 10.9 ± 2.8 IU/mL). Fibrin and fibrinogen degradation markers such as D-dimers and FPBβ 15 to 42 occurred in peak concentrations after neutralization of heparin by protamine. Only PRP patients showed baseline concentrations of coagulation parameters the next morning ( p < 0.05). Total postoperative blood loss within the first 24 hours reached 482 ± 273 mL (PRP group), 775 ± 256 mL (PPP group), and 948 ± 342 mL in the control group ( p < 0.05). None of the PRP patients received homologous blood, but one PPP patient received two units and five control patients received nine units of packed red cells ( p < 0.05). Conclusions: The results suggest that in cases of elective cardiac surgery, heparin cannot prevent generation of both thrombin and fibrin throughout CPB and postoperatively. The APP procedure had no negative effect on the quality of autologous plasma. APP-withdrawn PRP did not only increase platelet count effectively but also improved an altered hemostatic system after retransfusion. Because of the beneficial effects of APP-withdrawn PRP, it appears to be an attractive technique, especially in patients in whom withdrawal of autologous whole blood cannot be performed, as well as to reduce allogeneic blood usage and perioperative blood loss.

Increased number of thromboxane A 2-prostaglandin H 2 platelet receptors in active unstable angina and causative role of enhanced thrombin formation

The current study was designed to investigate the number and affinity of platelet thromboxane A 2/prostaglandin H2 ( TxA 2 PGH 2 ) receptors in patients with unstable angina and, if any, the role played by the increased thrombin formation that is a common finding in these patients. Measurements taken during active unstable angina but not those taken during inactive angina showed an increased number ( p < 0.001), without changes in affinity, of platelet TxA 2 PGH 2 receptors, evaluated as the binding capacity of iodine 125PTAOH, a stable TxA2 analogue. Moreover patients with active angina had higher plasma concentrations of fibrinopeptide A (FPA) ( p < 0.0001), which were significantly related to the number of platelet TxA 2 PGH 2 receptors ( r = 0.76; p < 0.01) Heparin infusion but not aspirin treatment promptly normalized the number of TxA 2 PGH 2 receptors and significantly reduced plasma FPA concentrations. In an in-vitro study thrombin in a concentration similar to that found in vivo significantly increased the number of platelet TxA 2 PGH 2 receptors ( p < 0.01), whereas heparin did not affect TxA 2 PGH 2 receptors. These results have important therapeutic implications and indicate the preferential use of heparin rather than aspirin during the acute phase of unstable angina.

The Effect of Insulin-Induced Hypoglycaemia on Factor VIII: C Concentrations and Thrombin Activity in Subjects with Type 1 (Insulin-Dependent) Diabetes

The effect of insulin-induced hypoglycaemia on plasma coagulant activity was studied in 11 subjects with well-controlled, uncomplicated type 1 diabetes. Thrombin generation was determined in plasma by a computer ex-vivo assisted chromogenic method and by the activated partial thromboplastin time (APTT). In addition, factor VIII:C, thrombin-antithrombin III (TAT) complex and fibrinopeptide A (FPA) levels were measured. Hypoglycaemia induced a rise in mean (SD) factor VIII:C concentrations from a baseline level of 1.13 (0.32) IU/ml to a peak 15 min after onset of symptoms and they remained increased at 90 min [1.54 (0.57) and 1.5 (0.54) IU/ml, p < 0.001 respectively]. A corresponding reduction in time to generate 50% maximal thrombin activity occurred from a pre-insulin value of 56 (6) s to a minimum reading of 46 (7) s at 15 min (p < 0.001) and remained low at 90 min [48 (6) s, p < 0.001]. APTT shortened from 43.3 (4.8) s to 40.1 (4.6) s at 30 min (p < 0.001) but did not fall below the normal range (37.6-42.7 s) and no significant changes in TAT or FPA levels were noted. Factor VIII:C correlated inversely with time to generate 50% maximal thrombin activity and APTT (r = -0.580, p < 0.001; r = -0.673, p < 0.001, n = 66, respectively). The results show that the rise in plasma factor VIII:C levels induced by hypoglycaemia is accompanied by accelerated rates of generation of thrombin in contact-activated plasma, though concentrations of FPA and TAT remain unchanged, although TAT complexes are not a sensitive marker of in vivo thrombin generation.

Prothrombin Fragment 1+2, Thrombin- Antithrombin III-Complexes and Fibrinopeptide A in Spontaneously Clotting Whole Blood In Vitro

Previous in vitro studies using spontaneously clotting whole blood revealed thrombin formation and high fibrinopeptide A (FPA) concentrations measured during incubation time. This occurred in spite of normal concentrations of thrombin antagonists present in the blood of the healthy subjects examined. However, there are several reports showing that in vivo increased thrombin-antithrombin III-complex (TAT) concentrations and relatively low FPA concentrations may occur e.g. in patients with (pre)thrombotic disorders. These in vivo findings indicate more effective thrombin inhibition by antithrombin III, with almost no fibrin formation. To find an explanation for the differences observed in vitro and in vivo, we extended the in vitro studies by measuring concentrations of prothrombin fragment 1 + 2 (F1 + 2), TAT and FPA at several time points until 30 min. Our goal was to test whether thrombin at least initially is neutralized by antithrombin III, resulting in a lack of fibrin formation, either in the absence or in the presence of heparin (0.2 and 0.5 U/ml whole blood, respectively). In the absence of heparin a simultaneous increase in the concentrations of F1 + 2, TAT and FPA was observed. Thrombin was only partially neutralized by antithrombin III and large amounts of fibrin were formed. The addition of heparin virtually suppressed thrombin formation since the F1 + 2 concentration remained low. Moreover, the small amounts of thrombin formed were neutralized by antithrombin III to a greater extent than in the absence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased thrombin-antithrombin III complexes after 1 h of physical exercise.

Concentrations of thrombin-antithrombin III (TAT) complexes in plasma were previously reported to be increased after a 100-km run, while fibrinopeptide A (FPA) concentration remained unchanged. Thus, antithrombin III appears to neutralize thrombin generated during running and prevents fibrin formation. To determine the clinical relevance of these findings, we compared the effects of exhaustive running (1 h, n = 10) on the plasma concentrations of prothrombin fragments F1 and F2, TAT, FPA, and beta-thromboglobulin with the effects of recreational jogging (1 h, n = 10) and exhaustive bicycling on an ergometer (1 h, n = 8). Prothrombin fragments F1 and F2 and TAT concentrations increased significantly in each group. The most significant increase in TAT concentration was measured in the running group (from 1.72 +/- 0.49 to 3.61 +/- 1.03 ng/ml, P < 0.001). The best correlation was found between the postexercise TAT and lactate concentrations (r = 0.62, n = 28, P < 0.001). Mean FPA concentrations after exercise did not exceed normal values in any of the three groups analyzed. An increase in beta-thromboglobulin concentration was measured in the running and in the cycling group. Thus, thrombin is formed, in particular, when associated with anaerobic metabolism, and platelets are activated during high-intensity exercise.

Plasma factor VII and thrombin-antithrombin III levels indicate increased tissue factor activity in sickle cell patients.

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasma in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin-antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D-dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme-linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0.70 +/- 0.19 U/ml versus 1.16 +/- 0.41 U/ml), whereas F(VII)z in the patients and controls were 0.47 +/- 0.15 U/ml and 1.15 +/- 0.33 U/ml respectively, P < 0.001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 +/- 44 pM versus 42 +/- 2 pM) (P < 0.001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12.85 +/- 1.96 ng/ml versus 4.22 +/- 0.37 ng/ml) (P < 0.001). The D-dimer levels were also higher in the HbSS than control plasmas (1029.6 +/- 58.6 ng/ml versus 224.3 +/- 27.6 g/ml) (P < 0.001), with the patients' values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso-occlusive processes associated with sickle cell painful episodes.

Persistent generation of thrombin after acute myocardial infarction.

During the acute phase of myocardial infarction, the generation of thrombin is reflected in the sudden rise of fibrinopeptide A (FPA) and the thrombin-antithrombin III (TAT) complex in blood. We have systematically determined the FPA and TAT plasma concentrations over a period of 14 days after acute myocardial infarction in 100 patients. Mean levels of both thrombin markers were the highest on admission, remained elevated over the following few days, and then gradually declined after day 5. Still, by the end of the first week two thirds of the patients had distinctly elevated TAT and FPA levels, and by the end of the second week such an abnormality was present in half of them. Continuous intravenous heparin infusion at a dose of 20,000 units/day, administered for 1 week to patients who had either received (n = 21) or not received (n = 17) streptokinase, led to a significant depression (p less than 0.05) of thrombin markers over the first 48 hours, an effect that did not persist over the subsequent days of treatment. In patients not assigned to heparin treatment, those in heart failure had significantly (p less than 0.05) higher mean TAT and FPA values on days 3, 5, and 7 compared with patients in whom heart failure was absent. Infarct extension, pulmonary embolism, and death were also associated with a rise in one or both thrombin markers, often preceding the onset of clinical symptoms. Thrombinogenesis was not accompanied by changes in mean plasma concentrations of prothrombin, antithrombin III, or alpha 2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Intra-arterial thrombin activity produced by percutaneous transluminal angioplasty eliminated by segmentally enclosed thrombolysis

We determined the specific marker of thrombin activity, fibrinopeptide A (FPA), in the vicinity of dilated sites during percutaneous transluminal angioplasty (PTA) for femoro-popliteal obstructions in 24 patients. Blood samples were drawn proximal to dilated segments from a 4F catheter inserted retrogradely in the common femoral artery and distal to dilated segments from the balloon catheter tip. Median +/- S.E. FPA concentration was 21.5 +/- 4.4ng ml-1 before PTA. Immediately after dilatation, FPA concentrations were increased to 970.0 +/- 836.9 ng ml-1 distal to dilated segments (p less than 0.00005) and to 48.5 +/- 11.4 ng ml-1 proximally (p less than 0.003). Segmentally enclosed thrombolysis (SET) was undertaken immediately after PTA, when a double balloon catheter was positioned with a balloon at each end of dilated segments. Both balloons were inflated and 5 mg recombinant tissue plasminogen activator (rt-PA) and 1000 IU heparin were enclosed in the segments for 30 min. Immediately after SET, FPA concentration distal to dilated segments was 34.0 +/- 14.2 ng ml-1 and not different from proximal concentrations found after PTA (p = 0.57). Intense fibrinolysis was indicated by significantly increased levels of cross-linked fibrin degradation products (D-dimer) for hours after SET, but FPA concentrations in peripheral blood remained near baseline values. This finding differed from increased thrombin activity found by others during systemic thrombolytic therapy. Early rethrombosis did not occur after PTA in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin

Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.

Fibrinopeptide A levels indicative of pulmonary vascular thrombosis in patients with primary pulmonary hypertension.

Although the mechanisms involved in the pathophysiology of primary pulmonary hypertension have not yet been delineated, thrombosis has been implicated. This study was designed to determine whether thrombin activity as reflected by plasma concentrations of fibrinopeptide A (FPA), a marker of the action of thrombin on fibrinogen, is increased in patients with primary pulmonary hypertension. To evaluate fibrinolytic activity, we measured plasma concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cross-linked fibrin degradation products. We studied 31 patients with primary pulmonary hypertension. Plasma FPA concentrations measured by radioimmunoassay, were elevated to 87.4 +/- 36.9 ng/ml (mean +/- SEM). Fifteen minutes after administration of heparin (5,000 U), FPA concentrations decreased to 6.8 +/- 1.4 ng/ml (p less than 0.001 compared with preheparin levels). In 21 of 30 patients (70%), FPA concentrations after heparin administration were less than half the preheparin levels, a response consistent with inhibition of thrombin by heparin and the short half-life of FPA. Despite evidence for marked thrombin activity, plasma concentrations of cross-linked fibrin degradation products were normal in all but four patients. Plasminogen activator inhibitor-1 activity was elevated in 19 of the 27 patients in whom it was measured, potentially limiting the fibrinolytic response. The elevations of FPA indicate that thrombin activity is increased in vivo in patients with primary pulmonary hypertension. Thus, sequential assays of plasma markers of thrombosis and fibrinolysis in vivo may help identify those patients who may benefit from treatment with anticoagulants.

Comparison of methods for detecting soluble fibrin in plasma. An in vitro study

The ability of the COA-SET Fibrin monomer (COA-SET FM) test to detect soluble fibrin was evaluated by comparing the results of the COA-SET FM test with fibrino-peptide A (FPA) determinations following thrombin incubation of plasma or whole blood. In addition, two semiquantitative tests (erytrocytes-agglutination test (FM-test) and ethanol gelation test (EGT)) were included in the study. Under the experimental conditions used, the COA-SET FM test proved less sensitive than the FPA-assay. There was a strong correlation between the results obtained by the two tests (r=0.86, p=0.0001). When solely regarding low levels of soluble fibrin, however, the correlation was weaker (r=0.59, p=0.0003). The FM-test was less sensitive than the COA-SET FM test, but more sensitive than EGT at normal and low fibrinogen concentrations. At high fibrinogen concentrations, however, EGT proved more sensitive than the FM-test. Knowing that 1–2 moles of FPA are released per mole of fibrin monomers formed, a discrepancy was observed between the FPA concentrations and the fibrin monomer concentrations as determined by the COA-SET FM test, the FPA levels being 2–25 times higher than the fibrin monomer levels. The discrepancy was greatest at incipient fibrinogen-fibrin transformation and at high plasma fibrinogen levels. This may suggest that fibrinogen in some way interfered with the stimulating effect of fibrin on the t-PA catalyzed activation of plasminogen, the principle upon which the COA-SET FM test is based. In addition, the way of preparing fibrin monomers might influence their properties as plasminogen activators and contribute to the discrepancy. Further studies are in progress to clarify these matters.

Inhibitory Effect of Heparin and/or Antithrombin III on Intraperitoneal Fibrin Formation in Continuous Ambulatory Peritoneal Dialysis

The intraperitoneal fibrin formation and its inhibition by intraperitoneal heparin and/or antithrombin III (AT III) were examined in 8 patients on continuous ambulatory peritoneal dialysis (CAPD). With 1,000 and 2,000 U/L of heparin added to inflow dialysate, the concentration of fibrinopeptide A (FPA) in plasma decreased from 39.43 +/- 5.30 (mean +/- SEM) to 8.00 +/- 2.20 and to 0.74 +/- 0.12 ng/ml, respectively. The FPA concentration in outflow dialysate decreased from 34.20 +/- 5.75 to 12.94 +/- 2.10 ng/ml (1,000 U/l of heparin) and to 4.54 +/- 0.79 ng/mg (2,000 U/l of heparin). The AT III concentration was 0.47 +/- 0.07 mg/dl in dialysate and that in plasma was 24.20 +/- 2.76 mg/dl. With 100 U/bag of AT III added to inflow dialysate, the AT III concentration increased from 0.47 +/- 0.07 to 3.36 +/- 0.17 mg/dl in outflow dialysate but did not increase in plasma. The inhibition of fibrin formation of intraperitoneal heparin was increased by addition of AT III without a systemic inhibitory effect on fibrin formation. These data suggest that intraperitoneal administration of heparin without AT III would be sufficient for the purpose of preventing fibrin formation in CAPD patients without any trouble, and additional AT III might increase inhibitory effect of heparin.

Alterations of Hemostatic Markers in Cardioembolic Stroke

To assess the state of thrombosis and fibrinolysis, we measured the plasma concentrations of fibrinopeptide A (FPA), fibrinopeptide Bβ15-42 (FPBβ15-42), D-dimer, protein C (PC) activity and antigen, and antithrombin III (AT III) in 17 patients of cardioembolic stroke. Patient group had significantly high concentrations of FPA and FPBβ15-42, and significantly low concentrations of PC and AT III compared with healthy control group (p<0.005-0.05). D-dimer was increased in 5 of 7 patients, and both FPA and FPBβ15-42 were increased in 15 of 16 patients. In contrast, PC activity, PC antigen and AT III were reduced only in 6 of 12, 4 of 12 and 5 of 15 patients, respectively. These findings suggest that FPA, FPBβ15-42 and D-dimer may be useful to detect the activities of thrombosis and fibrinolysis in patients with cardioembolic stroke.

Hyperglycemia may determine fibrinopeptide A plasma level increase in humans

The effects of hyperglycemia on plasma fibrinopeptide A (FPA) levels in normal subjects are reported. An increase of FPA concentration parallel to sustained hyperglycemia was observed; when the glycemia returned to basal values, FPA showed values in normal range. Heparin infusion was able to significantly decrease the hyperglycemia-induced augment of FPA levels. Isovolumic-isotonic NaCl solution infusion produced a slight (NS) increase in FPA levels; however, mild hyperglycemia, achieved by glucagon, was also able to produce a significant increase in FPA concentration. These data demonstrate the direct role of hyperglycemia in conditioning FPA level, and suggest that hyperglycemia, by itself, is a sufficient stimulus to produce thrombin activation in humans.

Hemostatic markers in acute stroke.

To assess the time course of thrombosis and fibrinolysis after acute stroke, we measured concentrations of fibrinopeptide A (FpA), B-beta 1-42 peptide (B-beta 1-42), B-beta 15-42 peptide (B-beta 15-42), and crosslinked D-dimer (XDP) in 31 patients at varying times following acute ischemic stroke and in 13 neurologically stable patients with chronic strokes. FpA levels were markedly elevated during the first week after stroke and declined slowly during the first month. Mean FpA levels were not significantly elevated in chronic stroke patients. Mean XDP levels were slightly elevated during the first week and increased during the next 2 weeks after stroke. B-beta 1-42 and B-beta 15-42 levels were not elevated at any time following acute stroke. Our data suggest that fibrin formation greatly exceeds endogenous fibrinolysis during the acute phase of ischemic stroke. Endogenous fibrinolysis develops slowly following stroke. Prolonged elevation of FpA concentration suggests that thrombin activity and fibrin formation continue for up to 4 weeks in some patients with ischemic stroke.