Haemostatic and inflammatory biomarkers in advanced chronic heart failure: role of oral anticoagulants and successful heart transplantation

Advanced chronic heart failure (CHF) is associated with abnormal haemostasis and inflammation, but it is not known how these abnormalities are related, whether they are modified by oral anticoagulants (OAT), or if they persist after successful heart transplantation. We studied 25 patients with CHF (New York Heart Association class IV, 10 of whom underwent heart transplantation) and 25 age- and sex-matched healthy controls by measuring their plasma levels of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin (TAT) complexes, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), D-dimer, factor VII (FVII), fibrinogen, von Willebrand factor (VWF), tumour necrosis factor (TNF), soluble TNF receptor II (sTNFRII), interleukin 6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelial-selectin (E-selectin) and thrombomodulin. CHF patients had higher plasma levels of TAT, D-dimer, t-PA, fibrinogen, VWF, TNF, IL-6, sTNFRII, sVCAM-1 (P = 0.0001), sICAM-1 (P = 0.003) and thrombomodulin (P = 0.007) than controls. There were significant correlations (r = 0.414-0.595) between coagulation, fibrinolysis, endothelial dysfunction and inflammation parameters, which were lower in those patients treated with OATs. Heart transplantation led to reductions in fibrinogen (P = 0.001), VWF (P = 0.05), D-dimer (P = 0.05) and IL-6 levels (P = 0.05), but all the parameters remained significantly higher (P = 0.01-0.0001) than in the controls. Advanced CHF is associated with coagulation activation, endothelial dysfunction and increased proinflammatory cytokine levels. Most of these abnormalities parallel each other, tend to normalize in patients treated with OATs and, although reduced, persist in patients undergoing successful heart transplantation, despite the absence of clinical signs of CHF.     

Relation of nocturnal blood pressure dipping to cellular adhesion, inflammation and hemostasis

BACKGROUND: Subjects who fail to dip their nocturnal blood pressure (BP) are at substantially increased risk for cardiovascular diseases. The pathogenetic mechanisms of this relationship have not been elucidated. We investigated whether non-dipping would relate to procoagulant and proinflammatory activity. DESIGN: Study participants were 76 unmedicated normotensive and hypertensive subjects (44 male, 32 female; 41 white, 35 black; mean age, 36 +/- 8 years) who underwent 24-h outpatient ambulatory BP monitoring. Based on whether their average nocturnal systolic BP relative to their average daytime systolic BP declined by less than 10%, 34 subjects were categorized as non-dippers. D-dimer, plasminogen activator inhibitor-1, von Willebrand factor, soluble intercellular adhesion molecule-1, and interleukin-6 were measured in plasma. RESULTS: Multivariate analyses showed that D-dimer (median/interquartile range, 242/162-419 ng/ml versus 175/132-254 ng/ml; P=0.041), plasminogen activator inhibitor-1 (36/19-61 ng/ml versus 17/6-44 ng/ml; P=0.010), von Willebrand factor (122/91-179% versus 92/66-110%; P=0.001), and soluble intercellular adhesion molecule-1(227/187-291 ng/ml versus 206/185-247 ng/ml; P=0.044) were all higher in non-dippers than in dippers. Adjustment for gender, ethnicity, age, body mass index, smoking status, hypertension status, and social class revealed independent effects of non-dipping. Non-dippers continued to have higher D-dimer (P=0.030) and von Willebrand factor (P=0.034) than dippers. A similar trend not reaching statistical significance emerged for soluble intercellular adhesion molecule-1 (P=0.055). In contrast, dipping status had no effect on interleukin-6. CONCLUSION: Nocturnal BP non-dipping is associated with elevated levels of molecules related to endothelial dysfunction and atherosclerosis. The finding provides one possible mechanism linking non-dipping with cardiovascular disease.     

The extrinsic coagulation activity assay

A chromogenic assay for the tissue factor-mediated thrombin generation was developed, the extrinsic coagulation activity assay: 50 microL citrated plasma is incubated with 5 microL tissue factor in 6% albumin and 250 mM CaCl2. After 1-minute (37 degrees C) coagulation reaction time, (extrinsic coagulation activity assay with 1-minute coagulation reaction time; generating normally about 1 IU/mL thrombin) 100 microL 2.5 M arginine is added to stop hemostasis activation. Generated thrombin is then chromogenically quantified. The normal extrinsic coagulation activity assay range is 100% +/- 20%. Extrinsic coagulation activity assay in plasma of patients on heparin or coumarines is about 10-fold lower. Advantages of extrinsic coagulation activity assay: normal range of extrinsic hemostasis is truly represented, patients prone to hyper-activated extrinsic pathway are detected, anticoagulants result in respective test inhibition, fibrinogen/fibrin concentration does not artefactually alters the test result, plasma matrix is not changed significantly in the assay, and assay results are IU/mL thrombin or % of normal, which can be measured by every normal photometer.     

Alteration of haemostasis in non-metastatic gastric cancer

BACKGROUND: Cancer is one of the most common acquired causes of venous thromboembolism. AIM: To evaluate haemostasis disorders in patients with non-metastatic gastric cancer. PATIENTS AND METHODS: We studied 11 patients with non-metastatic gastric cancer (9 males and 2 females, median age 54 years) and 20 healthy subjects (15 males and 5 females, median age 48 years) control. We measured prothrombin time, activated partial thromboplastin time, coagulation time, clot lysis time, fibrinogen, clotting factors (II, VII, VIII, IX, X), C protein, S protein, AT III, activated protein C resistance, prothrombin 1+2 fragment, tissue plasminogen activator and D-Dimer in all subjects. RESULTS: Fibrinogen plasma levels were significantly higher in patients with non-metastatic gastric cancer than in control group (505+/-24 mg/dl vs 336+/-30 mg/dl, p<0.001). We also found a significant increase in prothrombin 1+2 fragment plasma concentration compared with controls (3.8+/-0.6 nM vs 0.83+/-0.09 nM, p<0.001). Plasma D-dimer levels were 20-fold higher in patients with non-metastatic gastric cancer compared with controls (9.57+/-0.4 ng/dl vs 0.4+/-0.05 ng/dl, p<0.001). Also tissue plasminogen activator was significantly higher in gastric cancer patients than in controls (20.8+/-2.32 ng/ml vs 9.1+/-1.37 ng/ml, p<0.01). Finally clot lysis time was significantly accelerated in gastric cancer patients compared with control subjects (81+/-37 min vs 233+/-74 min, p<0.01). CONCLUSIONS: Patients with non-metastatic gastric cancer are at risk for thrombotic events due to the combined increase in fibrinogen plasma levels and thrombin formation.     

Specific determination of plasmatic thrombin activity.

Thrombin is the key enzyme of coagulation. Its activity can be determined via fibrinogen ? fibrin conversion or via cleavage of a chromogenic substrate. The latter method is easier than the first one, but in plasma it is hampered due to unspecific cleavage of the chromogenic substrate by thrombin-like enzymes of hemostasis, especially those of the contact phase. The concentration of the thrombin substrate (HD-CHG-Ala-Arg-pNA) was optimized, using final substrate concentrations of 0 to 5 mM, a final arginine concentration of 1.13 M, and samples of 10 mIU/mL purified thrombin in 7% human albumin or pooled normal citrated plasma without and with EDTA. Twenty microliters pooled normal citrated plasma (frozen/thawed) or factor II-deficient plasma (lyophilized) were incubated with 10 microL 0% to 0.5% Thromborel S (100% = 162 ng/mL tissue factor [TF]) in 6% BSA or with 10 microL 0% (physiol. NaCl) to 50% Pathromtin SL and with 20 microL 25 mM CaCl(2). After 0 to 22 minutes (37 degrees C), 20 microL 1.7 M arginine, pH 8.7 were added. Fifteen microliters 0.9 mM HD-CHGAla-Arg-pNA in 2.3 M arginine, pH 8.6, were added and the increase in absorbance (deltaA) at 405 nm was determined. Thrombin activity was standardized against the (3)A measured for 1 IU/mL thrombin in 7% human albumin (8.8 mA/min RT). The optimal final chromogenic substrate concentration to detect thrombin in this assay system is less than 0.6 mM. Higher substrate concentrations in a plasma milieu result in unspecific cleavage of the substrate. Using final concentrations of chromogenic substrate less than 0.4 mM (the approximate Km- value for thrombin) and final concentrations of arginine greater than 800 mM, in factor II-depleted plasma, when activated either by TF or by the contact phase, there is no significant thrombin generation. The circulating thrombin activity measured in EDTA plasma of 39 healthy donors is 100 +/- 20% of norm (mean value +/- 1 SD; 100% = 5.5 mIU/mL thrombin). This chromogenic assay detects thrombin activity independent of clotting seconds or fibrin mediated turbidity increases. This technique allows to standardize the thrombin activity generated in any biologic system in international thrombin units.     

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.     

The fibrinogen functional turbidimetric assay.

Hitherto, clinical fibrinogen methods were based on coagulation seconds, with assay conditions not similar to a plasma milieu. The fibrinogen functional turbidimetric assay included 50 microL citrated plasma + 100 microL 300 mIU/mL thrombin, 400 microg/mL polybrene, and 6% albumin-phosphate-buffered saline; an increase in absorbance at 405 nm/5 min at room temperature (or 2 minutes at 37 degrees C) was observed. In all, 6% albumin in the fibrinogen functional turbidimetric assay reagent abolishes falsely elevated fibrinogen to fibrin turbidity in hypoproteinemic plasma samples. This assay can detect fibrinogen activity of 250% to 300% of normal, the lower detection limit being 7% of normal (0.2 g/L). The normal range of this assay is 100% +/- 20% (mean value +/- 1 SD; coefficient of variations <4%). This assay imitates fibrinogen to fibrin conversion in clotting blood plasma; it is independent of plasmatic albumin or heparin and can be performed everywhere. This assay has a diagnostic value in pathology-disseminated intravascular coagulation and in assessing risk for atherothrombosis.     

The effects of perioperatively administered crystalloids and colloids on concentrations of molecular markers of activated coagulation and fibrinolysis.

To explore whether intravenous administration of routinely used crystalloid or colloid solutions differently affects the coagulation system, we investigated orthopaedic patients. Since crystalloid solutions might cause hypercoagulability, we here present our results on molecular markers of coagulation and fibrinolysis. Patients undergoing knee replacement surgery randomly received isovolemic amounts of lactated Ringer's solution, 6% hydroxyethyl starch 200/0.5 or 4% modified gelatine. Arterial blood samples for determination of specific molecular markers of activated coagulation (thrombin/antithrombin complex, D-dimer, prothrombin fragment F1 + 2), fibrinolysis (plasmin/alpha 2-antiplasmin complex, tissue plasminogen activator, plasminogen activator inhibitor-1), and concentrations of coagulation factor XIII were obtained at baseline, before tourniquet release, at the end of surgery and 2 h after operation. During the observation period, thrombin/antithrombin complex increased from 4.8 to 54.7 microg/l, D-dimer increased from 0.3 to 6.0 mg/ml, prothrombin fragment F1 + 2 increased from 1.7 to 5.9 nmol/l, tissue plasminogen activator decreased from 7.3 to 6.7 ng/ml, plasminogen activator inhibitor-1 increased from 68.4 to 71.0 ng/ml, plasmin/alpha 2-antiplasmin complex increased from 281.5 to 884 microg/l and factor XIII decreased from 89.0 to 58.5%. All parameters changed significantly but without any detectable difference in the response profile between the groups receiving different intravenous fluids. During knee replacement surgery a pronounced activation of the coagulation/fibrinolytic system was observed, regardless of whether patients received crystalloid or colloid fluids. Thus, these results cannot confirm the hypothesis that crystalloid fluids per se cause hypercoagulability in vivo.     

Thrombin generation and fibrinolytic activities among patients receiving reduced-dose alteplase plus abciximab or undergoing direct angioplasty plus abciximab for acute myocardial infarction

The purpose of this study was to determine the impact of these 2 reperfusion strategies (reduced-dose alteplase plus abciximab or direct angioplasty plus abciximab) on fibrinolytic and thrombin generation activities. The effect of reduced-dose alteplase plus abciximab and direct angioplasty plus abciximab on hemostatic factors is unknown. Of 70 patients with acute myocardial infarction of < or = 6 hours, 34 were randomized to reduced-dose alteplase (35 to 50 mg in 1 hour) and 36 to direct angioplasty. A standard bolus and infusion dose of abciximab was administered to all patients. Blood specimens were collected at baseline, and at 1, 4, 12, and 24 hours. The following parameters were assayed: fibrinogen, plasminogen and antiplasmin activities, tissue plasminogen activator antigen, D-dimer, prothrombin fragments F1 + 2, and thrombin/antithrombin III complexes. Among patients treated with reduced-dose alteplase plus abciximab, the fibrinogen level decreased by 28.4% in the first hour (11.7 +/- 3.4 vs 7.8 +/- 2.5 micromol/L, p <0.001). Correspondingly, plasminogen and antiplasmin activities decreased by 43.8% (p <0.001) and 59.1% (p <0.001), respectively. Prothrombin fragments F1 + 2 increased from 2.2 +/- 1.7 to 4.2 +/- 1.6 nmol/L (1 hour) (p <0.001) and thrombin/antithrombin III increased from 16.3 +/- 15.0 to 33.5 +/- 19.9 microg/L (1 hour) (p <0.001). Conversely, in the direct angioplasty group, there was a marginal elevation in fibrinogen level at 1 hour (10.2 +/- 2.4 vs 10.6 +/- 2.0 micromol/L, p = 0.064) despite a significant reduction in plasminogen and an increase in tissue plasminogen activator levels. There was no significant change in prothrombin fragments F1 + 2 and thrombin/antithrombin III levels. Thus, there was considerable fibrinolytic activity with reduced-dose alteplase plus abciximab; thrombin generation was not prevented. Among patients treated with direct angioplasty, there was some endogenous fibrinolytic activity, but there was no significant thrombin generation.     

Evolution of plasma D-dimer and fibrinogen in witnessed onset of paroxysmal atrial fibrillation

BACKGROUND: Although increased D-dimer and fibrinogen have been proved to be related with atrial fibrillation (AF), their evolution in the course of time remains unclear. METHODS: To elucidate the evolution of plasma D-dimer and fibrinogen in AF of different duration. 56 patients (group A) of 3,524 patients in whom the onset of AF had been witnessed in a chest pain clinic were enrolled for study. Plasma D-dimer and fibrinogen concentrations were checked within 30 min after the onset of AF and followed up as scheduled. Another 50 patients (group B) with chronic AF underwent the same protocol and served as controls. RESULTS: In group A, the D-dimer levels reached a plateau at the 18th hour (382 +/- 52 vs. 840 +/- 280 ng/ml, p < 0.001 by ANOVA) and the fibrinogen level increased gradually from 4.1 +/- 0.6 g/ml at baseline to 6.1 +/- 0.9 g/ml at the 48th hour (p < 0.001). No subjects had evidence of intra-cardiac thrombi by transesophageal echocardiography. There were significant differences in plasma D-dimer and fibrinogen levels between the two groups at each measurement. At the cut- off value of 500 ng/ml, plasma D-dimer had a sensitivity of 100%, a specificity of 93% in defining AF lasting for less than 12 h. The positive and negative predictive values were 52% and 100%, respectively. Plasma D-dimer increased above the normal range prior to the 12th hour after the onset of AF. CONCLUSION: The observations support its use as a screening tool for identifying patients with short duration AF capable of being safely converted.     

Inhibition of extrinsic hemostasis activation by low-molecular-weight heparin.

Low-molecular-weight heparins (LMWHs) are very important drugs; unfortunately, the routine global hemostasis assays activated partial thromboplastin time and prothrombin time are not sensitive to LMWHs. Here the 50% inhibitory concentration (IC(50)) values of heparin and LMWHs on extrinsic thrombin generation are determined. Pooled normal plasma was supplemented with 0-2 IU/ml unfractionated heparin, 0-2 IU/ml LMWH dalteparin, or 0-20 microg/ml pentosanpolysulfate in 5-ml polystyrole tubes (23 degrees C) and tested in the tissue-factor-triggered extrinsic coagulation activity assay (EXCA): 50 microl plasma + 5 microl tissue factor/CaCl(2), 1 and 2 min incubation time at 37 degrees C (coagulation reaction time for EXCA-1 and EXCA-2); + 100 microl of 2.5 mol/l arginine (pH 8.6), 20 min at room temperature; + 50 microl of 1 mmol/l CHG-Ala-Arg-pNA, 1.25 mol/l arginine; increase in absorbance/time at 23 degrees C; calibrator = 1 IU/ml bovine thrombin in 6.7% human albumin replacing the plasma sample; in EXCA-1, about 1 IU/ml thrombin is generated in pooled unfrozen normal citrated plasma. The IC(50) values in EXCA-1 are 0.1 IU/ml heparin, 0.02 IU/ml LMWH, and 4.7 microg/ml pentosanpolysulfate. In ECXA-2 the IC(50) values are 0.07 IU/ml, 0.01 IU/ml, and 4.6 microg/ml, respectively. The EXCA reflects the efficiency of anticoagulants on plasmatic coagulation. It is suggested to adjust the dosage of LMWH according to the EXCA value; about 30% of normal extrinsic thrombin generation might be the correct dose for prophylactic anticoagulation.     

Plasminogen activation by blood monocytes and alveolar macrophages in primary pulmonary hypertension.

The pathophysiology of primary pulmonary hypertension (PPH) remains poorly understood. Vascular wall remodeling and endothelial dysfunction reflected by modifications in plasma fibrinolytic proteins and von Willebrand factor have been well documented in PPH. We hypothesize that endothelial mediators, produced in excess in PPH patients, may stimulate migrating mononuclear cells and thereby modulate alveolar macrophage function; in particular, the plasminogen activation system. Components of the fibrinolytic system were therefore studied in plasma, blood monocytes and alveolar macrophages obtained from bronchoalveolar lavage in 10 patients with PPH and in four controls. Compared with controls, PPH patients had elevated plasma levels of tissue-type plasminogen activator (15.6 +/- 9.9 versus 5.5 +/- 3 ng/ml) and plasminogen activator inhibitor-1 (27.8 +/- 23 versus 16.4 +/- 12 ng/ml). In contrast, binding and activation of plasminogen by single-chain urokinase-type plasminogen activator (scu-PA) at the surface of blood monocytes and alveolar macrophages were not different from those of control values. Dissociation constants (K(d)) for binding of scu-PA and plasminogen to alveolar macrophages were similar in both PPH (4.7 +/- 1.5 and 0.88 +/- 0.3 micromol/l, respectively) and control (6.7 +/- 0.1 and 1.02 +/- 0.12 micromol/l, respectively) groups. These results indicate that in PPH patients the fibrinolytic activity of alveolar macrophages is normal, whereas endothelial fibrinolytic proteins are abnormally elevated in plasma.     

Postmenopausal hormone replacement therapy increases coagulation activity and fibrinolysis

Hormone replacement therapy (HRT) appears to be cardioprotective in postmenopausal women; however, concerns exist over its thrombogenic effects. To address the effects of combined HRT on coagulation and fibrinolysis, we have measured circulating markers of these processes in a double-blind placebo-controlled trial. Forty-two healthy postmenopausal women aged 50 to 75 years received continuous combined HRT with 2 mg estradiol+1 mg norethisterone or placebo for 6 weeks. Hormone profiles were measured at baseline, and lipid and hemostatic parameters were measured at baseline and after 6 weeks of therapy. Baseline characteristics were similar in the 2 groups. With change from baseline the main outcome measure, HRT increased the markers of coagulation (prothrombin fragments 1+2, 0.20+/-0.06 versus 0.06+/-0.04 nmol/L, P=0.0005; soluble fibrin, 2.3+/-0.4 versus 0.25+/-0.3 microgram/mL, P=0.0004), reduced plasma fibrinolytic inhibitory activity (plasminogen activator inhibitor-1, -0.67+/-0.16 versus 0.24+/-0.21 U/mL, P=0.002), and increased fibrinolysis (D-dimer, 24+/-12 versus -6+/-8 ng/mL, P=0.04) compared with placebo. Increases in soluble fibrin and D-dimer were positively correlated (r=0.59, P=0.02), but changes in plasminogen activator inhibitor-1 and D-dimer were unrelated. Although baseline hemostatic and lipid parameters were correlated, there were no associations between changes in hemostatic markers and lipids after treatment. Short-term oral combined continuous HRT (estradiol and norethisterone) increased thrombin and fibrin generation, reduced plasma fibrinolytic inhibitory activity, and increased fibrinolysis. Enhanced fibrinolysis was related to increased fibrin generation but not reduced plasma fibrinolytic inhibitory activity. Coagulation activation may partly explain the increases in venous thrombosis and cardiovascular events reported with the use of combined HRT.     

Tissue plasminogen activator levels change with plasma fibrinogen concentrations during pregnancy

To investigate the relationship between coagulation activities and the fibrinolytic system during normal pregnancy, we measured the plasma concentrations of coagulation factors, antithrombin III (AT III), D-dimer, tissue plasminogen activator (tPA), total protein S (TPS), and plasminogen activator inhibitor type 1 (PAI-1) in 436 apparently healthy pregnant, postpartum, and nonpregnant women. There were no significant changes in AT III, TPS, and factor XI concentrations during pregnancy and puerperium. However, factor VII, VIII, IX, and XII activities increased gradually as pregnancy progressed, reached maximum values in the third trimester, and returned to nonpregnant levels by 5-8 weeks postpartum. Plasma D-dimer levels in the third trimester of pregnancy were 1.23+/-0.42 micro g/ml, significantly higher than for the first trimester (0.34+/-0.16 micro g/ml, P<0.01). The tPA antigen levels averaged 1.8-fold higher in the late third trimester than in the first trimester; the plasma fibrinogen concentrations averaged 1.6-fold higher in the late third trimester than in the first trimester. Compared to the peak values during pregnancy, tPA levels averaged 39.8% lower and plasma fibrinogen concentrations averaged 40.0% lower at 5-8 weeks postpartum. The tPA levels correlated strongly with the plasma fibrinogen concentrations ( r=0.52, P<0.01). In short, this study shows that tPA levels change in parallel with plasma fibrinogen concentrations during and after normal pregnancy.     

RNAi targeting embryonic myosin heavy chain isoform inhibited bound thrombin-induced migration of vascular smooth muscle cells

To investigate the effect of bound thrombin, a complex of alpha-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell proliferation was measured by WST-1 reagent and migration was evaluated by counting migrated cells through pores of cell culture insert (8 mum size) after 48-hour treatment with bound thrombin (10 U/ml). To examine the role of an embryonic myosin heavy chain isoform (SMemb) in these effects by bound thrombin, the cells were subsequently treated for 48 h with an siRNA expression vector (ORF-2/pSilencer) directed against the open reading frame of SMemb mRNA. SMemb and plasminogen activator inhibitor-1 mRNA expressions were measured by Northern blot analysis. Bound thrombin significantly increased SMemb mRNA expression by 1.4 +/- 0.01-fold and significantly increased plasminogen activator inhibitor-1 mRNA expression by 2.65 +/- 0.69-fold (p < 0.01 vs. PBS treatment for each), which were abolished by treatment with ORF-2/pSilencer. Although bound thrombin had no effect on cell proliferation, bound thrombin significantly increased migration by 1.93 +/- 0.20-fold (p < 0.05). ORF-2/pSilencer treatment significantly reduced the bound thrombin-stimulated migration activity by 1.28 +/- 0.15-fold (p < 0.05). Thus, SMemb plays an important role in bound thrombin-induced cell migration activity of cultured vascular smooth muscle cells.     

Impact of the fibrinolytic enzyme system on prognosis and survival associated with non-small cell lung carcinoma.

Comprehensive studies of fibrinolysis in non-small cell lung carcinoma have been limited, and assignment of patients to high/low prognosis groups based on arbitrary cut-offs utilizing fibrinolytic measurements is unstandardized. This study was performed in 166 patients to examine the effects of cut-off values determined in three ways. Model 1 assigned patients to one of three equal groups (low, medium, high) based on fibrinolytic measurements made at diagnosis, Model 2 divided patients into low/high groups using median values, and Model 3 grouped according to the parameter being above/below normal range. In model 1, raised plasma fibrinogen, D-dimer and soluble fibrin were positively associated with poorer survival. In model 2, tissue plasminogen activator antigen was additionally related to poorer prognosis. Model 3 identified seven parameters as significantly related to survival, two not identified by the other models becoming significant [plasmin-antiplasmin, tissue plasminogen activator inhibitor-1 (PAI-1) antigen]. Using multivariate analysis, plasma fibrinogen level was the most uniformly significant parameter. Relative risk estimates indicated that raised plasma fibrinogen, soluble fibrin and D-dimer were associated with increased risk of death. Use of the normal/above normal cut-off is recommended to provide the maximum number of significant parameters relating to prognosis, and increased plasma D-dimer, PAI-1 antigen and fibrinogen were most closely related to survival/prognosis.     

A metabolic syndrome independent association between overweight, fibrinolysis impairment and low-grade inflammation in young women with venous thromboembolism.

It is known that overweight induces fibrinolysis impairment. Since this association has not been completely explored in patients with venous thromboembolism (VTE), we aimed to investigate its presence in young women with VTE and, if present, to determine its extent and the factors that influence it. Thirty women aged 23-49 years in the stable period after VTE were included [19 overweight (body mass index > or = 25) and 11 normal weight]; 52 healthy women (27 overweight and 25 normal weight) served as controls. The euglobulin clot lysis time (ECLT), plasminogen, D-dimer, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigen and activity, lipids, fasting plasma glucose, insulin, fibrinogen, interleukin-6 and sedimentation rate were compared between the groups. Overweight patients had more impaired fibrinolysis (delayed ECLT, higher PAI-1 and t-PA antigen) than overweight controls (and normal weight patients). There was no difference in levels of insulin, glucose, fibrinogen and interleukin-6, whereas the sedimentation rate was significantly elevated in overweight patients compared with overweight controls [16 (8-31) versus 8 (5-12), P < 0.05]. The sedimentation rate in overweight patients significantly correlated with body mass index, ECLT, t-PA and PAI-1 antigen, but not with fibrinogen or interleukin-6. We found that overweight VTE patients have more prominent fibrinolysis impairment than predictable from parameters of metabolic syndrome and that is associated with an elevated sedimentation rate. This association could represent a new thrombotic risk profile in overweight women.     

Increased immunoreactivity of plasma after fibrinolytic activation in an anti-DD ELISA system. Role of soluble crosslinked fibrin polymers

After addition of a low concentration of thrombin to normal plasma, a progressive and significant increase in crosslinked fibrin polymers was found by sodium dodecyl sulfate agarose gel electrophoresis, reaching 27% of total fibrinogen and fibrin before gel formation. As measured by enzyme-linked immunosorbent assay with a monoclonal antibody specific for an epitope near the gamma gamma crosslink site, increased immunoreactivity of plasma did not occur after adding thrombin despite formation of crosslinked fibrin polymers, which indicates that the antibody does not recognize the epitope in the polymers. Addition of tissue-type plasminogen activator (t-PA) to plasma resulted in a more rapid degradation of fibrin polymers than of fibrinogen, indicating that the fibrin specificity of t-PA is retained with soluble fibrin. Coincident with degradation of plasma crosslinked fibrin polymers, plasma DD immunoreactivity increased 70-fold from 50.3 +/- 4.5 (mean +/- SD) to 3,560 +/- 1,235 ng/ml. The presence of increased crosslinked fibrin polymers produced by adding thrombin to plasma significantly increased maximum immunoreactivity after t-PA-induced degradation to 18,500 +/- 11,780 ng/ml. The increase in DD immunoreactivity was dependent on t-PA concentration; no elevation occurred below 0.01 micrograms/ml, and maximal increases occurred above 100 micrograms/ml. Analysis of gel electrophoretic patterns of thrombin and t-PA-treated plasma samples suggests that the DD reactivity of t-PA-treated plasma is mainly due to degradation of soluble crosslinked fibrin polymers. Our findings indicate that plasmic degradation of soluble fibrin polymers in plasma may be an important source of fragment DD during thrombolytic therapy.     

Plasma t-PA/PAI-1 complex and blood coagulability in patients with coronary artery disease.

The t-PA/PAI-1 complex is a good indicator of the release of fibrinolysis activators and inhibitors from the vascular wall, but its clinical significance in chronic ischemic heart disease is unclear. The plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), and the t-PA/PAI-1 complex (including various coagulation factors) were assayed in 72 patients with coronary artery disease (CAD) and 29 control (C) subjects. The CAD patients were subdivided into 3 groups: single-vessel disease (G1, n = 30), double-vessel disease (G2, n = 20), and triple-vessel disease (G3, n = 22). The patients with triple-vessel disease had higher fibrinogen values (G3: 318 +/- 75 mg/dl, C: 263 +/- 56), factor VII activity (G3: 143 +/- 36%, C: 123 +/- 14), and t-PA antigen levels (G3: 4.7 +/- 0.8 ng/ml, C: 3.3 +/- 0.7) than controls. Patients with double- and triple-vessel disease also showed higher levels of factor VIII, vWF antigen, thrombin-antithrombin III complex (G1: 2.3 +/- 0.6 ng/ml, G2: 2.7 +/- 0.5, G3: 3.1 +/- 0.5, C: 2.0 +/- 0.5), and t-PA/PAI-1 complex (G1: 13.9 +/- 6.1 ng/ml, G2: 16.4 +/- 4.6, G3: 18.2 +/- 5.9, C: 10.7 +/- 4.9) than control subjects. The t-PA/PAI-1 complex levels were correlated significantly with the activities of factors VII and VIII and the thrombin-antithrombin III complex. These findings suggest that patients with CAD have greater blood coagulability than controls, and that this difference is related to the severity of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coagulation and fibrinolysis in preeclampsia and neonates.

Coagulation and fibrinolysis were determined in 67 Indonesian women admitted to the University Hospitals for delivery in Medan. They were diagnosed to be at term gestation (mean 39.3 +/- 1.1 weeks) with moderate and severe preeclampsia (n=32) and in labor, and 8 had preterm labor (gestation mean 33.5 +/- 2.6 weeks). Twenty-seven normal pregnant women in labor (gestation mean 39.7 +/- 1.0 weeks) served as controls. Cord blood from 23 neonates from normal pregnancy and 31 neonates from preeclampsia was also evaluated. Preeclamptic women in labor showed further enhanced coagulation activation (F(1+2)) with raised urokinase-like plasminogen activator (u-PA) activity and reduced plasminogen activator inhibitor-2 (PAI-2) levels. In preterm preeclampsia, significantly reduced antithrombin III (ATIII) and PAI-2 levels with further elevated tissue-type PA (t-PA) antigen and plasminogen activator inhibitor-1 (PAI-1) antigen were seen compared to normal pregnancy. These would suggest a state of enhanced thrombin generation with elevated fibrinolytic/inhibitor proteins in preterm preeclampsia. The reduced PAI-2 levels seen in preeclampsia have been suggested to be associated with reduced placental function. Neonates born to mothers of either normal pregnancy or preeclampsia at term showed similar hemostatic changes with reduced fibrinogen, ATIII, t-PA, u-PA antigen, PAI-1 levels, and coagulation activation compared to their respective maternal plasma levels. No significant differences in hemostatic parameters studied between the neonates of both cohorts were seen, and this would suggest that the neonates were protected from the adverse effects of preeclampsia and their hemostatic system was physiologically balanced.