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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

2型糖尿病患者糖耐量正常一级亲属血管内皮功能及炎症因子的研究

目的研究2型糖尿病患者一级亲属糖耐量正常者(FDR)的血管内皮功能、炎症因子水平及其影响因素。方法测定31例正常人、57例FDR的血管内皮功能、血浆纤溶酶原激活物抑制物1(PAI1)及血清可溶性血管细胞黏附分子1(VCAM1)水平,同时测定胰岛素水平,计算胰岛素敏感性(IAI)。结果与正常对照组比较,FDR内皮依赖性血管舒张功能减低[(1245±337)%比(503±034)%],IAI降低[(-379±057)比(-411±046)],血浆PAI1水平升高[(3046±1228)μg/L比(3925±654)μg/L],血清VCAM1水平升高[(63731±10732)μg/L比(74239±12431)μg/L],差异均有统计学意义(P值均<005)。结论糖耐量正常的2型糖尿病患者一级亲属胰岛素敏感指数下降、血管内皮功能受损、纤溶活性降低,且血管内皮功能失调与胰岛素抵抗密切相关。     

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.     

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.     

Plasma plasminogen activator inhibitor-1 activity in normoglycemic hypertriglyceridemic north Asian Indian subjects: a preliminary case-control study.

BACKGROUND: Recent evidence suggests that increased activity of plasma plasminogen activator inhibitor-1, an important component of the insulin resistance syndrome, plays a crucial role in the pathogenesis of atherosclerosis. METHODS AND RESULTS: In this case-control study, relationships between plasma plasminogen activator inhibitor-1 activity, serum triglyceride levels and hyperinsulinemia were explored in 40 non-diabetic patients with primary hypertriglyceridemia (Group 1) and 40 non-diabetic normotriglyceridemic controls (Group 2) matched for potential confounders like smoking and physical activity. Mean values of fasting serum insulin levels were increased in Group 1 (p>0.05). Hyperinsulinemia was observed in 14 (17.5%) individuals in Group 1 and 11 (13.8%) individuals in Group 2. Mean plasma plasminogen activator inhibitor-I activity in Group 1 (9.8+/-8.4 IU) was higher than in Group 2 (7.0+/-7.7 IU), though the difference was not significant (p>0.05). However, when only subjects with elevated levels of plasma plasminogen activator inhibitor-1 activity were taken into account, mean values were significantly higher in Group 1 (p<0.05). The plasma plasminogen activator inhibitor-1 activity was higher in subjects with body mass index >25 in both the groups, significantly so in males (p=0.05). Hyperinsulinemic subjects with a body mass index >25 and raised serum triglyceride levels had higher mean values of plasma plasminogen activator inhibitor-1 activity (18.42+/-11.15 IU) than subjects with similar characteristics and normal triglyceride levels (14.22+/-8.20 IU, p<0.05). CONCLUSIONS: Though in the current study a trend for hyperinsulinemia and high plasma plasminogen activator inhibitor-1 activity was observed in hypertriglyceridemic subjects, a larger study is needed to achieve significant differences and correlations. Obese male subjects, irrespective of their lipid profile, are at risk for thrombotic events in view of their significantly higher plasma plasminogen activator inhibitor-1 values. Procoagulant tendency is further enhanced if hypertriglyceridemia and hyperinsulinemia are added on to obesity.     

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.     

Type 2 diabetes: assessment of endothelial lesion and fibrinolytic system markers.

This study aimed to investigate whether endothelial cells are damaged and to evaluate fibrinolytic system function in patients with type 2 diabetes. For this proposal, plasma levels of von Willebrand factor (an endothelial marker of injury), homocysteine (an inductor of endothelial injury), D-dimer (a marker of coagulation cascade activation) and plasminogen activator inhibitor-1 (a fibrinolysis marker) were measured in individuals with both type 2 diabetes and high blood pressure, with type 2 diabetes, with high blood pressure and in healthy control individuals. No significant differences among groups were observed for von Willebrand factor and homocysteine plasma levels. The type 2 diabetes and high blood pressure group presented a significant difference to the other groups for D-dimer and also presented high values for plasminogen activator inhibitor-1. The high blood pressure group and type 2 diabetes group presented separately higher values of plasminogen activator inhibitor-1 compared with the control group. High levels of D-dimer and plasminogen activator inhibitor-1 in patients with type 2 diabetes and high blood pressure with normoalbuminuria therefore indicate a state of hypercoagulability and hypofibrinolysis, despite no evident microvascular injury supported by normal levels of von Willebrand factor and homocysteine.     

Increased thrombin levels during thrombolytic therapy in acute myocardial infarction. Relevance for the success of therapy

BACKGROUND. It has been suggested that thrombolysis in a feedback reaction may generate pro-coagulant activities. METHODS AND RESULTS. Fifty-five patients were treated with urokinase-preactivated prourokinase (n = 35) or tissue-type plasminogen activator (n = 20) for acute myocardial infarction and underwent coronary angiography at 90 minutes and at 24-36 hours into thrombolysis, and fibrinogen (Ratnoff-Menzie), D-dimer (ELISA) and thrombin-antithrombin III complex levels (ELISA) were measured. Primary patency was achieved in 39 patients (70.9%), 13 of whom (33.3%) suffered early reocclusion. Nonsignificant decreases in fibrinogen levels were observed while D-dimer levels increased +3,008 +/- 4,047 micrograms/l (p less than 0.01), differences not being significant in respect to the thrombolytic agents or to the clinical course. In contrast, while thrombin-antithrombin III complex levels decreased -4.4 +/- 13.0 micrograms/l in patients with persistent patency, they increased +7.5 +/- 13.6 micrograms/l in case of nonsuccessful thrombolysis (p less than 0.02) and +11.9 +/- 23.8 micrograms/l in case of early reocclusion (p less than 0.001). For patients with thrombin-antithrombin III complex levels greater than 6 ng/l 120 minutes into thrombolysis, the unfavorable clinical course was predicted with 96.2% sensitivity and 93.1% specificity. CONCLUSION. Generation of thrombin, occurring during thrombolysis, is a major determinant for the success of therapy and thrombin-antithrombin III levels may serve as predictors for the short-term prognosis.     

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.     

FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line.

We studied the effect of fibrinogen degradation products D, E, and D-dimer on a human promonocytic leukemia cell line, NOMO-1. After exposure to a 10(-5)-mol/L fragment D or D-dimer, the cells displayed macrophage-like characteristics, such as adherence to plastic surfaces, and showed approximately a twofold increase in response to the nitroblue tetrazolium reduction test. The secretion of interleukin-1 alpha (IL-1 alpha) into the medium was markedly stimulated by a 10(-5)-mol/L fragment D, E, and D-dimer, whereas a significant increase in IL-1 beta secretion was observed only in D-dimer-stimulated cells. In addition, D-dimer induced a rapid increase in urokinase-type plasminogen activator on day 1 (0.52 +/- 0.02 ng/mL v 0.07 +/- 0.01 ng/mL in the control culture) and a slow increase in plasminogen activator inhibitor-2 on day 5 (3.9 +/- 1.6 ng/mL v 1.2 +/- 0.2 ng/mL in the control culture). An increase in tissue factor (TF) was also demonstrated on the cell surface of NOMO-1 cells exposed to fragment D or D-dimer by indirect immunofluorescence using an anti-TF monoclonal antibody. Scatchard plot analysis showed that fragment D and D-dimer bound to the NOMO-1 cells with a kd of 3.3 nmol/L and 2.7 nmol/L, respectively. These results suggest that fragment D-dimer specifically stimulates cells of monocyte-macrophage lineage to secrete key substances that regulate blood coagulation, fibrinolysis, and inflammation.     

The intrinsic coagulation activity assay.

A new assay for the contact-phase-mediated generation of thrombin activity has been developed - the intrinsic coagulation activity assay (INCA). Citrated plasma (50 microl) is incubated with 5 microl SiO2, 250 mmol/l CaCl2 in polystyrole flat-bottom wells. After exactly 4 and 5 min (37 degrees C) coagulation reaction times (INCA-4 and INCA-5), 100 microl of 2.5 mol/l arginine, pH 8.6, is added to inhibit hemostasis activation in the important ascending part of the thrombin generation curve and to depolymerize fibrin. After 20 min, 50 microl of 1 mmol/l (final concentration 0.24 mmol/l) chromogenic thrombin substrate CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, is added. The increase in absorbance is determined at 405 nm using a microtiterplate photometer. The assay is calibrated against 1 IU/ml thrombin. The normal thrombin activity range of INCA-4 (main value) or INCA-5 (control value) is 100 +/- 30% of normal (mean value +/- 1 SD; 100% = 0.5 IU/ml for INCA-4 and 1.9 IU/ml for INCA-5). With the INCA the normal range of intrinsic hemostasis is reflected, low-molecular-weight heparins can be monitored, the plasma matrix is not changed significantly, and the assay results are a percentage of normal generated thrombin activity and not coagulation seconds.