BACKGROUND

Growth-differentiation factor-<em>1</em>5 (GDF-<em>1</em>5) has recently emerged as a risk predictor in patients with cardiac diseases. GDF-<em>1</em>5 is commonly related to cardiovascular risk factors, inflammatory activity and cardiac abnormalities. However, it is not clear whether it might be an indicator of vascular pathologies as well.

METHODS

Circulating levels of GDF-<em>1</em>5 were measured in <em>1</em>004 elderly community dwellers participating in the PIVUS study. The relations of GDF-<em>1</em>5 to biomarkers of endothelial activation (E-selectin, P-selectin, ICAM-<em>1</em>, VCAM-<em>1</em>), extracellular matrix degradation (MMP-9, TIMP-<em>1</em>), coagulatory activity (D-dimer, von Willebrand factor, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, factor VIIa), and fibrinolytic activity (PAI-<em>1</em> activity, tPA-antigen) were assessed by multiple linear regressions.

RESULTS

The median GDF-<em>1</em>5 level was <em>1</em><em>1</em>35 ng/L. By linear correlation analysis, GDF-<em>1</em>5 exhibited a moderate relation to von Willebrand factor (r = 0.30), and weak, albeit significant relations (r = 0.<em>1</em>3-0.<em>2</em>9) to E-selectin, P-selectin, ICAM-<em>1</em>, VCAM-<em>1</em>, MMP-9, TIMP-<em>1</em>, D-dimer, PAI-<em>1</em> activity and tPA-antigen. The relations to the assessed biomarkers of endothelial activation, TIMP-<em>1</em>, D-dimer and von Willebrand factor remained significant applying multiple linear regression models adjusted for clinical covariates and echocardiographic data. There were no significant relations between GDF-<em>1</em>5 and biomarkers solely reflecting coagulatory activity.

CONCLUSIONS

In the elderly, GDF-<em>1</em>5 reflects endothelial activation and vascular inflammation and thus, multiple pathways involved in the development and progression of atherosclerosis.

After a first episode of pulmonary embolism (PE), two major problems need to be considered: risk of recurrence when anticoagulation is stopped, and risk of chronic thromboembolic pulmonary hypertension (CTPH). We followed prospectively consecutive patients who survived a first episode of PE, with or without deep vein thrombosis, to assess the incidence of venous thromboembolism (VTE) recurrences and of symptomatic and asymptomatic CTPH. After 3-6 months of oral anticoagulant therapy (OAT) patients underwent transthoracic echocardiography for measuring transtricuspid (rV-rA) gradient. When rV-rA gradient was >35 mmHg further evaluations were performed to rule in or out CTPH. During follow-up patients who developed persistent dyspnea were re-evaluated. In patients who underwent OAT withdrawal D-dimer (DD), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), and thrombophilia were evaluated one month after warfarin discontinuation. Overall, <em>2</em>39 patients, <em>1</em><em>1</em>8 males, median age 59(<em>1</em>6-89) years, were followed up for a median time of 36(9-<em>1</em>9<em>2</em>) months. Nine patients had rV-rA gradient >30 mmHg and ≤35 mmHg, and one of 37 mmHg. Among patients with normal rV-rA gradient, one developed persistent dyspnea 55 months after the first event and CPTH was confirmed. Among <em>2</em>06 patients who stopped OAT, <em>2</em>3(<em>1</em><em>1</em>.<em>2</em>%) had VTE recurrence, <em>1</em><em>1</em> PE(48%). Elevated DD and F<em>1</em> + <em>2</em> levels after stopping OAT were significantly associated with recurrence. None of patients with recurrent VTE had elevated rV-rA gradient. In our series the incidence of CTPH after a first episode of PE was 0.4%. VTE recurrence and elevated DD and F<em>1</em> + <em>2</em> levels seemed not to be related to the development of CTPH.

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for <em>prothrombin</em> are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for <em>prothrombin</em> and for the prethrombin <em>1</em> <em>fragment</em> of <em>prothrombin</em> at <em>2</em>3.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.<em>1</em> M NaCl, 0.<em>1</em> mM benzamidine, and either <em>2</em> mM Ca<em>2</em>+ or <em>2</em> mM ethylenediaminetetraacetate (EDTA). Under these conditions a <em>1</em>:<em>1</em> complex of Vh with <em>prothrombin</em> is formed that is governed by a dissociation constant (Kd) of <em>1</em>0 microM, regardless of whether the buffer contains Ca<em>2</em>+ or EDTA. An identical Kd is observed when prethrombin <em>1</em> is substituted for <em>prothrombin</em>. This indicates that the <em>fragment</em> <em>1</em> portion of <em>prothrombin</em>, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin <em>1</em> for the bovine molecule also results in a <em>1</em>:<em>1</em> Vh-prethrombin <em>1</em> complex governed by a slightly weaker Kd (<em>2</em>7 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin <em>1</em> (Kd greater than <em>1</em> mM), without detectable change in the mass of the Vh.

The purpose of this study was to determine whether chronic thyroid hormone suppression therapy (THST) is prothrombotic. We obtained blood samples from <em>1</em>4 thyroid cancer patients while on THST and after they had become hypothyroid for radioiodine whole-body scanning and therapy. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, fibrinogen, factor VIII, antithrombin, tissue plasminogen activator antigen (tPA), plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>), PAI-<em>1</em>/tPA, and C-reactive protein were significantly (P < 0.05) higher in the hyper- than in the hypothyroid state, whereas protein C and plasmin-antiplasmin complexes were significantly lower during the hyperthyroid period. When the <em>1</em>0 female patients were hyperthyroid, their levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, fibrinogen, protein S, antithrombin, tPA, PAI-<em>1</em>, and PAI-<em>1</em>/tPA were significantly higher (P </= 0.05) than in healthy female controls, whereas when the female patients were hypothyroid, their antithrombin and plasmin-antiplasmin were lower and their protein S was higher than in controls. Factor II, plasminogen, and D-dimer were not significantly affected by the thyroid status in either assessment. In conclusion, we found evidence that the majority of patients treated with THST have a prothrombotic profile.

Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and <em>prothrombin</em>. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH<em>2</em>Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH<em>2</em>Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of <em>1</em><em>2</em>5I-<em>prothrombin</em> to <em>1</em><em>2</em>5I-<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and <em>1</em><em>2</em>5I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of <em>prothrombin</em> activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH<em>2</em>Cl also completely inhibited the intrinsic-pathway activation of <em>prothrombin</em> in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH<em>2</em>Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH<em>2</em>Cl. The inhibition of the activation of <em>prothrombin</em> by the three agents was also abolished with longer times with re-added Ca<em>2</em>+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of <em>1</em><em>2</em>5I-thrombin-antithrombin III and <em>1</em><em>2</em>5I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant effects of heparin and pentosan polysulphate are mediated primarily by their ability to inhibit the thrombin-dependent activation of Factor V, thereby inhibiting the formation of <em>prothrombin</em>ase complex, the physiological activator of <em>prothrombin</em>.

OBJECTIVE

In acute myocardial infarction (AMI), proinflammatory plasma C-reactive protein values are strongly associated with postinfarction morbidity and mortality. So far, the cause of these inflammatory changes is not well understood. Therefore, we sought to investigate the relationship between the activation of coagulation and subsequent systemic inflammatory changes in AMI.

RESULTS

Factor Xa (FXa) bound to tissue factor pathway inhibitor and <em>prothrombin</em> <em>fragments</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) were used as a measure for activated coagulation. To assess systemic inflammatory changes, plasma interleukin (IL)-6 and IL-8 concentrations were analyzed by immunoassay. Blood samples were taken from <em>2</em><em>1</em> patients with AMI and <em>2</em>0 patients with stable angina pectoris. In AMI, tissue factor pathway inhibitor FXa but not F<em>1</em>+<em>2</em> plasma levels were associated with circulating IL-8 (P=0.0<em>1</em>). In vitro experiments revealed that FXa stimulated IL-8 and monocyte chemoattractant protein-<em>1</em> release and RNA expression in endothelial cells and mononuclear leukocytes by activation of protease-activated receptor-<em>1</em>.

CONCLUSIONS

Our data suggest that coagulation FXa may contribute to proinflammatory changes in AMI by stimulation of IL-8 release. Therapeutic inhibition of the proinflammatory effects of FXa may improve the clinical course in AMI. This study investigates the relationship between the activation of coagulation and systemic inflammatory changes in acute myocardial infarction. Tissue factor pathway inhibitor factor Xa but not F<em>1</em>+<em>2</em> plasma levels were associated with circulating interleukin-8. In vitro factor Xa stimulated interleukin-8 and monocyte chemoattractant protein-<em>1</em> release and RNA expression by activation of protease-activated receptor <em>1</em> as an underlying mechanism.

Orthostatic stress causes significant plasma shift and raises transmural pressure in lower extremities, resulting in an increase in endothelial activation and plasma proteins concentrations, possibly including coagulation factors. This may lead to activation of the coagulation system during standing. To test this hypothesis, we recruited <em>1</em>8 healthy volunteers (9 females and 9 males; mean age: <em>2</em>5+/-<em>1</em>.<em>2</em> years; body mass index: <em>2</em><em>1</em>.7+/-0.5 kg/m(<em>2</em>)). Hemodynamics, plasma shift (extrapolated from sequential hematocrit concentration), plasma proteins, and coagulation tests, including procoagulants; fibrinogen, factor V, and factor VIII activity; <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>; and endothelial activation-related factors (tissue factor and von Willebrand factor), as well as protein C global pathway, were determined at rest supine and at <em>1</em>5 minutes, 30 minutes, and 60 minutes of still standing. Thirty minutes of standing caused a decrease in plasma volume by <em>1</em><em>2</em>.0+/-0.5% and an increase in plasma protein by <em>1</em>3.0+/-0.7%. Fibrinogen, factor V, and factor VIII activity rose by <em>1</em><em>2</em>.0+/-<em>1</em>.<em>2</em>%, <em>1</em>3.0+/-<em>1</em>.0%, and 40.0+/-6.0% (P<0.00<em>2</em> for all), respectively. <em>Prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> were elevated by <em>1</em>50.0+/-30.0%. Tissue factor and von Willebrand factor increased by 30.0+/-9.0% and <em>1</em>7.4+/-5<em>1</em>.0% (P<0.0<em>2</em> for both), respectively. However, protein C assay results decreased from 0.95+/-0.<em>2</em>0 to 0.83+/-0.<em>1</em>6 (P<0.00<em>1</em>). We hereby introduce a novel physiological mechanism, "orthostatic procoagulation," that should be considered during coagulation tests. Furthermore, it could be extrapolated to the pathophysiology of stasis and venous thromboembolism.

Postoperative complications associated with cardiopulmonary bypass (CPB) surgery and extracorporeal circulation (ECC) procedures are still a major clinical issue. Improving the hemocompatibility of blood contacting devices used for ECC procedures may ameliorate various postpump syndromes. In a simulated CPB model using human blood, we investigated the hemocompatibility, fibrinogen adsorption, and platelet receptor (GPIIb-IIIa) binding capacity of surface-modified membrane oxygenators (Jostra Quadrox). Three groups were compared: (i) biopassive protein coatings (SafeLine), (ii) bioactive heparin coatings (BioLine), and (iii) noncoated controls. During the <em>2</em> h recirculation period, plasma concentrations of activation markers for platelets (beta-thromboglobulin), inflammation (elastase), complement (C5a), and coagulation (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin III) were lower in the groups with biopassive and bioactive coatings compared to the noncoated group (p < 0.0<em>1</em>). These parameters did not significantly differ between the two surface-coated groups, except for complement activation: C5a levels were higher in the biopassive group compared to the bioactive group (p < 0.0<em>1</em>). Moreover, surface-coated oxygenators showed less fibrinogen adsorption, GPIIb-IIIa binding, and platelet/leukocyte adhesion (p < 0.0<em>1</em>). We assume that fewer fibrinogen and platelet receptor molecules bound to the surface-coated oxygenator surfaces results in fewer platelet adhesion and activation, which will significantly contribute to the improved hemocompatibility of the biopassive and bioactive oxygenators. Our results suggest that the application of bioactive oxygenators (BioLine) during CPB surgery may reduce postoperative complications for the patient more effectively than biopassive oxygenators (SafeLine).

OBJECTIVE

To determine whether pretreatment markers of coagulation and fibrinolysis are related to recanalization and functional outcome.

METHODS

The authors included patients treated with IV rt-PA with occlusion on baseline transcranial Doppler (Thrombolysis in Brain Ischemia [TIBI] criteria) in whom recanalization within 6 hours was monitored. At baseline, the authors recorded data about demographics, vascular risk factors, the NIH Stroke Scale (NIHSS) score, early CT signs, etiology, blood glucose, and time to rt-PA. The authors also measured plasmatic markers of coagulation (fibrinogen, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, Factor XIII, Factor VII) and fibrinolysis (alpha<em>2</em>-antiplasmin, Plasminogen Activator Inhibitor, Functional Thrombin Activatable Fibrinolysis Inhibitor [fTAFI]). A favorable outcome was defined as a modified Rankin score < <em>2</em> at 3 months.

RESULTS

The authors studied 63 patients with a mean age of 67.3 +/- <em>1</em><em>2</em>.5 years. The median NIHSS score was <em>1</em>6. Patients who recanalized had lower concentrations of alpha<em>2</em>-antiplasmin (87.5 +/- <em>1</em>8% vs 96.5 +/- <em>1</em><em>2</em>.5%, p = 0.0<em>2</em>3) and fTAFI (9<em>1</em>.7 +/- <em>2</em>6.7% vs <em>1</em>04.4 +/- <em>2</em><em>1</em>%, p = 0.039). A multivariant logistic regression analysis showed that the level of alpha<em>2</em>-antiplasmin was the only predictive variable of recanalization (OR 0.95, 95% CI 0.9<em>1</em>, 0.99, p = 0.038), while the NIHSS score was the only predictive variable of functional outcome (OR 0.8<em>1</em>, 95% CI 0.7<em>2</em>, 0.9<em>2</em>, p = 0.00<em>1</em>).

CONCLUSIONS

Baseline levels of alpha<em>2</em>-antiplasmin were predictive of recanalization but were not related to the long-term outcome in patients treated with rt-PA within the first 3 hours.

After an episode of unstable angina or myocardial infarction, a high proportion of patients show biochemical signs of coagulation activation, expressed as persistently elevated thrombin generation, in their blood. It is not known whether this has any influence on long-term outcome. In this prospective multicenter cohort study, we assessed the relation of persistently elevated thrombin generation to outcome in 3<em>1</em>9 consecutive patients with acute coronary syndromes enrolled in the Global Use of Strategies To Open occluded coronary arteries (GUSTO) IIb trial. Plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels, an index of "in vivo" thrombin generation, was measured during the acute phase and after <em>1</em>, 6, and <em>1</em><em>2</em> months, and its relation to outcome was assessed during a median <em>2</em>9-month follow-up period. The primary end point of cardiac death or myocardial (re)infarction occurred in 6<em>1</em> patients (<em>1</em>9%). There was a U-shaped relationship between plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels and the risk of developing the primary end point; intermediate levels (<em>1</em>.5-<em>1</em>.9 nM) were associated with the lowest risk, whereas both higher >> <em>1</em>.9 nM) and lower (< <em>1</em>.5 nM) values were associated with an increased risk (relative risk [RR] <em>1</em>.56 and 95% confidence interval [CI], <em>1</em>.<em>2</em>5-<em>2</em>.<em>2</em>8; RR, <em>1</em>.35 and 95% CI, <em>1</em>.<em>1</em><em>1</em>-<em>1</em>.86, respectively). After an episode of acute coronary syndrome, both high and low levels of thrombin generation are predictors of an increased risk of an unfavorable outcome.

The single nucleotide polymorphism (SNP) Ser<em>1</em><em>2</em>8Arg in the E-selectin gene is overrepresented in certain patient groups with atherosclerosis or restenosis. We hypothesized and tested whether it may affect cytokine-induced levels of soluble (s) E-selectin, or be associated with proinflammatory or procoagulant properties in a well-standardized inflammation model. Healthy male volunteers (n = <em>1</em>57) received a lipopolysaccharide (LPS) infusion and were genotyped for the S<em>1</em><em>2</em>8R SNP, and outcome parameters were measured by enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). The S<em>1</em><em>2</em>8R SNP had no pronounced effects on basal or inducible sE-selectin levels, or levels of tumor necrosis factor or interleukin-6. However, carriers of the S<em>1</em><em>2</em>8R SNP had <em>2</em>0% higher monocyte counts at <em>2</em>4 hours after LPS infusion. Importantly, the S<em>1</em><em>2</em>8R allele enhanced thrombin generation by 50% to 80%, as measured by <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) (P < .0<em>1</em>), and hence fibrin formation (D-dimer) <em>2</em>-fold (P = .0<em>1</em> to P = .00<em>2</em>). However, tissue factor (TF) mRNA levels were not affected. The S<em>1</em><em>2</em>8R E-selectin genotype is associated with procoagulant effects in a human model of endotoxin-induced, TF-triggered coagulation. This could contribute to its linkage with various thrombotic cardiovascular disorders.

Although exosites <em>1</em> and <em>2</em> regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite <em>1</em> with exosite <em>2</em> ligands, HD<em>2</em><em>2</em> (a DNA aptamer), gamma'-peptide (an analog of the COOH terminus of the gamma'-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite <em>2</em> ligands to inhibit thrombin binding to gamma(A)/gamma(A)-fibrin, an interaction mediated solely by exosite <em>1</em>. When gamma(A)/gamma(A)-fibrinogen was clotted with thrombin in the presence of HD<em>2</em><em>2</em>, gamma'-peptide, or <em>prothrombin</em> <em>fragment</em> <em>2</em> there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD<em>2</em><em>2</em> reduced the affinity of thrombin for gamma(A)/gamma(A)-fibrin 6-fold and accelerated the dissociation of thrombin from preformed gamma(A)/gamma(A)-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with gamma(A)/gamma(A)-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite <em>1</em> ligands, HD<em>1</em> (a DNA aptamer) or hirudin-(54-65) (an analog of the COOH terminus of hirudin), inhibited the exosite <em>2</em>-mediated interaction of thrombin with immobilized gamma'-peptide. These findings provide evidence for long range allosteric linkage between exosites <em>1</em> and <em>2</em> on thrombin, revealing further complexity to the mechanisms of thrombin regulation.

This study was planned for searching possible changes of the total coagulation and fibrinolysis system in inflammatory bowel disease (IBD) in order to obtain some clues for explaining the relation between IBD and hypercoagulability. A total of <em>2</em>4 patients with ulcerative colitis, <em>1</em><em>2</em> patients with Crohn disease, and <em>2</em>0 healthy controls were studied. Platelets; <em>prothrombin</em> time (PT); partial thromboplastin time (PTT); fibrinogen; D-dimer; fibrinogen degradation products; protein C; protein S; antithrombin; thrombin time; von Willebrand factor; coagulation factors V, VII, VIII, IX, XI, and XIII; plasminogen; antiplasmin; tissue plasminogen activator; plasminogen activator inhibitor <em>1</em>; and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> were studied. Most of the procoagulants (platelets, fibrinogen, von Willebrand factor, coagulation factor IX, and plasminogen activator inhibitor <em>1</em>) were found increased together with decreases in some anticoagulants (protein S and antithrombin) in IBD. Also the activation markers of coagulation (D-dimer, fibrinogen degradation products, and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>) were all increased. The parameters of the total coagulation-fibrinolysis system were increased in IBD, regardless of the form and the activity of the disease.

BACKGROUND

There is an increased risk of venous thrombosis after air travel, but the underlying mechanism is unclear. Our aim was to ascertain whether flying leads to a hypercoagulable state.

METHODS

We did a crossover study in 7<em>1</em> healthy volunteers (<em>1</em>5 men, 56 women), in whom we measured markers of activation of coagulation and fibrinolysis before, during, and after an 8-h flight. The same individuals participated in two control exposure situations (8-h movie marathon and daily life) to separate the effect of air travel on the coagulation system from those of immobilisation and circadian rhythm. To study the effect of risk factors for thrombosis, we included participants with the factor V Leiden mutation (n=<em>1</em><em>1</em>), those who took oral contraceptives (n=<em>1</em>5), or both (n=<em>1</em>5), as well as 30 individuals with no specific risk factors.

RESULTS

After the flight, median concentrations of thrombin-antithrombin (TAT) complex increased by 30.<em>1</em>% (95% CI <em>1</em><em>1</em>.<em>2</em>-63.<em>2</em>), but decreased by <em>2</em>.<em>1</em>% (-<em>1</em><em>1</em>.<em>2</em> to <em>1</em>4) after the cinema and by 7.9% (-<em>1</em>6.<em>2</em> to -<em>1</em>.<em>2</em>) after the daily life situation. We recorded a high response in TAT levels in <em>1</em>7% (<em>1</em><em>1</em> of 66) of individuals after air travel (3% [<em>2</em> of 68] for movie marathon; <em>1</em>% [<em>1</em> of 70] in daily life). These findings were most evident in the group with the factor V Leiden mutation who used oral contraceptives. We noted a high response in all variables (<em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em>, TAT, and D-dimer) in four of 63 (6.3%) volunteers after the flight, but in no-one after either of the control situations.

CONCLUSIONS

Activation of coagulation occurs in some individuals after an 8-h flight, indicating an additional mechanism to immobilisation underlying air travel related thrombosis.

Among 93 consecutive kidney-transplant patients who received prophylactic OKT3 <em>1</em>0 mg/day for <em>2</em> weeks, 9 had intragraft thromboses within <em>2</em> weeks of transplantation. The thromboses were in graft artery in <em>1</em> patient and veins in 3. The other 5 had thromboses in glomerular capillaries and thrombotic microangiopathy similar to that of haemolytic-uraemic syndrome. All attempted treatments failed, and the 9 grafts had to be removed. The finding that plasma concentrations of <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> were higher 4 h after the first OKT3 dose in OKT3 recipients than in transplant patients who received other prophylaxis (mean 5.88 [SEM 0.76] vs <em>2</em>.<em>2</em>5 [0.59] nmol/l, p less than 0.0<em>1</em>) confirms that OKT3 has procoagulant effects in vivo.

Pregnancy is considered as a hypercoagulable state and an increased incidence of thromboembolic phenomena has been reported in pregnant women. Relevant changes in the hemostatic mechanism have been reported during physiological pregnancy: briefly, increased levels of coagulation factors, enhanced thrombin generation and suppression of fibrinolysis are commonly found in women with uncomplicated pregnancy. We recently described progressive increases in fibrinogen and D-dimer plasma levels during normal pregnancy. The increase in D-dimer levels makes difficult their interpretation for the exclusion of thromboembolic phenomena in pregnancy. The behavior of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) levels during physiological pregnancy is scarcely known. The aim of this preliminary study was to establish range values of F<em>1</em>+<em>2</em> plasma levels for different periods of normal pregnancy.

Two automated turbidimetric D-dimer assays (BC D-dimer Plus, Dade Behring, Marburg, Germany and Auto-Dimer, Biopool, Umeå, Sweden) were compared to two enzyme-linked immunosorbent assays (ELISAs) (Enzygnost D-dimer micro, Dade Behring and Asserachrome D-dimer, Diagnostica Stago, Asnières, France) and two rapid D-dimer assays (SimpliRed, Agen Biomedical, Brisbane, Australia and Minutex, Biopool) in out-patients with suspected deep vein thrombosis (DVT). In addition, the performance of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complex (TAT), and soluble adhesion molecules VCAM-<em>1</em> and P-selectin for DVT diagnosis was assessed. One hundred and thirty-five consecutive out-patients with suspected DVT of the lower limb were included, and in 5<em>2</em> (39%), DVT was confirmed by compression ultrasound. All D-dimer assays investigated reliably excluded DVT in those patients without DVT irrespective of their pre-test clinical probability of DVT. One D-dimer ELISA (Dade Behring) gave the highest area under the receiver operating characteristic (ROC) curve compared to other assays, and therefore, this was the most accurate assay in differentiating patients with from patients without DVT. The diagnostic performance of one automated turbidimetric assay (Auto Dimer, Biopool) was similar to ELISA and its convenience close to rapid latex agglutination assays. Most patients with a high pre-test clinical probability of DVT had positive D-dimer regardless of the presence or absence of DVT, which decreased the specificity of the tests and made D-dimer determination less useful for this group of patients. Because the diagnostic accuracy [sensitivity, specificity, negative (NPV) and positive predictive value (PPV)] of F<em>1</em>+<em>2</em>, TAT, VCAM-<em>1</em> and P-selectin was inferior to D-dimer assay, these assays could not substitute or supplement D-dimer testing in diagnosis of DVT. Levels of VCAM-<em>1</em> and P-selectin were increased in patients with DVT and should therefore be investigated further to clarify their role in DVT.

OBJECTIVE

The impact of infection-associated antiphospholipid antibodies (APA) on endothelial cell activation, blood coagulation and fibrinolysis was evaluated in patients with infective endocarditis with and without major embolic events.

BACKGROUND

An embolic event is a common and severe complication of infective endocarditis. Despite the fact that APAs are known to be associated with infectious diseases, their pathogenic role in infective endocarditis has not been clearly defined.

METHODS

The relationship among the occurrence of major embolic events, echocardiographic vegetation size, endothelial cell activation, thrombin generation, fibrinolysis and APA was examined in 9<em>1</em> patients with definite infective endocarditis, including <em>2</em>6 patients with embolic events and 65 control subjects without embolic events.

RESULTS

Overall, <em>1</em>4.3% of patients exhibited elevated APA levels. Embolic events occurred more frequently in patients with elevated levels of APA than in patients without (6<em>1</em>.5% vs. <em>2</em>3.<em>1</em>%; p = 0.008). Patients with elevated levels of APA showed higher levels of <em>prothrombin</em>-<em>fragment</em> F<em>1</em> +<em>2</em> (p = 0.005), plasminogen-activator inhibitor <em>1</em> (p = 0.000<em>2</em>), von Willebrand factor (p = 0.00<em>2</em>) and lower levels of activated protein C (p = 0.00<em>1</em>) than patients with normal levels of APA. Thrombin generation and endothelial cell activation were both positively correlated with levels of APA. The occurrence of elevated APA levels was frequently associated with structural valve abnormalities (p = 0.0<em>1</em>) and vegetations>><em>1</em>.3 cm (p = 0.00<em>2</em>).

CONCLUSIONS

Infection-associated elevated APA levels in patients with infective endocarditis are related to endothelial cell activation, thrombin generation and impairment of fibrinolysis. This may contribute to the increased risk for major embolic events in these patients.

BACKGROUND

In inflammatory bowel disease (IBD), gut microvascular thrombosis as well as thromboembolic complications have repeatedly been observed. We examined the long-term course of markers of coagulation and fibrinolysis in relation to clinical disease activity.

METHODS

In a prospective study, <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> (F<em>1</em>.<em>2</em>), thrombin-antithrombin complex (TAT), antithrombin, D-dimer, plasmin-alpha(<em>2</em>)-antiplasmin complex (PAP) and plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) were measured in <em>2</em>0 patients with Crohn's disease (CD), <em>1</em>8 with ulcerative colitis (UC), and <em>1</em>9 with giant cell arteritis during active and inactive disease, as well as in 5<em>1</em> controls without inflammation.

RESULTS

Levels of F<em>1</em>.<em>2</em>, TAT, D-dimer, PAP and PAI-<em>1</em> were significantly higher in active versus inactive CD and UC. However, even after <em>1</em><em>2</em> months of follow-up, in CD and UC the mean levels of F<em>1</em>.<em>2</em>, D-dimer and PAP were significantly higher than the levels of the controls.

CONCLUSIONS

Levels of F<em>1</em>.<em>2</em>, D-dimer and PAP were markedly raised for a long time in clinically inactive IBD, underlining a chronic state of hypercoagulation and enhanced fibrinolysis.

BACKGROUND

Although chronic urticaria (CU) is often regarded as autoimmune in nature, only less than 50% of sera from CU patients contain histamine-releasing autoantibodies. This suggests that other factors may contribute to its pathogenesis. We evaluated the possible involvement of vascular endothelial growth factor (VEGF), one of the major mediators of vascular permeability, in CU.

METHODS

Eighty consecutive adult patients with CU and 53 healthy subjects were studied. VEGF and <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) were measured by enzyme immunoassays. Autologous plasma skin test (APST) was performed in CU patients and, in six of them, skin biopsy specimens were taken from wheals to evaluate the immunohistochemical expression of VEGF and eosinophil cationic protein (ECP).

RESULTS

Plasma VEGF concentrations were higher in CU patients (8.00 +/- 0.90 pmol/l) than in controls (0.54 +/- 0.08 pmol/l) (P = 0.000<em>1</em>) and tended to parallel both the severity of CU and to correlate with F(<em>1</em>+<em>2</em>) levels. APST was positive in 85.<em>1</em>% of patients. VEGF concentration was significantly higher in APST-positive than in APST-negative patients (P = 0.0003). Immunohistochemically, all specimens from patients with CU showed a strong expression of VEGF (P = 0.00<em>2</em>) that colocalized with ECP, a classic eosinophil marker.

CONCLUSIONS

VEGF plasma levels are elevated in CU and parallel the disease severity. This supports a possible role of this molecule in CU pathophysiology. Eosinophils are the main cellular source of VEGF in CU lesional skin.

A genetic variation in the <em>prothrombin</em> gene, the G->>A transition at nucleotide <em>2</em>0<em>2</em><em>1</em>0, is a risk factor for venous thrombosis in heterozygotes and is associated with increased <em>prothrombin</em> activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included <em>2</em> homozygous and 5 heterozygous carriers of the <em>2</em>0<em>2</em><em>1</em>0 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in <em>2</em><em>2</em> unrelated carriers of the <em>2</em>0<em>2</em><em>1</em>0 A allele by measuring (<em>1</em>) <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) as an index of ongoing thrombin generation and (<em>2</em>) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F<em>1</em>+<em>2</em> levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the <em>2</em>0<em>2</em><em>1</em>0A allele compared with the controls (5<em>2</em>7.8+/-<em>1</em><em>1</em>4.9 versus 387+/-50.<em>1</em> nmol/L x min, P<0.000<em>1</em>). In the <em>2</em> homozygotes, the ETP was almost twice (639 and 75<em>1</em> nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G<em>2</em>0<em>2</em><em>1</em>0A mutation in the <em>prothrombin</em> gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the <em>2</em>0<em>2</em><em>1</em>0 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.

Malignancy is a risk factor for thromboembolism and anti-cancer chemotherapy can increase this risk. Prophylaxis of thrombosis with very-low-dose warfarin given concurrently with chemotherapy has a significantly reduced rate of thromboembolism in a randomized trial in women with stage IV breast cancer. In a group of 3<em>2</em> patients randomized in one center (<em>1</em>6 subjects on warfarin and <em>1</em>6 on placebo), we have prospectively studied the plasma levels of: <em>1</em>. Markers of 'in vivo' clotting activation (thrombin-antithrombin complex [TAT], <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>] and D-dimer), <em>2</em>. Factor VII (FVII), and 3. Natural anticoagulants (protein C [PC] and antithrombin [AT]). The aims of this study were: <em>1</em>. to examine whether laboratory tests predicted those patients who developed thrombosis, and <em>2</em>. to evaluate the effect of very-low-dose warfarin on hemostatic variables. The patients' hemostatic parameters were evaluated before entry into the study and after starting chemotherapy +/- prophylaxis, before each course for nine courses. Before-treatment results were compared to those of a sex and age-matched non-cancer control group. There was a significant elevation of plasma levels of TAT (p <0.00<em>1</em>), F<em>1</em>+<em>2</em> (p <0.00<em>1</em>), D-dimer (p <0.000<em>1</em>) and FVIIa (p <0.05), as well as an increase of FVII proteolysis (p <0.05), whereas plasma PC and AT concentrations were not different from controls. After starting chemotherapy, markers of clotting activation were progressively lower in the group receiving warfarin prophylaxis compared to the group on placebo. Differences between the groups became statistically significant (p <0.0<em>1</em>) after the 4th course of chemotherapy. Deep vein thrombosis occurred in two patients in the placebo arm. The results of this study indicate that before therapy, an hypercoagulable state is present in stage IV breast cancer, and after starting chemotherapy, abnormalities of hypercoagulation markers persist, however they are reduced by very-low-dose-warfarin. None of the laboratory variables could predict thrombosis in the single patient.

OBJECTIVE

We investigated the safety and pharmacodynamics of escalating doses of recombinant nematode anticoagulant protein c<em>2</em> (rNAPc<em>2</em>) in patients undergoing elective coronary angioplasty.

BACKGROUND

Recombinant NAPc<em>2</em> is a potent inhibitor of the tissue factor/factor VIIa complex, which has the potential to reduce the risk of thrombotic complications in coronary artery disease.

METHODS

In a randomized, double-blinded, dose-escalation, multicenter trial, <em>1</em>54 patients received placebo or rNAPc<em>2</em> at doses of 3.5, 5.0, 7.5, and <em>1</em>0.0 microg/kg body weight as a single subcutaneous administration <em>2</em> to 6 h before angioplasty. All patients received aspirin, unfractionated heparin during angioplasty, and clopidogrel in case of stent implantation.

RESULTS

Minor bleeding rates for the doses 3.5 to 7.5 microg/kg were comparable to that with placebo (6.7%), whereas an incidence of <em>2</em>6.9% was observed at the <em>1</em>0.0 microg/kg dose level (p < 0.0<em>1</em>). Major bleedings occurred in the 5.0 microg/kg (n = 3) and 7.5 microg/kg (n = <em>1</em>) dose groups. The three patients in the 5.0 microg/kg dose group also received a glycoprotein IIb/IIIa receptor inhibitor at the moment of major bleeding. Systemic thrombin generation, as measured by <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F(<em>1</em>+<em>2</em>)), was suppressed in all rNAPc<em>2</em> dose groups to levels below pretreatment values for at least 36 h. In the placebo group, a distinct increase of F(<em>1</em>+<em>2</em>) levels was observed following cessation of heparin.

CONCLUSIONS

Inhibition of the tissue factor/factor VIIa complex with rNAPc<em>2</em>, at doses up to 7.5 microg/kg, in combination with aspirin, clopidogrel, and unfractionated heparin appears to be a safe and effective strategy to prevent thrombin generation during coronary angioplasty.

This study was performed to determine the accuracy of D-Dimer fibrin derivatives, thrombin-antithrombin III (TAT) complexes and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>) determinations for the diagnosis of deep vein thrombosis (DVT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected DVT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal DVT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of DVT was confirmed in 34 and excluded in 8<em>2</em>. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the D-Dimer was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of DVT was the D-Dimer ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The D-Dimer latex, TAT complexes and F <em>1</em> + <em>2</em> were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of the 3 markers, their association did not improve their overall accuracy for detecting DVT. Therefore, with the exception of the D-Dimer ELISA, these markers were of little value for the diagnosis of DVT in this specific population.