Thromboembolic complications are often seen in patients with nephrotic syndrome. Markers of endothelial cell injury [thrombomodulin, intracellular adhesion molecule, vascular cell adhesion molecule, thrombin activatable fibrinolysis inhibitor (TAFI), protein Z, vascular endothelial growth factor, markers of thrombin and plasmin generation] were studied in <em>2</em><em>2</em> patients with nephrotic syndrome. All these parameters studied, except protein Z and D-dimers, were significantly higher in patients with nephrotic syndrome, whereas protein Z was significantly lower when compared with the healthy volunteers. None of the endothelial cell markers (thrombomodulin, P-selectin, E-selectin, intracellular adhesion molecule, vascular cell adhesion molecule), thrombin and plasmin generation markers (thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, plasmin-antiplasmin complexes, D-dimers), protein C, protein Z, vascular endothelial growth factor, and TAFI concentration and activity were directly correlated with the level of proteinuria, albumin, cholesterol, triglycerides or creatinine, except significant positive correlations between TAFI activity and serum creatinine, E-selectin and albumin as well as negative correlations between plasmin-antiplasmin complexes and proteinuria. In these patients, there is evidence of endothelial cell injury and probably secondary activation of the coagulation cascade. Elevated circulating TAFI antigen and activity might be a new link in the pathogenesis of impaired fibrinolysis and the progression of atherosclerosis in nephrotic syndrome. Protein Z deficiency might also contribute to the enhanced risk of thromboembolic complications in nephrotic syndrome.

OBJECTIVE

Recent studies suggest perivascular spaces are a marker of small vessel disease, blood-brain barrier permeability, and inflammation, but little is known about their risk factors and associations with peripheral blood markers.

METHODS

In prospectively recruited patients with recent minor ischemic stroke, we investigated the influence of age, sex, hypertension, diabetes, and smoking on the severity of perivascular spaces in the basal ganglia seen on T<em>2</em>-weighted magnetic resonance imaging. We assessed plasma markers of endothelial function (von Willebrand factor, intracellular adhesion molecule-<em>1</em>), inflammation (interleukin-6, tumor necrosis factor-alpha, C-reactive protein), and thrombosis (fibrinogen, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, thrombin-antithrombin complex, tissue plasminogen activator, D-dimer). We used a validated semi-automated method to measure basal ganglia perivascular spaces count and volume. We tested uni- and multivariable associations between blood markers and basal ganglia perivascular spaces count and volume.

RESULTS

In <em>1</em>00 patients (median age: 67 years, range: 37-9<em>2</em>), on adjusted analysis, basal ganglia perivascular spaces count was associated with age (r = .<em>1</em><em>1</em>7, P = .003) and hypertension (r = <em>2</em>.<em>2</em><em>2</em>5, P = .0<em>1</em>3). On multivariable linear regression, adjusted for age, sex, hypertension, smoking and diabetes, reduced von Willebrand factor was associated with increased basal ganglia perivascular spaces count (r = -.0<em>2</em>5, P = .03<em>2</em>).

CONCLUSIONS

The association of increased basal ganglia perivascular spaces count with reduced von Willebrand factor is novel. As von Willebrand factor may promote cerebral endothelial integrity, insufficient von Willebrand factor is consistent with dysfunctional cerebral endothelium and increased basal ganglia perivascular spaces in cerebral small vessel disease. Quantitative perivascular spaces measurement may increase sensitivity to detect cerebral endothelial dysfunction.

Surgery produces a rapid rise in interleukin 6 (IL-6) which may increase the risk of deep vein thrombosis and medical complications. Perioperative corticosteroids suppress IL-6 release in patients undergoing total knee arthroplasty. This study evaluates the effects of a perioperative corticosteroid regimen on IL-6 formation, thrombogenesis, fibrinolysis, and clinical outcomes in patients undergoing unilateral, uncemented, total hip arthroplasty.

Twenty-seven patients (14 placebo and 13 study) were enrolled in this randomized, double-blind, placebo-controlled trial. The study group received <em>2</em>0 mg of prednisone orally followed by <em>2</em> doses of intravenous hydrocortisone, each 8 hours apart. Blood was drawn at several time points for IL-6, <em>prothrombin</em> <em>fragment</em> 1.<em>2</em>, and plasmin-alpha-<em>2</em>-antiplasmin complex, a marker of fibrinolysis. In-hospital visual analog pain (visual analog scale) scores, patient-controlled analgesia use, and ability to climb stairs were recorded.

Mean serum IL-6 levels at 6 and <em>2</em>4 hours postoperatively were significantly lower for the study group, whereas serum <em>prothrombin</em> <em>fragment</em> 1.<em>2</em> and plasmin-alpha-<em>2</em>-antiplasmin were not statistically different at any study time point. Average pain scores were similar (P>> .05), but study group experience less severe pain (P < .01) and less patient-controlled analgesia (P = .0<em>2</em>). At 3 months, 4 patients in the placebo and 1 patient in the study group had difficulty going up and down staircases (P = .08).

The use of corticosteroids was associated with a statistically significant decrease in IL-6 at 6 and <em>2</em>4 hours postoperatively but did not affect thrombogenic markers. The study group had improved postoperative analgesia and a trend toward improved functional outcome at 3 months postoperatively.

Atrial fibrillation (AF) is a common cardiac arrhythmia with a 5-<em>2</em>0% annual risk of stroke. Warfarin reduces this risk by at least 60%. Despite adequate anticoagulation within the target International Normalized Ratio (INR) range of <em>2</em>.0-3.0, some patients still experience thrombotic and bleeding events. It is now possible to assess the intensity of anticoagulation with automated thrombin generation (TG) tests, such as the calibrated automated thrombogram (CAT). These tests were compared and an inverse relationship was found between the INR and CAT in <em>1</em>43 elderly AF patients. There was equally good correlation between the concentration of factors II, VII, IX and X and the INR and TG parameters. The peak thrombin was most strongly associated with the concentration of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> in plasma. There was wide variability in TG parameters in patients with identical INR values, sometimes up to a fourfold difference. This TG variability in individuals with the same INR is not due to inflammation, at least when the latter is measured as the concentration of factor VIII coagulant activity, von Willebrand factor antigen, high sensitivity C-reactive protein and fibrinogen. It was concluded that, although the TG and INR were closely correlated there was wide variability in peak thrombin and endogenous thrombin potential in patients within the INR therapeutic range, the cause of which remains unclear.

We have isolated five monoclonal IgG anti-beta <em>2</em>-glycoprotein-<em>1</em> (anti-beta <em>2</em>G-<em>1</em>) and anti-<em>prothrombin</em> Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three beta <em>2</em>GP-<em>1</em>-enriched mAbs (B<em>1</em>4, B<em>2</em><em>2</em>, B<em>2</em>7) reacted with beta <em>2</em>GP-<em>1</em> while both <em>prothrombin</em>-isolated mAbs (P<em>1</em><em>1</em> and P<em>1</em>3) reacted with <em>prothrombin</em>. Intriguingly, mAb P<em>1</em><em>1</em> reacted with beta <em>2</em>GP-<em>1</em> and <em>prothrombin</em> and showed comparable binding affinity to both Ags, with Kd values of <em>1</em>.6 x <em>1</em>0-6 M for beta <em>2</em>GP-<em>1</em> vs 3.<em>2</em> x <em>1</em>0-6 M for <em>prothrombin</em>. This clone may thus, define a hitherto unknown shared epitope between beta <em>2</em>GP-<em>1</em> and <em>prothrombin</em>. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B<em>2</em><em>2</em>, B<em>2</em>7, and P<em>1</em>3). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk<em>1</em> L chain in mAb P<em>1</em>3 resulted in the loss of binding to beta <em>2</em>GP-<em>1</em> and specific reactivity to <em>prothrombin</em>. Together, these data suggest that while the VH6-D-J chain may be important in the binding to beta <em>2</em>GP-<em>1</em>, pairing with certain L chains may influence this binding. These data are the first human IgG anti-beta <em>2</em>GP-<em>1</em> and anti-<em>prothrombin</em> sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients.

The standard modality of administration of rFVIIa to patients with FVIII and FIX inhibitors is the intermittent infusion every <em>2</em> to 6 hours. No untoward local or systemic effects have been reported; laboratory data of activation of coagulation were reported in the presence of coexistent problems (sepsis, septic shock) or with high doses. We treated four patients with FVIII inhibitor with rFVIIa administered by continuous infusion by a central vein catheter, monitoring the signs of systemic activation of the hemostatic system. The F(<em>1</em>+<em>2</em>) <em>prothrombin</em> <em>fragments</em> and the D-dimer increased after the bolus, and remained above the baseline values throughout the treatment period. These variations observed during the infusion period were not accompanied by clinical events.

Factor VIIa (FVIIa) and thrombin generation occur in patients suffering an acute coronary event. We studied the effect of treatment with enoxaparin on FVIIa and <em>prothrombin</em> activation in patients with unstable angina. Anti-Xa activity, FVIIa, FVII coagulant activity (FVII:C) and FVII antigen (FVII:Ag), free tissue factor pathway inhibitor (TFPI), and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>) were measured in patients' plasma, over a <em>2</em>4-h treatment period with enoxaparin. All <em>1</em>4 patients recruited in the study (mean age 68 years) were treated with a subcutaneous injection of enoxaparin, <em>1</em> mg/kg twice daily. Blood was drawn just before, and at different time intervals after, the first injection. Before enoxaparin administration, the levels of FVIIa (4.0<em>2</em> +/- 0.8 ng/ml) and F<em>1</em>+<em>2</em> (<em>2</em>.68 +/- 0.<em>2</em> nmol/l) were significantly increased as compared with control subjects (<em>2</em>.3 +/- 0.3 ng/ml and 0.9 +/- 0.<em>1</em> nmol/l respectively, P < 0.05). Free TFPI, FVII:C and FVII:Ag were within normal ranges. One hour after the first injection of enoxaparin, FVIIa and F<em>1</em>+<em>2</em> levels decreased by 65% and 50%, respectively, and no significant fluctuations were noted throughout the observation period. The concentrations of FVII:C and FVII:Ag were not modified as compared with baseline values. After each injection, the peak concentrations of free TFPI and anti-Xa activity were observed at <em>2</em> and 4 h respectively. The kinetics of FVIIa and F<em>1</em>+<em>2</em> inhibition did not follow those of anti-Xa activity and TFPI release.

Peripheral arterial disease is diagnosed by measuring the ankle-brachial index. Values lower than 0.90 define the disease being usually related to its severity. Patients with peripheral arterial disease may show a hypercoagulability state. The aim of this study was to assess hemostatic variables and to correlate them with the presence of peripheral arterial disease and its severity as assessed by ankle-brachial index values. Plasma levels of D dimer, plasminogen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, plasminogen activator inhibitor and thrombomodulin were measured in 36 patients with peripheral arterial disease (group <em>1</em>) and 30 without disease (group <em>2</em>). Significant differences for D dimer, plasminogen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and plasminogen activator inhibitor type <em>1</em> between the <em>2</em> groups were found (P<0.05). Significant and inverse correlations were also observed (Pearson correlation, P<0.05) between ankle-brachial index values and levels of both plasminogen and plasminogen activator inhibitor type <em>1</em>. Although there was no significant correlation between ankle-brachial index and levels of D dimer, higher D dimer values were observed in patients with lower ankle-brachial index values. The results confirm a trend to hypercoagulability and hypofibrinolysis in patients with peripheral arterial disease. Increased levels of plasminogen activator inhibitor type <em>1</em> seem to be associated with the severity of the disease, considering the inverse correlation between this inhibitor and ankle-brachial index.

BACKGROUND

Thrombus growth under low blood flow velocity plays an important role in the development of deep venous thrombosis (DVT). Increased plasma levels and activities of coagulation factor VIII (FVIII) comprise risk factors for DVT and pulmonary thromboembolism.

OBJECTIVE

To localize FVIII in human venous thrombi of DVT and to determine whether FVIII contributes to thrombus formation under low shear conditions.

METHODS

The localization of FVIII in venous thrombi obtained from patients with DVT was examined by immunohistochemistry. The role of FVIII in thrombus formation was investigated using a flow chamber system. Venous blood from healthy volunteers were incubated with an anti-FVIII monoclonal antibody (VIII-3776) or non-immunized mouse IgG(<em>1</em>). Blood samples were perfused on immobilized type III collagen at wall shear rates of 70/s and 400/s and then the surface area covered by platelets and fibrin was morphometrically evaluated. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (PF<em>1</em>+<em>2</em>) generation was measured before and after perfusion.

RESULTS

Venous thrombi of DVT comprised a mixture of platelets, fibrin and erythrocytes. Factor VIII appeared to be colocalized with glycoprotein IIb/IIIa, fibrin and von Willebrand factor in the thrombi. VIII-3776 specifically recognized the light chain of FVIII and prolonged the activated partial thromboplastin time (aPTT), but not prothrombin time (PT). The antibody significantly reduced platelets and fibrin covering, as well as PF<em>1</em>+<em>2</em> generation at wall shear rates of 70/s and 400/s.

CONCLUSIONS

These results suggest that FVIII contributes to platelet aggregation and fibrin formation via thrombin generation under low shear conditions.

BACKGROUND

Patients with familial hypercholesterolaemia (FH) are characterized by high total and LDL cholesterol. Pregnant women with FH have higher absolute levels of total and LDL cholesterol, and a more pro-coagulant pattern compared with healthy pregnant women. Maternal hypercholesterolaemia has been shown to affect early atherosclerosis formation in the offspring. The aim of the present study was to investigate whether maternal FH leads to differences in plasma or serum levels of haemostatic and fibrinolytic markers in children with and without FH born of mothers with FH compared to control children born of non-FH mothers.

RESULTS

Children with (n=9) and without (n=7) FH born of mothers with FH, as well as control children (n=<em>1</em>6) born of non-FH mothers were included in the study. The concentrations of tissue plasminogen activator, plasminogen activator inhibitor (PAI-<em>1</em>), tissue factor (TF), TF pathway inhibitor (TFPI), thrombomodulin, fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and von Willebrand Factor were measured. Our findings show i) higher levels of PAI-<em>1</em> and TFPI in children with and without FH born of mothers with FH compared with control children, ii) lower levels of thrombomodulin in children with FH compared with control children, and iii) significant correlations between maternal PAI-<em>1</em> levels during pregnancy and PAI-<em>1</em> levels in the offspring.

CONCLUSIONS

We found that maternal FH may confer an unfavourable phenotype by affecting haemostatic and fibrinolytic markers in offspring independent of the children's FH status. However, the association between maternal hypercholesterolaemia and haemostatic risk markers in the offspring needs to be further elucidated.

Increased regional left atrial (LA) coagulation activity has recently been implicated in the pathophysiology of LA thrombus and systemic embolism in mitral stenosis (MS). Anticoagulation with warfarin reduces the risk of such thromboembolism, but the effect of warfarin on LA coagulation activity is unknown. We have addressed this question in MS patients with normal or prolonged clotting times. Peripheral venous and LA coagulation activities were measured in MS patients on long-term oral anticoagulation, who were predisposed to increased LA coagulation activity because of the presence of LA spontaneous echo contrast. Patients ceased warfarin 4 days before percutaneous balloon mitral valvuloplasty, and had either a normal (n = <em>1</em>5) or prolonged (n = 8) International Normalized Ratio (INR) at valvuloplasty. Coagulation activity was assessed during the valvuloplasty procedure, but before valve dilation, by measuring levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), a marker of thrombin generation. The LA F<em>1</em> + <em>2</em> level exceeded the peripheral venous level in patients with a normal INR (p <0.00<em>1</em>), but these levels were similar in patients with a prolonged INR (p = 0.<em>1</em>6). Moreover, the LA (p <0.005) and peripheral venous (p <0.03) F<em>1</em> + <em>2</em> levels, as well as the LA-peripheral venous F<em>1</em> + <em>2</em> difference (p <0.03) were lower in patients with a prolonged INR. These results suggest that anticoagulation with warfarin in MS not only reduces systemic coagulation activity but is associated with a greater reduction in LA coagulation activity. The latter may contribute to the reduced risk of LA thrombus formation that accompanies warfarin therapy in MS.

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A<em>2</em>3<em>1</em>87, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca<em>2</em>+ dose-response curves of both activities in intact platelets, obtained by stimulation with A<em>2</em>3<em>1</em>87 in the presence of Ca<em>2</em>+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for <em>prothrombin</em>ase activity in the presence of saturating concentrations of factors Xa, Va, and <em>prothrombin</em>. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their <em>fragments</em> as judged from protein patterns after gel electrophoresis. Platelet stimulation by A<em>2</em>3<em>1</em>87 resulted in a fast increase in <em>prothrombin</em>ase activity, reaching its maximum level after about <em>2</em>0 seconds. Filamin and talin were completely hydrolysed within <em>1</em>5 s, and myosin was partly degraded between <em>1</em>5 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, <em>prothrombin</em>ase activity was generated with a sigmoid time course, the steepest increase being observed between <em>1</em> and <em>2</em> min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and <em>1</em>.5 min after platelet activation, while degradation of myosin became visible after <em>2</em> to <em>2</em>.5 min. Dibucaine and tetracaine were both found to be potent stimulators of <em>prothrombin</em>ase activity, with half-maximal activities obtained at 0.7 and <em>2</em>.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of <em>prothrombin</em>ase activity closely paralleled that of calpain activity over a time course of <em>1</em> hour. Ca<em>2</em>+ titration of intact platelets using A<em>2</em>3<em>1</em>87 and Ca<em>2</em>+/HEDTA buffers, revealed half-maximal response at about <em>1</em>5 microM free Ca<em>2</em>+ for both calpain and <em>prothrombin</em>ase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.

Urine samples obtained from 37 normal individuals have been screened for the presence of <em>prothrombin</em> activation products using radioimmunoassays developed for <em>fragment</em> <em>1</em>, <em>fragment</em> <em>2</em> and <em>prothrombin</em> derivatives bearing the thrombin region. The cross-reacting materials detected in urine were isolated by affinity chromatography on insolubilized antibodies, and analysed by SDS polyacrylamide gel electrophoresis. The only <em>prothrombin</em> derivatives detected were <em>fragment</em> <em>1</em> (37/37) and <em>fragment</em> <em>2</em> (<em>2</em><em>2</em>/37). The mean values of daily urinary <em>fragment</em> <em>1</em> and <em>fragment</em> <em>2</em> excretion were respectively <em>1</em>3.4 nM and <em>1</em>.5 nM. The excretion of <em>prothrombin</em> derivatives has been quantitated in <em>1</em>4 normal pregnant women, during the third trimester of gestation. The mean values of urinary excretion were 47.<em>2</em> nM per day for <em>fragment</em> <em>1</em> (P less than 0.005) and 6.4 nM per day for <em>fragment</em> <em>2</em> (P less than 0.05). The significant increase in <em>fragment</em> <em>1</em> and <em>fragment</em> <em>2</em> excretion observed in a condition known to be associated with the so-called hypercoagulable state suggest that the measurement of <em>prothrombin</em> derivatives in urine could be a useful tool for the non-invasive detection of thromboembolic diseases or prethrombotic states.

BACKGROUND

A frequent complication of orthopaedic procedures is venous thromboembolism (VTE ). Hyperglycaemia has been shown to activate the coagulation system and is associated with postoperative morbidity and mortality. Therefore, we hypothesised that glucose levels increase during orthopaedic surgery and are associated with an activation of the coagulation system.

METHODS

Nine adult patients undergoing elective hip replacement were included. Venous blood samples were taken before, during and after surgery. Plasma glucose levels, factor VIII clotting activity (fVIII:c), von Willebrand ristocetin cofactor activity, von Willebrand factor antigen and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> were measured.

RESULTS

Immediately after induction of anaesthesia, plasma glucose levels started to increase until the second day postoperatively (peak 8.0 mmol/l). After seven weeks glucose values had returned to baseline (6.<em>1</em> mmol/l), p<0.00<em>1</em> with ANOVA. All coagulation parameters increased during surgery, subsequent to the rise in glucose. The change in mean FVIII:c and von Willebrand ristocetin cofactor activity was significantly correlated with mean glucose values.

CONCLUSIONS

These observations indicate that total hip replacement surgery causes an increase in glucose levels that precedes the proportional rise of the measured coagulation parameters. This suggests a possible role of glucose in the activation of the coagulation system during hip surgery.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), <em>prothrombin</em> G<em>2</em>0<em>2</em><em>1</em>0A, or HFE C<em>2</em>8<em>2</em>Y mutation using a standard method (polymerase chain reaction [PCR]-restriction <em>fragment</em> length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was <em>1</em>00% concordance between the genotypes ascertained by the <em>2</em> methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C<em>2</em>8<em>2</em>Y homozygotes (n = 74) had significantly higher (P < .000<em>1</em>) transferrin saturation levels (74% +/- <em>2</em>5%) than did nonhomozygotes (n = 340; 5<em>1</em>.4% +/- <em>2</em>8%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = <em>2</em>55; APCR = <em>2</em>.59 +/- 0.<em>2</em>6), <em>1</em> (n = 84; APCR = <em>1</em>.6<em>1</em> +/- 0.<em>1</em>3), or <em>2</em> (n = 5; APCR = <em>1</em>.<em>1</em>6 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

Essentials Glucocorticoids are associated with an increased risk of thrombosis. Healthy volunteers received dexamethasone or placebo in an endotoxin lung instillation model. Dexamethasone suppressed thrombin generation in bronchoalveolar lavage. Glucocorticoids inhibit endotoxin induced pulmonary coagulopathy.

Background Activation of local and systemic coagulation is a common finding in patients with pneumonia. There is evidence that glucocorticoids have procoagulant activity in the circulation, particularly in the context of inflammation. The effects of glucocorticoids on local pulmonary coagulation have not yet been investigated. Objective To use a human model of lung inflammation based on the local instillation of endotoxin in order to investigate whether glucocorticoids alter pulmonary coagulation. Methods Twenty-four healthy volunteers were randomized to receive either dexamethasone or placebo in a double-blind trial. Endotoxin was instilled via bronchoscope into right or left lung segments, followed by saline into the contralateral site. Six hours later, a bilateral bronchoalveolar lavage (BAL) was performed and coagulation parameters were measured. Results Endotoxin induced activation of coagulation in the bronchoalveolar compartment: the level of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em> ) was increased three-fold (<em>2</em>48 pmol L-<em>1</em> , 95% confidence interval [CI] 43-454 versus 743 pmol L-<em>1</em> , 95% CI 437-<em>1</em>050) and the level of thrombin-antithrombin complex (TATc) was increased by ~ 50% (3<em>1</em> μg L-<em>1</em> , 95% CI <em>1</em>8-45 versus 49 μg L-<em>1</em> , 95% CI 36-6<em>1</em>) as compared with saline-challenged segments. Dexamethasone reduced F<em>1</em> + <em>2</em> (<em>2</em>84 pmol L-<em>1</em> , 95% CI 34-534) and TATc (9 μg L-<em>1</em> , 95% CI 0.7-<em>1</em>7) levels almost to those measured in BAL fluid from the saline-instilled segments in the placebo group. Dexamethasone even profoundly reduced F<em>1</em> + <em>2</em> levels (80%) in saline-instilled lung segments (50 pmol L-<em>1</em> , 95% CI <em>1</em><em>2</em>-87). In contrast, dexamethasone had no effect on systemic F<em>1</em> + <em>2</em> levels. Conclusions Dexamethasone inhibits endotoxin-induced coagulopathy in lungs. This trial is the first to provide insights into the effects of glucocorticoids on pulmonary coagulation in response to endotoxin.

OBJECTIVE

To compare the effects of intranasal and oral administration of <em>1</em>7beta-estradiol (E<em>2</em>) and norethisterone(acetate) [NET(A)] in healthy postmenopausal women on activated protein C (APC) resistance and other hemostatic parameters associated with venous thrombosis.

RESULTS

In this <em>2</em>-center, randomized, double-blind, <em>1</em>-year trial, 90 postmenopausal women (56.6+/-4.7 years of age) received daily either an intranasal spray with <em>1</em>75 microg/<em>2</em>75 microg E<em>2</em>/NET (n=47) or <em>1</em> mg/0.5 mg oral E<em>2</em>/NETA (n = 43). Normalized APC sensitivity ratios (nAPCsr) were determined with a thrombin generation-based APC resistance test. After <em>1</em> year, the increase in nAPCsr was smaller in the intranasal than in the oral group: <em>1</em><em>1</em>% (95% CI, <em>1</em>% to <em>2</em><em>2</em>%) versus 53% (95% CI, 37% to 7<em>2</em>%). Overall, the decrease in antithrombin and increase in <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) were smaller and the decrease in free protein S larger in the intranasal compared with the oral group after <em>1</em> year. In both groups, the decreases in protein C and <em>prothrombin</em>, and the increase in d-dimer were similar.

CONCLUSIONS

Compared with oral E<em>2</em>/NETA therapy, intranasal administration of E<em>2</em>/NET had less effect on APC resistance and on a number of other parameters associated with venous thrombosis. This observation suggests the possibility of a lower venous thrombosis risk for intranasal E<em>2</em>/NET compared with oral therapy.

BACKGROUND

<em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> is excreted in urine (uF<em>1</em> + <em>2</em>) as a result of thrombin generation and, therefore, may be a useful marker of coagulation status.

OBJECTIVE

To assess uF<em>1</em> + <em>2</em> levels after total hip replacement (THR) in patients with venous thromboembolism (VTE) and bleeding events.

METHODS

This study was conducted in parallel with a prospective, dose-finding study evaluating the efficacy and safety of different doses of rivaroxaban (Xarelto, Bayer HealthCare AG, Wuppertal, Germany) for thromboprophylaxis, relative to enoxaparin. Deep vein thrombosis was diagnosed by mandatory venography performed 5-9 days after THR, or earlier if symptomatic. Symptomatic pulmonary embolism was diagnosed by objective testing. Bleeding complications were registered and stratified into major bleeding, clinically relevant, non-major bleeding, and minor bleeding, using predefined criteria.

RESULTS

Eighty-four patients had a VTE and 57 patients had a bleeding event (n = 7<em>2</em><em>2</em>). Significantly higher median uF<em>1</em> + <em>2</em> levels were observed in the VTE group on day 3 after THR (P = 0.03), compared with control. Median uF<em>1</em> + <em>2</em> levels were lower in the bleeding group on day 3 after THR (P = 0.005) and on the day of venography (P = 0.36), compared with control. Comparisons between the VTE and bleeding groups showed significantly lower median uF<em>1</em> + <em>2</em> levels in the bleeding group on day 3 after THR and on the day of venography (P < 0.000<em>1</em> and P = 0.006, respectively).

CONCLUSIONS

Measurement of uF<em>1</em> + <em>2</em> could provide a simple clinical test to evaluate non-invasively the intensity of coagulation activation after THR. However, further studies are required to confirm these encouraging preliminary results.

Twenty-four children with juvenile rheumatoid arthritis (JRA) and <em>1</em>0 children with postinfectious arthropathies were investigated for markers of blood coagulation and fibrinolytic activity: <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complex (TAT), and D-Dimer were measured using solid phase enzyme linked immunosorbent assays (ELISA). Results were compared with clinical and conventional laboratory signs of disease activity. F<em>1</em>+<em>2</em>, TAT, D-Dimer, and fibrinogen were significantly elevated in children with JRA as compared with healthy children and children with postinfectious arthropathies. F<em>1</em>+<em>2</em>, TAT, and D-Dimer correlated significantly with disease activity, assessed by determination of the joint index score and C-reactive protein (CRP). The study demonstrates a subclinical activation of the haemostatic system in children with JRA correlating with disease activity, which might be caused by the action of several immunomediators on cells (monocytes, endothelial cells) playing a role in the regulation of blood coagulation activity.

We have investigated the influence of blood flow on thrombin generation, fibrin formation, and fibrin deposition on procoagulant and nonprocoagulant surfaces. Nonanticoagulated human blood was drawn for 5 minutes directly from an antecubital vein over stimulated endothelial cells expressing tissue factor and over human type III collagen fibrils, positioned in parallel-plate perfusion chambers. The shear rates at these surfaces were 50, 650, and <em>2</em>,600 s-<em>1</em>. Deposition of platelets and fibrin was measured by morphometry. Thrombin and fibrin formation was determined by measuring <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>), thrombin-antithrombin III complexes, (T-AT) and fibrinopeptide A (FPA) in blood effluent from the perfusion chamber at the end of the 5-minute perfusion period. On procoagulant endothelial cells, the thrombi were primarily composed of fibrin. The fibrin deposition (8<em>1</em>%, <em>2</em><em>1</em>%, and <em>2</em>% at 50, 650, and <em>2</em>,600 s-<em>1</em>, respectively) and plasma levels of F <em>1</em> + <em>2</em>, T-AT and FPA were shear rate dependent and highest at 50 s-<em>1</em>. There was a positive correlation between F <em>1</em> + <em>2</em> and T-AT and the fibrin deposition (P < .0<em>1</em>). In contrast, the collagen surface triggered primarily thrombi that were composed of platelets. The platelet thrombi and plasma levels of F <em>1</em> + <em>2</em> and T-AT were also dependent on the shear rate, but highest at 650 and <em>2</em>,600 s-<em>1</em>. F <em>1</em> + <em>2</em> and T-AT reached the same level as observed with procoagulant endothelial cells at the higher shear rates. There was a positive correlation between F <em>1</em> + <em>2</em> and T-AT and the platelet thrombus formation (P < .05), confirming the predominant role of platelets in thrombin generation. Thus, thrombin formation is strongly influenced by the blood flow, and this effect depends on the composition of the thrombogenic surface.

Hyperlipidemic patients with coronary heart disease were treated with atorvastatin, and its effects on hemostatic and inflammatory parameters were assessed. After 3 months of therapy, the plasma levels of plasminogen activator inhibitor, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, highly sensitive C-reactive protein, von Willebrand factor, and fibrinogen were significantly reduced; no significant reductions were observed in lipoprotein(a) and tissue plasminogen activator antigen levels.

Patients with sickle cell trait (STr) are usually considered to be asymptomatic. However, complications, including hypercoagulability, increased risk of venous thromboembolism and the exertional exercise syndrome with rhabdomyolysis and sudden death, have been described. The exact cause of these adverse events is unclear. We have investigated two patients, a set of monozygotic twins with STr, to establish their procoagulant activity status as a potential indicator of thrombotic risk. In-vivo thrombin generation was assessed by the measurement of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and thrombin-antithrombin complexes (TAT). D-dimer was used as a marker of fibrinolytic activity. The potential to generate thrombin was determined using an ex-vivo thrombin generation test (TGT). The impact of red blood cell (RBC)-derived microparticle shedding and RBC rheology were examined. TAT (>60 μg/l) and F<em>1</em> + <em>2</em> (948 pmol/l) were markedly elevated in patient <em>2</em> but within the normal reference range in patient <em>1</em> (TAT = <em>2</em>.5 μg/l; F<em>1</em> + <em>2</em> = <em>1</em>38 pmol/l). D-dimer levels (0.9 mg/l FEU) were similarly elevated in both patients. TGT peak thrombin and endogenous thrombin potential (ETP) were elevated to similar degrees in both patients. Flow cytometric analysis for RBC-derived microparticles showed that both patients had elevated levels on two occasions. RBC deformability, blood viscosity and RBC aggregation were normal and similar in both patients. The results demonstrated different coagulation activity in the patients with one patient in a prothrombotic state, suggesting that there may be two levels of hypercoagulability in STr. Measurement of such differences would allow for separation of high and low-risk patients from serious complications.

Thrombin generation (TG) is an important pathogenic factor in acute coronary syndromes including acute myocardial infarction (AMI). Since the diagnostic utility of TG remains uncertain we sought to determine whether markers of TG may triage patients presenting to the Emergency Department with chest pain. Soluble plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F(<em>1</em>+<em>2</em>)), and thrombin/antithrombin III complexes (TAT) were determined by ELISA in 80 patients presenting with chest pain to the Emergency Department and compared with <em>2</em>0 controls. There were no differences in TG markers between patients with non-cardiac chest pain and healthy controls. Patients with unstable angina (UA), and congestive heart failure (CHF) did not differ from controls with respect to F(<em>1</em>+<em>2</em>), and TAT was elevated in UA patients (6.05 +/- <em>1</em>.<em>1</em>5 ng/ml, p = 0.033) when compared with controls (3.34 +/- 0.<em>2</em>0 ng/ml). Contrary to expectations, TAT levels at presentation with AMI were well below the concentrations observed in patiens with UA and CHF. Moreover, plasma F(<em>1</em>+<em>2</em>) levels were significantly lower than in healthy controls (0.84 +/- 0.<em>1</em>0 ng/ml versus <em>1</em>.<em>2</em><em>2</em> +/- 0.<em>1</em><em>1</em>, p = 0.0<em>2</em>6). At the time of presentation to the Emergency Department, F(<em>1</em>+<em>2</em>) and TAT failed to suitably triage patients with chest pain. The surprisingly low levels of TG markers in AMI patients before applying intensive therapy and reperfusion strategies deserves further investigation.

Spinal cord injured patients are at increased risk of developing deep vein thrombosis (DVT). Whether these patients have increased blood levels of prothrombotic markers remains to be clarified. In general, the risk of developing DVT is highest in the morning hours. In healthy humans, several haemostatic and fibrinolytic parameters exhibit circadian variations, but it is not known whether this also applies to those with spinal cord injury. The aim of the present study was to examine possible circadian variations in prothrombotic markers in tetraplegic patients. We studied six patients with complete tetraplegia and eight control subjects with repetitive blood sampling over a <em>2</em>4 h period. While the control subjects showed marked circadian variations in factor VIII activity, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> and D-dimer levels, the tetraplegic patients did not (P < 0.05). Circadian variation in plasminogen activator inhibitor type-<em>1</em> was present in both groups, being most marked (P < 0.05) in tetraplegia. We conclude that the circadian variations of several factors of the haemostatic and fibrinolytic systems are impaired in spinal cord injury. This could possibly reflect a deregulated autonomic nervous system, leading to a dysfunctional link between central and peripheral circadian oscillators.