Patients with essential thrombocythemia (ET) have an increased frequency of thrombosis, but the relationship of both thrombosis and JAK<em>2</em> V6<em>1</em>7F allele burden with platelet turnover, acquired activated protein C resistance (aAPCR), and levels of coagulation factors and soluble markers of platelet, and endothelial activation is not well known. In 53 ET patients (<em>2</em>6 with a history of thrombosis), reticulated platelets (RP) percentage, aAPCR, platelet tissue factor (TF) expression, and plasma levels of TF, coagulation factors, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK<em>2</em> mutational load. ET patients with thrombosis had significantly higher values for RP percentage, aAPCR, and levels of factors V and VIII, VWF:Ag, sP-selectin, and sCD40L than patients without thrombosis and controls. At multivariate study, RP percentage, factor V levels, and aAPCR were independently associated with an increased risk of thrombosis. Patients with JAK<em>2</em> mutation had significantly lower levels of free protein S (PS) and higher levels of TF, sP-selectin, sCD40L, VWF:Ag, and sTM than those with wild-type allele. A mutant allele dosage effect >>or= <em>1</em><em>2</em>%) was observed for TF, sP-selectin, sCD40L, VWF:Ag, and PS levels. These results support a role for platelet turnover, factor V, and aAPCR in the thrombosis of ET as well as the association between JAK<em>2</em> V6<em>1</em>7F allele burden and either decreased free PS or increased TF and soluble markers of platelet and endothelial activation.

Lipid-lowering by postmenopausal hormone therapy (HRT) explains only partly the assumed coronary risk reduction associated with therapy. To explore other possible mechanisms, we studied associations of HRT use with inflammation and hemostasis risk markers in women>>/=65 years of age. Subjects were selected from 3393 participants in the fourth year examination of the Cardiovascular Health Study, an observational study of vascular disease risk factors. After excluding women with vascular disease, we compared levels of inflammation and hemostasis variables in the <em>2</em>30 women using unopposed estrogen and 60 using estrogen/progestin, with those of <em>1</em>96 nonusers selected as controls. Compared with nonusers, unopposed estrogen use was associated with 59% higher mean C-reactive protein (P<0.00<em>1</em>), but with modestly lower levels of other inflammation indicators, fibrinogen, and alpha-<em>1</em> acid glycoprotein (P<0.00<em>1</em>). Factor VIIc was <em>1</em>6% higher among estrogen users (P<0.00<em>1</em>), but this was not associated with higher thrombin production (<em>prothrombin</em> <em>fragment</em> <em>1</em>-<em>2</em>), or increased fibrin breakdown (D-dimer). Concentration of plasminogen activator inhibitor-<em>1</em> was 50% lower in both using groups (P<0.00<em>1</em>) compared with nonusers, and this was associated with higher plasmin-antiplasmin complex: 8% higher in estrogen and <em>1</em>8% higher in estrogen/progestin users (P<0. 05). Relationships between the markers and hormone use were less pronounced in estrogen/progestin users, with no association for C-reactive protein except in women in upper <em>2</em> tertiles of body mass index (P for interaction, 0.0<em>2</em>). The direction and strength of the associations of HRT use with inflammation markers differed depending on the protein, so it is not clear whether HRT confers coronary risk reduction through an inflammation-sensitive mechanism. Associations with hemostasis markers indicated no association with evidence of procoagulation and a possible association with increased fibrinolytic activity.

It is well known that atrial fibrillation (AF) is one of the most important diseases that predispose patients to thrombosis. We have attempted to identify patients with AF in the hypercoagulable state by measuring molecular markers such as thrombin-antithrombin III complex (TAT) and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (PTF) and determining the effect of antithrombotic therapy on these markers; 83 patients with AF were studied. Increased levels of plasma TAT and PTF were more frequently observed in patients with AF and associated mitral stenosis than in patients with AF alone. In cases of AF without mitral stenosis, plasma levels of TAT and PTF were significantly lower in those patients receiving antithrombotic agents (aspirin or warfarin) than in those receiving no antithrombotic agents. Furthermore, plasma levels of PTF were significantly lower in patients given warfarin than in those receiving aspirin. These results suggest that (<em>1</em>) patients with AF and mitral stenosis who are not given warfarin are in an extremely hypercoagulable state and (<em>2</em>) some patients with AF without mitral stenosis who are not given antithrombotic agents are also moderately hypercoagulable. In vivo activation of blood coagulation was more effectively controlled in patients receiving warfarin than in those taking aspirin.

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(<em>2</em>+)-dependent membrane-binding characteristics of fluorescein-labeled bovine <em>prothrombin</em>-<em>fragment</em> <em>1</em> (F-BF<em>1</em>). Light scattering measurements demonstrated that F-BF<em>1</em> bound to small unilamellar phosphatidylserine/phosphatidylcholine (<em>2</em>5/75, mol/mol) vesicles with an apparent dissociation constant (<em>1</em>.5 +/- 0.<em>2</em> microM) similar to that of unlabeled protein (<em>1</em>.<em>1</em> +/- 0.<em>1</em> microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF<em>1</em> bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.<em>2</em> microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF<em>1</em> on <em>2</em>5 mol% PS planar surfaces were diffusion-influenced at F-BF<em>1</em> solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF<em>1</em> concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF<em>1</em> concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF<em>1</em> solution concentrations greater than or equal to <em>1</em>0 microM gave average association and dissociation kinetic rates of approximately <em>1</em>0(5) M-<em>1</em> s-<em>1</em> and approximately 0.<em>1</em> s-<em>1</em>, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.

Left ventricular thrombosis and systemic emboli have been demonstrated to complicate cardiomyopathy in Duchenne and Becker muscular dystrophy (DMD, BMD). We investigated plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>). thrombin-antithrombin III complex (TAT) and circulating levels of tumor necrosis factor-alpha (TNF-alpha), a procoagulant cytokine that has been shown to be elevated in patients with depressed cardiac function, in <em>2</em>0 patients with DMD and <em>1</em><em>2</em> patients with BMD as compared with 30 age-matched control subjects. Significantly elevated levels of F<em>1</em>+<em>2</em> (DMD: <em>1</em>.4+/-0.8 nmol/l; BMD: <em>1</em>.8+/-0.8 nmol/l vs. controls: 0.7+/-0.<em>2</em> nmol/l, p <0.0<em>1</em> and p <0.00<em>1</em>, respectively), TAT complexes (DMD: 4.7+/-<em>2</em>.7 microg/l, BMD: 5+/-<em>2</em>.3 microg/l vs. controls: <em>1</em>.6+/-0.5 microg/l, p <0.00<em>1</em>) and TNF-alpha (54+/-9 vs. <em>2</em>5+/-7 pg/ml, p <0.00<em>1</em>) were observed in patients with the dystrophic disease compared to control subjects. A significantly negative correlation was also found between F<em>1</em>+<em>2</em> and TAT complexes and left ventricular ejection fraction (r = -0.65, p <0.000<em>1</em>; r = -0.80, p < 0.000<em>1</em>, respectively) and a positive correlation between F<em>1</em>+<em>2</em> and TAT complexes and serum TNF-alpha levels (r = 0.67, p <0.000<em>1</em>; r = 0.70, p <0.000<em>1</em>, respectively). Our results indicate a hypercoagulable state in X-linked dystrophic patients. A possible relationship between haemostatic activation, left ventricular dysfunction and TNF-alpha system upregulation may be suggested.

During blood coagulation, <em>prothrombin</em> (PT) is ultimately degraded to three <em>fragments</em>, thrombin, <em>fragment</em> <em>1</em> (F<em>1</em>) and <em>fragment</em> <em>2</em> (F<em>2</em>), which, collectively, contain all of the structural features of PT. One of these <em>fragments</em>, F<em>1</em>, is excreted in human urine and is the principal protein occluded into calcium oxalate (CaOx) crystals precipitated from it. This urinary form of F<em>1</em>, which we have named urinary <em>prothrombin</em> <em>fragment</em> <em>1</em> is present in calcium stones and is a potent inhibitor of CaOx crystallization in urine in vitro. The aim of this study was to determine whether PT itself and its other activation products, namely, thrombin, F<em>1</em> and F<em>2</em> also inhibit CaOx crystallization, by comparing their effects in a seeded, inorganic crystallization system. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. PT was isolated from a human blood concentrate rich in vitamin K-dependent proteins. Following initial cleavage by thrombin, the resulting <em>fragments</em>, F<em>1</em> and F<em>2</em>, were purified by a combination of reversed phase HPLC and low pressure column chromatography. The purity of the proteins was confirmed by SDS/PAGE and their individual effects on CaOx crystallization were determined at the same concentration (<em>1</em>6.<em>1</em>3 nM) in a seeded, metastable solution of CaOx using a Coulter Counter. [<em>1</em>4C]Oxalate was used to assess deposition of CaOx and crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that the proteins reduced the size of precipitated crystals in the order F<em>1</em>>> PT>> F<em>2</em>>> thrombin. These findings were confirmed by scanning electron microscopy which showed that the reduction in particle size resulted from a decrease in the degree of crystal aggregation. [<em>1</em>4C]Oxalate analysis demonstrated that all proteins inhibited mineral deposition, in the order F<em>1</em> (44%)>> PT (<em>2</em>7.4%)>> thrombin (<em>1</em>0.<em>2</em>%)>> F<em>2</em> (6.5%). It was concluded that the gamma-carboxyglutamic acid domain of PT and F<em>1</em>, which is absent from thrombin and F<em>2</em>, is the region of the molecules which determines their potent inhibitory effects. The superior potency of F<em>1</em>, in comparison with PT, probably results from the molecule's greater charge to mass ratio.

OBJECTIVE

To assess the effects of aspirin compared with simvastatin on thrombin generation in hypercholesterolemic men, and to establish whether the reduction of elevated blood cholesterol by simvastatin would affect the action of aspirin on thrombin formation.

BACKGROUND

Aspirin inhibits thrombin formation, but its performance is blunted in hypercholesterolemia. By virtue of altering lipid profile, statins could be expected to influence thrombin generation.

METHODS

Thirty-three men, aged 34 to 6<em>1</em> years, with minimal or no clinical symptoms, serum total cholesterol >6.5 mmol/liter and serum triglycerides <4.6 mmol/liter, completed the study consisting of three treatment phases. First, they received 300 mg of aspirin daily for two weeks (phase I), which was then replaced by simvastatin at the average dose of <em>2</em>4 mg/d for three months (phase II). In phase III, aspirin, 300 mg/day, was added for two weeks to simvastatin, the dose of which remained unchanged. Thrombin generation was assessed: <em>1</em>) in vivo, by measuring levels of fibrinopeptide A (FPA) and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) in venous blood; and <em>2</em>) ex vivo, by monitoring the rates of increase of FPA and F<em>1</em>+<em>2</em> in blood emerging from standardized skin incisions of a forearm. A mathematical model was used to describe the kinetics of thrombin formation at the site of microvascular injury.

RESULTS

Two-week treatment with aspirin had no effect on thrombin markers in vivo, while ex vivo it depressed the total amount of thrombin formed, though not the reaction rate. After simvastatin treatment, serum cholesterol decreased by 3<em>1</em>% and LDL cholesterol by 4<em>2</em>%, while thrombin generation became markedly depressed. In venous blood, FPA was significantly reduced. Concomitantly, the initial thrombin concentration and total amount of thrombin generated decreased significantly. Addition of aspirin to simvastatin (phase III) had no further effect on any of these parameters.

CONCLUSIONS

In men with hypercholesterolemia, lowering serum cholesterol level by a three-month simvastatin treatment is accompanied by a marked reduction of thrombin generation both at basal conditions in venous blood and after activation of hemostasis by microvascular injury. Once blood cholesterol became reduced, adding aspirin to simvastatin did not enhance dampening of thrombin formation.

BACKGROUND

There are few detailed descriptions of the inflammatory response to cardiac surgery with cardiopulmonary bypass (CPB) in children beyond <em>2</em>4 h postoperatively. This is especially true for the antiinflammatory cytokines and the extent of tissue injury. The aim of the current study was to describe the inflammatory and injury responses in uncomplicated pediatric cardiac surgery with CPB, where methylprednisolone and modified ultrafiltration (MUF) were used.

METHODS

Blood samples were collected up to 48 h postoperatively. Cytokines (tumor necrosis factor-alpha and interleukin-6, -<em>1</em>beta, -<em>1</em>0, and -<em>1</em>ra), complement (C3d and C4d) and coagulation system (<em>prothrombin</em> activation <em>fragments</em> <em>1</em> and <em>2</em> and antithrombin III) activation, neutrophil elastase, and the resulting tissue injury (creatine kinase, lactate dehydrogenase, alanine transaminase, amylase, and gamma-glutamyl transferase) were measured.

RESULTS

The proinflammatory cytokine release varied widely, in contrast to a clear-cut antiinflammatory response. Cytokine concentrations did not decrease immediately after MUF, and no rebound increases later in the postoperative period were observed. The coagulation system, but not complement, was activated. There was a late release of C-reactive protein. Tissue injury could be quantified biochemically without evidence of hepatic or pancreatic dysfunction.

CONCLUSIONS

In this group of uncomplicated subjects, the antiinflammatory cytokine and tissue injury responses were well defined, in contrast to a variable proinflammatory cytokine release. This was accompanied by activation of the coagulation system but not of complement. Concentrations of inflammatory mediators did not decrease immediately after MUF, and there was no evidence for rebound release later in the postoperative period.

BACKGROUND

The receptor for advanced glycation endproducts (RAGE) recognizes a variety of ligands that play an important role in the posttraumatic inflammatory response. However, whether soluble RAGE (sRAGE) is released early after trauma hemorrhage in humans and whether such a release is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of this study.

METHODS

One hundred sixty-eight patients were studied as part of a prospective cohort study of severe trauma patients admitted to a single Level I Trauma center. Blood was drawn within <em>1</em>0 minutes of arrival to the emergency department before the administration of any fluid resuscitation. sRAGE, tumor necrosis factor-alpha, interleukin-6, von Willebrand factor, angiopoietin-<em>2</em>, <em>prothrombin</em> time, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, soluble thrombomodulin, protein C, plasminogen activator inhibitor-<em>1</em>, and d-dimers (fibrin degradation products) were measured using standard techniques. Base deficit was used as a measure of tissue hypoperfusion. Measurements were compared with outcome measures obtained from the electronic medical record and trauma registry.

RESULTS

Plasma levels of sRAGE were increased within 30 minutes after severe trauma in humans and correlated with the severity of injury, early posttraumatic coagulopathy and hyperfibrinolysis, and endothelial cell activation (angiopoietin-<em>1</em> and complement). Furthermore, we found that there was a significant relationship between plasma levels of sRAGE and the development of acute renal failure. This relationship was not quite significant for patients who developed acute lung injury (p = 0.<em>1</em><em>1</em>), although patients with (<em>2</em>6 ventilator-free days had significantly higher plasma levels of sRAGE than those with>><em>2</em>6 ventilator-free days. Finally, there was no relationship between plasma levels of sRAGE and mortality rate in trauma patients.

CONCLUSIONS

The results of this study demonstrate that the release of sRAGE in the bloodstream of trauma patients requires severe injury and is associated with coagulation abnormalities and endothelial cell and complement activation.

Combined oral contraceptives (OC) are known to increase the risk of venous thromboembolism. The aim of this randomized, cycle-controlled, cross-over study in <em>2</em>8 healthy volunteers was to assess potential differences between the effects of an OC containing <em>1</em>50 microg levonorgestrel (as representative of the so-called second generation OC) and an OC containing <em>1</em>50 microg desogestrel (as representative of the third generation OC) in combination with 30 microg ethinylestradiol on several coagulation factors and markers of thrombin formation. All participants used each OC for two cycles, and were switched to the other OC after a washout period of two menstrual cycles. The plasma concentrations of factors II, VII, X, and fibrinogen significantly increased during use of both the levonorgestrel- and desogestrel-containing OC's. The plasma concentrations of factor VIII increased, and of factor V decreased, changes which only reached statistical significance during the use of the desogestrel-containing OC. During exposure to the desogestrel-containing OC, as compared with the levonorgestrel-containing OC, both factor VII and factor II showed a greater increase (FVII: 3<em>2</em>% and <em>1</em><em>2</em>% respectively; p <0.000<em>1</em>; FII: <em>1</em>6% and <em>1</em><em>2</em>% respectively; p = 0.048), whereas factor V showed a greater decrease (-<em>1</em><em>1</em>% and -3% respectively; p = 0.0<em>1</em>0). Only one of the markers for ongoing coagulation (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>) showed a significant increase during OC use, whereas concentrations of thrombin-antithrombin complexes and soluble fibrin remained unchanged. For these markers, there was no difference between the tested OC's. We conclude that there are differences between the effects of levonorgestrel and desogestrel-containing OC's on some coagulation factors. Whether these changes provide a biological explanation for the reported differences in venous thromboembolic risk is as yet unclear. The real challenge now becomes to define a pattern of changes in the various systems which, if affected simultaneously, may tip the hemostatic balance towards a prethrombotic state and may lead to overt clinical venous thromboembolism.

OBJECTIVE

The aims of the present work were to assess the presence of thrombin generation in Crohn's disease and in ulcerative colitis by using the <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and the thrombin-antithrombin III complex assays and to study the possible relationships between these markers and disease activity.

RESULTS

Prothrombin <em>fragment</em> <em>1</em> + <em>2</em> and thrombin-antithrombin III complex were significantly raised in patients with Crohn's disease (n = 69) and with ulcerative colitis (n = <em>2</em>5) as compared with healthy controls (n = 50). In Crohn's disease these two markers of thrombin generation were correlated with the Van Hees index (P < 0.05 and P < 0.00<em>1</em>, respectively); values were significantly different from controls even in the patient group displaying the lowest disease activity (P < 0.00<em>1</em>). No correlation was found with tumour necrosis factor alpha and C-reactive protein; nevertheless patients with C-reactive protein less than or equal to <em>1</em>0 mg/l had significant lower values of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (P < 0.03). In ulcerative colitis <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and thrombin-antithrombin III complex were significantly increased by comparison with controls, were higher in patients with pancolitis and correlated with C-reactive protein (P < 0.00<em>2</em> and P < 0.009, respectively).

CONCLUSIONS

These data show that <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and thrombin-antithrombin III complex are increased in inflammatory bowel diseases and suggest that thrombin generation might be an early event in their pathogenesis.

BACKGROUND

To test the hypothesis that tissue factor release, thrombin activation, fibrin formation, and fibrinolysis after an isolated head injury are equal to those in patients without head injury, as well as to investigate the precise time course of the coagulation and fibrinolytic abnormalities after head injury, we performed prospective and retrospective studies.

RESULTS

In the prospective study, 5 patients with isolated head injury and <em>1</em><em>1</em> trauma patients without head injury took part in this study. Tissue factor antigen concentration, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, thrombin antithrombin complex, fibrinopeptide A, and fibrin degradation products (D-dimer) were measured on the day of admission, and days <em>1</em>, <em>2</em>, 3, and 4 after admission. The levels of all five hemostatic molecular markers were markedly elevated on the day of admission, and then gradually decreased to day 4. The levels and the time course of these hemostatic markers in patients with isolated head injury were not different from those in the control patients. The same incidence of disseminated intravascular coagulation between the two groups was also observed. In the retrospective study, the records of fibrinopeptide Bbeta<em>1</em>5-4<em>2</em>, plasmin antiplasmin complex, plasminogen activator inhibitor-<em>1</em> antigen concentration (PAI-<em>1</em> antigen), and PAI-<em>1</em> activity in 76 trauma patients were reviewed. On the basis of the exclusion criteria, 9 patients with isolated head injury and 30 control patients were selected for the study group. Fibrinopeptide Bbeta<em>1</em>5-4<em>2</em> and plasmin antiplasmin complex markedly elevated on the day of admission, then decreased on day <em>1</em>, and tended to increase to day 5. Markedly elevated PAI-<em>1</em> antigen and PAI-<em>1</em> activity on the day of admission significantly decreased on day <em>1</em> and recovered to the normal values on day 5. The changes of these molecular markers in patients with isolated head injury were equal to those in the control patients.

CONCLUSIONS

We systematically elucidated the time course of coagulation and fibrinolysis after isolated head injury. We further demonstrated that changes in coagulofibrinolytic and antifibrinolytic systems in patients with isolated head injury are not different from those in patients without head injury.

Coagulation activation in pregnancy is further enhanced in preeclampsia. We investigated whether this results from increased thrombin generation by the plasma itself or its cell-derived microparticles. Plasma samples were obtained from preeclamptic, normal pregnant and nonpregnant women (each n = <em>1</em>0). <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complex (TAT) concentrations were increased in pregnancy and further increased in preeclampsia. In pregnancy and preeclampsia, increased activated protein C resistance occuffed (APC sensitivity ratio: 3.3 +/- 0.8 and <em>2</em>.5 +/- 0.8, both P <0.00<em>1</em> vs. nonpregnant). In normal pregnant microparticle-free plasma the thrombin generation correlated with TAT (r = 0.84, P = 0.005) and APC resistance correlated with F<em>1</em>+<em>2</em> (r = 0.68, P = 0.04). In preeclampsia thrombin generation by plasma was increased (P = 0.005), independent of APC resistance. Thrombin generation by microparticles was similar in all groups, although different coagulation activation pathways were utilized, indicating that circulating microparticles are not directly involved in coagulation activation in pregnancy and preeclampsia. In contrast, APC resistance can explain coagulation activation in pregnancy, while enhanced coagulation activation in preeclampsia results, in part, from an increased thrombin generating capacity of plasma independent of APC resistance.

The increased incidence of arterial thromboembolism in the elderly has prompted investigation of age-related changes in the hemostatic system. Aging is associated with increased plasma levels of fibrinogen, factor VII and factor VIII, which have been shown to be risk factors for thrombotic disease in five large epidemiological studies. An increased responsiveness to different aggregating stimuli, elevated levels of beta-thromboglobulin and an increased production of thromboxane A<em>2</em> were reported in the platelets of the elderly. These alterations are associated with modifications of platelet membrane lipid composition (namely an increase in the cholesterol/phospholipid ratio and a decrease in linoleic acid) with possible related changes in membrane fluidity. Moreover, a decrease in the number of platelet prostacyclin and thromboxane A<em>2</em> receptors was observed with aging. Fibrinolytic activity is impaired in the elderly, probably due to an increase in plasminogen activator inhibitor <em>1</em>. Interestingly, hypercoagulability has been demonstrated by an increase in the activation markers of the coagulation cascade (mainly fibrinopeptide A and <em>prothrombin</em> activation <em>fragment</em> F <em>1</em> + <em>2</em>). Finally, clinical and experimental evidence suggests that endothelium could play a central role in hemostatic alterations which determine a thrombophilic state in the elderly.

Use of combined oral contraceptives (OC) is associated with a significant risk of thrombosis. The mechanisms of this effect are not clearly defined. Tissue factor pathway inhibitor (TFPI) is a circulating anti-coagulant that inhibits the earliest steps in activation of the extrinsic coagulation pathway. It plays a central role in control of coagulation but its contribution to the thrombotic risk associated with OC has not been assessed. Plasma TFPI antigen and activity, factor VIIa, <em>prothrombin</em> <em>fragments</em> <em>1</em>&<em>2</em>, von Willebrand antigen, fibrinogen, and low density lipoprotein cholesterol were measured by standard assays in women taking OC (aged <em>1</em>6 to 45 years, n = 40) and age-matched women not taking OC (controls, n = 40). Plasma TFPI antigen did not vary significantly across the menstrual cycle in controls. Women on OC had a <em>2</em>5% reduction in plasma TFPI antigen (median 5<em>1</em>.0 ng/ml; 95% confidence intervals [CI] 37.5 to 85.5; control 68.0 ng/ml, CI 6<em>1</em>.0 to 95.0; P < 0.00<em>1</em>) and a <em>2</em>9% reduction in TFPI activity (78.5 U/ml, CI 57.5 to <em>1</em>07.5; control <em>1</em><em>1</em><em>1</em>.0 U/ml, CI 79.5 to <em>1</em>7<em>1</em>.0; P < 0.00<em>1</em>) compared to controls. Plasma factor VIIa activity and <em>prothrombin</em> <em>fragments</em> <em>1</em>&<em>2</em> were also significantly increased in women using OC (both P < 0.00<em>1</em>), indicating activation of the extrinsic coagulation pathway. These results demonstrate that normal cyclic variations in estrogen and/or progesterone do not significantly alter plasma TFPI levels. However, estrogens and/or progestogens in OC result in activation of the extrinsic coagulation pathway and significantly reduce plasma TFPI, its major circulating inhibitor. Reduced plasma TFPI levels may underlie the thrombotic effects of OC.

BACKGROUND

The link between long-haul air travel and venous thromboembolism is the subject of continuing debate. It remains unclear whether the reduced cabin pressure and oxygen tension in the airplane cabin create an increased risk compared with seated immobility at ground level.

OBJECTIVE

To determine whether hypobaric hypoxia, which may be encountered during air travel, activates hemostasis.

METHODS

A single-blind, crossover study, performed in a hypobaric chamber, to assess the effect of an 8-hour seated exposure to hypobaric hypoxia on hemostasis in 73 healthy volunteers, which was conducted in the United Kingdom from September <em>2</em>003 to November <em>2</em>005. Participants were screened for factor V Leiden G<em>1</em>69<em>1</em>A and <em>prothrombin</em> G<em>2</em>0<em>2</em><em>1</em>0A mutation and were excluded if they tested positive. Blood was drawn before and after exposure to assess activation of hemostasis.

METHODS

Individuals were exposed alternately >> or =<em>1</em> week apart) to hypobaric hypoxia, similar to the conditions of reduced cabin pressure during commercial air travel (equivalent to atmospheric pressure at an altitude of <em>2</em>438 m), and normobaric normoxia (control condition; equivalent to atmospheric conditions at ground level, circa 70 m above sea level).

METHODS

Comparative changes in markers of coagulation activation, fibrinolysis, platelet activation, and endothelial cell activation.

RESULTS

Changes were observed in some hemostatic markers during the normobaric exposure, attributed to prolonged sitting and circadian variation. However, there were no significant differences between the changes in the hypobaric and the normobaric exposures. For example, the median difference in change between the hypobaric and normobaric exposure was 0 ng/mL for thrombin-antithrombin complex (95% CI, -0.30 to 0.30 ng/mL); -0.0<em>2</em> [corrected] nmol/L for <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (95% CI, -0.03 to 0.0<em>1</em> nmol/L); <em>1</em>.38 ng/mL for D-dimer (95% CI, -3.63 to 9.7<em>2</em> ng/mL); and -<em>2</em>.00% for endogenous thrombin potential (95% CI, -4.00% to <em>1</em>.00%).

CONCLUSIONS

Our findings do not support the hypothesis that hypobaric hypoxia, of the degree that might be encountered during long-haul air travel, is associated with prothrombotic alterations in the hemostatic system in healthy individuals at low risk of venous thromboembolism.

OBJECTIVE

The goal of this study was to investigate whether omega-3 polyunsaturated fatty acids (n-3 PUFA) are able to alter plasma fibrin clot properties and reduce thrombin formation in stable coronary artery disease patients undergoing percutaneous coronary intervention (PCI).

RESULTS

In an investigator-initiated, prospective, double-blind, placebo-controlled, randomized study, patients undergoing PCI who received standard pharmacotherapy were assigned to the treatment with <em>1</em> g/day n-3 PUFA (n = 30) or placebo (n = <em>2</em>4) for <em>1</em> month. Plasma fibrin clot permeability (K(s)); lysis time (t(50%)); <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em>; and peak thrombin generation from automated thrombogram, 8-isoprostaglandin F(<em>2</em>α) (8-iso-PGF(<em>2</em>α), an oxidative stress marker), and C-reactive protein were determined at baseline, 3 to 5 days after randomization, and 30 days after randomization. At baseline, both treatment groups did not differ significantly. A <em>1</em>-month treatment with n-3 PUFA compared with placebo was associated with <em>1</em>5.3% higher K(s), indicating larger pores in the fibrin network (P = 0.0005); <em>1</em>4.3% shorter t(50%), indicating increased susceptibility to fibrinolysis (P<0.000<em>1</em>); 33.8% lower <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (P = 0.00<em>1</em>3); <em>1</em>3.4% lower peak thrombin generation (P = 0.04); and <em>1</em>3.<em>1</em>% lower 8-iso-PGF(<em>2</em>α) (P = 0.009). Treatment with n-3 PUFA had no effect on fibrinogen and C-reactive protein. After <em>1</em> month of treatment, fibrinogen (r = -0.53, P<0.000<em>1</em>), treatment assignment (r = 0.<em>2</em>9, P = 0.006) and 8-iso-PGF(<em>2</em>α) (r = -0.<em>2</em>7, P = 0.0<em>1</em>5) were independently associated with clot permeability (P<0.000<em>1</em>, R(<em>2</em>) = 0.66).

CONCLUSIONS

Adding n-3 PUFA to standard therapy in stable patients undergoing PCI significantly decreases thrombin formation and oxidative stress and favorably alters fibrin clot properties. These findings indicate novel antithrombotic effects induced by n-3 PUFA in humans.

BACKGROUND

Bullous pemphigoid (BP) is a blistering skin disease caused by autoantibodies to hemidesmosomal proteins, with eosinophils participating in blister formation. Eosinophils are a source of tissue factor (TF), an initiator of blood coagulation.

OBJECTIVE

To evaluate the local and systemic activation of coagulation in BP.

METHODS

We studied <em>2</em>0 patients with active BP (eight re-evaluated during remission) and 40 controls. The coagulation markers <em>prothrombin</em> <em>fragment</em> F1+<em>2</em> and d-dimer were measured in the plasma of all subjects and in both plasma and blister fluid of patients with BP. TF was evaluated immunohistochemically in skin specimens from the <em>2</em>0 patients and in <em>2</em>0 normal samples.

RESULTS

F1+<em>2</em> and d-dimer levels were higher in plasma of patients with BP (649 +/- 96 pmol L(-1) and 18.5<em>2</em> +/- 3.44 nmol L(-1), respectively) than in plasma of controls (157 +/- 7 pmol L(-1) and 1.4<em>2</em> +/- 0.06 nmol L(-1); P = 0.0001), and were very high in blister fluid (40 449 +/- 3491 pmol L(-1) and 153<em>2</em>.3<em>2</em> +/- <em>2</em>6<em>2</em>.81 nmol L(-1); P = 0.0001). Plasma and blister fluid F1+<em>2</em> and d-dimer levels paralleled blood and tissue eosinophilia and disease severity. In the eight patients re-evaluated during remission, there was a marked reduction in F1+<em>2</em> (from 11<em>2</em>7 +/- 144 to <em>2</em>87 +/- 5<em>2</em> pmol L(-1); P = 0.005) and d-dimer (from <em>2</em>4.03 +/- 4.08 to 4.69 +/- 1.51 nmol L(-1); P = 0.0<em>2</em>9). Immunohistochemistry revealed strong TF reactivity in BP skin (P = 0.0001), and colocalization studies confirmed eosinophils as a source of TF.

CONCLUSIONS

The coagulation cascade is activated in BP and correlates with the severity of the disease and with eosinophilia, indicating that eosinophils play a role in coagulation activation via TF. The hypercoagulability may contribute to inflammation, tissue damage, blister formation and possibly thrombotic risk in BP.

The Oxford Cholesterol Study is a randomized placebo-controlled trial designed primarily to assess the effects of simvastatin on blood cholesterol levels and side-effects in preparation for a large, long-term trial of the effects of cholesterol-lowering drug therapy on mortality. At present there is only limited evidence from randomized comparisons of the effects of HMG-CoA reductase inhibitors, such as simvastatin, on thrombogenic, as distinct from atherogenic, pathways in coronary heart disease. The present sub-study was carried out to assess the effects of simvastatin on a range of haemostatic variables, as well as on free fatty acids and on lipoprotein fractions not studied in detail previously. At an average of about <em>2</em> years after starting study treatment, non-fasting blood samples were obtained from a sequential sample of <em>1</em>6<em>2</em> participants who had been randomly allocated to receive 40 mg (54 patients) or <em>2</em>0 mg (57 patients) daily simvastatin or matching placebo treatment (5<em>1</em> patients). Only patients who reported taking their study treatment and who were not known to be diabetic or to be taking some other lipid lowering treatment were to be included. The principal comparisons were to be of those allocated simvastatin (i.e. <em>2</em>0 and 40 mg doses combined) vs those allocated placebo. Among patients allocated simvastatin, marginally significant lower factor VII antigen levels (<em>1</em><em>2</em>.<em>1</em>0% +/- 6.08 of standard; <em>2</em>P < 0.05) and non-significantly lower factor VII coagulant activity (8.<em>2</em>4% +/- 4.99 of standard) and fibrinogen concentrations (0.<em>1</em>0 +/- 0.08 g. l-<em>1</em>) were observed. In contrast, plasminogen activator inhibitor activity was significantly higher (<em>2</em>.6<em>2</em> +/- <em>1</em>.03 IU; <em>2</em>P < 0.0<em>1</em>) among patients allocated simvastatin. No significant differences were seen in the other haemostatic factors studied (e.g. <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em>, factor XII and C<em>1</em> inhibitor). Total free fatty acid concentration was marginally significantly reduced (<em>2</em>P = 0.0<em>2</em>) with simvastatin, but none of the reductions in individual free fatty acids was significant. Lipoprotein fractions were only measured among patients allocated 40 mg daily simvastatin or placebo. Compared with placebo, simvastatin produced significant decreases not only in LDL cholesterol (<em>1</em>.74 +/- 0.<em>1</em>5 mmol.<em>1</em>(-<em>1</em>): <em>2</em>P < 0.000<em>1</em>) but also in VLDL cholesterol (0.<em>2</em>8 +/- 0.08 mmol.<em>1</em>(-<em>1</em>); <em>2</em>P < 0.00<em>1</em>) and IDL cholesterol (0.<em>1</em>7 +/- 0.03 mmol.<em>1</em>(-<em>1</em>); <em>2</em>P < 0.000<em>1</em>). There were also lower triglyceride levels associated with LDL (0.07 +/- 0.0<em>1</em> mmol.<em>1</em>(-<em>1</em>); <em>2</em>P < 0.000<em>1</em>), IDL (0.03 +/- 0.0<em>1</em> mmol.<em>1</em>(-<em>1</em>); <em>2</em>P < 0.0<em>1</em>) and VLDL (0.<em>2</em>7 +/- 0.<em>1</em>4; <em>2</em>P = 0.05). The effects of simvastatin on haemostatic variables appear to be far less marked than its lipid effects. Given the associations of haemostatic factors with coronary heart disease incidence, larger randomized comparisons of the HMG-CoA reductase inhibitors (and of the newer fibrates which may produce greater effects) are needed to provide more reliable estimates of the extent to which they influence these variables.

<em>Prothrombin</em> is the zymogen precursor of the clotting enzyme thrombin, which is generated by two sequential cleavages at R<em>2</em>7<em>1</em> and R3<em>2</em>0 by the <em>prothrombin</em>ase complex. The structure of <em>prothrombin</em> is currently unknown. Prethrombin-<em>1</em> differs from <em>prothrombin</em> for the absence of <em>1</em>55 residues in the N-terminal domain and is composed of a single polypeptide chain containing <em>fragment</em> <em>2</em> (residues <em>1</em>56-<em>2</em>7<em>1</em>), A chain (residues <em>2</em>7<em>2</em>-3<em>2</em>0), and B chain (residues 3<em>2</em><em>1</em>-579). The X-ray crystal structure of prethrombin-<em>1</em> solved at <em>2</em>.<em>2</em>-Å resolution shows an overall conformation significantly different (rmsd = 3.6 Å) from that of its active form meizothrombin desF<em>1</em> carrying a cleavage at R3<em>2</em>0. <em>Fragment</em> <em>2</em> is rotated around the y axis by <em>2</em>9° and makes only few contacts with the B chain. In the B chain, the oxyanion hole is disrupted due to absence of the I<em>1</em>6-D<em>1</em>94 ion pair and the Na(+) binding site and adjacent primary specificity pocket are highly perturbed. A remarkable feature of the structure is that the autolysis loop assumes a helical conformation enabling W<em>1</em>48 and W<em>2</em><em>1</em>5, located <em>1</em>7 Å apart in meizothrombin desF<em>1</em>, to come within 3.3 Å of each other and completely occlude access to the active site. These findings suggest that the zymogen form of thrombin possesses conformational plasticity comparable to that of the mature enzyme and have significant implications for the mechanism of <em>prothrombin</em> activation and the zymogen → protease conversion in trypsin-like proteases.

OBJECTIVE

To assess the influence of tourniquet inflation-deflation as well as desmopressin and tranexamic acid (TA) administration on prothrombin fragment 1.2, fibrinogen, plasmin antiplasmin complex, and D-dimer concentrations during total knee replacement.

METHODS

Randomized, placebo-controlled study.

METHODS

Large referral hospital.

METHODS

30 ASA physical status I, II, and III patients undergoing total knee replacement.

METHODS

Patients were randomized to one of three treatment groups. Patients received either tranexamic acid, desmopressin, or an equal volume of saline, intravenously.

RESULTS

Cubital blood was drawn immediately before induction of anesthesia, 1 hour after tourniquet application, and 2 and 15 minutes after tourniquet deflation. Fibrinogen and D-dimer levels were measured using the Clauss Method and latex agglutination, respectively. Plasmin antiplasmin complex and prothrombin fragment 1.2 levels were measured by enzyme-linked immunosorbent assay (ELISA). All assays were performed in duplicate, and intra-assay variability was documented. No statistically significant difference in fibrinogen, D-dimer, plasmin antiplasmin complex, or prothrombin fragment 1.2 levels was demonstrated among the groups. Similarly, within each group there were no statistically significant differences in the variables studied. However, despite the lack of statistical significance, when compared with their levels during tourniquet application, an increase in D-dimer and plasmin antiplasmin complex levels was observed in all three groups at 2 and 15 minutes after tourniquet release. In contrast, no increase in prothrombin fragment 1.2 generation was noted. Significantly more allogeneic blood was transfused in the Control and Desmopressin Groups when compared with the tranexamic acid group (p< 0.02).

CONCLUSIONS

No evidence of tourniquet-induced fibrinolysis or thrombin generation was demonstrated in the systemic circulation. Desmopressin and tranexamic acid had no significant effect on the variables measured.

BACKGROUND

High-grade gliomas (HGGs) are among the most prothrombotic of malignancies.

METHODS

We performed a prospective study to investigate <em>1</em><em>1</em> potential biomarkers for prediction of venous thromboembolism (VTE) in newly diagnosed HGG patients who had undergone a neurosurgical intervention. In addition, we tested <em>2</em> VTE risk assessment models (RAMs). The strongest predictors of VTE, which were identified by statistical forward selection, were used for the first RAM. The parameters used for the second RAM were both predictive of VTE and available in routine clinical practice.

RESULTS

One hundred forty-one HGG patients were included in this study, and <em>2</em>4 (<em>1</em>7%) of them developed VTE during follow-up. An association with the risk of future VTE was found for the following parameters: leukocyte count, platelet count, sP-selectin, <em>prothrombin</em>-<em>fragment</em> <em>1</em> + <em>2</em>, FVIII activity, and D-dimer. The first RAM included low platelet count ((<em>2</em>5th percentile of the study population) and elevated sP-selectin (≥75th percentile). The cumulative VTE probability after <em>1</em><em>2</em> months was 9.7% for score 0 (n = 76), <em>1</em>8.9% for score <em>1</em> (n = 59), and 83.3% for score <em>2</em> (n = 6). The second RAM included low platelet count ((<em>2</em>5th percentile), elevated leukocyte count, and elevated D-dimer (≥75th percentile). The probability of VTE was 3.3% for score 0 (n = 63), <em>2</em>3.0% for score <em>1</em> (n = 53), and 37.7% for score <em>2</em> (n = <em>2</em><em>2</em>) or score 3 (n = 3).

CONCLUSIONS

We identified biomarkers suitable for assessing the VTE risk in newly diagnosed HGG patients. The application of <em>2</em> RAMs allowed identification of patients at high risk of developing VTE. We could also define patients at low risk of VTE, who would most probably not benefit from extended primary thromboprophylaxis.

BACKGROUND

Atrial fibrillation (AF) is associated with cognitive impairment and dementia, perhaps through encouraging a prothrombotic state and cardioembolism.

OBJECTIVE

We wished to test the hypotheses that hemostatic function is altered in subjects with AF who develop dementia, and that long-term warfarin anticoagulation is protective against this complication.

METHODS

Recruitment was from an observational cohort study of AF. Baseline assessment included measurement of plasma fibrinogen, fibrin D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complexes (TAT), von Willebrand factor and tissue plasminogen activator. We assessed cognitive function after 3 years' follow-up using the <em>1</em>3-item modified Telephone Interview for Cognitive Status (TICSm) and the short form of the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE).

RESULTS

Of the <em>2</em><em>1</em>8 subjects assessed, <em>1</em>45 (66%) were prescribed warfarin. Forty-nine (<em>2</em><em>2</em>%) met TICSm/IQCODE criteria for dementia. D-dimer, F<em>1</em>+<em>2</em> and TAT levels were higher in AF subjects with dementia compared with those without (medians 8<em>1</em> vs. 60 ng mL(-<em>1</em>), P = 0.008; 0.76 vs. 0.49 nmol L(-<em>1</em>), P = 0.006; and <em>1</em>.78 vs. <em>1</em>.44 microg L(-<em>1</em>), P = 0.003, respectively). These associations became of borderline statistical significance following adjustment for age. Logistic regression showed a trend towards warfarin use being independently associated with reduced prevalence of dementia (odds ratio 0.5<em>2</em>, P = 0.08).

CONCLUSIONS

We found evidence of increased thrombin generation and fibrin turnover in subjects with AF and dementia compared with those without dementia. Long-term warfarin use may be protective against the development of dementia in subjects with AF.

This cross-sectional study was conducted to determine the incidence of autoantibodies to phospholipids and coagulation proteins in children with acute varicella zoster virus (VZV) infection. Study groups included children with VZV alone or complicated by purpura fulminans and/or thromboembolism. VZV naïve children and children who had VZV>><em>1</em> y before sample collection formed a control group. Blood was assayed for the following: free protein S (PS), protein C, antithrombin, and <em>prothrombin</em>; antibody binding to these proteins; lupus anticoagulant; anticardiolipin antibody; antiphospholipid antibodies; and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>. Data regarding coinfections was collected. Forty-three VZV-infected children showed an increased frequency of lupus anticoagulant, anticardiolipin antibody, antiphospholipid antibodies, and autoantibodies to PS, protein C, <em>prothrombin</em>, and antithrombin in comparison to 5<em>2</em> children without acute VZV (p < 0.000<em>1</em>). Seventeen children with VZV and purpura fulminans and/or thromboembolism showed a statistically significant decrease in free PS, significantly increased PS IgG antibody, and significantly increased <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (p < 0.000<em>1</em>) compared with the group without acute VZV and the group with uncomplicated VZV. Twenty-six children with uncomplicated VZV showed increased PS IgG antibody (p < 0.00<em>1</em>) compared with the children without acute VZV. For all groups combined, elevated PS IgG antibody showed negative correlation with free PS (p < 0.000<em>1</em>) and positive correlation with <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (p = 0.000<em>2</em>). Autoantibodies were transient. Transient antiphospholipid and coagulation protein autoantibodies were common with VZV infection, but were not predictive of thrombotic complications.