Inflammation, angiogenesis, and coagulation are linked to the development of cancer. In glioblastoma, microvascular proliferation is a hallmark, and lymphocytic infiltration is a common finding. Thromboses are frequent in patients with glioblastoma. The objective of this study was to assess presurgical levels of circulating markers of inflammation, angiogenesis, and coagulation in a prospective series of patients with glioblastoma, and to explore their correlations and possible associations with clinical findings. Angiogenesis markers included were vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor-receptor <em>1</em> (sVEGFR-<em>1</em>), and thrombospondin-<em>1</em> (TSP-<em>1</em>). Inflammatory markers included were C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and sialic acid (SA). Coagulation markers included were fibrinogen (Fg), endogen thrombin generation (ETG), <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), and tissue factor (TF). Forty-seven patients and 60 healthy subjects were included in the study. Signs of tumor necrosis in presurgical MRI were associated with shorter survival (P < 0.0<em>1</em>). All inflammation markers, F<em>1</em> + <em>2</em>, ETG, VEGF and sVEGFR-<em>1</em>, were significantly elevated in glioblastoma patients. Correlations were found between ETG and Fg (r = 0.44, P < 0.0<em>1</em>). Sialic acid correlated with Fg (r = 0.63, P < 0,00<em>1</em>); CPR correlated with SA (r = 0.60, P < 0.00<em>1</em>), Fg (r = 0.76, P < 0.00<em>1</em>), TNFα (r = 0.56, P < 0.00<em>1</em>), and IL-6 (r = 0.65, P < 0.00<em>1</em>); and IL-6 also correlated positively with TNFα (r = 0.40, P < 0.0<em>2</em>) and Fg (r = 0.45, P < 0.0<em>1</em>). Vascular endothelial growth factor inversely correlated with sVEGFR-<em>1</em> (r = -0.35, P < 0.0<em>2</em>). No associations were found between marker levels and survival or progression-free survival.

OBJECTIVE

Early clinical progression of ischemic stroke is common and is associated with increased risk of death and dependency. We hypothesized that activation of the coagulation system is an important contributor in some cases of deterioration. We aimed to characterize alterations in circulating hemostatic markers in patients with progressing stroke.

METHODS

Consecutive acute ischemic stroke admissions were recruited. Progressing stroke was defined by deterioration in components of the Scandinavian Stroke Scale. Hemostatic markers (coagulation factors VIIc, VIIIc, and IXc, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>], thrombin-antithrombin complexes [TAT], D-dimer, fibrinogen, von Willebrand factor [vWF] and tissue plasminogen activator) were measured within <em>2</em>4 hours of symptom recognition.

RESULTS

Fifty-four (<em>2</em>5%) of the <em>2</em><em>1</em>9 patients met criteria for progressing stroke. F<em>1</em>+<em>2</em> (median <em>1</em>.<em>2</em>8 versus <em>1</em>.06 nmol/L, P=0.0<em>1</em>), TAT (5.<em>2</em>8 versus 4.07 microg/L, P<0.0<em>1</em>), D-dimer (443 versus <em>1</em>94 ng/mL, P<0.00<em>1</em>) and vWF (<em>2</em><em>1</em>6 versus <em>1</em>98 IU/dL, P<0.05) levels were higher in these patients than in stable/improving patients. In logistic regression analysis, with all important clinical and laboratory variables included, only natural log D-dimer (odds ratio [OR]: <em>1</em>.87; 95% confidence interval [CI]: <em>1</em>.38 to <em>2</em>.54; P=0.000<em>1</em>) and mean arterial blood pressure (OR: <em>1</em>.<em>2</em>6 per <em>1</em>0 mm Hg change; 95% CI: <em>1</em>.05 to <em>1</em>.5<em>1</em>; P=0.0<em>1</em>) remained independent predictors of progressing stroke.

CONCLUSIONS

There is evidence of excess thrombin generation and fibrin turnover in patients with progressing ischemic stroke. Measurement of D-dimer levels can identify patients at high risk for stroke progression. Further research is required to determine whether such patients benefit from acute interventions aimed at modifying hemostatic function.

The aim of the present study was to investigate aspects of coagulation and fibrinolysis during knee arthroplasties in order to find out. <em>1</em>. whether an increased fibrinolysis is correlated to an increased blood loss <em>2</em>. whether there is a difference in markers for coagulation and fibrinolysis in peripheral venous blood compared to those in blood from the wounds 3. whether the administration of tranexamic acid modifies the fibrinolytic response. Twenty-four patients were included. Twelve patients were given tranexamic acid intravenously at the end of the operation. The dose was repeated three hours later. The other <em>1</em><em>2</em> patients were given an equivalent amount of placebo. The administration was randomised and double-blind. Levels of <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, D-dimers, plasminogen, alpha <em>2</em>-antiplasmin, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI-<em>1</em>) in venous blood were investigated just before the operation, at the end of the operation and three hours later. At the end of the operation blood for analysis was also drawn from the wound. Coagulation and fibrinolysis was activated during and after surgery. The activation was significantly higher in blood from the wounds than in peripheral venous blood. We found no direct correlation between the degree of fibrinolysis and blood loss. The administration of tranexamic acid reduced fibrinolysis in the wounds but not in peripheral venous blood. The postoperative blood loss was reduced by half.

A report is presented of a young, otherwise apparently healthy, woman who had three pregnancies which for some unknown reason terminated in intrauterine death (macerated foetuses). During the third pregnancy a coagulation defect was diagnosed, which was characterized by prolonged coagulation times and prolonged one-stage <em>prothrombin</em> time. This defect disappeared after the end of the pregnancy, but returned during the fourth pregnancy. This time a circulating anticoagulant was found, which inhibited the action of thromboplastin. The values found for the various coagulation factors were normal. The anticoagulant titre rose during the pregnancy from <em>1</em>/<em>2</em> to <em>1</em>/<em>1</em>0. Leucocyte agglutinating as well as lymphocytotoxic antibodies directed against the husband's cells were demonstrated in the patient during the pregnancy. In this case, by passage of cell <em>fragments</em> and thromboplastic substances to the mother, the foetus had probably induced the development of antibodies against the foetal tissues. The foetus may be regarded as an incompatible transplant. The fourth pregnancy was terminated by caesarean section in the 34th week. The child weighed <em>1</em>440 g and, after three exchanges of blood, did very well. The placenta was severely infarcted. It is postulated that the development of antithromboplastin during pregnancy may be a contributory cause of intrauterine death.

Both human and bovine <em>prothrombin</em> <em>fragment</em> <em>2</em> (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.<em>1</em>55 and 0.<em>1</em>57, respectively) at 3.3-A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle <em>2</em>. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K<em>1</em> and K4 and TPA K<em>2</em>. The anionic motif is DGDEE in <em>prothrombin</em> kringle <em>2</em>. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of <em>fragment</em> <em>2</em> is different from that of <em>prothrombin</em> kringle <em>1</em> and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin beta-turn of the second inner loop pivots at Val64 and Asp70 by 60 degrees. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the <em>fragment</em> <em>2</em>-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since <em>fragment</em> <em>2</em> has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K<em>2</em> and the urokinase kringle. The movement of the Val64-Asp70 beta-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The <em>fragment</em> <em>2</em>-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca<em>2</em>+ <em>prothrombin</em> <em>fragment</em> <em>1</em> with a Gla domain and a kringle.

Crystal polymorphism is exhibited by calcium oxalates in nephrolithiasis, and we have proposed that a shift in the preferred crystalline form of calcium oxalate (CaOx) from monohydrate (COM) to dihydrate (COD) induced by urinary macromolecules reduces crystal attachment to epithelial cell surfaces, thus potentially inhibiting a critical step in the genesis of kidney stones. We have tested the validity of this hypothesis by studying both the binding of monohydrate and dihydrate crystals to renal tubule cells and the effect of macromolecular urinary solutes on crystal structure. Renal tubule cells grown in culture bound 50% more CaOx monohydrate than dihydrate crystals of comparable size. The effects of macromolecules on the spontaneous nucleation of CaOx were examined in HEPES-buffered saline solutions containing Ca<em>2</em>+ and C<em>2</em>O4(<em>2</em>-) at physiologic concentrations and supersaturation. Many naturally occurring macromolecules known to be inhibitors of crystallization, specifically osteopontin, nephrocalcin and urinary <em>prothrombin</em> <em>fragment</em> <em>1</em>, were found to favor the formation of calcium oxalate dihydrate in this in vitro system, while other polymers did not affect CaOx crystal structure. Thus, the natural defense against nephrolithiasis may include impeding crystal attachment by an effect of macromolecular inhibitors on the preferred CaOx crystal structure that forms in urine.

Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates <em>prothrombin</em> (ProT) without proteolysis. The SC.(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC <em>fragment</em> containing residues <em>1</em>-3<em>2</em>5 (SC-(<em>1</em>-3<em>2</em>5)) bound to human prethrombin <em>2</em> showed previously that SC inserts its Ile(<em>1</em>)-Val(<em>2</em>) N terminus into the Ile(<em>1</em>6) pocket of prethrombin <em>2</em>, inducing a functional active site in the cognate zymogen conformationally. Exosite I of alpha-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain <em>2</em> of SC-(<em>1</em>-3<em>2</em>5). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(<em>1</em>-3<em>2</em>5).ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(<em>1</em>-3<em>2</em>5).ProT complex, resulting in formation of an (SC-(<em>1</em>-3<em>2</em>5).ProT)(<em>2</em>).Fbg pentameric complex with a dissociation constant of 8-34 nm. In both crystal structures, the SC-(<em>1</em>-3<em>2</em>5).(pre)thrombin complexes form dimers, with both proteinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(<em>1</em>-3<em>2</em>5).thrombin)(<em>2</em>).Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC.(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis.

Thromboembolic complications are common in patients with malignant disease. We studied the activation of coagulation in <em>1</em>06 patients with solid tumours and 7<em>2</em> healthy volunteers by measuring plasma levels of tissue factor, factor VIIa, factor XIIa, thrombin-antithrombin complex, and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>. Tissue factor was 67% higher in cancer patients (median 58<em>2</em> vs 349 pg/mL, p = 0.0006) and factor VIIa was 46% higher (<em>1</em>00 vs 69 mU/mL, p = 0.000<em>2</em>), indicating extrinsic pathway activation. Modest activation of the intrinsic pathway (elevated factor XIIa) was seen only in patients with advanced disease or those receiving chemotherapy. Excess thrombin generation was manifested by elevations in thrombin-antithrombin complex and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>. Tissue factor pathway is clearly implicated in the hypercoagulable state of cancer.

Insulin resistance is associated with a low chronic inflammatory state. In this study we investigated the relationship between impaired insulin sensitivity and selected markers of inflammation and thrombin generation in obese healthy women. We examined 3<em>2</em> healthy obese women (body mass index>> or = <em>2</em>8), with normal insulin sensitivity (NIS, n = <em>1</em>4) or impaired insulin sensitivity (n = <em>1</em>8), and <em>1</em>0 nonobese women (body mass index < <em>2</em>5). Impaired insulin sensitivity patients had significantly higher levels of C-reactive protein (CRP), TGF-beta <em>1</em>, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), activated factor VII (VIIa), and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) compared with either control subjects or NIS patients. On the other hand, NIS patients had higher CRP, TGF-beta <em>1</em>, PAI-<em>1</em>, and factor VIIa, but not F<em>1</em> + <em>2</em>, levels than controls. Significant inverse correlations were observed between the insulin sensitivity index and TGF-beta <em>1</em>, CRP, PAI-<em>1</em>, factor VIIa, and F<em>1</em> + <em>2</em> levels. Moreover, significant direct correlations were noted between TGF-beta <em>1</em> and CRP, PAI-<em>1</em>, factor VIIa, and F<em>1</em> + <em>2</em> concentrations. Finally, multiple regressions revealed that TGF-beta <em>1</em> and the insulin sensitivity index were independently related to F<em>1</em> + <em>2</em>. Our results are the first to document an in vivo relationship between insulin sensitivity and coagulative activation in obesity. The elevated TGF-beta <em>1</em> levels detected in the obese population may provide a biochemical link between insulin resistance and an increased risk for cardiovascular disease.

OBJECTIVE

Low high-density lipoprotein (HDL) cholesterol is a strong independent cardiovascular risk factor, which has been attributed to its role in reverse cholesterol transport. Whereas HDL also has potent antiinflammatory effects, the relevance of this property remains to be established in humans. In the present study, we evaluated whether there is a relation between HDL and sensitivity toward a low-dose endotoxin challenge.

RESULTS

Thirteen healthy men with genetically determined isolated low HDL cholesterol (averaging 0.7+/-0.<em>1</em> mmol/L) and <em>1</em>4 age- and body weight-matched healthy men with normal/high HDL cholesterol levels (<em>1</em>.9+/-0.4 mmol/L) were challenged with low-dose endotoxin intravenously (<em>1</em> ng/kg body weight). The incidence and severity of endotoxin-associated clinical symptoms was increased in the low HDL group. Accordingly, both the inflammatory response (tumor necrosis factor-alpha, IL-<em>1</em>beta, IL-6, IL-8, and monocyte chemoattractant protein-<em>1</em>) as well as thrombin generation (<em>prothrombin</em> activation <em>fragments</em> F(<em>1</em>+<em>2</em>)) were significantly increased in the low HDL group on endotoxin challenge.

CONCLUSIONS

Low HDL in healthy males is associated with increased sensitivity toward inflammatory stimuli as reflected by enhanced inflammatory and coagulation responses on endotoxin challenge. These antiinflammatory effects of HDL in humans may lend further support to HDL-increasing interventions, particularly in proinflammatory conditions, such as acute coronary syndromes.

We compared the effects of oral estradiol (<em>2</em> mg), transdermal estradiol (50 microg), and placebo on measures of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in <em>2</em>7 postmenopausal women at baseline and after <em>2</em> and <em>1</em><em>2</em> weeks of treatment. Oral and transdermal estradiol induced similar increases in serum free estradiol concentrations. Oral therapy increased the plasma concentrations of factor VII antigen (FVIIag) and activated factor VII (FVIIa), and the plasma concentration of the <em>prothrombin</em> activation marker <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>). Oral but not transdermal estradiol therapy significantly lowered plasma plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) antigen and tissue-type plasminogen activator (tPA) antigen concentrations and PAI-<em>1</em> activity, and increased D-dimer concentrations, suggesting increased fibrinolysis. The concentration of soluble E-selectin decreased and serum C-reactive protein (CRP) increased significantly in the oral but not in the transdermal or placebo groups. In the oral but not in the transdermal or placebo estradiol groups low-density-lipoprotein (LDL) cholesterol, apolipoprotein B and lipoprotein (a) concentrations decreased while high-density-lipoprotein (HDL) cholesterol, apolipoprotein AI and apolipoprotein All concentrations increased significantly. LDL particle size remained unchanged. In summary, oral estradiol increased markers of fibrinolytic activity, decreased serum soluble E-selectin levels and induced potentially antiatherogenic changes in lipids and lipoproteins. In contrast to these beneficial effects, oral estradiol changed markers of coagulation towards hypercoagulability, and increased serum CRP concentrations. Transdermal estradiol or placebo had no effects on any of these parameters. These data demonstrate that oral estradiol does not have uniformly beneficial effects on cardiovascular risk markers and that the oral route of estradiol administration rather than the circulating free estradiol concentration is critical for any changes to be observed.

Tumor necrosis factor (TNF) is considered to be a pivotal mediator of endotoxin-induced lethality. To assess the intermediate role of TNF in specific systemic inflammatory responses known to contribute to tissue injury in endotoxemia, eight healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In four of these animals the administration of endotoxin was followed immediately by a bolus intravenous injection of an anti-TNF monoclonal antibody (<em>1</em>5 mg/kg). Treatment with anti-TNF completely prevented the endotoxin-induced increase in serum TNF activity, and profoundly reduced the appearance of interleukin-6 and -8 (both P < .05). Neutrophilia and lymphopenia were not affected by anti-TNF, whereas neutrophil degranulation, as measured by the plasma concentrations of elastase-alpha <em>1</em>-antitrypsin complexes, was only slightly reduced (peak levels after endotoxin alone 3<em>1</em>.0 +/- 3.4 ng/mL, versus <em>2</em>5.5 +/- 3.4 ng/mL after endotoxin with anti-TNF; P < .05). Anti-TNF did not influence endotoxin-induced activation of the coagulation system, as reflected by unchanged increases in the plasma concentrations of the <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> and thrombin-antithrombin III complexes. In contrast, anti-TNF strongly attenuated the activation of the fibrinolytic system, ie, peak plasma levels of plasmin-alpha <em>2</em>-antiplasmin were 33.8 +/- <em>1</em><em>1</em>.<em>1</em> nmol/L after endotoxin alone and <em>1</em>7.0 +/- <em>2</em>.9 nmol/L after endotoxin with anti-TNF (P < .05). These results suggest that TNF is not the common mediator of systemic inflammatory changes in low-grade endotoxemia. Moreover, the finding that in this mild model anti-TNF specifically inhibited fibrinolysis suggests that treatment with anti-TNF potentially may enhance the tendency towards microvascular thrombosis in sepsis.

BACKGROUND

Paroxysmal nocturnal hemoglobinuria (PNH) is associated with an increased risk of thrombosis through unknown mechanisms.

METHODS

We studied <em>2</em>3 patients with PNH, before and after five and <em>1</em><em>1</em> weeks of treatment with eculizumab. We examined markers of thrombin generation and reactional fibrinolysis (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), D-dimers, and plasmin antiplasmin complexes (P-AP), and endothelial dysfunction tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-<em>1</em>), soluble thrombomodulin (sTM), intercellular adhesion molecule <em>1</em> (sICAM-<em>1</em>), vascular cell adhesion molecule (sVCAM-<em>1</em>), endothelial microparticles (EMPs), and tissue factor pathway inhibitor (TFPI).

RESULTS

At baseline, vWF, sVCAM-<em>1</em>, the EMP count, and F<em>1</em>+<em>2</em> and D-dimer levels were significantly elevated in the patients, including those with no history of clinical thrombosis. Treatment with eculizumab was associated with significant decreases in plasma markers of coagulation activation (F<em>1</em>+<em>2</em>, P=0.0<em>1</em><em>2</em>, and D-dimers, P=0.0<em>1</em>), and reactional fibrinolysis (P-AP, P=0.000<em>2</em>). Eculizumab treatment also significantly reduced plasma markers of endothelial cell activation (t-PA, P=0.0005, sVCAM-<em>1</em>, P<0.000<em>1</em>, and vWF, P=0.0047) and total (P=0.0008) and free (P=0.00<em>1</em>3) TFPI plasma levels.

CONCLUSIONS

Our results suggest a new understanding of the contribution of endothelial cell activation to the pathogenesis of thrombosis in PNH. The terminal complement inhibitor, eculizumab, induced a significant and sustained decrease in the activation of both the plasma hemostatic system and the vascular endothelium, likely contributing to the protective effect of eculizumab on thrombosis in this setting.

OBJECTIVE

Increased cardiovascular (CV) risk is a rheumatoid arthritis (RA) hallmark and it has been mainly related to chronic systemic inflammation. Since inflammation is linked to coagulation perturbation, both may play a role in increasing CV risk. Treatment with tumor necrosis factor (TNF)-alpha blocking agents is effective in RA and reduces local and systemic inflammation but there is little information on its effect on coagulation. We therefore investigated inflammation and coagulation plasma biomarkers before and after infliximab treatment in RA patients.

METHODS

We studied <em>2</em>0 patients with active RA and 40 healthy controls. Patients were treated with: a stable dose of methotrexate (<em>1</em>0mg/week), and infliximab (3mg/kg) at weeks 0, <em>2</em>, 6 and <em>1</em>4. At baseline and week <em>1</em>4, we determined: disease activity score (DAS-<em>2</em>8), visual analogue scale pain, erythrocyte sedimentation rate (ESR), and plasma levels of C-reactive protein (CRP), TNF-alpha, interleukin (IL)-6, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and D-dimer. The same inflammation and coagulation parameters were evaluated <em>1</em>h after infliximab infusion in <em>1</em>0 patients.

RESULTS

At baseline, ESR, CRP, TNF-alpha, IL-6, F<em>1</em>+<em>2</em> and D-dimer levels were significantly higher in RA patients than in controls (P=0.000<em>1</em>). After <em>1</em>4weeks of infliximab treatment, there was a significant clinical improvement and ESR and CRP, IL-6, F<em>1</em>+<em>2</em> and D-dimer level decrease (P=0.00<em>1</em>-P=0.008). The levels of TNF-alpha, IL-6, F<em>1</em>+<em>2</em> and D-dimer significantly decreased <em>1</em>h after infliximab infusion (P=0.005).

CONCLUSIONS

Infliximab decreases inflammation and coagulation biomarkers in RA patients. Such a combined effect may be pivotal in reducing the whole thrombotic risk in these patients.

BACKGROUND

Several cardiovascular risk factors are present in patients with chronic renal failure (CRF), among which are systemic inflammation and hyperhomocysteinemia. Increased oxidative stress, endothelial activation/dysfunction, and coagulation activation are considered integral components of the inflammatory response, but have also been proposed as mediators of plasma homocysteine (tHcy)-induced cell damage. Using correlation analysis, we assessed the relative contributions of inflammation and hyperhomocysteinemia in the abnormal oxidative stress, endothelial activation/dysfunction, and hemostasis activation in patients with CRF.

METHODS

The relationships of inflammatory proteins and tHcy with plasma markers of these processes were studied in 64 patients with CRF (serum creatinine 5<em>2</em>6 +/- 3<em>1</em>9 micromol/L) on conservative treatment, comparing the results with healthy controls (N = <em>1</em>5 to 40, depending on the measured variable) of similar sex and age.

RESULTS

Patients had significant increases in inflammatory cytokines (TNF-alpha and IL-8) and acute-phase proteins (C-reactive protein, fibrinogen and alpha<em>1</em>-antitrypsin). tHcy was increased in 87.5% of patients (mean = <em>2</em>7.<em>1</em> micromol/L, range 6.5 to <em>1</em><em>1</em>8). Patients had significant increases in (<em>1</em>) indices of oxidative stress: TBARS (thiobarbituric acid-reactive species), a marker of lipid peroxidation and AOPP (advanced oxidation protein products), a marker of protein oxidation; (<em>2</em>) endothelial cell markers such as von Willebrand factor (vWF:Ag), soluble ICAM-<em>1</em> and soluble thrombomodulin (sTM); (3) markers of intravascular thrombin generation: thrombin-antithrombin complexes (TAT) and <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) (PF(<em>1</em>+<em>2</em>)); and (4) indices of activation of fibrinolysis: plasmin-antiplasmin complexes (PAP), fibrin degradation products (FnDP) and fibrinogen degradation products (FgDP). tHcy was significantly correlated with plasma creatinine (r = 0.<em>2</em>9, P < 0.0<em>1</em>8) and with serum folate (r = -0.38, P < 0.00<em>2</em>). However, no significant correlations were observed between tHcy and TBARS, AOPP, vWF:Ag, sICAM-<em>1</em>, sTM, TAT, F(<em>1</em>+<em>2</em>), sTF, PAP, FnDP, and FgDP. Conversely, acute-phase proteins showed significant, positive correlations with most markers of oxidative stress, endothelial dysfunction and hemostatic activation.

CONCLUSIONS

Systemic inflammation, which is closely associated with augmented oxidative stress, endothelial cell dysfunction and hemostatic activation, emerges as a major cardiovascular risk factor in CRF. tHcy is unrelated to these events. Thus, alternative mechanisms through which hyperhomocysteinemia could predispose to vascular lesion and thrombotic events in CRF needs to be investigated.

BACKGROUND

Thrombelastography (TEG) is a whole blood assay to evaluate the viscoelastic properties during blood clot formation and clot lysis. Rotation thrombelastography (e.g. ROTEM) has overcome some of the limitations of classical TEG and is used as a point-of-care device in several clinical settings of coagulation disorders. Endotoxemia leads to systemic activation of the coagulation system and fibrinolysis in humans.

OBJECTIVE

We validated whether ROTEM is sensitive to endotoxin induced, tissue factor-triggered coagulation and fibrinolysis and if its measures correlate with biohumoral markers of coagulation and fibrinolysis.

METHODS

Twenty healthy male volunteers participated in this randomized placebo-controlled trial. Volunteers received either <em>2</em> ng kg(-<em>1</em>) National Reference Endotoxin or saline.

RESULTS

Endotoxemia significantly shortened ROTEM clotting time (CT) by 36% (CI 0.<em>2</em>6-0.46; P < 0.05) with a strong inverse correlation with the peak plasma levels of prothrombin fragments (F(<em>1</em> + <em>2</em>)) (r = -0.83, P < 0.05). Additionally, endotoxin infusion enhanced maximal lysis (ML) 3.9-fold (CI: <em>2</em>.5-5.<em>2</em>) compared with placebo or baseline after <em>2</em> h (P < 0.05). Peak ML and peak tissue plasminogen activator (t-PA) values correlated excellently (r = 0.8<em>2</em>, P < 0.05). ROTEM parameters clot formation time and maximal clot firmness were not affected by LPS infusion, whereas platelet function analyzer (PFA-<em>1</em>00) closure times decreased.

CONCLUSIONS

Rotation thrombelastography (ROTEM) detects systemic changes of in vivo coagulation activation, and importantly it is a point of care device, which is sensitive to changes in fibrinolysis in humans. The ex vivo measures CT and ML correlate very well with established in vivo markers of coagulation activation (F(<em>1</em> + <em>2</em>)) and fibrinolysis (t-PA), respectively.

OBJECTIVE

Major depressive disorder (MDD) is associated with low-grade inflammation, and it is considered a risk factor for coronary artery disease (CAD). CD40 ligand (CD40L) plays an important role in inflammation, platelet activation, and clotting system activation. We investigated soluble CD40L (sCD40L) expression in MDD and assessed whether it may represent a molecular mechanism that links inflammation and a prothrombotic state and whether this condition may be modified by selective serotonin reuptake inhibitor (SSRI) therapy.

METHODS

Levels of sCD40L, interleukin-<em>1</em>beta (IL-<em>1</em>beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), soluble P-selectin (sP-selectin), activated factor VII (FVIIa), and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) were measured in 46 drug-naïve, first-episode MDD patients without conventional CAD risk factors and in 46 matched healthy controls. Participants were screened between March <em>2</em>00<em>2</em> and November <em>2</em>005. Twenty of the 46 MDD patients were then randomly assigned to either sertraline <em>1</em>00 mg/day (N = <em>1</em>0) or citalopram <em>2</em>0 mg/day (N = <em>1</em>0); the aforementioned variables were measured at baseline and after 6 weeks of treatment.

RESULTS

Compared with control subjects, MDD patients had higher baseline levels of sCD40L, IL-<em>1</em>beta, IL-6, TNF-alpha, sP-selectin, FVIIa, and F<em>1</em>+<em>2</em>. In the clinical group, sCD40L levels, HAM-D total scores, and proinflammatory markers were strongly intercorrelated. In contrast, there were no significant correlations in the control group. Mood improvement achieved with SSRI therapy was associated with significant reduction in sCD40L, proinflammatory markers, and prothrombotic markers expression. (All p values < .000<em>1</em>.)

CONCLUSIONS

This pilot study shows that CD40/ CD40L pathway up-regulation in MDD patients relates increased levels of sCD40L to a prothrombotic state and, preliminarily, indicates that SSRI therapy may significantly reduce sCD40L and CD40L levels associated with proinflammatory and prothrombotic states.

Interleukin-<em>1</em>0 (IL-<em>1</em>0) has been found to inhibit lipopolysaccharide (LPS)-induced tissue factor expression by monocytes in vitro. To determine the effects of IL-<em>1</em>0 on LPS-induced activation of the hemostatic mechanisms in vivo, we performed a placebo-controlled, cross-over study of human endotoxemia. Two groups of eight volunteers were challenged with LPS (4 ng/kg) on two occasions: once in conjunction with placebo, and once with recombinant human IL-<em>1</em>0 (rhIL-<em>1</em>0; <em>2</em>5 microg/kg). In group <em>1</em>, placebo or rhIL-<em>1</em>0 was given <em>2</em> minutes before LPS challenge, group <em>2</em> received placebo or rhIL-<em>1</em>0 <em>1</em> hour after LPS administration. Pretreatment with rhIL-<em>1</em>0 reduced both LPS-induced activation of the fibrinolytic system (plasma concentrations of tissue type plasminogen activator, plasmin-alpha<em>2</em>-antiplasmin complexes, and D-dimer), and inhibition of fibrinolysis (plasma levels of plasminogen activator inhibitor <em>1</em>), whereas posttreatment only inhibited the latter response. Both IL-<em>1</em>0 pre- and posttreatment attenuated activation of the coagulation system (plasma levels of <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> and thrombin-antithrombin complexes). These results indicate that rhIL-<em>1</em>0, besides its well-described inhibitory effects on cytokine release, potently modulates the fibrinolytic system and inhibits the coagulant responses during endotoxemia.

C-reactive protein (CRP) has been suggested to exert direct adverse effects on the vasculature in experimental setups, including endothelial dysfunction and proinflammatory changes. Here, we assessed the consequences of <em>1</em>.<em>2</em>5 mg/kg highly purified recombinant human CRP, administered as an intravenous bolus, in six patients with familial hypercholesterolemia (FH) and six normocholesterolemic subjects. Endothelium-dependent and -independent vasoreactivity to serotonin and nitroprusside, respectively, were assessed using venous occlusion plethysmography before and after CRP infusion. For biochemical analyses, blood was drawn at different time points. At baseline, FH patients showed blunted endothelium-dependent vasodilation (maximum, 89.<em>2</em> +/- 30.0% vs. <em>1</em><em>1</em>7.7 +/- <em>1</em>3.<em>1</em>% in normolipidemic subjects; P = 0.037). Procoagulant activity was also higher in FH patients, illustrated by increased <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F(<em>1</em>+<em>2</em>)) levels (P = 0.030) and plasminogen activator inhibitor type-<em>1</em> (PAI-<em>1</em>) activity (P = 0.0<em>1</em>6). Upon CRP challenge, endothelium-dependent vasodilator capacity further deteriorated in FH patients (P = 0.0<em>2</em>9), whereas no change in vascular reactivity was observed in normolipidemic subjects. Additionally, coagulation activation was augmented in FH patients compared with normolipidemic subjects (P = 0.009 for F(<em>1</em>+<em>2</em>) levels; P = 0.0<em>1</em>8 and P = 0.003 for PAI-<em>1</em> antigen and activity, respectively). No difference in inflammatory responses was observed between groups. In hypercholesterolemic patients, CRP aggravates endothelial dysfunction and also evokes augmented procoagulant responses. These findings suggest that particularly in hypercholesterolemia, CRP-lowering strategies should be considered in addition to LDL reduction.

BACKGROUND

Severe community-acquired pneumonia (sCAP) is a leading cause of death worldwide. Adjunctive therapies for sCAP are needed to further improve outcome. A systemic inhibitor of coagulation, tifacogin (recombinant human tissue factor pathway inhibitor) seemed to provide mortality benefit in the sCAP subgroup of a previous sepsis trial.

OBJECTIVE

Evaluate the impact of adjunctive tifacogin on mortality in patients with sCAP.

METHODS

A multicenter, randomized, placebo-controlled, double-blind, three-arm study was conducted from July <em>2</em>005 to June <em>2</em>008 at <em>1</em>88 centers in North and South America, Europe, South Africa, Asia, Australia, and New Zealand. Adults with sCAP were randomized to receive a continuous intravenous infusion of tifacogin 0.0<em>2</em>5 mg/kg/h, tifacogin 0.075 mg/kg/h, or matching placebo over 96 hours.

RESULTS

Severity-adjusted <em>2</em>8-day all-cause mortality. Of <em>2</em>,<em>1</em>38 randomized patients, 946, <em>2</em>38, and 9<em>1</em>8 received tifacogin 0.0<em>2</em>5 mg/kg/h, tifacogin 0.075 mg/kg/h, and placebo, respectively. Tifacogin 0.075 mg/kg/h was discontinued after the first interim analysis according to prespecified futility criterion. The <em>2</em>8-day all-cause mortality rates were similar between the 0.0<em>2</em>5 mg/kg/h (<em>1</em>8%) and placebo groups (<em>1</em>7.9%) (P = 0.56). Greater reduction in <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and thrombin antithrombin complexes levels relative to baseline throughout the first 96 hours was found with tifacogin 0.0<em>2</em>5 mg/kg/h than with placebo. The incidence of adverse events and serious adverse events were comparable between the tifacogin 0.0<em>2</em>5 mg/kg/h and placebo groups.

CONCLUSIONS

Tifacogin showed no mortality benefit in patients with sCAP despite evidence of biologic activity.

GP IIb/IIIa antagonists are qualitatively different from classical antiplatelet agents, such as aspirin or clopidogrel. They do not inhibit platelet activation, i.e. intraplatelet signal generation or conduction but primarily act outside the platelet by competing with ligand (e.g. fibrinogen) binding that is essential for platelet bridging and aggregate formation. Three compounds are in clinical use: abciximab, an antibody <em>fragment</em> and two low-molecular weight compounds, tirofiban and eptifibatide. In comparison to the low-molecular weight compounds, abciximab has a substantially longer platelet half-life (4 h), i.e. slow off-rate and a short plasma half-life (<em>2</em>0-30 min) without significant distribution into the extravascular space. The plasma half-life of tirofiban and eptifibatide is about <em>2</em> h and parallels the antiplatelet effect. The off-rate from the platelet GP IIb/IIIa receptor is much faster and there is a significant distribution into the extravascular space. These pharmacokinetic variables might influence the competition between the antagonists and fibrinogen for GP IIb/IIIa binding. Other pharmacological variables are a partial agonistic activity, facilitation of thrombolysis, modification of other integrin-related actions, including inflammatory responses, effects on vascular cells and apoptosis. Importantly, GP IIb/IIIa antagonists might also interfere with <em>prothrombin</em> binding to the platelet surface and, thus, might influence the coagulation pathway. There is no clear evidence that the biological activity of the agents is modified by gene polymorphism (HPA-<em>1</em>). All three compounds may cause thrombocytopenia, possibly related to drug-induced antibodies. There is no clear data suggesting that these pharmacological differences transfer into significant differences in clinical outcome, for example in patients with acute coronary syndromes (ACS) subjected to acute percutaneous coronary interventions (PCI). The only head-to-head comparison of all three clinically used parenteral compounds did not demonstrate differences in major adverse cardiac effects (MACE) at 30 days although those have been described in particular with long-term use of oral antagonists. The inherent problems with all GP IIb/IIIa antagonists are the narrow therapeutic range because the same mechanisms are involved in hemostasis and thrombosis and their inability to inhibit platelet activation.

BACKGROUND

Patients with chronic urticaria (CU) frequently show signs of thrombin generation as a result of the activation of the extrinsic pathway of coagulation and signs of fibrinolysis as shown by slightly increased mean D-dimer plasma levels. Here, we studied patients with severe CU to see whether the activation of coagulation and fibrinolysis parallels the severity of the disease.

METHODS

Eight consecutive patients with severe exacerbations of CU and <em>1</em>3 with slight CU were studied. Plasma <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) as well as D-dimer were measured by ELISA. Serum histamine-releasing activity was assessed by basophil histamine release assay. Seventy-four normal subjects were used as controls.

RESULTS

In patients with severe CU, median levels of both D-dimer (<em>1</em><em>1</em>.<em>2</em>0 nmol/l) and F(<em>1</em>+<em>2</em>) (59<em>2</em> pmol/l) largely exceeded those found in patients with slight CU [D-dimer: <em>2</em>.66 nmol/l (P = 0.00<em>1</em>) and F(<em>1</em>+<em>2</em>): <em>2</em><em>2</em>8 pmol/l (P = 0.003)] and in normal subjects [D-dimer: <em>1</em>.4<em>1</em> nmol/l (P = 0.000<em>1</em>) and F(<em>1</em>+<em>2</em>): <em>1</em>59 pmol/l (P = 0.000<em>1</em>)]. Sera from <em>2</em>5% of patients with severe CU and 3<em>1</em>% of those with slight CU, but from none of normal subjects, showed in vitro histamine-releasing activity. D-dimer and F(<em>1</em>+<em>2</em>) levels were significantly correlated each other (r = 0.64, P = 0.00<em>2</em>) and with CU severity score (r = 0.80-0.90, P = 0.000<em>1</em>), but no correlation was observed between serum histamine-releasing activity and coagulation parameters or severity score.

CONCLUSIONS

Severe exacerbations of CU are associated with a strong activation of coagulation cascade and fibrinolysis. Whether this activation is the cause of CU or acts as an amplification system is still a matter of debate.

OBJECTIVE

To obtain systematic information on the extrinsic coagulation pathway, as well as to investigate the time course of the coagulation abnormalities in sepsis.

METHODS

Prospective observational study.

METHODS

General intensive care unit.

METHODS

Nineteen patients with the diagnosis of severe sepsis or septic shock and nine control patients.

METHODS

None.

RESULTS

Tissue factor antigen concentration (tissue factor antigen), <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, thrombin antithrombin III complex, fibrinopeptide A, D-dimer, and antithrombin III concentrations were measured on the day of diagnosis of severe sepsis and septic shock, and on days <em>1</em>, <em>2</em>, 3, and 4 after diagnosis. The concentrations of tissue factor antigen, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, fibrinopeptide A, and D-dimer were significantly increased in patients with severe sepsis and septic shock compared with control subjects. However, the concentrations of thrombin antithrombin III complex showed no statistical differences between the septic patients and the control subjects. Significantly, low antithrombin III concentrations were observed in the septic patient groups compared with control subjects. With the exception of D-dimer, the concentrations of the hemostatic markers were similar between severe sepsis and septic shock patients. Significant correlations were noted between tissue factor antigen and the disseminated intravascular coagulation score (r<em>2</em>=.<em>2</em>36, p< .000<em>1</em>) and the number of dysfunctioning organs (r<em>2</em>=.<em>2</em><em>2</em>9, p=.035).

CONCLUSIONS

We systematically elucidated coagulation disorders in newly defined sepsis. The extrinsic coagulation pathway is activated in patients with severe sepsis and septic shock. In these patients, enhanced thrombin generation and activation, and fibrin formation were demonstrated when compared with the control subjects. Furthermore, the thrombin generated appears not to be fully neutralized by antithrombin III.

OBJECTIVE

To clarify developmental changes in the pharmacokinetics and dynamics of warfarin enantiomers to establish rational pediatric dosage.

METHODS

Plasma concentrations of unbound warfarin enantiomers, vitamin K<em>1</em> and vitamin K-dependent proteins (that is, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, protein C, and the protein-induced by vitamin K absence) and international normalized ratio were measured in 38 prepubertal (<em>1</em> to <em>1</em><em>1</em> years), <em>1</em>5 pubertal (<em>1</em><em>2</em> to <em>1</em>8 years), and 8<em>1</em> adult (37 to 76 years) patients given long-term warfarin therapy. Unbound oral clearance values for warfarin enantiomers and its body weight-, body surface area-, and liver weight-normalized values, as well as the pharmacodynamic parameters, were compared among the groups.

RESULTS

The prepubertal, pubertal, and adult patients exhibited comparable mean plasma concentrations of unbound warfarin enantiomers for pharmacologically more active (S)-warfarin. Although the unbound oral clearance of (S)-warfarin for the prepubertal patients was significantly (P < .0<em>1</em>) less than that for the adult group (346 versus 637 mL/min), the body weight-normalized unbound oral clearance for the prepubertal patients was significantly (P < .0<em>1</em>) greater than that for the adults and showed a negative correlation (P < .05) with age. In contrast, no differences were observed in the liver weight-normalized unbound oral clearance for (S)-warfarin between the prepubertal and adult groups. The prepubertal patients showed significantly (P < .0<em>1</em> or .05) lower plasma concentrations of protein C and <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> and greater international normalized ratio and international normalized ratio/dose than the adults. In contrast, the pubertal patients showed largely similar pharmacokinetic and pharmacodynamic properties to adults.

CONCLUSIONS

Liver weight may be a better parameter than body weight for estimating the warfarin doses for prepubertal patients on the basis of the corresponding adult values. Augmented responses to warfarin in children should also be taken into account for estimating warfarin doses for children.