OBJECTIVE

Treatment options are limited for advanced pancreatic cancer progressive after gemcitabine therapy. The vascular endothelial growth factor pathway is biologically important in pancreatic cancer, and docetaxel has modest antitumor activity. We evaluated the role of the anti-vascular endothelial growth factor antibody bevacizumab as second-line treatment for patients with metastatic pancreatic cancer.

METHODS

Patients with metastatic adenocarcinoma of the pancreas who had progressive disease on a gemcitabine-containing regimen were randomized to receive bevacizumab alone or bevacizumab in combination with docetaxel.

RESULTS

Thirty-two patients were enrolle<em>d</em>; 16 to bevacizumab alone (Arm A) an<em>d</em> 16 to bevacizumab plus <em>d</em>ocetaxel (Arm B). Toxicities were greater in Arm B with the most common gra<em>d</em>e 3/4 nonhematologic toxicities inclu<em>d</em>ing fatigue, <em>d</em>iarrhea, <em>d</em>ehy<em>d</em>ration, an<em>d</em> anorexia. No confirme<em>d</em> objective responses were observe<em>d</em>. At 4 months, <em>2</em> of the 16 patients in Arm A an<em>d</em> 3 of the 16 patients in Arm B were free from progression. The stu<em>d</em>y was stoppe<em>d</em> accor<em>d</em>ing to the early stopping rule for futility. Me<em>d</em>ian progression-free survival an<em>d</em> overall survival were 43 <em>d</em>ays an<em>d</em> 165 <em>d</em>ays in Arm A an<em>d</em> 48 <em>d</em>ays an<em>d</em> 1<em>2</em>5 <em>d</em>ays in Arm B. Elevate<em>d</em> <em>d</em>-<em>dimer</em> levels an<em>d</em> thrombin-antithrombin complexes were associate<em>d</em> with <em>d</em>ecrease<em>d</em> survival an<em>d</em> increase<em>d</em> toxicity.

CONCLUSIONS

Bevacizumab with or without docetaxel does not have antitumor activity in gemcitabine-refractory metastatic pancreatic cancer. Baseline and on-treatment d-dimer and thrombin-antithrombin complex levels are associated with increased toxicity and decreased survival.

OBJECTIVE

The combination of D-dimer and Wells score can exclude, but not confirm, the diagnosis of deep venous thrombosis (DVT). Since thrombosis and inflammation are interrelated, we evaluated the combination of soluble P-selectin (sPsel) with other inflammatory biomarkers for the diagnosis of DVT.

METHODS

Sixty-two positive and one hundred and sixteen patients with negative DVT, by duplex scan, were prospectively evaluated for sPsel, D-dimer, C-reactive protein (CRP), microparticles (MPs; total, leukocyte, and platelet-derived and tissue factor positive microparticles), and clinical Wells score.

RESULTS

Biomarkers and clinical scores that differentiated DVT positives from negatives were sPsel (87.3 vs 53.4 ng/mL, P < .0001), D-dimer (5.8 vs 2.1 mg/ L, P < .0001), CRP (2.1 vs 0.8 μg/mL, P < .0005), and Wells score (3.2 vs 2.0, P < .0001). For MP analysis, platelet-derived MPs were found to differentiate DVT from negatives. Using multivariable logistic regression, a combination of sPsel and Wells score could establish the diagnosis of DVT (cut point ≥ 90 ng/mL + Wells ≥ 2), with a specificity of 96% and positive predictive value (PPV) of 100%, and could exclude DVT diagnosis (cut point ≤ 60 ng/mL and Wells <2) with a sensitivity of 99%, a specificity of 33%, and a negative predictive value (NPV) of 96%.

CONCLUSIONS

This study establishes a biomarker and clinical profile combination that can both confirm and exclude the diagnosis of DVT.

BACKGROUND

The level of soluble vascular endothelial growth factor receptor 1 (sVEGFR1) is increased in sepsis and strongly associated with disease severity and mortality. Endothelial activation and damage contribute to both sepsis and trauma pathology. Therefore, this study measured sVEGFR1 levels in trauma patients upon hospital admission hypothesizing that sVEGFR1 would increase with higher injury severity and predict a poor outcome.

METHODS

Prospective observational study of 80 trauma patients admitted to a Level I Trauma Centre. <em>D</em>ata on demography, biochemistry, Injury Severity Score (ISS), transfusions and 30-day mortality were recorded and plasma/serum (sampled a median of 68 min (IQR 48-88) post-injury) was analyzed for sVEGFR1 and biomarkers reflecting sympathoadrenal activation (adrenaline, noradrenaline), tissue injury (histone-complexed <em>D</em>NA fragments, hc<em>D</em>NA), endothelial activation and damage (von Willebrand Factor Antigen, Angiopoietin-<em>2</em>, soluble endothelial protein C receptor, syndecan-1, soluble thrombomodulin (sTM)), coagulation activation/inhibition and fibrinolysis (prothrombinfragment 1 + <em>2</em>, protein C, activated Protein C, tissue-type plasminogen activator, plasminogen activator inhibitor-1, <em>D</em>-<em>dimer</em>) and inflammation (interleukin-6). Spearman correlations and regression analyses to identify variables associated with sVEGFR1 and its predictive value.

RESULTS

Circulating sVEGFR1 correlated with injury severity (ISS, rho = 0.46), shock (SBE, rho = -0.38; adrenaline, rho = 0.47), tissue injury (hc<em>D</em>NA, rho = 0.44) and inflammation (IL-6, rho = 0.54) (all p < 0.01) but by multivariate linear regression analysis only lower SBE and higher adrenaline and IL-6 were independent predictors of higher sVEGFR1. sVEGFR1 also correlated with biomarkers indicative of endothelial glycocalyx degradation (syndecan-1, rho = 0.67), endothelial cell damage (sTM, rho = 0.66) and activation (Ang-<em>2</em>, rho = 0.31) and hyperfibrinolysis (tPA, rho = 0.39; <em>D</em>-<em>dimer</em>, rho = 0.58) and with activated protein C (rho = 0.31) (all p < 0.01). High circulating sVEGFR1 correlated with high early and late transfusion requirements (number of packed red blood cells (RBC) at 1 h (rho = 0.<em>2</em>7, p = 0.016), 6 h (rho = 0.<em>2</em>7, p = 0.017) and <em>2</em>4 h (rho = 0.31, p = 0.004) but was not associated with mortality.

CONCLUSIONS

sVEGFR1 increased with increasing injury severity, shock and inflammation early after trauma but only sympathoadrenal activation, hypoperfusion, and inflammation were independent predictors of sVEGFR1 levels. sVEGFR1 correlated strongly with other biomarkers of endothelial activation and damage and with RBC transfusion requirements. Sympathoadrenal activation, shock and inflammation may be critical drivers of endothelial activation and damage early after trauma.

A novel Ca(<em>2</em>+)-binding protein, tentatively designated calgizzarin, has been purified to apparent homogeneity from chicken gizzard smooth muscle by W-7 (N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide))-Sepharose affinity chromatography and ion-exchange chromatography. Application of W-7-Sepharose affinity chromatography to various tissues revealed that calgizzarin-like proteins were abundant in bovine aorta and rabbit lung. Using the same procedure, we could purify a calgizzarin-like protein from rabbit lung. Calgizzarin has a Mr of 13,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 30,000 as determined by gel filtration on a TSK G 3000SW high performance liquid chromatography column, suggesting that calgizzarin seems to be a rodlike protein. The isoelectric point of calgizzarin was found to be pH 5.8. Calgizzarin can exist as a <em>dimer</em> by forming a disulfide bridge. The 45Ca autoradiographic technique showed that the protein binds to Ca<em>2</em>+. On an alkaline/urea gel, calgizzarin migrated faster in the presence of EGTA than in the presence of CaCl<em>2</em>, thereby indicating a Ca(<em>2</em>+)-dependent conformational change in this protein. The partial amino acid sequence (65 amino acid residues) of calgizzarin was seen to be SLLAVFQRYAGREG<em>D</em>NLKLSKKEFRTFMNTELASFTKNQK<em>D</em>PAVV<em>D</em>RMMKRL<em>D</em>INS<em>D</em>GQL<em>D</em>FQEF, and two putative Ca(<em>2</em>+)-binding sites (GREG<em>D</em>NLKLSKKE and <em>D</em> INS<em>D</em>GQL<em>D</em>FQE) were detected. So far as the obtained 65-amino acid sequence is concerned, calgizzarin has approximately a 50% sequence homology with S-100 alpha, 47% with S-100 beta, and 39% with pEL-98 protein.

A new class of protein phosphatases has emerged in the study of bacterial/archaeal chemotaxis, the CheC-type phosphatases. These proteins are distinct and unrelated to the well-known CheY-P phosphatase CheZ, though they have convergently evolved to dephosphorylate the same target. The family contains a common consensus sequence <em>D</em>/S-X(3)-E-X(<em>2</em>)-N-X(<em>2</em><em>2</em>)-P that defines the phosphatase active site, of which there are often two per protein. Three distinct subgroups make up the family: CheC, FliY and CheX. Further, the CheC subgroup can be divided into three classes. Bacillus subtilis CheC typifies the first class and might function as a regulator of Che<em>D</em>. Class II CheCs likely function as phosphatases in systems other than chemotaxis. Class III CheCs are found in the archaeal class Halobacteria and might function as class I CheCs. FliY is the main phosphatase in the B. subtilis chemotaxis system. CheX is quite divergent from the rest of the family, forms a <em>dimer</em> and some may function outside chemotaxis. A model for the evolution of the family is discussed.

The coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) has been spreading worldwide. Severe cases quickly progressed with unfavorable outcomes. We aim to investigate the clinical features of COVI<em>D</em>-19 and identify the risk factors associated with its progression. <em>D</em>ata of confirmed SARS-CoV-<em>2</em>-infected patients and healthy participants were collected. Thirty-seven healthy people and 79 confirmed patients, which include 48 severe patients and 31 mild patients, were recruited. COVI<em>D</em>-19 patients presented with dysregulated immune response (decreased T, B, and NK cells and increased inflammatory cytokines). Also, they were found to have increased levels of white blood cell, neutrophil count, and <em>D</em>-<em>dimer</em> in severe cases. Moreover, lymphocyte, C<em>D</em>4⁺ T cell, C<em>D</em>8⁺ T cell, NK cell, and B cell counts were lower in the severe group. Multivariate logistic regression analysis showed that C<em>D</em>4⁺ cell count, neutrophil-to-lymphocyte ratio (NLR) and <em>D</em>-<em>dimer</em> were risk factors for severe cases. Both CT score and clinical pulmonary infection score (CPIS) were associated with disease severity. The receiver operating characteristic (ROC) curve analysis has shown that all these parameters and scores had quite a high predictive value. Immune dysfunction plays critical roles in disease progression. Early and constant surveillance of complete blood cell count, T lymphocyte subsets, coagulation function, CT scan and CPIS was recommended for early screening of severe cases.

Vitamin <em>D</em> production requires UVB. In turn, we have shown that vitamin <em>D</em> compounds reduce UV-induced damage, including inflammation, sunburn, thymine <em>dimers</em>, the most frequent type of cyclobutane pyrimidine <em>dimer</em>, immunosuppression, and photocarcinogenesis. Our previous studies have shown most of the photoprotective effects by 1α,<em>2</em>5-dihydroxyvitamin <em>D</em>3 (1,<em>2</em>5(OH)<em>2</em><em>D</em>3) occurred through the nongenomic pathway because similar protection was seen with an analog, 1α,<em>2</em>5-dihydroxylumistrol3 (JN), which has little ability to alter gene expression and also because a nongenomic antagonist of 1,<em>2</em>5(OH)<em>2</em><em>D</em>3 abolished protection. In the current study, we tested whether this photoprotective effect would extend to other types of <em>D</em>NA damage, and whether this could be demonstrated in human ex vivo skin, as this model would be suited to pre-clinical testing of topical formulations for photoprotection. In particular, using skin explants, we examined a time course for thymine <em>dimers</em> (T<em>D</em>s), the most abundant <em>D</em>NA photolesion, as well as 8-oxo-7,8-dihydro-<em>2</em>'-deoxyguanosine (8-oxodG), which is a mutagenic <em>D</em>NA base lesion arising from UV-induced oxidative stress, and 8-nitroguanosine (8-NG). Nitric oxide products, known markers for chronic inflammation and carcinogenesis, are also induced by UV. This study showed that 1,<em>2</em>5(OH)<em>2</em><em>D</em>3 significantly reduced T<em>D</em> and 8-NG as early as 30min post UV, and 8-oxodG at 3h post UV, confirming the photoprotective effect of 1,<em>2</em>5(OH)<em>2</em><em>D</em>3 against <em>D</em>NA photoproducts in human skin explants. At least in part, the mechanism of photoprotection by 1,<em>2</em>5(OH)<em>2</em><em>D</em>3 is likely to be through the reduction of reactive nitrogen species and the subsequent reduction in oxidative and nitrosative damage. This article is part of a Special Issue entitled 'Vitamin <em>D</em> Workshop'.

BACKGROUND

D-dimer concentrations have not been evaluated extensively as a predictor of increased venous thromboembolism (VTE) risk in acutely ill, hospitalized medical patients.

OBJECTIVE

To analyze the relationships between D-dimer concentration, VTE and bleeding in the MAGELLAN trial (NCT00571649).

METHODS

This was a multicenter, randomized, controlled trial. Patients aged ≥ 40 years, hospitalized for acute medical illnesses with risk factors for VTE received subcutaneous enoxaparin 40 mg once daily for 10 ± 4 days then placebo up to day 35, or oral rivaroxaban 10 mg once daily for 35 ± 4 days. Patients (n = 7581) were grouped by baseline D-dimer ≤ 2 × or>> 2 × the upper limit of normal. VTE and major plus non-major clinically relevant bleeding were recorded at day 10, day 35, and between days 11 and 35.

RESULTS

The frequency of VTE was 3.5-fold greater in patients with high D-dimer concentrations. Multivariate analysis showed that D-dimer was an independent predictor of the risk of VTE (odds ratio 2.29 [95% confidence interval 1.75-2.98]), and had a similar association to established risk factors for VTE, for example cancer and advanced age. In the high D-dimer group, rivaroxaban was non-inferior to enoxaparin at day 10 and, unlike the low D-dimer group, superior to placebo at day 35 (P < 0.001) and days 11-35 (P < 0.001). In both groups, bleeding outcomes favored enoxaparin/placebo.

CONCLUSIONS

Elevated baseline D-dimer concentrations may identify acutely ill, hospitalized medical patients at high risk of VTE for whom extended anticoagulant prophylaxis may provide greater benefit than for those with low D-dimer concentrations.

BACKGROUND

P-selectin antagonism has been shown to decrease thrombogenesis and inflammation in animal models of deep venous thrombosis (DVT).

OBJECTIVE

To determine the effectiveness of P-selectin inhibitors versus saline and enoxaparin in venous thrombus resolution in nonhuman primate models of venous thrombosis.

METHODS

Studies reporting vein re-opening, inflammation expressed as Gadolinium enhancement and coagulation parameters were searched in the literature and pooled into a meta-analysis using an inverse variance with random effects.

RESULTS

Five studies were identified comparing P-selectin/ PSGL-1 inhibitors versus saline or enoxaparin regarding venous thrombosis resolution. Vein re-opening was significantly higher on P-selectin/ PSGL-1 compounds, when compared to saline (Inverse Variance [IV] 95% CI; 44.37 [17.77-70.96], p=0.001, I(<em>2</em>)=97%) and similar to enoxaparin (IV 95% CI; 5.03 [-8.88-18.95], p=0.48, I(<em>2</em>)=41%). Inflammation, reflected as Gadolinium enhancement at magnetic resonance venography (MRV), was significantly decreased in the P-selectin treated group when compared to saline (IV 95% CI; -17.84 [-14.98-(-8.30)], p<0.00001, I(<em>2</em>)=80%). No significant differences on vein wall inflammation were observed between P-selectin/ PSGL-1 inhibitors and enoxaparin treated animals (IV95% CI; -3.59 [-10.67-3.48], p=0.3<em>2</em>, I(<em>2</em>)=66%). In addition, there was no differences in the coagulation parameters (aPTT, TCT, BT, <em>D</em>-<em>Dimer</em>, fibrinogen, platelets) between P-selectin/ PSGL-1 inhibitors and enoxaparin (IV 95% CI; -1.1<em>2</em>[-<em>2</em>.36-0.11], p=0.07, I(<em>2</em>)=9<em>2</em>%), although there was a trend showing less of a prolongation in TCT with P-selectin/PSGL-1 inhibitors compared to enoxaparin (p<0.0001).

CONCLUSIONS

P-selectin antagonism successfully paralleled the low-molecular-weight-heparin enoxaparin, for the treatment of DVT in nonhuman primate models, by decreasing both thrombus burden and inflammation without causing any bleeding complications and without increasing coagulation times.

Ornithine aminotransferase (OAT), a pyridoxal-5'-phosphate dependent enzyme, catalyses the transfer of the <em>delta</em>-amino group of L-ornithine to <em>2</em>-oxoglutarate, producing L-glutamate-gamma-semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate, and L-glutamate. The crystal structure determination of human recombinant OAT is described in this paper. As a first step, the structure was determined at low resolution (6 A) by molecular replacement using the refined structure of dialkylglycine decarboxylase as a search model. Crystallographic phases were then refined and extended in a step-wise fashion to <em>2</em>.5 A by cyclic averaging of the electron density corresponding to the three monomers within the asymmetric unit. Interpretation of the resulting map was straightforward and refinement of the model resulted in an R-factor of 17.1% (Rfree=<em>2</em>4.3%). The success of the procedure demonstrates the power of real-space molecular averaging even with only threefold redundancy. The alpha6-hexameric molecule is a trimer of intimate <em>dimers</em> with a monomer-monomer interface of 5500 A<em>2</em> per subunit. The three <em>dimers</em> are related by an approximate 3-fold screw axis with a translational component of 18 A. The monomer fold is that of a typical representative of subgroup <em>2</em> aminotransferases and very similar to those described for dialkylglycine decarboxylase from Pseudomonas cepacia and glutamate-1-semialdehyde aminomutase from Synechococcus. It consists of a large domain that contributes most to the subunit interface, a C-terminal small domain most distant to the <em>2</em>-fold axis and an N-terminal region that contains a helix, a loop and a three stranded beta-meander embracing a protrusion in the large domain of the second subunit of the <em>dimer</em>. The large domain contains the characteristic central seven-stranded beta-sheet (agfedbc) covered by eight helices in a typical alpha/beta fold. The cofactor pyridoxal-5'-phosphate is bound through a Schiff base to Lys<em>2</em>9<em>2</em>, located in the loop between strands f and g. The C-terminal domain includes a four-stranded antiparallel beta-sheet in contact with the large domain and three further helices at the far end of the subunit. The active sites of the <em>dimer</em> lie, about <em>2</em>5 A apart, at the subunit and domain interfaces. The conical entrances are on opposite sides of the <em>dimer</em>. In the active site, R180, E<em>2</em>35 and R413 are probable substrate binding residues. Structure-based sequence comparisons with related transaminases in this work support that view. In patients suffering from gyrate atrophy, a recessive hereditary genetic disorder that can cause blindness in humans, ornithine aminotransferase activity is lacking. A large number of frameshift and point mutations in the ornithine aminotransferase gene have been identified in such patients. Possible effects of the various point mutations on the structural stability or the catalytic competence of the enzyme are discussed in light of the three-dimensional structure.

OBJECTIVE

This study was designed to investigate whether or not combination aspirin-clopidogrel therapy would reduce markers of thrombogenesis and platelet activation in atrial fibrillation (AF), in a manner similar to warfarin.

BACKGROUND

<em>D</em>ose-adjusted warfarin is beneficial as thromboprophylaxis in AF, but potentially serious side effects and regular monitoring leave room for alternative therapies. METHO<em>D</em>S; We randomized 70 patients with nonvalvular AF who were not on any antithrombotic therapy to either dose-adjusted warfarin (international normalized ratio <em>2</em> to 3) (Group I) or combination therapy with aspirin 75 mg and clopidogrel 75 mg (Group II). Plasma indices of thrombogenesis (fibrin <em>D</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em>) and platelet activation (beta-thromboglobulin [TG] and soluble P-selectin) were quantified, along with platelet aggregation responses to standard agonists, at baseline (pretreatment) and at six weeks posttreatment. RESULTS; Pretreatment levels of fibrin <em>D</em>-<em>dimer</em> (p = 0.001), beta-TG (p = 0.01) and soluble P-selectin (p = 0.03) were raised in patients with AF, whereas plasma prothrombin fragment 1+<em>2</em> levels and platelet aggregation were not significantly different compared with controls. <em>D</em>ose-adjusted warfarin reduced plasma levels of fibrin <em>D</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em> and beta-thromboglobulin levels at six weeks (all p < 0.001), enhanced plasma levels of soluble P-selectin (p < 0.001) and had no significant effect on platelet aggregation. Aspirin-clopidogrel combination therapy made no difference to the plasma markers of thrombogenesis or platelet activation (all p = NS), but the platelet aggregation responses to adenosine diphosphate (p < 0.001) and epinephrine (p = 0.0<em>2</em>) were decreased.

CONCLUSIONS

Aspirin-clopidogrel combination therapy failed to reduce plasma indices of thrombogenesis and platelet activation in AF, although some aspects of ex vivo platelet aggregation were altered. Anticoagulation with warfarin may be superior to combination aspirin-clopidogrel therapy as thromboprophylaxis in AF.

BACKGROUND

Tissue injury increases blood levels of extracellular histones and nucleic acids, and these may influence hemostasis, promote inflammation and damage the endothelium. Trauma-induced coagulopathy (TIC) may result from an endogenous response to the injury that involves the neurohumoral, inflammatory and hemostatic systems.

OBJECTIVE

To study the contribution of extracellular nucleic constituents to TIC, inflammation and endothelial damage.

METHODS

Prospective observational study.

METHODS

We investigated histone-complexed DNA fragments (hcDNA) along with biomarkers of coagulopathy, inflammation and endothelial damage in plasma from 80 trauma patients admitted directly to the Trauma Centre from the scene of the accident. Blood was sampled a median of 68 min (IQR 48-88) post injury. Trauma patients with hcDNA levels>>median or ≤median were compared.

RESULTS

Trauma patients with high plasma hcDNA had higher Injury Severity Score (ISS) and level of sympathoadrenal activation (higher adrenaline and noradrenaline) and a higher proportion of prolonged activated partial thromboplastin time (APTT) and higher D-dimer, tissue-type plasminogen activator (tPA), Annexin V and soluble CDD (all P < 0.05), all indicative of impaired thrombin generation, hyperfibrinolysis and platelet activation. Furthermore, patients with high hcDNA had enhanced inflammation and endothelial damage evidenced by higher plasma levels of terminal complement complex (sC5b-9), IL-6, syndecan-1, thrombomodulin and tissue factor pathway inhibitor (all P < 0.05).

CONCLUSIONS

Excessive release of extracellular histones and nucleic acids seems to contribute to the hypocoagulability, inflammation and endothelial damage observed early after trauma.

P-selectin inhibition has been shown to decrease thrombogenesis in multiple animal species. In this study, we show that a novel oral small-molecule inhibitor of P-selectin, PSI-697, promotes thrombus resolution and decreases inflammation in a baboon model of venous thrombosis. Experimental groups consisted of the following: 1) primates receiving a single oral dose of PSI-697 (30 mg/kg) daily starting three days pre-iliac vein balloon occlusion, and continued for six days; <em>2</em>) primates receiving a single treatment dose of a low-molecular-weight-heparin (LMWH) (1.5 mg/kg) daily starting one day pre-iliac balloon occlusion, and continued for six days; and 3) primates receiving a single oral dose of a vehicle control daily starting three days pre-iliac vein balloon occlusion, and continued for six days. Animals receiving PSI-697, although thrombosed after balloon deflation, demonstrated greater than 80% vein lumen opening over time, with no opening (0%) for vehicle control (p < 0.01). LMWH opening evident after balloon deflation slightly deteriorated over time compared to PSI-697. PSI-697 therapy also significantly decreased vein wall inflammation determined by magnetic resonance venography (MRV). Importantly, this beneficial opening occurred without measured anticoagulation. Animals receiving PSI-697 demonstrated significantly increased plasma <em>D</em>-<em>dimer</em> levels versus LMWH and control animals six hours post thrombus induction (p < 0.01). This study is the first to demonstrate the effectiveness of oral P-selectin inhibition to modify venous thrombogenesis, increase vein lumen opening, and decrease inflammation in a large animal model.

The wheat (Triticum aestivum) and rye (Secale cereale) beta-<em>D</em>-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three c<em>D</em>NAs encoding beta-<em>D</em>-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48- and 96-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and heterohexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 A. The N-terminal region was located at the <em>dimer</em>-<em>dimer</em> interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for <em>D</em>IMBOA-glucose (<em>2</em>-O-beta-<em>D</em>-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over <em>D</em>IBOA-glucose (7-demethoxy-<em>D</em>IMBOA-glucose).

Acute pancreatitis (AP) is an inflammatory <em>d</em>isease with varie<em>d</em> severity, ranging from mil<em>d</em> local inflammation to severe systemic involvement resulting in substantial mortality. Early pathologic events in AP, both local an<em>d</em> systemic, are associate<em>d</em> with vascular <em>d</em>erangements, inclu<em>d</em>ing en<em>d</em>othelial activation an<em>d</em> injury, <em>d</em>ysregulation of vasomotor tone, increase<em>d</em> vascular permeability, increase<em>d</em> leukocyte migration to tissues, an<em>d</em> activation of coagulation. The purpose of the review was to summarize current evi<em>d</em>ence regar<em>d</em>ing the interplay between inflammation, coagulation an<em>d</em> en<em>d</em>othelial <em>d</em>ysfunction in the early phase of AP. Practical aspects were emphasize<em>d</em>: (1) we summarize<em>d</em> available <em>d</em>ata on <em>d</em>iagnostic usefulness of the markers of en<em>d</em>othelial <em>d</em>ysfunction an<em>d</em> activate<em>d</em> coagulation in early pre<em>d</em>iction of severe AP; (<em>2</em>) we reviewe<em>d</em> in <em>d</em>etail the results of experimental stu<em>d</em>ies an<em>d</em> clinical trials targeting coagulation-inflammation interactions in severe AP. Among laboratory tests, <em>d</em>-<em>dimer</em> an<em>d</em> angiopoietin-<em>2</em> measurements seem the most useful in early pre<em>d</em>iction of severe AP. Although most clinical trials evaluating anticoagulants in treatment of severe AP <em>d</em>i<em>d</em> not show benefits, they also <em>d</em>i<em>d</em> not show significantly increase<em>d</em> blee<em>d</em>ing risk. Promising results of human trials were publishe<em>d</em> for low molecular weight heparin treatment. Several anticoagulants that prove<em>d</em> beneficial in animal experiments are thus worth testing in patients.

OBJECTIVE

To investigate the role of perfusion defect (PD) size on dual energy CT pulmonary blood volume assessment as predictor of right heart strain and patient outcome and its correlation with d-dimer levels in acute pulmonary embolism (PE).

METHODS

53 patients with acute PE who underwent DECT pulmonary angiography were retrospectively analyzed. Pulmonary PD size caused by PE was measured on DE iodine maps and quantified absolutely (VolPD) and relatively to the total lung volume (RelPD). Signs of right heart strain (RHS) on CT were determined. Information on d-dimer levels and readmission for recurrent onset of PE and death was collected.

RESULTS

D-dimer level was mildly (r = 0.43-0.47) correlated with PD size. Patients with RHS had significantly higher VolPD (215 vs. 73 ml) and RelPD (9.9 vs. 2.9%) than patients without RHS (p < 0.003). There were 2 deaths and 1 readmission due of PE in 18 patients with >5% RelPD, while no such events were found for patients with <5% RelPD.

CONCLUSIONS

Pulmonary blood volume on DECT in acute PE correlates with RHS and appears to be a predictor of patient outcome in this pilot study.

OBJECTIVE

(1) To study the changes in the haemostatic variables during normal pregnancy, and (<em>2</em>) to compare them with the corresponding variables in a control group of nonpregnant women.

METHODS

University Hospital.

METHODS

The study involves two groups of women: pregnant women (in 35th-40th gestational weeks (GW)) (N = 35) and a control group of nonpregnant women (N = 35).

METHODS

We examined the global tests, the variables relating to coagulation and fibrinolysis and the group of natural inhibitors of coagulation, and compared them between the two groups.

RESULTS

The pregnant women had statistically significantly higher values for: prothrombin time (PT) (P < 0.0001), thrombin time (TT) (P < 0.0001), fibrinogen (P < 0.0001), activity of factor VII (P < 0.0001), factor X (P < 0.0001) and alpha<em>2</em>-antiplasmin (P < 0.00<em>2</em>), plasma concentration of D-dimer (plsDD) (P < 0.0001) and activity of heparin cofactor II (HCII) (P < 0.00<em>2</em>). They had statistically significantly lower activity of protein C (PrC) (P < 0.0001) and of total protein S (TPrS) (P < 0.0001).

CONCLUSIONS

During normal pregnancy the activation of coagulation is counterbalanced by the activation of fibrinolysis, which maintains the haemostatic balance.

The natural history of calf deep-vein thrombosis (<em>D</em>VT) is still uncertain and it is debated whether it warrants to be diagnosed and treated. We aimed to investigate the complication rate of untreated isolated calf <em>D</em>VT (IC<em>D</em>VT). Symptomatic outpatients were prospectively managed with serial compression ultrasonography (SCUS). Those without proximal <em>D</em>VT and with likely pre-test clinical probability (PCP) or altered <em>D</em>-<em>dimer</em> received immediate subsequent complete examination of calf deep veins (CCUS) by a different operator. The result of CCUS was kept blind both to the managing doctor and the patient and disclosed after three months. Primary outcome was the rate of venous thromboembolism at three months. We examined 431 subjects (196 males; median age 68.0 years) in whom five outcomes were recorded (1.<em>2</em>%; 95% confidence intervals [CI]: 0.4-<em>2</em>.7). If CCUS results had been available, outcomes would have been recorded in 3/4<em>2</em>4 patients (0.7%; 95% CI: 0.<em>2</em>-<em>2</em>.1) with two events in subjects negative at both serial and complete CUS. IC<em>D</em>VT was diagnosed in 65 subjects (15.3%; 95% CI: 1<em>2</em>-19); of whom 59 remained uneventful (one was lost to follow-up). A significant higher rate of outcomes was recorded in subjects with than without IC<em>D</em>VT (5/64; 7.8%; 95% CI: 3-17 vs. 3/351; 0.8%; 95% CI: 0-<em>2</em>; p=0.003). However, after excluding two events picked at serial CUS in subjects with IC<em>D</em>VT, the difference became barely significant (3/64; 4.7%; 95% CI: 1-13; p=0.049). Thrombotic evolution of untreated IC<em>D</em>VT in high-risk subjects may be relevant. Larger studies are needed to address this issue.

<em>D</em>eep venous thrombosis and pulmonary embolism represent two expressions of a similar clinical pathological process traditionally referred to as venous thromboembolism. Several population studies evidence venous thromboembolism as a leading healthcare problem worldwide, highlighting the need for early and reliable diagnosis to enable appropriate triage of affected patients and to optimize outcome. There is still debate, however, on which thrombotic markers to use, as well as their most suitable position within diagnostic algorithms. This article aims to review the pathophysiology and clinical usefulness of past, present and future markers of thrombosis, including soluble fibrin monomers, fibrin/fibrinogen degradation products, thrombin-antithrombin complex, plasmin-antiplasmin complex, fibrinopeptide A and B, prothrombin fragments 1 + <em>2</em>, thrombus precursor protein, <em>D</em>-<em>dimer</em>, activated protein C-protein C inhibitor complex, myeloperoxidase, thrombin generation assays and proteomic analysis. Several lines of evidence now attest that the global diagnostic performances of some <em>D</em>-<em>dimer</em> assays largely outperform those of any other coagulation or fibrinolytic marker proposed thus far, and a "negative" <em>D</em>-<em>dimer</em> measured with rapid enzyme linked fluorescent immunoassay is now considered the biochemical gold standard for ruling out an acute episode of venous thromboembolism in a patient with a low pretest probability for venous thromboembolism, so that additional testing can be safely omitted. However, to further improve clinical outcomes, the diagnostic efficiency of combining <em>D</em>-<em>dimer</em> testing with other markers covering different pathophysiological aspects of thrombosis such as continuous and progressive thrombin generation (e.g., activated protein C-protein C inhibitor complex) or neutrophil activation (i.e., myeloperoxidase) merits further investigation. Proteomic analysis, which would help to characterize the structure and function of each protein and the complexities of protein-protein interactions in physiological and pathological hemostasis, also holds promise for identifying novel markers and developing effective diagnostic protocols in the future.

Using a <em>dimer</em>ic bZIP protein, we have <em>d</em>esigne<em>d</em> a leucine zipper that becomes more stable after a serine in the e position is phosphorylate<em>d</em> by protein kinase A (<em>delta</em> <em>delta</em> GP = -1.4 kcal mol-1 <em>dimer</em>-1 or -0.7 kcal mol-1 resi<em>d</em>ue-1). Mutagenesis stu<em>d</em>ies in<em>d</em>icate that three arginines form a network of inter-helical (i,i' + 5; i, i' + <em>2</em>) an<em>d</em> intra-helical (i, i + 4) attractive interactions with the phosphorylate<em>d</em> serine. When the arginines are replace<em>d</em> with lysines, the stabilizing effect of serine phosphorylation is re<em>d</em>uce<em>d</em> (<em>delta</em> <em>delta</em> GP = -0.5 kcal mol-1 <em>dimer</em>-1). The hy<em>d</em>rophobic interface of the leucine zipper nee<em>d</em>s a glycine in the <em>d</em> position to obtain an increase in stability after phosphorylation. The phosphorylate<em>d</em> protein bin<em>d</em>s DNA with a 15-fol<em>d</em> higher affinity. Using a transient transfection assay, we <em>d</em>ocument a PKA <em>d</em>epen<em>d</em>ent four-fol<em>d</em> activation of a reporter gene. Phosphorylation of a threonine in the same e position <em>d</em>ecreases the stability by <em>delta</em> <em>delta</em> GP = +1.<em>2</em> kcal mol-1 <em>dimer</em>-1. We present circular <em>d</em>ichroism (CD) thermal <em>d</em>enaturations of 15 bZIP proteins before an<em>d</em> after phosphorylation. These <em>d</em>ata provi<em>d</em>e insights into the structural <em>d</em>eterminants that result in stabilization of a coile<em>d</em> coil by phosphorylation.

Accurate diagnostic assessment of suspected ipsilateral recurrent deep vein thrombosis (<em>D</em>VT) is a major clinical challenge because differentiating between acute recurrent thrombosis and residual thrombosis is difficult with compression ultrasonography (CUS). We evaluated noninvasive magnetic resonance direct thrombus imaging (MR<em>D</em>TI) in a prospective study of 39 patients with symptomatic recurrent ipsilateral <em>D</em>VT (incompressibility of a different proximal venous segment than at the prior <em>D</em>VT) and 4<em>2</em> asymptomatic patients with at least 6-month-old chronic residual thrombi and normal <em>D</em>-<em>dimer</em> levels. All patients were subjected to MR<em>D</em>TI. MR<em>D</em>TI images were judged by <em>2</em> independent radiologists blinded for the presence of acute <em>D</em>VT and a third in case of disagreement. The sensitivity, specificity, and interobserver reliability of MR<em>D</em>TI were determined. MR<em>D</em>TI demonstrated acute recurrent ipsilateral <em>D</em>VT in 37 of 39 patients and was normal in all 4<em>2</em> patients without symptomatic recurrent disease for a sensitivity of 95% (95% CI, 83% to 99%) and a specificity of 100% (95% CI, 9<em>2</em>% to 100%). Interobserver agreement was excellent (κ = 0.98). MR<em>D</em>TI images were adequate for interpretation in 95% of the cases. MR<em>D</em>TI is a sensitive and reproducible method for distinguishing acute ipsilateral recurrent <em>D</em>VT from 6-month-old chronic residual thrombi in the leg veins.

OBJECTIVE

Clinical studies suggest that platelet P<em>2</em>Y1<em>2</em> inhibitors reduce mortality from sepsis, although the underlying mechanisms have not been clearly defined in vivo. We hypothesized that P<em>2</em>Y1<em>2</em> inhibitors may improve survival from sepsis by suppressing systemic inflammation and its prothrombotic effects. We therefore determined whether clopidogrel and the novel, more potent P<em>2</em>Y1<em>2</em> inhibitor, ticagrelor, modify these responses in an experimental human model.

RESULTS

We randomized 30 healthy volunteers to ticagrelor (n=10), clopidogrel (n=10), or no antiplatelet medication (controls; n=10). We examined the effect of P<em>2</em>Y1<em>2</em> inhibition on systemic inflammation, which was induced by intravenous injection of Escherichia coli endotoxin. Both P<em>2</em>Y1<em>2</em> inhibitors significantly reduced platelet-monocyte aggregate formation and peak levels of major proinflammatory cytokines, including tumor necrosis factor α, interleukin-6, and chemokine (C-C motif) ligand <em>2</em>. In contrast to clopidogrel, ticagrelor also significantly reduced peak levels of IL-8 and growth colony-stimulating factor and increased peak levels of the anti-inflammatory cytokine IL-10. In addition, ticagrelor altered leukocyte trafficking. Both P<em>2</em>Y1<em>2</em> inhibitors suppressed <em>D</em>-<em>dimer</em> generation and scanning electron microscopy revealed that ticagrelor also suppressed prothrombotic changes in fibrin clot ultrastructure.

CONCLUSIONS

Potent inhibition of multiple inflammatory and prothrombotic mechanisms by P<em>2</em>Y1<em>2</em> inhibitors demonstrates critical importance of platelets as central orchestrators of systemic inflammation induced by bacterial endotoxin. This provides novel mechanistic insight into the lower mortality associated with P<em>2</em>Y1<em>2</em> inhibitors in patients with sepsis in clinical studies.

The fatty acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions, such as innate immunity, energy balance, and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G protein-coupled receptors (e.g., cannabinoid receptors) and nuclear receptors (e.g., PPAR-α). Here, we report the crystal structure of human NAPE-hydrolyzing phospholipase <em>D</em> (NAPE-PL<em>D</em>) at <em>2</em>.65 Å resolution, a membrane enzyme that catalyzes FAE formation in mammals. NAPE-PL<em>D</em> forms homo<em>dimer</em>s partly separated by an internal ∼ 9-Å-wide channel and uniquely adapted to associate with phospholipids. A hydrophobic cavity provides an entryway for NAPE into the active site, where a binuclear Zn(<em>2</em>+) center orchestrates its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing <em>dimer</em> assembly and enabling catalysis. These elements offer multiple targets for the design of small-molecule NAPE-PL<em>D</em> modulators with potential applications in inflammation and metabolic disorders.

BACKGROUND

Psychological stress and anxiety have been shown to produce an activation of coagulation and fibrinolysis. Resulting hypercoagulability is a risk factor for cardiovascular diseases, and could therefore contribute to an increased prevalence of coronary artery disease in anxiety patients. However, hemostasis function has not yet been studied in patients with clinically relevant anxiety disorders.

METHODS

A group of anxiety patients (panic disorder with agoraphobia or social phobia) and a healthy control group (each n = <em>2</em>9) completed some questionnaires [SCL-K9 (a short form of the SCL-90-R), State Trait Anxiety Inventory, A<em>D</em>S (general depression scale)], and had blood drawn after a 15-min rest period. To assess the reaction of the hemostatic system by global entities, sum scores were computed from parameters of coagulation and fibrinolysis (fibrinogen, FVII, FVIII, vWF, F1 + <em>2</em>, TAT, <em>D</em>-<em>dimer</em>, alpha(<em>2</em>)-AP, PAP, tPA, PAI-1). Interfering variables, such as age, gender, alcohol consumption and smoking status, were controlled.

RESULTS

Anxiety patients scored higher in a composite hemostatic score and a sum score of fibrinolysis in comparison to the control group, with a predominant activation of inhibitors in fibrinolysis. However, the psychological variable with the closest association to hemostasis was not trait anxiety, but self-perceived worry about blood drawing before blood sampling was performed.

CONCLUSIONS

The coagulation and fibrinolysis system is activated in the direction of a hypercoagulable state in patients with severe phobic anxiety, triggered by fear of blood drawing. This could be one mediating factor for the increased risk of cardiovascular diseases in this population. Acute situational phobic anxiety should be monitored closely when studying the association between anxiety and hemostasis.