UreE is a homo<em>dimer</em>ic metallo-chaperone that assists the insertion of Ni(<em>2</em>+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(<em>2</em>+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of <em>2</em> Ni(<em>2</em>+) ions to the functional <em>dimer</em>, with an overall dissociation constant K(<em>D</em>) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(<em>2</em>+) ions in BpUreE-Ni(<em>2</em>), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(<em>2</em>+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(<em>2</em>+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(<em>2</em>+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(<em>2</em>+) ion bound to one His(100) from each monomer, and the second Ni(<em>2</em>+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.

Dimeric <em>2</em>-amino-1,8-naphthyri<em>d</em>ine selectively bin<em>d</em>s to a G-G mismatch with high affinity (K(<em>d</em>) = 53 nM). We have investigate<em>d</em> a bin<em>d</em>ing mechanism of naphthyri<em>d</em>ine <em>dimer</em> <em>2</em> to a G-G mismatch by spectroscopic stu<em>d</em>ies, thermo<em>d</em>ynamic analysis, an<em>d</em> structure-activity stu<em>d</em>ies for the thermal stabilization of the mismatch. 1H NMR spectra of a complex of <em>2</em> with 9-mer <em>d</em>uplex <em>d</em>(CATCGGATG)<em>2</em> containing a G-G mismatch showe<em>d</em> that all hy<em>d</em>rogens in two naphthyri<em>d</em>ine rings of <em>2</em> were observe<em>d</em> upfiel<em>d</em> compare<em>d</em> to those of <em>2</em> in a free state. The <em>2</em>D-NOESY experiments showe<em>d</em> that each naphthyri<em>d</em>ine of <em>2</em> bin<em>d</em>s to a guanine in the G-G mismatch within the pi-stack. In CD spectra, a large conformational change of the G-G mismatch-containing <em>d</em>uplex was observe<em>d</em> upon complex formation with <em>2</em>. Isothermal calorimetry titration of <em>2</em> bin<em>d</em>ing to the G-G mismatch showe<em>d</em> that the stoichiometry for the bin<em>d</em>ing is about 1:1 an<em>d</em> that the bin<em>d</em>ing is enthalpy-controlle<em>d</em>. It is clarifie<em>d</em> by structure-activity stu<em>d</em>ies that show (i) the linker connecting two naphthyri<em>d</em>ine rings was essential for the stabilization of the G-G mismatch, (ii) the bin<em>d</em>ing efficiency was very sensitive to the linker structure, an<em>d</em> (iii) the bin<em>d</em>ing of two naphthyri<em>d</em>ines to each one of two Gs in the G-G mismatch is essential for a strong stabilization. These results strongly supporte<em>d</em> the intercalation of both naphthyri<em>d</em>ine rings of <em>2</em> into DNA base pairs an<em>d</em> the formation of a hy<em>d</em>rogen bon<em>d</em>e<em>d</em> complex with the G-G mismatch.

The purification of hepatic <em>delta</em>-aminolevulinic acid synthase (EC <em>2</em>.3.1.37) was accomplished from chick embryo liver mitochondria, which had been treated with the combination of drugs, <em>2</em>-allylisopropylacetamide and 1,4-dihydro-3,5-dicarbethoxycollidine to produce a high starting level of enzyme activity. After extraction from the mitochondria by sonication, the enzyme was purified to a final specific activity of over 10,000 nmol of aminolevulinate formed/30 min/mg of protein/37 degrees, using the techniques of Sephadex chromatography, ammonium sulfate fractionation, affinity chromatography for pyridoxal phosphate, and preparative isoelectric focusing. An isoelectric point of 7.0 and a molecular weight of 87,000 were obtained for the native enzyme. The subunit molecular weight of 49,000, obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggested it was a <em>dimer</em>. The enzyme was inhibited by p-chloromercuribenzoate and N-ethylmaleimide, stimulated by cations and exhibited an ultraviolet absorption spectrum characteristic of pyridoxal 5'-phosphate enzymes with absorption maxima at 3<em>2</em>5 and 4<em>2</em>0 nm.

Women with premature menopause are at high risk for vascular compications associated with thrombogenesis and atherogenesis. The use of hormone-replacement therapy (HRT), however, may protect against these complications. Hemostatic abnormalities and endothelial function are closely related to the processes of thrombogenesis and atherogenesis. The purpose of the study was to evaluate the effects of premature menopause on markers of hemostasis, platelet function, and endothelial function and the effects of starting HRT. This is a prospective longitudinal study of premenopausal women undergoing surgical menopause in whom estrogen HRT is started. We measured sequential changes in plasma levels of the hemostatic factors (fibrinogen, fibrin <em>D</em>-<em>dimer</em>, and plasminogen activiator inhibitor [PAI]), markers of platelet function (soluble leukocyte adhesion molecule P-selectin) and endothelial function (von Willebrand factor [vWf], soluble thrombomodulin [sTM], and tissue plasminogen activator [TPA]), and serum lipid levels, including lipoprotein A. Twenty-seven premenopausal women (mean age 43.6 +/- 6.5 years) undergoing hysterectomy and bilateral salpingo-oophrectomy were studied. In the postsurgical menopausal state (visit <em>2</em>), there was a significant elevation in sTM levels (paired Wilcoxon test, p = 0.008). There was also a trend toward higher median soluble P-selectin, PAI, and mean TPA levels and lower vWf levels. After 6 weeks of HRT (visit 3), there was a significant reduction in mean vWf (paired Wilcoxon test, p = 0.00<em>2</em>6), sTM (p = 0.039), and TPA levels (p = 0.0<em>2</em>) compared with premenopausal levels. There were no significant changes in plasma fibrinogen, fibrin <em>D</em>-<em>dimer</em>, and PAI levels at visit <em>2</em> or visit 3 compared with premenopausal levels. There was a significant increase in serum lipoprotein A (paired Wilcoxon test, p = 0.008), cholesterol, and triglyceride levels after surgical menopause (paired t test, p < 0.01). Lipoprotein A and cholesterol levels after HRT (visit 3) were not significantly different from prehysterectomy levels, although triglyceride levels were increased further. HRT results in a significant reduction in vWf, sTM, and TPA levels, suggesting beneficial effects on endothelial function and atherogenesis. Although there was a significant increase in serum lipoprotein A and cholesterol levels after surgical menopause, lipoprotein A and cholesterol levels after HRT were not significantly different from presurgery levels. These observations are consistent with the beneficial effects of HRT in cardiovascular hemodynamics and cardiovascular disease.

Fructokinase from beef liver has been purified <em>2</em>300-fold by acid and heat treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-100, <em>D</em>EAE- and CM-cellulose. The purified enzyme is homogeneous by all criteria examined, has a molecular weight of 56 000, and is a <em>dimer</em> of equal molecular weight subunits. The isoelectric point is 5.7. The Michaelis constant for activation by K+ is 15 mM, and the enzyme is also activated by Na+, Rb+, Cs+, NH4+, and TL+. The kinetic mechanism has been determined at pH 7.0, <em>2</em>5 degrees C. The initial velocity, product, and dead-end inhibition patterns for CrATP, CrA<em>D</em>P, and 1-deoxy-<em>D</em>-fructose are consistent with a random kinetic mechanism with the formation of two dead-end complexes. Substrates for fructokinase include: <em>D</em>-fructose, L-sorbose, <em>D</em>-tagatose, <em>D</em>-psicose, <em>D</em>-xylulose, L-ribulose, <em>D</em>-sedoheptulose, L-galactoheptulose, <em>D</em>-mannoheptulose, 5-keto-<em>D</em>-fructose, <em>D</em>-ribose, <em>2</em>,5-anhydro-<em>D</em>-mannitol, <em>2</em>,5-anhydro-<em>D</em>-glucitol, <em>2</em>,5-anhydro-<em>D</em>-mannose, <em>2</em>,5-anhydro-<em>D</em>-lyxito.l, and <em>D</em>-ribono-gamma-lactone. 5-Thio-<em>D</em>-fructose was not a substate, but was a competitive inhibitor vs. <em>D</em>-fructose. Thus the minimum molecular for substrate activity seems to be (<em>2</em>R)-<em>2</em>-hydroxy-methyl-3,4-dihydroxytetrahydrofuran. The configuration of the substituents at carbons 3, 4, and 5 appears not to be critical, but the hydroxymethyl group must have the configuration corresponding to beta-<em>D</em>-(or alpha-L-) keto sugars. The anomeric hydroxyl on carbon <em>2</em> is not required (although it contributes to binding), and a wide variety of groups may be present at carbon 5.

The monomeric Escherichia coli Rep protein un<em>d</em>ergoes a DNA-in<em>d</em>uce<em>d</em> <em>dimer</em>ization upon bin<em>d</em>ing either single-stran<em>d</em>e<em>d</em> (ss) or <em>d</em>uplex DNA with the <em>dimer</em> being the active form of the Rep helicase. Using stoppe<em>d</em>-flow fluorescence, we have <em>d</em>etermine<em>d</em> a minimal kinetic mechanism for this reaction in which Rep monomer (P) bin<em>d</em>s to ss oligo<em>d</em>eoxynucleoti<em>d</em>es (<em>d</em>N(pN)15) (S) by a two-step mechanism to form PS*, which can then <em>dimer</em>ize with P to form P<em>2</em>S as in<em>d</em>icate<em>d</em>: [reaction in text]. This minimal mechanism is supporte<em>d</em> by four in<em>d</em>epen<em>d</em>ent stu<em>d</em>ies in which the kinetics were monitore<em>d</em> by changes in fluorescence intensity of three <em>d</em>ifferent probes: the intrinsic Rep tryptophan fluorescence, the fluorescence of <em>d</em>(T5(<em>2</em>-AP)T4(<em>2</em>-AP)T5), containing the fluorescent base, <em>2</em>-aminopurine (<em>2</em>-AP), an<em>d</em> <em>d</em>T(pT)15 labele<em>d</em> at its 3'-en<em>d</em> with fluorescein (3'-F-<em>d</em>T(pT)15). Simultaneous (global) analysis of the time courses of <em>d</em>(T5(<em>2</em>-AP)T4(<em>2</em>-AP)T5) (100 nM) bin<em>d</em>ing to a range of Rep monomer concentrations (<em>2</em>5-400 nM) yiel<em>d</em>s the following rate constants: k1 = (3.3 +/- 0.5) x 10(7) M-1 s-1; k-1 = 1.4 +/- 0.4 s-1; k<em>2</em> = <em>2</em>.7 +/- 0.9 s-1; k-<em>2</em> = 0.<em>2</em>1 +/- 0.06 s-1; k3 = (4.5 +/- 0.3) x 10(5) M-1 s-1; k-3 = 0.00<em>2</em>7 +/- 0.0008 s-1 [<em>2</em>0 mM Tris-HCl, pH 7.5, 6 mM NaCl, 5 mM MgCl<em>2</em>, 5 mM <em>2</em>-mercaptoethanol, an<em>d</em> 10% (v/v) glycerol, 4.0 <em>d</em>egrees C]. This mechanism provi<em>d</em>es <em>d</em>irect evi<em>d</em>ence that Rep monomers can bin<em>d</em> ss DNA an<em>d</em> that ss DNA bin<em>d</em>ing in<em>d</em>uces a conformational change in the Rep monomer that is probably require<em>d</em> for Rep <em>dimer</em>ization. This conformational change is likely to be large an<em>d</em> global since it is <em>d</em>etecte<em>d</em> by all three fluorescence probes. The apparent bimolecular rate constant for Rep monomer bin<em>d</em>ing to 3'-F-<em>d</em>T(pT)15 [k1(app) = (6.0 +/- 0.7) x 10(7) M-1 s-1] is slightly larger than measure<em>d</em> with <em>d</em>(T5(<em>2</em>-AP)T4(<em>2</em>-AP)T5) bin<em>d</em>ing. The apparent rate constant for <em>d</em>issociation of <em>d</em>(T5(<em>2</em>-AP)T4(<em>2</em>-AP)T5) (S) from the half-ligate<em>d</em> Rep <em>dimer</em>, P<em>2</em>S, increases with increasing concentration of a nonfluorescent competitor ss DNA (<em>d</em>(T5-AT4AT5)) (C), in<em>d</em>icating transient formation of a <em>d</em>oubly ligate<em>d</em> P<em>2</em>SC interme<em>d</em>iate. However, the apparent bimolecular rate constant for bin<em>d</em>ing of C to P<em>2</em>S is extremely slow >> or = <em>2</em>50 M-1 s-1), suggesting the occurrence of a multistep process before <em>d</em>issociation of ss DNA. In the absence of competitor DNA, <em>d</em>issociation of ss DNA from P<em>2</em>S occurs only after slow <em>d</em>issociation of the Rep <em>dimer</em> to form PS* + P. The implications of these results for Rep-catalyze<em>d</em> DNA unwin<em>d</em>ing are <em>d</em>iscusse<em>d</em>.

CHIP<em>2</em>8 occurs naturally in glycosylated and nonglycosylated forms. The purpose of this study was to determine the role of glycosylation in CHIP<em>2</em>8 structure and function. A new purification procedure based on phenylboronic acid-agarose (PBA) affinity chromatography was developed to isolate CHIP<em>2</em>8. In purified native CHIP<em>2</em>8 from erythrocytes, approximately 50% of CHIP<em>2</em>8 molecules were glycosylated; each mole of glycosylated CHIP<em>2</em>8 contained 5.4 k<em>D</em>a of monosaccharides consisting of <em>2</em> mol of Fuc, 8 mol of Gal, 1 mol of GalN, 13 mol of GlcN, 3 mol of Man, and 1 mol of Neu5Ac. The proportions of each monosaccharide and the sensitivity to endo-beta-galactosidase indicated that CHIP<em>2</em>8 contained polylactosaminyl oligosaccharides. Glycosylated and nonglycosylated CHIP<em>2</em>8 remained tightly associated when solubilized in octyl beta-<em>D</em>-glucoside (OG) and could not be separated by conventional chromatographic procedures. To remove the sugar moiety, CHIP<em>2</em>8 was enzymatically deglycosylated by PNGase F and purified by Q-Sepharose anion-exchange and Erythrina cristagalli lectin chromatography. High-performance size-exclusion chromatography revealed that native CHIP<em>2</em>8 eluted as an apparent <em>dimer</em>, whereas deglycosylated CHIP<em>2</em>8 eluted as an apparent monomer. In reconstituted proteoliposomes, deglycosylated CHIP<em>2</em>8 had a single channel water permeability (pf) of 3.1 x 10(-14) cm3/s (10 degrees C), not different from that of 3.<em>2</em> x 10(-14) cm3/s for native CHIP<em>2</em>8. Circular dichroism of native and deglycosylated CHIP<em>2</em>8 in OG revealed 45% and 48% alpha-helix, respectively; intrinsic tryptophan fluorescence showed no effects of glycosylation on tryptophan environment. Freeze-fracture electron microscopy with rotary shadowing indicated that native and deglycosylated CHIP<em>2</em>8 assembled as tetramers in reconstituted proteoliposomes.(ABSTRACT TRUNCATE<em>D</em> AT <em>2</em>50 WOR<em>D</em>S)

BACKGROUND

Growth-differentiation factor-15 (GDF-15) has recently emerged as a risk predictor in patients with cardiac diseases. GDF-15 is commonly related to cardiovascular risk factors, inflammatory activity and cardiac abnormalities. However, it is not clear whether it might be an indicator of vascular pathologies as well.

METHODS

Circulating levels of G<em>D</em>F-15 were measured in 1004 elderly community dwellers participating in the PIVUS study. The relations of G<em>D</em>F-15 to biomarkers of endothelial activation (E-selectin, P-selectin, ICAM-1, VCAM-1), extracellular matrix degradation (MMP-9, TIMP-1), coagulatory activity (<em>D</em>-<em>dimer</em>, von Willebrand factor, prothrombin fragment 1 + <em>2</em>, factor VIIa), and fibrinolytic activity (PAI-1 activity, tPA-antigen) were assessed by multiple linear regressions.

RESULTS

The median G<em>D</em>F-15 level was 1135 ng/L. By linear correlation analysis, G<em>D</em>F-15 exhibited a moderate relation to von Willebrand factor (r = 0.30), and weak, albeit significant relations (r = 0.13-0.<em>2</em>9) to E-selectin, P-selectin, ICAM-1, VCAM-1, MMP-9, TIMP-1, <em>D</em>-<em>dimer</em>, PAI-1 activity and tPA-antigen. The relations to the assessed biomarkers of endothelial activation, TIMP-1, <em>D</em>-<em>dimer</em> and von Willebrand factor remained significant applying multiple linear regression models adjusted for clinical covariates and echocardiographic data. There were no significant relations between G<em>D</em>F-15 and biomarkers solely reflecting coagulatory activity.

CONCLUSIONS

In the elderly, GDF-15 reflects endothelial activation and vascular inflammation and thus, multiple pathways involved in the development and progression of atherosclerosis.

OBJECTIVE

Increased frequency of hyperfibrinolytic activity was reported in patients with cirrhosis. However, the incidence, clinical presentation, and the parameters related to hyperfibrinolysis remain largely unknown in these patients. By utilizing euglobulin lysis time (ELT) and other clinical coagulation tests, the present study investigated the incidence of and clinical parameters related to hyperfibrinolytic activity, and assessed predicting factors to epsilon-aminocaproic acid (EACA) treatment in cirrhotic patients with hyperfibrinolysis in a liver unit.

METHODS

The study included 86 consecutive patients who were referred and admitted to a referral liver unit for various liver diseases. The mean age was 50.0 yr, with a male: female ratio of 60:<em>2</em>6. Sixty-six patients (76.7%) were Hispanic and 75 (87.<em>2</em>%) were cirrhotic. The etiologies of liver diseases included alcoholic liver disease (n = 68, 79.1%), hepatitis B (n = <em>2</em>, <em>2</em>.3%), hepatitis C (n = 6, 7.0%), autoimmune hepatitis (n = 3, 3.5%), cryptogenic liver disease (n = 4, 4.7%), and hepatocellular carcinoma (n = 3, 3.5%). Coagulation studies included ELT, PT, PTT, fibrinogen, <em>D</em>-<em>dimer</em>, and fibrin degradation product levels.

RESULTS

Hyperfibrinolytic activity as reflected by shortened ELT was present in <em>2</em>7/75 cirrhotic (31.3%) but 0/11 noncirrhotic patients, which was significantly correlated with higher Child-Pugh (C-P) class, abnormal levels of PT, PTT, fibrinogen, platelet count, and total bilirubin. Shortened ELT was more frequently seen in patients with hepatic decompensation and mucocutaneous bleeding, although these relationships were not statistically significant. In <em>2</em>7 patients with hyperfibrinolysis, five (18.5%) required EACA treatment for progressive mucocutaneous bleeding and/or hematoma. EACA treatment was significantly associated with higher C-P scores; greatly shortened ELT (< or =50% of normal value); and abnormal levels of fibrinogen, total bilirubin, and PT, indicating that these factors may serve as predictors for EACA treatment.

CONCLUSIONS

Hyperfibrinolytic activity was seen in 31.3% of patients with cirrhosis, which is correlated with higher C-P scores; abnormal PT, PTT, fibrinogen level, and platelet count; and hyperbilirubinemia. Patients who received EACA treatment usually have a more severe hyperfibrinolytic activity as indicated by shortened ELT and low level of fibrinogen, and more severe liver disease as indicated by higher C-P scores and hyperbilirubinemia.

After a first episode of pulmonary embolism (PE), two major problems need to be considered: risk of recurrence when anticoagulation is stopped, and risk of chronic thromboembolic pulmonary hypertension (CTPH). We followed prospectively consecutive patients who survived a first episode of PE, with or without deep vein thrombosis, to assess the incidence of venous thromboembolism (VTE) recurrences and of symptomatic and asymptomatic CTPH. After 3-6 months of oral anticoagulant therapy (OAT) patients underwent transthoracic echocardiography for measuring transtricuspid (rV-rA) gradient. When rV-rA gradient was >35 mmHg further evaluations were performed to rule in or out CTPH. <em>D</em>uring follow-up patients who developed persistent dyspnea were re-evaluated. In patients who underwent OAT withdrawal <em>D</em>-<em>dimer</em> (<em>D</em><em>D</em>), prothrombin fragment 1 + <em>2</em> (F1 + <em>2</em>), and thrombophilia were evaluated one month after warfarin discontinuation. Overall, <em>2</em>39 patients, 118 males, median age 59(16-89) years, were followed up for a median time of 36(9-19<em>2</em>) months. Nine patients had rV-rA gradient >30 mmHg and ≤35 mmHg, and one of 37 mmHg. Among patients with normal rV-rA gradient, one developed persistent dyspnea 55 months after the first event and CPTH was confirmed. Among <em>2</em>06 patients who stopped OAT, <em>2</em>3(11.<em>2</em>%) had VTE recurrence, 11 PE(48%). Elevated <em>D</em><em>D</em> and F1 + <em>2</em> levels after stopping OAT were significantly associated with recurrence. None of patients with recurrent VTE had elevated rV-rA gradient. In our series the incidence of CTPH after a first episode of PE was 0.4%. VTE recurrence and elevated <em>D</em><em>D</em> and F1 + <em>2</em> levels seemed not to be related to the development of CTPH.

Activation of hemostasis during surgery was investigated in 30 elective cases, who underwent either gastric (group G) or hepatic (group H) resection by a serial determination of various molecular markers such as fibrinopeptide A (FPA), fibrinopeptide B beta 15-4<em>2</em> (B beta 15-4<em>2</em>) <em>D</em>-<em>dimer</em>, thrombin-antithrombin III complex (TAT) and plasmin-alpha <em>2</em> plasmin inhibitor complex (PIC). In both groups, the values of FPA and TAT were significantly elevated intraoperatively, indicating an occurrence of hypercoagulable state. The degree of the elevation was more marked in group H, probably due to greater tissue damage during hepatic resection. Also in both groups, the values B beta 15-4<em>2</em> and PIC were significantly increased during surgery, while the amount of <em>D</em>-<em>dimer</em> was within normal range in most cases, indicating the occurrence of the primary fibrinolysis. These findings are compatible with our previous observations on the postoperative changes in hemostasis. There were statistically significant but variable correlations between the values of fibrinopeptides and the enzyme-inhibitor complexes. The absolute values of the molecular markers of fibrinolysis were always higher than those of coagulation, suggesting that a considerable amount of plasmin, rather than thrombin, is released by surgical tissue damages.

The purpose of this study was to determine whether chronic thyroid hormone suppression therapy (THST) is prothrombotic. We obtained blood samples from 14 thyroid cancer patients while on THST and after they had become hypothyroid for radioiodine whole-body scanning and therapy. Prothrombin fragment 1 + <em>2</em>, fibrinogen, factor VIII, antithrombin, tissue plasminogen activator antigen (tPA), plasminogen activator inhibitor 1 (PAI-1), PAI-1/tPA, and C-reactive protein were significantly (P < 0.05) higher in the hyper- than in the hypothyroid state, whereas protein C and plasmin-antiplasmin complexes were significantly lower during the hyperthyroid period. When the 10 female patients were hyperthyroid, their levels of prothrombin fragment 1 + <em>2</em>, fibrinogen, protein S, antithrombin, tPA, PAI-1, and PAI-1/tPA were significantly higher (P </= 0.05) than in healthy female controls, whereas when the female patients were hypothyroid, their antithrombin and plasmin-antiplasmin were lower and their protein S was higher than in controls. Factor II, plasminogen, and <em>D</em>-<em>dimer</em> were not significantly affected by the thyroid status in either assessment. In conclusion, we found evidence that the majority of patients treated with THST have a prothrombotic profile.

Aspergillus and Penicillium species produce extracellular polysaccharides which are immunologically active. Methyl beta-<em>D</em>-galactofuranoside interferes with the reaction between the polysaccharide antigens and the antibodies raised in rabbits. Of the different interlinked <em>dimers</em> of beta-<em>D</em>-galactofuranosides (1----<em>2</em>; 1----3; 1----5; 1----6) the (1----5) interlinked beta-<em>D</em>-galactofuranoside gave the highest inhibition. An increasing inhibitory effect of di-, tri-, tetra-, penta-, hexa-, and heptamer of (1----5) interlinked beta-<em>D</em>-galactofuranosides was observed. It was noticed that the penta-, hexa- and heptamer of (1----5) interlinked beta-<em>D</em>-galactofuranosides were able to link antibodies raised against the extracellular polysaccharides produced by Penicillium species. The tetramer molecule was able to neutralize the binding of antibodies, which are naturally present in human sera, to the polysaccharides produced by Penicillium and Aspergillus species.

This study was undertaken to evaluate the relationship between serum tumor necrosis factor (TNF) and coagulopathy in patients with prostate cancer. TNF levels in 104 sera obtained from 101 prostate cancer patients were determined using an enzyme immunoassay. Serum levels of fibrin/fibrinogen degradation product E fragment (F<em>D</em>P) and plasma levels of fibrin degradation product <em>D</em>-<em>dimer</em> in patients with elevated serum TNF levels were 1<em>2</em><em>2</em>1.95 +/- 375.94 ng/ml and <em>2</em>7.34 +/- 9.81 micrograms/ml, which were significantly higher than those (F<em>D</em>P, 94.35 +/- 13.17 ng/ml; <em>D</em>-<em>dimer</em>, 1.03 +/- 0.<em>2</em>0 micrograms/ml) in patients with undetectable serum TNF levels (P < 0.01). In addition, patients with elevated serum TNF levels showed significant increases in plasma levels of thrombin-antithrombin-III complex and plasmin-alpha <em>2</em>-antiplasmin inhibitor complex and a significantly higher incidence of positive plasma soluble fibrin monomer complex than did those with undetectable serum TNF levels. The percentage of prothrombin time was significantly decreased in the group with elevated serum levels of TNF. Serum levels of TNF were significantly elevated in patients with serum F<em>D</em>P levels of>> or = <em>2</em>00 ng/ml than in those with serum F<em>D</em>P levels of < <em>2</em>00 ng/ml (3.91 +/- 0.45 versus <em>2</em>.17 +/- 0.08 units/ml) and in patients with plasma <em>D</em>-<em>dimer</em> levels of>> or = <em>2</em> micrograms/ml than in those with plasma <em>D</em>-<em>dimer</em> levels of < <em>2</em> micrograms/ml (3.8<em>2</em> +/- 0.48 versus <em>2</em>.10 +/- 0.06 units/ml). These results suggest that TNF may be one of the pathogenetic factors that could explain the occurrence of coagulopathy in patients with prostate cancer.

Multiorgan dysfunction ensuing from severe heatstroke includes hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. We attempted to assess whether human umbilical cord blood-derived C<em>D</em>34+ cell therapy improves survival during experimental heatstroke by attenuating multiorgan dysfunction. Anesthetized rats, immediately after the onset of heatstroke, were divided into <em>2</em> major groups and given C<em>D</em>34- or C<em>D</em>34+ cells (1 x 10(5)-5 x 10(5)/mL/kg body weight) i.v. They were exposed to ambient temperature of 43 degrees C to induce heatstroke. Another group of rats were exposed to room temperature (<em>2</em>6 degrees C) and used as normothermic controls. Hypotension, hepatic and renal failure (evidenced by increased serum urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels in plasma), hypercoagulable state (evidenced by increased prothrombin time, activated partial thromboplastin time, and <em>D</em>-<em>dimer</em>, and decreased platelet count and protein C in plasma), activated inflammation (evidence by increased TNF-alpha levels in serum), and cerebral dysfunction (evidenced by intracranial hypertension, cerebral hypoperfusion and hypoxia, and cerebral ischemia and injury) were monitored. When the C<em>D</em>34- cell-treated or untreated rats underwent heat stress, their survival time values were found to be 19 to <em>2</em>3 min. Resuscitation with C<em>D</em>34+ cells significantly improved survival time (duration, 63-<em>2</em>91 min). As compared with normothermic controls, all C<em>D</em>34- cell-treated heatstroke animals displayed hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. However, C<em>D</em>34+ cell therapy significantly caused attenuation of all the above-mentioned heatstroke reactions. In addition, the levels of IL-10 in plasma and glial cell line-derived neurotrophic factors in brain were all significantly increased after C<em>D</em>34+ cell therapy during heatstroke. Our data indicate that C<em>D</em>34+ cell therapy may resuscitate persons who had a heatstroke by reducing multiorgan dysfunction or failure.

Timely analysis of the laboratory characteristics associated with <em>2</em>019 novel coronavirus pneumonia (COVI<em>D</em>-19) can assist with clinical diagnosis and prognosis. This study is a collection of clinical data from 54 hospitalized adult patients diagnosed with COVI<em>D</em>-19 in the Zhongfa Xincheng district of China at Tongji Hospital of Huazhong University of Science and Technology from January <em>2</em>8, <em>2</em>0<em>2</em>0 to February 11, <em>2</em>0<em>2</em>0. The average age of the patients was 61.8 ± 14.5 years, and the predominant age group was 50-79. The proportion of critical-type patients with comorbidities was higher than that of severe-type patients. Lymphocyte counts were significantly reduced in routine bloodwork for all patients, but significantly lower in critical-type patients than that in severe-type patients. Prolongation of prothrombin times (PT) and elevation of fibrinogen degradation products (F<em>D</em>Ps) and <em>D</em>-<em>dimers</em> (<em>D</em>-<em>D</em>s) were detected in coagulation function tests, and more significant changes were observed in critical-type patients compared to severe-type patients. Serum ferritin levels were sensitive to severe acute respiratory syndrome coronavirus <em>2</em> (SARS-CoV-<em>2</em>) infection but could not be used for disease assessment. In addition, levels of two inflammatory factors, soluble interleukin-<em>2</em> receptor (sIL-<em>2</em>R) and interleukin-6 (IL-6) were significantly increased in all patients, but higher in critical-type patients than in severe-type patients. Moreover, kidney injury was the second-most common organ affected by COVI<em>D</em>-19 followed by heart and liver. Kidney and heart injury were more severe in critical-type patients than in severe-type patients. All of the 31 severe-type patients recovered. Of the critical-type patients, six died and 17 recovered. The length of hospital stay for critical-type patients was significantly longer for severe-type patients. In summary, increased lymphocyte counts, prolonged PT, secondary increases in fibrinolytic activity and increases in sIL-<em>2</em>R and IL-6 are typical features of COVI<em>D</em>-19 and are associated with disease severity.

Objective: To delineate the clinical characteristics of critically ill COVID-19 patients co-infected with influenza.

Methods: In this study, we included adult patients with laboratory-confirmed COVID-19 form Tongji Hospital (Wuhan, China), with or without influenza, and compared their clinical characteristics.

<strong class="sub-title">Results:</strong> Among 93 patients, 44 died and 49 were discharged. Forty-four (47.3%) were infected with influenza virus A and <em>2</em> (<em>2</em>.<em>2</em>%) with influenza virus B. Twenty-two (50.0%) of the non-survivors and <em>2</em>4 (49.0%) of the survivors were infected with the influenza virus. Critically ill COVI<em>D</em>-19 patients with influenza were more prone to cardiac injury than those without influenza. For the laboratory indicators at admission, white blood cell counts, neutrophil counts, levels of tumor necrosis factor-α, <em>D</em>-<em>dimer</em> value, and proportion of elevated creatinine were higher in non-survivors with influenza than in those without influenza.

Conclusion: The results showed a high proportion of COVID-19 patients were co-infected with influenza in Tongji Hospital, with no significant difference in the proportion of co-infection between survivors and non-survivors. The critically ill COVID-19 patients with influenza exhibited more severe inflammation and organ injury, indicating that co-infection with the influenza virus may induce an earlier and more frequently occurring cytokine storm.

Keywords: COVID-19; co-infection; cytokine storm; influenza; organ injury.

Human glucose 6-phosphate <em>d</em>ehy<em>d</em>rogenase, purifie<em>d</em> after overexpression in E. coli, was shown to contain one molecule/subunit of aci<em>d</em>-extractable "structural" NADP+ an<em>d</em> no NADPH. This tightly boun<em>d</em> NADP+ was re<em>d</em>uce<em>d</em> by G6P, presumably following migration to the catalytic site. Gel-filtration yiel<em>d</em>e<em>d</em> apoenzyme, <em>d</em>evoi<em>d</em> of boun<em>d</em> NADP+ but, surprisingly, still fully active. Mr of the main component of "strippe<em>d</em>" enzyme by gel filtration was approximately 100,000, suggesting a <em>dimer</em>ic apoenzyme (subunit Mr = 59,000). Holoenzyme also containe<em>d</em> tetramer molecules an<em>d</em>, at high protein concentration, a <em>d</em>ynamic equilibrium gave an apparent interme<em>d</em>iate Mr of 150 kDa. Fluorescence titration of the strippe<em>d</em> enzyme gave the K <em>d</em> for structural NADP+ as 37 nM, <em>2</em>00-fol<em>d</em> lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 <em>d</em>egrees C, strippe<em>d</em> enzyme, much less stable than holoenzyme, inactivate<em>d</em> irreversibly within <em>2</em> <em>d</em>. Inactivation at 4 <em>d</em>egrees C was partially reverse<em>d</em> at room temperature, especially with a<em>d</em><em>d</em>e<em>d</em> NADP+. Apoenzyme was imme<em>d</em>iately active, without any visible lag, in rapi<em>d</em>-reaction stu<em>d</em>ies. Human G6PD thus forms active <em>dimer</em> without structural NADP+. Apparently, the true role of the secon<em>d</em>, tightly boun<em>d</em> NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD <em>d</em>eficiency affecting the long-live<em>d</em> non-nucleate erythrocyte. The K<em>d</em> values for two class I mutants, G488S an<em>d</em> G488V, were <em>2</em>73 nM an<em>d</em> 480 nM, respectively (seven- an<em>d</em> 13-fol<em>d</em> elevate<em>d</em>), matching the structural pre<em>d</em>iction of weakene<em>d</em> structural NADP+ bin<em>d</em>ing, which woul<em>d</em> explain <em>d</em>ecrease<em>d</em> stability an<em>d</em> consequent <em>d</em>isease. Preparation of native apoenzyme an<em>d</em> measurement of K<em>d</em> constant for structural NADP+ will now allow quantitative assessment of this <em>d</em>efect in clinical G6PD mutations.

BACKGROUND

The diagnostic work-up of patients with suspected pulmonary embolism (PE) has been optimized and simplified by the use of clinical decision rules (CDR), D-dimer (DD) testing and spiral computed tomography (s-CT). Whether this strategy is equally safe and efficient in specific subgroups of patients is evaluated in this study.

METHODS

A diagnostic strategy including a CDR, DD test and s-CT was evaluated in patients with malignancy, previous venous thromboembolism (VTE), chronic obstructive pulmonary disease or heart failure and in older patients. PE was ruled out by either an unlikely CDR and a normal DD or a s-CT negative for PE. The safety of these tests was assessed by the 3-month incidence rate of symptomatic VTE in those without PE at baseline. The efficiency was evaluated by calculating the numbers needed to test for the different subgroups.

RESULTS

The venous thromboembolic incidence rate after the combination of an unlikely CDR and a normal DD varied from 0% (95% CI: 0-7.9%) in the 482 patients older than 75 years of age to 2% (95% CI: 0.05-10.9%) in the 474 patients with a malignancy. For s-CT these incidences varied from 0.3% to 1.8%. The number needed to test in order to rule out one patient from PE with the studied strategy was highest in cancer patients and in the elderly patients (approximately 10).

CONCLUSIONS

It appears to be safe to rule out PE by either the combination of an unlikely CDR and a normal DD or by a negative s-CT in various subgroups of patients with suspected PE. However, the clinical usefulness of the CDR in combination with the DD as the initial step in the diagnostic process varied among these patient groups.

The haemostatic system and the use of heparin during cardiopulmonary bypass (CPB) have been studied extensively in adults but not in children. Results from adult trials cannot be extrapolated to children because of age-dependent physiologic differences in haemostasis. We studied <em>2</em><em>2</em> consecutive paediatric patients who underwent CPB at The Hospital for Sick Children, Toronto. Fibrinogen, factors II, V, VII, VIII, IX, XII, prekallikrein, protein C, protein S, antithrombin (AT), heparin cofactor II, alpha <em>2</em>-macroglobulin, plasminogen, alpha <em>2</em>-antiplasmin, tissue plasminogen activator (tPA), plasminogen activator inhibitor, thrombin-AT complexes (TAT), <em>D</em>-<em>dimer</em>, heparin (by both anti-factor Xa assay and protamine titration) and activated clotting time (ACT) were assayed perioperatively. The timing of the sampling was: pre heparin, post heparin, after initiation of CPB, during hypothermia, post hypothermia, post protamine reversal and <em>2</em>4 h post CPB. Plasma concentrations of all haemostatic proteins decreased by an average of 56% immediately following the initiation of CPB due to haemodilution. <em>D</em>uring CPB, the majority of procoagulants, inhibitors and some components of the fibrinolytic system (plasminogen, alpha <em>2</em> AP) remained stable. However, plasma concentrations of TAT and <em>D</em>-<em>dimers</em> increased during CPB showing that significant activation of the coagulation and fibrinolytic systems occurred. Mechanisms responsible for the activation of haemostasis are likely complex. However, low plasma concentrations of heparin (< <em>2</em>.0 units/ml in 45% of patients) during CPB were likely a major contributing etiology. ACT values showed a poor correlation (r = 0.38) with heparin concentrations likely due to concurrent haemodilution of haemostatic factors, activation of haemostatic system, hypothermia and activation of platelets. In conclusion, CPB in paediatric patients causes global decreases of components of the coagulation and fibrinolytic systems, primarily by haemodilution and secondarily by consumption.

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-<em>dimer</em> subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average <em>2</em>0-fold weakening of tetramer-<em>dimer</em> interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The <em>dimer</em>-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the <em>dimer</em>, although tetramer formation reciprocally influences the amount of <em>dimer</em> available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The <em>dimer</em> strengths increase in the order Hb Portland-<em>2</em> (zeta(<em>2</em>)beta(<em>2</em>)) < Hb Portland-1 (zeta(<em>2</em>)gamma(<em>2</em>)) approximately equal Hb Gower-1 (zeta(<em>2</em>)epsilon(<em>2</em>)) < Hb Gower-<em>2</em> (alpha(<em>2</em>)epsilon(<em>2</em>)) < HbF(1) < HbF (alpha(<em>2</em>)gamma(<em>2</em>)) < HbA(<em>2</em>) (alpha(<em>2</em>)<em>delta</em>(<em>2</em>)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic ->> fetal ->> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

<strong class="sub-title"> Objectives: </strong> One of the defining features of the novel coronavirus disease <em>2</em>019 infection has been high rates of venous thromboses. The present study aimed to describe the prevalence of venous thromboembolism in critically ill patients receiving different regimens of prophylactic anticoagulation.

<strong class="sub-title"> Design: </strong> Single-center retrospective review using data from patients with confirmed severe acute respiratory syndrome coronavirus <em>2</em> requiring intubation.

Setting: Tertiary-care center in Indianapolis, IN, United States.

<strong class="sub-title"> Patients: </strong> Patients hospitalized at international units Health Methodist Hospital with severe acute respiratory syndrome coronavirus <em>2</em> requiring intubation between March <em>2</em>3, <em>2</em>0<em>2</em>0, and April 8, <em>2</em>0<em>2</em>0, who underwent ultrasound evaluation for venous thrombosis.

Interventions: None.

<strong class="sub-title"> Measurements and main results: </strong> A total of 45 patients were included. Nineteen of 45 patients (4<em>2</em>.<em>2</em>%) were found to have deep venous thrombosis. Patients found to have deep venous thrombosis had no difference in time to intubation (p = 0.97) but underwent ultrasound earlier in their hospital course (p = 0.0<em>2</em>). Sequential Organ Failure Assessment scores were similar between the groups on day of intubation and day of ultrasound (p = 0.44 and p = 0.07, respectively). D-dimers were markedly higher in patients with deep venous thrombosis, both for maximum value and value on day of ultrasound (p < 0.01 for both). Choice of prophylactic regimen was not related to presence of deep venous thrombosis (p = 0.35). Ultrasound evaluation is recommended if D-dimer is greater than <em>2</em>,000 ng/mL (sensitivity 95%, specificity 46%) and empiric anticoagulation considered if D-dimer is greater than 5,500 ng/mL (sensitivity 53%, specificity 88%).

<strong class="sub-title"> Conclusions: </strong> Deep venous thrombosis is very common in critically ill patients with coronavirus disease <em>2</em>019. There was no difference in incidence of deep venous thrombosis among different pharmacologic prophylaxis regimens, although our analysis is limited by small sample size. D-dimer values are elevated in the majority of these patients, but there may be thresholds at which screening ultrasound or even empiric systemic anticoagulation is indicated.

BACKGROUND

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) can be fatal, and abnormalities in the coagulation system of patients with AE-IPF have been reported. Recombinant human soluble thrombomodulin (rhTM) forms a complex with thrombin to inactivate coagulation. It also inhibits high-mobility group box protein 1 (HMGB-1), which results in the suppression of inflammation.

OBJECTIVE

We aimed to evaluate the effectiveness of rhTM for the treatment of AE-IPF.

METHODS

We retrospectively reviewed the medical records of 41 patients with AE-IPF who were admitted to our institution during the period <em>2</em>006-<em>2</em>013. The clinical features and outcomes of 16 patients treated with rhTM (rhTM group) were compared with those of <em>2</em>5 patients treated with conventional therapy (control group). Patients were treated with corticosteroid (CS) pulse therapy for 3 days, followed by maintenance treatment with a tapered dose of CS. Patients in the rhTM group also received rhTM (0.06 mg/kg/day) for 6 days as an initial treatment, in combination with CS.

RESULTS

Except for <em>D</em>-<em>dimer</em> level, there were no significant differences in the baseline characteristics of the <em>2</em> groups. When compared with the control group, the rhTM group had a significantly higher survival rate at 3 months (40 vs. 69%, p = 0.048). A univariate Cox proportional hazards regression model showed that the predictive factors for survival were lactate dehydrogenase level and rhTM treatment. Regarding adverse events, 1 patient in the rhTM group developed mild bleeding events.

CONCLUSIONS

rhTM as an add-on to conventional treatment may improve survival in patients with AE-IPF.

BACKGROUND

A rapid laboratory test for diagnosis of acute aortic dissection (AAD) has not been available. We performed this prospective study to determine the utility of a rapid bedside D-dimer (DD) assay for detection of AAD.

RESULTS

Patients with suspected AAD were recruited and their DD levels were measured by rapid bedside assay. They were divided into 2 groups according to enhanced computed tomography findings: an AAD group (n = 30) and a non-AAD group (n = 48). The median DD level was higher in the AAD group (1.80 microg/ml) than in the non-AAD group (0.42 microg/ml) (p = 0.000). The rapid bedside DD assay showed 100% sensitivity, 54% specificity, 58% positive predictive value and 100% negative predictive value for detection of AAD with a normal DD level of up to 0.5 microg/ml. The combination of DD level >0.5 microg/ml and systolic blood pressure>> or = 180 mmHg showed 86% positive predictive value for detection of AAD. Conclusions We conclude that the rapid bedside DD assay is a highly sensitive method for early exclusion of AAD in patients with chest and/or back pain suggestive of AAD. Acute aortic dissection is highly probable if a rapid DD assay shows the elevated DD level with systolic blood pressure>> or = 180 mmHg on admission.