Objectives: To describe the clinical characteristics and predictors of major outcomes in patients treated with tocilizumab (TCZ) for severe COVID-19 pneumonia.

<strong class="sub-title"> Patients and methods: </strong> Case series of all sequential patients with severe COVID-19 pneumonia treated with TCZ at an Academic Spanish hospital (March 1<em>2</em> - May <em>2</em>, <em>2</em>0<em>2</em>0). Clinical outcomes: death, length of hospital stay. An early clinical response to TCZ (48-7<em>2</em> h after the administration) was assessed by variations in respiratory function markers, Brescia COVID Respiratory Severity Scale (BCRSS), inflammatory parameters, and patients' and physicians' opinion. Associations were tested by multiple logistic regression.

<strong class="sub-title"> Results: </strong> From a cohort of <em>2</em>36 patients, 77 patients treated with TCZ were included (median age 6<em>2</em> years (IQR 53.0-7<em>2</em>.0), 64.9% were males), 4<em>2</em>.9% had Charlson index ≥3; hypertension (41.6%), obesity (34.7%), and diabetes (<em>2</em>0.8%). Median follow-up was 83.0 days (78.0-86.5), no patient was readmitted. ICU admission was required for 4<em>2</em> (54.5%), invasive mechanical ventilation in 38 (49.4%) and 10 patients died (1<em>2</em>.9% global, <em>2</em>3.8% at ICU admitted). After multivariate adjustment, TCZ response by BCRSS (OR 0.03 (0.01-0.68), p = 0.0<em>2</em>8), and Charlson index (OR 3.54 (1.<em>2</em>0-10.44), p = 0.0<em>2</em><em>2</em>) has been identified as independent factors associated with mortality. Median of hospital stay was 16.0 days (11.0-<em>2</em>3.0); BCRSS, physician subjective and D-dimer response were associated with shorter hospitalization stay.

<strong class="sub-title"> Conclusions: </strong> In a Mediterranean cohort, use of tocilizumab for severe COVID-19 show 1<em>2</em>.9% of mortality. Early TCZ-response by BCRSS and low comorbidity were associated with increased survival. Early TCZ-response was related to shorter median hospital stay.

Keywords: COVID19 pneumonia; Case series; Mechanical invasive ventilation; Mortality; Tocilizumab.

BACKGROUND

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

METHODS

We performed a prospective study to investigate 11 potential biomarkers for prediction of venous thromboembolism (VTE) in newly diagnosed HGG patients who had undergone a neurosurgical intervention. In addition, we tested <em>2</em> VTE risk assessment models (RAMs). The strongest predictors of VTE, which were identified by statistical forward selection, were used for the first RAM. The parameters used for the second RAM were both predictive of VTE and available in routine clinical practice.

RESULTS

One hundred forty-one HGG patients were included in this study, and <em>2</em>4 (17%) of them developed VTE during follow-up. An association with the risk of future VTE was found for the following parameters: leukocyte count, platelet count, sP-selectin, prothrombin-fragment 1 + <em>2</em>, FVIII activity, and D-dimer. The first RAM included low platelet count ((<em>2</em>5th percentile of the study population) and elevated sP-selectin (≥75th percentile). The cumulative VTE probability after 1<em>2</em> months was 9.7% for score 0 (n = 76), 18.9% for score 1 (n = 59), and 83.3% for score <em>2</em> (n = 6). The second RAM included low platelet count ((<em>2</em>5th percentile), elevated leukocyte count, and elevated D-dimer (≥75th percentile). The probability of VTE was 3.3% for score 0 (n = 63), <em>2</em>3.0% for score 1 (n = 53), and 37.7% for score <em>2</em> (n = <em>2</em><em>2</em>) or score 3 (n = 3).

CONCLUSIONS

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

<strong class="sub-title"> Background: </strong> Coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) is a novel infectious viral disease, which lacks well-established diagnostic laboratory parameters that could be used to evaluate disease severity, thromboembolism or cardiovascular events and to predict clinical prognosis. Coagulation cascade and platelet functions have not been well studied in the COVI<em>D</em>-19 patients.

Methods: A total of 178 patients enrolled in Wuhan Huoshenshan Hospital were included for the study. Blood platelets and coagulation functions were analyzed in COVID-19 patients with non-severe and severe subgroups. Other biochemical laboratory parameters were also analyzed.

<strong class="sub-title"> Results: </strong> Forty-nine (<em>2</em>7.5%) out of 178 patients were diagnosed with severe disease in this study, and 1<em>2</em>9 patients with non-severe disease. Severe disease group had significant lower platelet count 186.00 (103.50-<em>2</em>49.00) ×10⁹/L than <em>2</em>51.00 (<em>2</em>0<em>2</em>.00-317.00) ×10⁹/L of non-severe group, <i>p </i>= 0.000. Severe group also had significantly abnormal coagulation parameters than non-severe group: prothrombin time (PT) 14.55 (13.40-16.53) s vs. 1<em>2</em>.70 (1<em>2</em>.15-13.59) s, <i>p </i>= 0.000; international normalized ratio (INR) 1.<em>2</em>1 (1.13-1.36) vs. 1.06 (1.01-1.13), <i>p </i>= 0.000; thrombin time (TT) 16.35 (15.69-17.47) s vs. 15.68 (14.79-16.69) s, <i>p </i>= 0.011; <em>D</em>-<em>Dimer</em> 1.05 (0.68-5.90) mg/L vs. 0.4<em>2</em> (0.<em>2</em>8-0.79) mg/L, <i>p </i>= 0.000; While the liver function parameter alanine aminotransferase (ALT) and aspartate aminotransferase (AST) didn't show significance between two groups, ALT 30.80 (19.00-58.30) IU/L vs. <em>2</em>8.80 (15.75-50.15) IU/L, <i>p </i>= 0.487; AST <em>2</em>7.80 (19.30-40.55) IU/L vs. <em>2</em><em>2</em>.6 (16.7-3<em>2</em>.03) IU/L, <i>p </i>= 0.10<em>2</em>. <em>D</em>isseminated intravascular coagulation (<em>D</em>IC) rate was 6.1% in severe group while 0% in non-severe group. Survival rate of severe disease group was worse than non-severe group, 85.7% vs. 100%, <i>p </i>= 0.000. Thrombocytopenia correlated with coagulation function, <em>D</em>IC rate and survival. Six out of 7 death case had thrombocytopenia during hospitalization, and platelet count decreased subsequently until death. Thrombocytopenia occurred within 1 week after admission in 6 recovered patients. And increased platelet levels followed by positive SARS-CoV-<em>2</em> IgM/IgG and negative coronavirus nucleic acid tested in 8 recovered patients.

Conclusions: Low platelet count is associated with abnormal coagulation function and increased risk of DIC, severe disease manifestation and increased mortality in patients with COVID-19.

<strong class="sub-title"> Keywords: </strong> COVI<em>D</em>-19; Coagulation; <em>D</em>IC; SARS-CoV-<em>2</em>; Thrombocytopenia.

<strong class="sub-title"> Backgroun<em>d</em>: </strong> The COVID-19 outbreak challenges the Spanish health system since March <em>2</em>0<em>2</em>0. Some available therapies (antimalarials, antivirals, biological agents) were groun<em>d</em>e<em>d</em> on clinical case observations or basic science <em>d</em>ata. The aim of this stu<em>d</em>y is to <em>d</em>escribe the characteristics an<em>d</em> impact of <em>d</em>ifferent therapies on clinical outcomes in a cohort of severe COVID-19 patients.

Methods: In this retrospective, single-center, observational study, we collected sequential data on adult patients admitted to Hospital Universitario Quironsalud Madrid. Eligible patients should have a microbiological (positive test on RT-PCR assay from a nasal swab) or an epidemiological diagnosis of severe COVID-19. Demographic, baseline comorbidities, laboratory data, clinical outcomes, and treatments were compared between survivors and non-survivors. We carried out univariate and multivariate logistic regression models to assess potential risk factors for in-hospital mortality.

<strong class="sub-title"> Fin<em>d</em>ings: </strong> From March 10th to April 15th, <em>2</em>0<em>2</em>0, 607 patients were inclu<em>d</em>e<em>d</em>. Me<em>d</em>ian age was 69 years [interquartile range, {IQR} <em>2</em><em>2</em>; 65% male). The most common comorbi<em>d</em>ities were hypertension (<em>2</em>76 [46·94%]), <em>d</em>iabetes (95 [16·16%]), chronic car<em>d</em>iac (133 [<em>2</em><em>2</em>·6<em>2</em>%]) an<em>d</em> respiratory (114 [19·39%]) <em>d</em>iseases. 141 patients (<em>2</em>3·<em>2</em>%) <em>d</em>ie<em>d</em>. In the multivariate mo<em>d</em>el the risk of <em>d</em>eath increase<em>d</em> with ol<em>d</em>er age (o<em>d</em><em>d</em>s ratio, for every year of age, 1·15, [95% CI 1·11 - 1·<em>2</em>]), tocilizumab therapy (<em>2</em>·4, [1·13 - 5·11]), C-reactive protein at a<em>d</em>mission (1·07, per 10 mg/L, [1·04 - 1·10]), <em>d</em>-<em>dimer</em> > <em>2</em>·5 μg/mL (1·99, [1·03 - 3·86]), <em>d</em>iabetes mellitus (<em>2</em>·61, [1·19 - 5·73]), an<em>d</em> the PaO_<em>2</em>/FiO_<em>2</em> at a<em>d</em>mission (0·99, per every 1 mmHg, [0·98 - 0·99]). Among the prescribe<em>d</em> therapies (tocilizumab, glucocorticoi<em>d</em>s, lopinavir/ritonavir, hy<em>d</em>roxychloroquine, cyclosporine), only cyclosporine was associate<em>d</em> with a significant <em>d</em>ecrease in mortality (0·<em>2</em>4, [0·1<em>2</em> - 0·46]; <i>p</i><0·001).

Interpretation: In a real-clinical setting, inhibition of the calcineurin inflammatory pathway, NF-κΒ, could reduce the hyperinflammatory phase in COVID-19. Our findings might entail relevant implications for the therapy of this disease and could boost the design of new clinical trials among subjects affected by severe COVID-19.

Funding: Hospital Universitario Quironsalud Madrid. Own fundings for COVID-19 research.

<strong class="sub-title"> Keywor<em>d</em>s: </strong> Acute respiratory insufficiency; Cyclosporine a; Hyperinflammation state; Immunosuppressants; SARS-CoV-<em>2</em> infection; Severe COVID-19.

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive and life-threatening disease characterized by complement-mediated chronic hemolysis, resulting in serious life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody that inhibits terminal complement activation, has been shown to reduce hemolysis in PNH patients. The pivotal open-label, 1<em>2</em>-week phase II registration study (AEGIS) was designed to evaluate the efficacy and safety of eculizumab in Japanese patients with PNH. This trial achieved its primary endpoint of reducing intravascular hemolysis with high statistical significance. Twenty-seven of the <em>2</em>9 patients responded to eculizumab treatment, resulting in an 87% reduction in hemolysis (P < 0.0001) and subsequent improvement in anemia (P = 0.0003) despite reduction in transfusion requirements (P = 0.006). Fatigue and dyspnea significantly improved within 1-<em>2</em> weeks of eculizumab treatment and the improvement was independent of changes in hemoglobin. Chronic kidney disease (CK<em>D</em>) was common (66%) and eculizumab treatment improved CK<em>D</em> in 41% of patients at 1<em>2</em> weeks (P < 0.001). Elevated thrombotic risk was evident in Japanese PNH patients and eculizumab treatment normalized <em>D</em>: -<em>dimer</em> levels in 45% of patients with elevated <em>D</em>: -<em>dimers</em> at baseline (P < 0.001). The AEGIS results demonstrate that eculizumab is effective, safe and well tolerated in Japanese patients with PNH.

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (<em>D</em> EGTA, <em>D</em>-<em>dimer</em>, Y) and also CNBr fragment <em>2</em> from fibrinogen have this property. CNBr fragment <em>2</em> consists of amino acid residues A alpha (148-<em>2</em>07), B beta (191-<em>2</em><em>2</em>4) + (<em>2</em><em>2</em>5-<em>2</em>4<em>2</em>) + (<em>2</em>43-305) and gamma 95-<em>2</em>65, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment <em>2</em> after reduction and carboxymethylation, and found that only A alpha 148-<em>2</em>07 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in <em>D</em> EGTA and <em>D</em>-<em>dimer</em> also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.

BACKGROUND

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

OBJECTIVE

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

METHODS

Recruitment was from an observational cohort study of AF. Baseline assessment included measurement of plasma fibrinogen, fibrin <em>D</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em> (F1+<em>2</em>), thrombin-antithrombin complexes (TAT), von Willebrand factor and tissue plasminogen activator. We assessed cognitive function after 3 years' follow-up using the 13-item modified Telephone Interview for Cognitive Status (TICSm) and the short form of the Informant Questionnaire on Cognitive <em>D</em>ecline in the Elderly (IQCO<em>D</em>E).

RESULTS

Of the <em>2</em>18 subjects assessed, 145 (66%) were prescribed warfarin. Forty-nine (<em>2</em><em>2</em>%) met TICSm/IQCO<em>D</em>E criteria for dementia. <em>D</em>-<em>dimer</em>, F1+<em>2</em> and TAT levels were higher in AF subjects with dementia compared with those without (medians 81 vs. 60 ng mL(-1), P = 0.008; 0.76 vs. 0.49 nmol L(-1), P = 0.006; and 1.78 vs. 1.44 microg L(-1), P = 0.003, respectively). These associations became of borderline statistical significance following adjustment for age. Logistic regression showed a trend towards warfarin use being independently associated with reduced prevalence of dementia (odds ratio 0.5<em>2</em>, P = 0.08).

CONCLUSIONS

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

Human erythrocyte spectrin is an antiparallel hetero<em>dimer</em> comprise<em>d</em> of a <em>2</em>80 kDa alpha subunit an<em>d</em> a <em>2</em>46 kDa beta subunit which further associates into tetramers in the re<em>d</em> cell membrane cytoskeleton. Lateral association of the flexible ro<em>d</em>like monomers involves a multiple-step process that is initiate<em>d</em> by a high affinity association near the actin-bin<em>d</em>ing en<em>d</em> of the molecule (<em>dimer</em> nucleation site). In this stu<em>d</em>y, recombinant alpha an<em>d</em> beta proteins comprising two or four "spectrin type" motifs with an<em>d</em> without a<em>d</em>jacent, terminal nonhomologous <em>d</em>omains were evaluate<em>d</em> for their relative contributions to <em>dimer</em> initiation, an<em>d</em> the thermo<em>d</em>ynamic properties of these hetero<em>dimer</em> complexes were measure<em>d</em>. Se<em>d</em>imentation equilibrium stu<em>d</em>ies showe<em>d</em> that in the absence of the heterologous subunit, in<em>d</em>ivi<em>d</em>ual recombinant proteins forme<em>d</em> weak homo<em>dimer</em>s (K(<em>d</em>)>> 0.3 mM). When <em>2</em>-motif (alpha<em>2</em>0-<em>2</em>1 an<em>d</em> beta1-<em>2</em>) an<em>d</em> 4-motif (alpha18-<em>2</em>1 an<em>d</em> beta1-4) recombinants lacking the terminal nonhomologous <em>d</em>omains were paire<em>d</em> with the complementary protein, high affinity hetero<em>dimer</em>s were forme<em>d</em> in se<em>d</em>imentation equilibrium analysis. Both the alpha<em>2</em>0-<em>2</em>1/beta1-<em>2</em> complex an<em>d</em> the alpha<em>2</em>0-<em>2</em>1EF/betaABD1-<em>2</em> complex showe<em>d</em> stoichiometric bin<em>d</em>ing with similar bin<em>d</em>ing affinities (K(<em>d</em>) approximately 10 nM) using isothermal titration calorimetry. The alpha<em>2</em>0-<em>2</em>1/beta1-<em>2</em> complex showe<em>d</em> an enthalpy of -10 kcal/mol, while the alpha<em>2</em>0-<em>2</em>1EF/betaABD1-<em>2</em> complex showe<em>d</em> an enthalpy of -13 kcal/mol. Pull-<em>d</em>own assays using alpha spectrin GST fusion proteins showe<em>d</em> strong associations between all hetero<em>dimer</em> complexes in physiological buffer, but all hetero<em>dimer</em> complexes were <em>d</em>estabilize<em>d</em> by the presence of Triton X-100 an<em>d</em> other <em>d</em>etergents. Complexes lacking the nonhomologous <em>d</em>omains were <em>d</em>estabilize<em>d</em> to a greater extent than complexes that inclu<em>d</em>e<em>d</em> the nonhomologous <em>d</em>omains. The <em>d</em>etergent effect appears to be responsible for the apparent essential role of the nonhomologous <em>d</em>omains in prior reports. Taken together, our results in<em>d</em>icate that the terminal nonhomologous <em>d</em>omains <em>d</em>o not contribute to <em>dimer</em> initiation nor are they require<em>d</em> for formation of high affinity spectrin hetero<em>dimer</em>s in physiological buffers.

OBJECTIVE

In a phase 3 trial, recombinant human activated protein C (drotrecogin alfa [activated]) significantly reduced mortality in adult patients with severe sepsis. We have now performed a preliminary analysis of the safety, pharmacokinetics, and pharmacodynamics of drotrecogin alfa (activated) in pediatric patients with severe sepsis.

METHODS

Open-label, nonrandomized, sequential, <em>2</em>-part study conducted in 11 medical centers in the United States and United Kingdom.

METHODS

Eighty-three pediatric patients with severe sepsis aged term newborn >>or=38 weeks' gestation) to <18 years old.

METHODS

In part 1, drotrecogin alfa (activated) was administered as escalating doses of 6, 1<em>2</em>, <em>2</em>4, and 36 micro g/kg per hour for 6 hours for each patient (n = <em>2</em>1). In part <em>2</em>, drotrecogin alfa (activated) was infused at a rate of <em>2</em>4 micro g/kg per hour for 96 hours in 6<em>2</em> patients.

METHODS

Plasma clearance, plasma concentration, D-dimer, protein C, and antithrombin levels were measured, and adverse events were monitored.

RESULTS

The trial enrolled 83 pediatric patients with severe sepsis, aged term newborn >>or=38 weeks' gestation) to <18 years. In part 1, a dose of <em>2</em>4 micro g/kg per hour produced steady-state plasma concentrations of activated protein C similar to those attained in equivalently dosed adult severe sepsis patients. For all pediatric patients dosed at <em>2</em>4 micro g/kg per hour, the median weight-normalized clearance was 0.45 L/hour/kg and the median steady-state concentration was 51.3 ng/mL. The mean plasma half-life was 30 minutes. Weight-normalized clearance in pediatric and adult patients did not differ significantly with age or weight. D-dimer levels decreased <em>2</em>6% from baseline to end of infusion. Baseline levels of protein C and antithrombin increased 79% and <em>2</em>4%, respectively, over the 96-hour treatment period in part <em>2</em>. The incidence of serious bleeding during infusion and during the entire study period was <em>2</em>.4% and 4.8%, respectively.

CONCLUSIONS

Pediatric patients with severe sepsis manifest sepsis-induced coagulopathy including protein C deficiency comparable to that seen in adults with severe sepsis. The pharmacokinetics, pharmacodynamic effects, and safety profile of drotrecogin alfa (activated) in pediatric patients are similar to those previously published for adult patients. A large, phase 3, randomized, placebo-controlled study is ongoing to confirm these results and formally assess the safety and efficacy of drotrecogin alfa (activated) in children.

BACKGROUND

Recommended management of attacks of hereditary angioedema (HAE) due to C1 esterase inhibitor (C1-INH) deficiency (C1-INH-HAE) includes therapy with exogenous C1INH. Thrombotic/thromboembolic events (TEE) have been reported with plasma-derived C1INH, but so far none with recombinant human C1INH (rhC1INH). This phase III, randomized, placebo (saline)-controlled study evaluated the safety of rhC1INH 50 IU/kg for the treatment of acute attacks in 74 patients with C1-INH-HAE.

METHODS

Monitoring for TEE and assessment of risk of deep vein thrombosis (<em>D</em>VT) by the Wells prediction rule were performed, and levels of fibrin degradation products (plasma <em>D</em>-<em>dimers</em>) were assessed before study drug administration (baseline), <em>2</em> h, and 7 days posttreatment.

RESULTS

Plasma <em>D</em>-dimer levels were elevated in 80% of the patients (median [<em>2</em>5th-75th percentiles]: <em>2</em>149 [480-5105] μg/l; normal ≤<em>2</em>50 μg/l) and were higher in patients with submucosal (abdominal, oropharyngeal-laryngeal) attacks (3095 [890-10000] μg/l; n = <em>2</em>9) compared with subcutaneous (peripheral, facial) attacks (960 [450-4060] μg/l; n = 35). Median plasma <em>D</em>-dimer levels were comparable across treatment groups at baseline (1874 [475-4568] μg/l rhC1INH; <em>2</em><em>2</em>59 [586-7533] μg/l saline) and <em>2</em> h postinfusion (<em>2</em>389 [760-4974] μg/l rhC1INH; <em>2</em>550 [310-8410] μg/l saline); median plasma <em>D</em>-dimer levels were decreased by <em>D</em>ay 7 in both groups (4<em>2</em>5 [<em>2</em>3<em>2</em>-3<em>2</em>40] μg/l rhC1INH; 418 [<em>2</em>46-<em>2</em>318] μg/l saline). No increased risk of <em>D</em>VT was identified, nor any TEE reported in rhC1INH treated or controls.

CONCLUSIONS

Elevated plasma D-dimer levels were associated with acute C1-INH-HAE attacks, particularly with submucosal involvement. However, rhC1INH therapy was not associated with thrombotic events.

The lymphatic en<em>d</em>othelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 an<em>d</em> the nature of its interaction with ligan<em>d</em> are obscure. Here we present new evi<em>d</em>ence that Lyve-1 is functionally <em>d</em>istinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an exten<em>d</em>e<em>d</em> HA-bin<em>d</em>ing unit, comprising elements flanking the N an<em>d</em> C termini of the consensus lectin-like Link mo<em>d</em>ule, bri<em>d</em>ge<em>d</em> by a thir<em>d</em> conserve<em>d</em> <em>d</em>isulfi<em>d</em>e linkage that is critical for HA bin<em>d</em>ing. In a<em>d</em><em>d</em>ition, we i<em>d</em>entify six essential resi<em>d</em>ues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, an<em>d</em> Lys-108 that <em>d</em>efine a compact HA-bin<em>d</em>ing surface on Lyve-1, encompassing the epitope for an a<em>d</em>hesion-blocking monoclonal antibo<em>d</em>y 3A, in an analogous position to the HA-bin<em>d</em>ing surface in CD44. The overtly electrostatic character of HA bin<em>d</em>ing in Lyve-1 an<em>d</em> its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast marke<em>d</em>ly with CD44 (IC(50)>> <em>2</em> m NaCl) in which HA bin<em>d</em>ing is me<em>d</em>iate<em>d</em> by hy<em>d</em>rogen bon<em>d</em>ing an<em>d</em> hy<em>d</em>rophobic interactions. In a<em>d</em><em>d</em>ition, unlike the exten<em>d</em>e<em>d</em> Link mo<em>d</em>ule in CD44, which bin<em>d</em>s HA efficiently when expresse<em>d</em> as a soluble monomer (K(<em>d</em>) = 65.7 mum), that of Lyve-1 requires artificial <em>d</em>imerization, although the full ecto<em>d</em>omain is active as a monomer (K(<em>d</em>) = 35.6 mum). Finally, full-length Lyve-1 <em>d</em>i<em>d</em> not form stable <em>dimers</em> in bin<em>d</em>ing-competent <em>2</em>93T transfectants when assesse<em>d</em> using bioluminescent resonance energy transfer. These results reveal that elements a<em>d</em><em>d</em>itional to the exten<em>d</em>e<em>d</em> Link mo<em>d</em>ule are require<em>d</em> to stabilize HA bin<em>d</em>ing in Lyve-1 an<em>d</em> in<em>d</em>icate important structural an<em>d</em> functional <em>d</em>ifferences with CD44.

Thrombin-catalyzed activation of hetero<em>dimer</em>ic factor VIII occurs by limited proteolysis, yielding subunits A1 and A<em>2</em> derived from the heavy chain (HC) and A3-C1-C<em>2</em> derived from the light chain (LC). The roles of these cleavages in the function of procoagulant activity are poorly understood. To determine whether LC cleavage contributes to the potentiation of factor VIII activity, factor VIII hetero<em>dimer</em>s were reconstituted from native HC and either thrombin-cleaved LC (A3-C1-C<em>2</em>) or intact LC and purified by Mono S chromatography. The reconstituted factor VIII form containing the A3-C1-C<em>2</em> subunit had a specific activity (<em>2</em> units/micrograms) that was approximately 3-fold greater than that of the reconstituted factor VIII form containing native LC (0.6 units/microgram). Factor Xa generation assays using the hybrid hetero<em>dimer</em> showed an initial rate that was unaffected by the presence of von Willebrand factor and a reduced lag time when compared with the native hetero<em>dimer</em>. The A1/A3-C1-C<em>2</em> <em>dimer</em> was dissociated by chelation, and the purified A1 subunit was reacted with either the A3-C1-C<em>2</em> subunit or the LC in the presence of Mn<em>2</em>+ to reconstitute the <em>dimer</em>. Factor VIIIa heterotrimers were reconstituted from either A1/A3-C1-C<em>2</em> or A1/LC plus the A<em>2</em> subunit. The authentic factor VIIIa heterotrimer (A1/A3-C1-C<em>2</em>/A<em>2</em>) had 3-fold greater activity than the form containing the LC. However, upon reaction with thrombin, the activity of the latter form was increased to that of the factor VIIIa form containing native subunits. The incremental increase in fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa was markedly greater in the presence of HC/A3-C1-C<em>2</em> (<em>delta</em> r = 0.037) compared with HC/LC (<em>delta</em> r = 0.011) and approached the value obtained with factor VIIIa (<em>delta</em> r = 0.051). These results suggest that cleavage of factor VIII LC directly contributes to the potentiation of coagulant activity by modulating the conformation of the factor IXa active site.

The nonnucleoside inhibitor binding pocket is a well-defined region in the p66 palm domain of the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). This binding pocket opens toward the interface of the p66/p51 hetero<em>dimer</em> and we have investigated whether ligand binding at or near this site induces structural changes that have an impact on the <em>dimer</em>ic structure of HIV-1 RT. 1-[<em>2</em>',5'-bis-O-(tert-butyldimethylsilyl]-3'-spiro-5' '-(4' '-amino-1' ',<em>2</em>' '-oxathiole-<em>2</em>' ',<em>2</em>' '-dioxide)-3-ethylthymine (TSAOe(3)T) was found to destabilize the subunit interactions of both the p66/p51 hetero<em>dimer</em> and p66/p66 homo<em>dimer</em> enzymes. The Gibbs free energy of <em>dimer</em> dissociation (<em>D</em>eltaG(<em>D</em>)(H)<em>2</em>(O)) is decreased with increasing concentrations of TSAOe(3)T, resulting in a loss in <em>dimer</em> stability of 4.0 and 3.<em>2</em> kcal/mol for the p66/p51 and p66/p66 HIV-1 RT enzymes, respectively. This loss of energy is not sufficient to induce the dissociation of the subunits in the absence of denaturant. This destabilizing effect seems to be unique for TSAOe(3)T, since neither the tight-binding inhibitor UC781 nor nevirapine showed any effects on the stability of HIV-1 RT <em>dimers</em>. TSAOe(3)T was unable to destabilize the subunit interactions of the E138K mutant enzyme, which exhibits significant resistance to TSAOe(3)T inhibition. Molecular modeling of TSAOm(3)T into the nonnucleoside inhibitor binding pocket of wild-type RT suggests that it makes significant interactions with the p51 subunit of the enzyme, a feature that has not been observed with other types of nonnucleoside inhibitors. The observed destabilization of the <em>dimer</em>ic HIV-1 RT may result from structural/conformational perturbations at the reverse transcriptase subunit interface.

Although coagulopathy is known to be the major contributor to a poor outcome of traumatic brain injury (TBI), the mechanisms that trigger coagulation abnormalities have not been studied in detail. We undertook a prospective observational study at a neurosurgical ICU (NICU) in a university hospital. We examined 11 patients with severe isolated TBI, at admittance to the hospital and during the next 3 days. We collected cerebrovenous blood samples from a jugular bulb catheter, arterial blood, and cerebrospinal fluid (CSF) samples. We measured concentrations of thrombin-antithrombin complex (TAT), fibrin <em>D</em>-<em>dimer</em> (<em>D</em><em>D</em>), prothrombin fragment 1 + <em>2</em> (F1 + <em>2</em>), interleukin-6 (IL-6), and complement complex (C5b-9). All patients had some degree of consumption coagulopathy at the study start and a tendency to thrombocytopenia during the next few days. Levels of <em>D</em><em>D</em> (3.6 +/- <em>2</em>.7 mg/L), TAT (86 +/- 7<em>2</em> microg/L) and F1 + <em>2</em> (5.9 +/- 6.8 nmol/L) were significantly increased shortly after the trauma compared to reference values, with considerable transcranial gradients for TAT (49 microg/L) and F1 + <em>2</em> (3.<em>2</em> nmol/L). Compared to controls, IL-6 levels were increased more than a hundredfold in both blood (<em>2</em>83 +/- 19<em>2</em> ng/L) and CSF (4<em>2</em>4 +/- 355 ng/L) samples, with a transcranial gradient at the study start (107 ng/L). C5b-9 levels were moderately increased in blood samples, <em>2</em>70 +/- 114 microg/L, versus controls, 184 +/- 39 (p < 0.05). We conclude that activation of the coagulation system takes place during the passage of blood through the damaged brain, and is already evident hours after the trauma. IL-6 and activation of the complement system (C5b-9) co-vary with hemostatic parameters in TBI patients.

The structure of Kaposi's sarcoma-associate<em>d</em> herpesvirus protease (KSHV Pr), at <em>2</em>.<em>2</em> A resolution, reveals the active-site geometry an<em>d</em> <em>d</em>efines multiple possible target sites for <em>d</em>rug <em>d</em>esign against a human cancer-pro<em>d</em>ucing virus. The catalytic tria<em>d</em> of KSHV Pr, (Ser114, His46, an<em>d</em> His157) an<em>d</em> transition-state stabilization site are arrange<em>d</em> as in other structurally characterize<em>d</em> herpesviral proteases. The <em>d</em>istal histi<em>d</em>ine-histi<em>d</em>ine hy<em>d</em>rogen bon<em>d</em> is solvent accessible, unlike the situation in other classes of serine proteases. As in all herpesviral proteases, the enzyme is active only as a weakly associate<em>d</em> <em>dimer</em> (K(<em>d</em>) approximately <em>2</em> microM), an<em>d</em> inactive as a monomer. Therefore, both the active site an<em>d</em> <em>dimer</em> interface are potential targets for antiviral <em>d</em>rug <em>d</em>esign. The <em>dimer</em> interface in KSHV Pr is compare<em>d</em> with the interface of other herpesviral proteases. Two conserve<em>d</em> arginines (Arg<em>2</em>09), one from each monomer, are burie<em>d</em> within the same region of the <em>dimer</em> interface. We propose that this conserve<em>d</em> arginine may provi<em>d</em>e a <em>d</em>estabilizing element contributing to the tune<em>d</em> micromolar <em>d</em>issociation of herpesviral protease <em>dimers</em>.

The purpose of this prospective study was to evaluate the safety of salvage and reinfusion of postoperative sanguineous wound drainage using the ConstaVac Blood Conservation System (Stryker, Kalamazoo, MI). A prospective analysis of 135 primary total hip and total knee arthroplasties was carried out. The collection time for reinfusion was limited to 6 hours, and suction pressure was kept to a minimum by using the lowest setting on the device. For all patients, no citrate-phosphate-dextrose anticoagulant was added to the reservoir. To evaluate the effect of reinfusion on hemostasis and the blood coagulation system, antithrombin III, fibrinogen, and <em>D</em>-<em>dimer</em> levels of 40 of 135 patients were measured before surgery and on the first and seventh days after the operation. The mean volume of reinfusion of postoperative drainage was 437 mL for the patients with total hip arthroplasties, 883 mL for those with total knee arthroplasties, and 1,713 mL for those with bilateral total knee arthroplasties. Ninety-nine of 135 patients underwent operations without homologous blood replacement. Transient chills with mild fever were seen in <em>2</em> patients during reinfusion. No complications related to air embolism, coagulopathy, renal failure, or sepsis were recognized in any of the patients. This study suggests that postoperative blood salvage and reinfusion appear to be safe and effective in patients undergoing primary total hip and knee arthroplasties.

We assessed the predictive value of <em>D</em>-<em>dimer</em> levels in combination with residual venous obstruction (RVO) for recurrent venous thromboembolism (VTE) in a prospective cohort of outpatients after oral anticoagulant therapy (OAT) suspension for a first episode of idiopathic proximal deep vein thrombosis of the lower limbs during a <em>2</em>-year follow-up. Patients (n=400) were enrolled on the day of OAT suspension when RVO was determined by compression ultrasonography (present in 48.6% of patients). <em>D</em>-<em>dimer</em> (cut-off value: 500 ng/mL) was measured 30+/-10 days afterwards (abnormal in 56.4% of patients). The overall recurrence rate was 16.7% (67/400; 95% confidence intervals - CI -: 13-<em>2</em>1 %). The multivariate hazard ratio (HR) for recurrence was 3.3<em>2</em> (95% CI: 1.78-6.75; p<0.0001) for abnormal <em>D</em>-<em>dimer</em> compared to normal <em>D</em>-<em>dimer</em> and 1.<em>2</em> (95% CI:0.7<em>2</em>-<em>2</em>.07; p>0.05) for RVO compared to absent RVO. The recurrence rate was 5.7% (95% CI:<em>2</em>-13%) and 10.4% (95% CI:6-18%), respectively, for normal <em>D</em>-<em>dimer</em> either without or with RVO, <em>2</em><em>2</em>.9% (95% CI: 14-33%) and <em>2</em>5.9% (95% CI: 18-35%), respectively, for abnormal <em>D</em>-<em>dimer</em>, either without or with RVO. When compared with normal <em>D</em>-<em>dimer</em> without RVO, the multivariate HR for recurrence was similar for abnormal <em>D</em>-<em>dimer</em> either with RVO (4.76 - 95% CI:1.78-1<em>2</em>.8) or without RVO (4.3-95%:1.56-11.88). Abnormal <em>D</em>-<em>dimer</em> at one month after OAT withdrawal is an independent risk factor for recurrent VTE, while RVO at the time of OAT withdrawal, either with normal or abnormal <em>D</em>-<em>dimer</em> after one month, does not influence the risk of recurrence.

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(<em>2</em>+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(<em>2</em>+) and Mg(<em>2</em>+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and <em>dimer</em>ization. Mg(<em>2</em>+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(<em>D</em>) = <em>2</em>0 μM). Unmyristoylated VILIP-1 binds two Ca(<em>2</em>+) sequentially at EF<em>2</em> and EF3 (K(EF3) = 0.1 μM and K(EF<em>2</em>) = 1-4 μM), whereas myristoylated VILIP-1 binds two Ca(<em>2</em>+) with lower affinity (K(<em>D</em>) = 1.<em>2</em> μM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(<em>2</em>+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(<em>2</em>+)-dependent chemical shifts and NOE patterns consistent with Ca(<em>2</em>+)-induced extrusion of the myristate. VILIP-1 forms a <em>dimer</em> in solution independent of Ca(<em>2</em>+) and myristoylation. The <em>dimer</em>ization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 <em>dimer</em> and a Ca(<em>2</em>+)-myristoyl switch to provide structural insights into Ca(<em>2</em>+)-induced trafficking of nicotinic acetylcholine receptors.

Thermal unfolding/refolding studies of the three tropomyosin <em>dimers</em>, alpha alpha, alpha beta, and beta beta, from chicken gizzard muscle were performed to explain the preferential assembly of alpha- and beta-tropomyosin subunits into hetero<em>dimers</em>, alpha beta [Lehrer, S. S., & Qian, Y. (1989) J. Biol. Chem. <em>2</em>65, 1134]. Circular dichroism measurements showed that all three <em>dimers</em> unfolded in cooperative reversible transitions with T1/<em>2</em> = 40.0 degrees C and <em>delta</em> H degrees = 16<em>2</em> kcal/mol for alpha alpha and with T1/<em>2</em> = 4<em>2</em>.6 degrees C and <em>delta</em> H degree = 98 kcal/mol for beta beta at 0.4-0.5 microM concentrations. Fluorescence measurements on pyrenyliodoacetamide-labeled tropomyosin showed that (i) excimer fluorescence decreases in parallel with unfolding of homo<em>dimers</em>, (ii) at physiological temperature, hetero<em>dimers</em> are formed from micromolar mixtures of homo<em>dimers</em> over a period of minutes, and (iii) hetero<em>dimers</em> unfold/refold with temperature without appreciable formation of homo<em>dimers</em>. To understand the preferential formation of alpha beta, we calculated the concentrations of all species present as a function of temperature for equal total amounts of alpha and beta, using the measured thermodynamic constants of the unfolding/dissociation equilibria for alpha alpha and beta beta. Values for <em>delta</em> H degrees = <em>2</em><em>2</em>5 kcal/mol and T1/<em>2</em> = 43 degrees C for unfolding of alpha beta at 0.5 microM concentration were obtained from the best fit of the calculations to the measured helical content vs temperature of alpha beta.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-<em>2</em>, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin <em>dimer</em> pool and part of the remaining tau and MAP-<em>2</em>. When the remaining cytoskeletal pellet was treated with cytochalasin <em>D</em> to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.

The xeroderma pigmentosum group A complementing protein (XPA) and eukaryotic replication protein A (RPA) are among the major damage-recognition proteins involved in the early stage of nucleotide excision repair (NER). XPA and RPA are able to bind damaged <em>D</em>NA independently, although RPA interaction stimulates XPA binding to damaged <em>D</em>NA [Li, L., Lu, X., Peterson, C. A., and Legerski, R. J. (1995) Mol. Cell. Biol. 15, 5396-540<em>2</em> (1); Stigger, E., <em>D</em>rissi, R., and Lee, S.-H. (1998) J. Biol. Chem. <em>2</em>73, 9337-9343 (<em>2</em>)]. In this study, we used surface plasmon resonance (SPR) analysis to investigate the interaction of XPA and RPA with two major types of UV-damaged <em>D</em>NA: the (6-4) photoproduct and the cis-syn cyclobutane <em>dimer</em> of thymidine. Both XPA and RPA preferentially bind to (6-4) photoproduct-containing duplex <em>D</em>NA over cis-syn cyclobutane <em>dimer</em>-containing <em>D</em>NA. The binding of XPA to (6-4) photoproduct was weak (K(<em>D</em>) = <em>2</em>.13 x 10(-)(8) M), whereas RPA showed a very stable interaction with (6-4) photoproduct (K(<em>D</em>) = <em>2</em>. 0<em>2</em> x 10(-)(10) M). When XPA and RPA were incubated together, the stability of the XPA-damaged <em>D</em>NA interaction was significantly enhanced by wild-type RPA. On the other hand, mutant RPA (RPA:p34<em>D</em>elta33C) defective in its interaction with XPA failed to stabilize XPA-damaged <em>D</em>NA complex. Taken together, our results suggest that a role for RPA in UV-damage recognition is to stabilize XPA-damaged <em>D</em>NA complex through protein-protein interaction.

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene (phaZ) was identified near the PHA synthase gene (phaC) of P. denitrificans. Cell extract of Escherichia coli carrying lacZ--phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly <em>D</em>(--)-3-hydroxybutyrate (3HB) <em>dimer</em> and 3HB oligomer. <em>D</em>iisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg(<em>2</em>+) ion inhibited PHB degradation. However, the inhibitory effect by Mg(<em>2</em>+) ion was not observed using the cell extract of P. denitrificans.

It has been previously demonstrated that hyperglycaemia activates haemostasis; diabetes mellitus is considered a thrombosis-prone state. Acarbose, by inhibiting dietary carbohydrate absorption, reduces post-meal hyperglycaemia. In this study we evaluated the effect of post-meal hyperglycaemia on two markers of coagulation activation: prothrombin fragments 1 + <em>2</em> and <em>D</em>-<em>dimer</em>. Seventeen non-insulin-dependent diabetic patients maintained on diet therapy alone were randomly assigned to receive- with a cross-over study design-acarbose (100 mg orally) or placebo before a standard meal. Blood samples for measurement of plasma glucose, insulin, prothrombin fragments 1 + <em>2</em> and <em>D</em>-<em>dimer</em> were drawn at 0, 60, 1<em>2</em>0 and <em>2</em>40 min. After both placebo and acarbose, hyperglycaemia and hyperinsulinaemia which followed a standard meal were accompanied by a significant increase of plasma concentration of prothrombin fragments 1 + <em>2</em> and <em>D</em>-<em>dimer</em> in comparison to their baseline values. Acarbose administration significantly reduced the rise of glucose, insulin, prothrombin fragments 1 + <em>2</em> and <em>D</em>-<em>dimer</em> from 0 to <em>2</em>40 min in comparison to placebo. We conclude that post-meal hyperglycaemia, at the level reached by many diabetic patients on diet therapy alone, induces a coagulation activation. Acarbose, by decreasing post-meal hyperglycaemia, may be useful in reducing meal-induced activation of haemostasis in diabetic patients.

3-Acetami<em>d</em>o-3,6-<em>d</em>i<em>d</em>eoxy-alpha-<em>d</em>-glucose or Quip3NAc is an unusual <em>d</em>eoxyamino sugar foun<em>d</em> in the O-antigens of some Gram-negative bacteria an<em>d</em> in the S-layers of Gram-positive bacteria. It is synthesize<em>d</em> in these organisms as a <em>d</em>TDP-linke<em>d</em> sugar via the action of five enzymes. The focus of this investigation is on Q<em>d</em>tB from Thermoanaerobacterium thermosaccharolyticum E<em>2</em>07-71, a PLP-<em>d</em>epen<em>d</em>ent aminotransferase that catalyzes the penultimate step in the pro<em>d</em>uction of <em>d</em>TDP-Quip3NAc. For this analysis, the enzyme was crystallize<em>d</em> in the presence of its pro<em>d</em>uct, <em>d</em>TDP-Quip3N, an<em>d</em> the structure was solve<em>d</em> an<em>d</em> refine<em>d</em> to <em>2</em>.15 A resolution. Q<em>d</em>tB is a <em>dimer</em>, an<em>d</em> its overall fol<em>d</em> places it into the well-characterize<em>d</em> aspartate aminotransferase superfamily. Electron <em>d</em>ensity correspon<em>d</em>ing to the boun<em>d</em> pro<em>d</em>uct reveals the presence of a Schiff base between C-4' of the PLP cofactor an<em>d</em> the amino nitrogen of the sugar. Those amino aci<em>d</em> si<em>d</em>e chains involve<em>d</em> in bin<em>d</em>ing the <em>d</em>TDP-sugar into the active site inclu<em>d</em>e Tyr 183, His 309, an<em>d</em> Tyr 310 from subunit 1 an<em>d</em> Lys <em>2</em>19 from subunit <em>2</em>. Notably there is a <em>d</em>eci<em>d</em>e<em>d</em> lack of interactions between the pyranosyl C-4' hy<em>d</em>roxyl of the <em>d</em>TDP-sugar an<em>d</em> the protein. In keeping with this observation, we show that Q<em>d</em>tB can also turn over <em>d</em>TDP-3-acetami<em>d</em>o-3,6-<em>d</em>i<em>d</em>eoxy-alpha-<em>d</em>-galactose. This investigation represents the first structural analysis of a sugar-mo<em>d</em>ifying aminotransferase with a boun<em>d</em> pro<em>d</em>uct in its active site that functions at the C-3' rather than the C-4' position of the hexose.